Citation
Proto-oncogene expression in human chondrosarcoma and malignant fibrous histiocytoma

Material Information

Title:
Proto-oncogene expression in human chondrosarcoma and malignant fibrous histiocytoma
Creator:
Gibson, Jane Carolyn Strandberg, 1962-
Publication Date:
Language:
English
Physical Description:
ix, 182 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Actins ( jstor )
Cell lines ( jstor )
Cells ( jstor )
Chondrosarcoma ( jstor )
DNA ( jstor )
Genomics ( jstor )
myc genes ( jstor )
Oncogenes ( jstor )
RNA ( jstor )
Tumors ( jstor )
Chondrosarcoma ( mesh )
Dermatofibroma ( mesh )
Dissertations, Academic -- Pathology -- UF ( mesh )
Pathology thesis Ph.D ( mesh )
Proto-Oncogenes ( mesh )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 160-181).
Additional Physical Form:
Also available online.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jane Carolyn Strandberg Gibson.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
024265279 ( ALEPH )
20896329 ( OCLC )

Downloads

This item has the following downloads:


Full Text
















PROTO-ONCOGENE EXPRESSION IN HUMAN
CHONDROSARCOMA AND MALIGNANT FIBROUS HISTIOCYTOMA












By


JANE CAROLYN STRANDBERG GIBSON


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSTIY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1989




























Copyright 1989

by

Jane Carolyn Strandberg Gibson
























To my husband Ron, to my grandparents, to Karen and

Ken, and to Mom and Dad; whose love, support, and belief in

me will always be my inspiration.










ACKNOWLEDGEMENTS


I would like to thank the members of my committee, Dr.

Byron Croker, Dr. Warren Ross, Dr. Lindsey Hutt-Fletcher, Dr.

Linda Smith, and Dr. Harry Ostrer, for their assistance and

advice. I would especially like to thank Dr. Croker for his

faith in my abilities, his guidance, encouragement, and

friendship.

I would also like to thank Dr. Susan Chrysogelos for all

her help during the past 18 months. I am most appreciative

of all she has taught me. Special thanks go to Dr. Cheryl

Zack and Herb Houck for all their help and encouragement, to

Jerry Phipps for his assistance in obtaining surgical

specimens, and to my fellow graduate students for their help

and encouragement. In addition, I am most grateful to my

friends who have been very supportive, particularly Gail

Waldman, Patty Leginus, and Patty DeHaan.

Lastly, I would like to thank my husband Ron, my

grandparents, Karen, and Ken for all their much needed love,

support and encouragement. I have no words which could

adequately express my gratitude to my Mom and Dad. Without

them none of this would have ever been possible. They have

taught me so many things, but I think the most important

lesson I have learned from them is that family is one of

life's greatest treasures. I will never forget that. I will

never forget everything they have done for me.

iv













TABLE OF CONTENTS


page

ACKNOWLEDGEMENTS ....................................... iv

ABSTRACT ............................ ................... vii

CHAPTERS

1 INTRODUCTION........................................ 1

Mechanisms of Tumor Development.................... 1
Multistep Carcinogenesis ........................ 7
The Neoplastic Phenotype and Steps of Tumor
Progression.............................. ......... 12
Specific Questions Addressed During the Course
of This Project................................. 17

2 REVIEW OF THE LITERATURE ........................ 20

Differentiation of Mesenchyme ................... 20
Chondrosarcoma and Malignant Fibrous Histio-
cytoma ........................................ 22
Proto-Oncogenes................................... 29
Biochemistry of Oncogene Products................ 34
Cytoplasmic Kinases ........ .................... .. 34
Ras Proteins.................................... 38
Growth Factors and Their Receptors............. 40
Nuclear Proteins............................... 42
Molecular, Biological, and Physiological Char-
acteristics of Proto-Oncogenes Examined in
This Study................................... 44
Growth Factor Related ......................... 44
Protein Kinases................................ 47
Nuclear Related Proto-Oncogenes................ 50
Chromatin Structure Analysis of the C-myc
Gene .......................................... 59
Relevance To This Project ....................... 66

3 MATERIALS AND METHODS ........................... 67

Slot-Blotting of RNA and DNA .................... 67
Preparation of Total Cellular RNA.............. 67
Preparation of Genomic DNA .................... 68
RNA Slot-Blotting.............................. 69
DNA Slot-Blotting.............................. 70













Preparation of Radiolabeled Probes............... 71
Nick Translated Probes ............... ......... 71
Probes Labeled by Random Primer Extension..... 77
Hybridization of Slot-Blots ................... 77
Southern Blot Analysis .......................... 78
Northern Blot Analysis ......................... 79
Chromatin Structure Analysis .................... 80
Cell Lines Used in Chromatin Structure
Analysis............ ............................. 80
Preparation of Nuclei ......................... 81
Isolation of Genomic DNA From DNAse I Treated
Nuclei........................ ................... 82
Chromatin Structure/Fibroblast Cell Synchrony
Experiment..................... ................. 83
Strategy for Fine Mapping DNAse I Hyper-
sensitive Sites 3' to C-myc Exon 1........... 85
Polyacrylamide Gel Electrophoresis (PAGE) ....... 88
Western Blotting and Immunoperoxidase Assay..... 89

4 RESULTS .......................... ............... 91

Quantitation of Moderately Degraded RNA Using
the Slot-Blotting Technique.................... 91
RNA Slot-Blot Results ........................... 96
DNA Slot-Blot Results and Determination of C-myc
Gene Copy Number......................... ........ 109
Regions Contained in the C-myc Amplicon.......... 123
Chromatin Structure Analyses .................... 125
C-myc Gene Copy Number and Transcript Levels
in P3C, UR HCL 1, HFF and ST 486 Cell
Lines.......................... ........ .... 125
Locations of DNAse I Hypersensitve Sites in
P3C, UR HCL 1, HFF, and ST 486 Cell Lines... 126
Chromatin Structure of the C-myc Gene During
the GO/G1 Transition in the HFF Normal Human
Fibroblast Cell Line ...................... 134
Fine Mapping Analysis of DNAse I Hyper-
sensitive Sites in P3C Cells From a 5'
Direction..................................... 139
C-myc Protein Levels in the P3C, UR HCL 1, HFF
and ST 486 Cell Lines ....................... 143

5 DISCUSSION........................................... 146

REFERENCES ............................................. 160

BIOGRAPHICAL SKETCH...................................... 182



















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

PROTO-ONCOGENE EXPRESSION IN HUMAN
CHONDROSARCOMA AND MALIGNANT FIBROUS HISTIOCYTOMA

By

Jane Carolyn Strandberg Gibson

May, 1989

Chairman: Dr. Byron P. Croker
Major Department: Pathology and Laboratory Medicine

Total cellular RNA and genomic DNA were extracted from

20 chondrosarcomas, 23 malignant fibrous histiocytomas (MFH),

9 muscle, and 6 bone marrow specimens. Levels of RNA and

gene copy numbers of c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-

1, v-src, thymidine kinase (TK) and actin were quantified

densitometrically from slot-blot analysis. C-myc, c-Ha-ras,

and c-fos transcript levels are undetectable in muscle. Mean

c-myc:TK ratios do not differ significantly among groups of

bone marrows, chondrosarcomas and 17 MFHs (p 0.05). Six

MFHs have a mean c-myc:TK ratio of 2.0 which is significantly

higher than the other groups (p 0.05). Intergroup

comparisons between bone marrows, chondrosarcomas, and


vii














MFHs show no significant differences in expression of c-Ha-

ras, c-fos, v-erb-B-1, and v-src. C-sis RNA levels are 2-to

3- fold greater in MFHs. DNA analysis shows c-myc to be a

single copy gene in all tissues except 6 MFHs which have

between 2 and 11 copies. C-myc amplicons were found to be

large, extending at least 50 kb 5' to the c-myc promoter and

slightly 3' of exon 3. These same tumors have increased

levels of c-myc transcript as determined from RNA analysis.

C-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-src are single copy

genes in all tissues.

Chromatin structure studies show that amplified c-myc

in P3C cells does not contain a DNAse I hypersensitive site

near the PO promoter region 5' to exon 1, known to be

involved in maintaining c-myc transcript production in HL-

60 cells. Additionally, a new site is present in a region

known to contain a block to transcription elongation in

Burkitt lymphomas. These changes are not seen during normal

upregulation of c-myc in GO serum released fibroblasts (GO/G1

transition).

These data suggest that increased levels of c-myc

expression are due to gene dosage, while those of c-sis


viii















are due to some unknown mechanism other than gene

amplification. Additionally, differences in chromatin

structure between amplified and single copy c-myc in

MFH cells may represent a compensatory response to increased

c-myc transcript production. Increased levels of c-myc

protein provide further evidence that c-myc may be an

oncogene in these cells.


















CHAPTER 1
INTRODUCTION


Mechanisms of Tumor Development


Fundamental requirements for successful prevention and

sometimes treatment of cancer are knowledge and understanding

of its causitive factors. This task is not an easy one by

any means. Agents having abilities to contribute to or

cause cancer are called carcinogens. Studies to determine

the roles these agents play in neoplastic processes have

focused on three general classes of carcinogens: chemical,

physical, and biological.

Carcinogenic chemicals and ionizing radiation are known

to affect DNA at a structural level and to be mutagenic

under certain conditions. Therefore, one of the long-

standing theories of carcinogenesis has been that cancer is

caused by genetic mutations.

Evidence that chemicals can induce cancer has been

reported for more than two centuries. The first observations

of chemically induced cancer were made in humans. The first

of these was in 1761 when Hill noticed that nasal cancer was














more common in people who frequently used snuff (117). In

1775, Pott reported a high incidence of scrotal cancer in

men who were chimney sweeps (117). More discoveries of this

nature were made in subsequent years, leading to attempts to

induce cancer in animals with chemicals. One of the first

successful attempts was made in 1915 by Yamagiwa and Ichikawa

who induced skin carcinomas by the repeated application of

coal tar to the ears of rabbits (117). Subsequent studies

focused on identifying the actual carcinogenic chemicals in

the compounds which could induce cancer. Today, the list of

known carcinogenic chemicals is quite extensive and includes

a wide variety of different chemicals. Examples of

carcinogenic substances include industrial chemicals such as

aromatic hydrocarbons, halogenated hydrocarbons,

nitrosamines, intercalating agents, alkylating agents, nickel

and chromium compounds, asbestos, vinyl chloride,

diethylstilbesterol, and certain naturally occurring

substances such as aflatoxins and radon gas.

Chemical carcinogens are capable of interacting with a

wide variety of cellular macromolecules. This usually

involves the alkylation of nucleophilic groups on nucleic

acids or the reaction of electrophilic groups of the

carcinogen with proteins (142). Some chemical carcinogens














are known to react with cellular RNA as in the case of

dimethylnitrosamine (105), but most react with DNA (142).

Reaction of chemical carcinogens with DNA can facilitate the

induction of heritable changes in cells and may lead to

malignant transformation. Thus, it is generally believed

that this is the most likely mechanism for chemical

initiation of carcinogenesis. Representative agents from

virtually all classes of chemical carcinogens have been shown

to affect DNA in some way. The actions of many of these have

been found to result in the formation of base-adducts. The

potential biological consequences of these are are several:

Base adducts may stabilize intercalation reactions. For

example, if the flat planar rings of a polycyclic hydrocarbon

were stably integrated between the stacked bases of double

helical DNA, the helix would be distorted. This could lead

to a frame-shift mutation which would occur during DNA

replication past the point of intercalation (86).

Many of the base adducts formed by carcinogens involve

modification of N-3 or N-7 positions on purines. This

induces an instability in the glycosidic bond between the

purine base and deoxyribose. The destabilized structure can

then undergo cleavage by DNA glycosylase, resulting in loss

of the base, and creation of an apurinic site in the DNA.














This open space can then be filled by any base, resulting in

a base transition (purine-pyrimidine base change) (110).

Interaction with some carcinogens has been shown to

favor a conformational transition of DNA from its usual

double-helical B form to a Z DNA form (125). This could

alter the ability of certain genes to be transcribed, since

B-Z conformational transitions are thought to be involved in

regulating chromatin structure (142).

Both X-rays and ultraviolet radiation also produce

damage to DNA. As with chemical carcinogens, this damage

induces DNA repair processes, some of which are error prone

and lead to mutations. Studies have shown that the

development of malignant transformations in cultured cells

after irradiation requires fixation of the initial damage

into a heritable change. This is experimentally accomplished

by allowing clonal proliferation and expression of the

transformed phenotypes (109).

In addition to chemical and physical carcinogens,

biological carcinogens exist as well. It was long suspected

that various forms of cancer, particularly certain lymphomas

and leukemias, were caused or at least cocaused by

transmissable viruses. The known carcinogenic effects of

certain chemicals, irradiation, chronic irritation and














hormones did not fit with the notion of an infectious origin

of cancer. Early studies attempted to transmit malignant

disease by inoculation of filtered extracts prepared from

diseased tissues. It was later demonstrated by Ellerman and

Bang in 1908 that chicken leukemia could be transmitted by

cell-free filtered extracts (50). They were among the first

to demonstrate the viral etiology of this disease. An example

of a virus thought to cause cancer in humans is seen with

Human T-cell leukemia virus 1 (HTLV-1), a transmissible

virus thought to cause leukemia (133).

Other oncogenic RNA viruses are capable of participating

in transformation. Understanding of the molecular mechanisms

involved in cellular transformation by these viruses is based

on the Nobel prize winning work of Baltimore and Temin (5,

173). In the early 1960s, Temin demonstrated that mutations

in the Rous sarcoma virus (RSV) genome of RSV-infected

chicken cells could be induced at a high rate. It was also

shown that mutation of an RSV gene present in an infected

cell often changed the morphology of the cell, and the virus

genome was stably inherited by progeny cells (173). This led

to the notion that virus genetic information was contained in

a regularly inherited structure of the host cell as a

"provirus" integrated into the host cell's genome. The















problem with the provirus hypothesis was that there was no

known way for the tumor virus RNA to be converted into DNA

and integrated. Temin and Baltimore independently

demonstrated the presence of a virus coded, RNA directed, DNA

polymerase activity now known as reverse transcriptase (4,

174). As a result of this work, Temin proposed the

"protovirus" theory in which he postulated that genomes of

oncogenic viruses arose during evolution from normal

cellular DNA altered by some exogeneous carcinogen (173).

The normal cellular homologues of viral oncogenes (v-

onc) are known as proto-oncogenes (c-onc). These are thought

to have been evolutionarily conserved in the genomes of most

animal cells over a long period of time. They seem to be

involved in control of cellular growth and proliferation. It

is likely that their activations to oncogenic states occur

from one or more rare events such as translocations,

amplifications, point mutations or other aberrations of key

nucleotide sequences (102). Highly oncogenic viruses

presumably arose from genetic recombination events between

viruses of low oncogenicity and proto-oncogenes. The

combination of these two elements seems to have produced

highly transforming viral genomes. Many of these viruses are

replication defective, and do not form complete viruses














unless coinfected with a "helper" virus. Recombination

between replication-competent helper viruses and cellular

genes also may have produced highly oncogenic virus strains.

The "oncogene" hypothesis of Huebner and Todaro (88)

postulates that the cells of most or all vertebrates contain

"virogenes". These genes include sequences responsible for

transformation and are transmitted vertically form parent to

offspring. In this hypothesis, the occurrence of cancer may

be determined by the derepression of endogenous viral

oncogenes. Activation of repressed genes could result from

exposure of cells to chemical carcinogens, irradiation,

normal aging, or a combination thereof. This theory provides

an explanation for the known vertical transmission of certain

animal viruses. It also explains the observed necessity of

synergistic interactions between chemical carcinogens and

irradiation for transformation by some oncogenic viruses.



Multistep Carcinoqenesis



The idea that development of cancer is a multistage

process arose from early studies of virus induced tumors, and

from discovery of cocarcinogenic effects of croton oil.

Rous discovered that certain virus induced skin papillomas in














rabbits regressed after a period of time. The papillomas

could be made to reappear if the skin was stressed by

punching holes in it or treating it with irritants such as

turpentine or chloroform (142). These experiments led to

conclusions that tumor cells could exist in a latent or

dormant state, and tumor induction processes and subsequent

growth of the tumor involved different mechanisms. These

mechanisms are known as initiation and promotion (62).

Studies of events involved in the initiation and

promotion phases of carcinogenesis were greatly aided by

isolation and identification of initiating agents such as

urethane, and the purification of the components of croton

oil which had promoting activities. The promoting substances

were found to be diesters of the ditepene alcohol; phorbol

(84). Of these, 12-O-tetradecanoylphorbol-13-acetate (TPA)

is the most potent promoter (11).

Initiation of transformation in normal cells by a

carcinogenic agent involves a permanent, heritable change in

gene expression. This could occur by direct genotoxic or

mutational events, where the carcinogenic agent reacts with

DNA directly. It may also occur via indirect "epigenetic"

events which regulate gene expression without direct

interaction with DNA sequences. Many feel initiating events















have a direct impact on DNA itself. According to Ruddon

(142), this theory depends on three kinds of evidence.

1. Agents which damage DNA are frequently carcinogenic.

It has been shown that chemical carcinogens are usually

activated to generate electrophilic agents. These form

specific reaction products with DNA. In some cases, as with

alkyl 0-6-guanine, the extent of product formation has been

shown to correlate with mutagenicity and carcinogenicity of

the agent (142).

2. Most carcinogenic agents are mutagens. A number of

in vitro test systems using mutational events in

microorganisms have been developed to rapidly screen the

mutagenic potential of various chemical agents. One of the

best known is the Ames test. Ames and his colleagues have

shown that about 90 percent of all carcinogens are also

mutagenic (114). Very few noncarcinogens showed significant

mutagenicity in this test system.

3. The incidence of cancer in patients with DNA-repair

deficiencies is increased. In individuals with certain

recessively inherited disorders, the prevalence of cancer is

significantly higher than in the general population (103,

153). The common characteristic shared between these

disorders is the inability to repair some kinds of physical














or chemical damage to DNA. Such examples include, xeroderma

pigmentosum (deficiency in excision repair), ataxia

telangiectasia (greater sensitivity to X-irradiation, more

prone to leukemia and other cancers), Fanconi's syndrome

(deficiency in repair of cross-linked bases, repair of X-ray

or UV induced damage), and Bloom's syndrome (increased

propensity to develop cancer, high genetic instability of

chromosomes). The high incidence of cancer in patients with

these diseases constitutes the best available evidence for a

causal relationship between mutagenicity and carcinogenicity

in humans (168, 169, 185).

Tumor initiating agents most likely interact with DNA to

induce mutations, rearrangements or amplifications, producing

a genotypically altered cell. The initiated cell then

undergoes clonal expansion influenced by promoting agents

which act as mitogens for the transformed cell (142). It has

been suggested that promoting activities may be mediated by

cellular membrane events. Direct action of promoters on DNA

has also been proposed (142). As a result, multiple clones

of cells are likely to be initiated by a DNA damaging agent.

Then, through a rare second event, one or a small number of

these clones progresses to malignant cancer.














Tumor promotion is itself a multistage process

sometimes labeled collectively as "tumor progression". Tumor

promotion is thought to be a stage of cell proliferation and

clonal expansion induced by mitogenic stimuli. The

progression phase is the evolution of genotypically and

phenotypically altered cells resulting from genetic

instability (128). During tumor progression which can take

years in humans, individual tumors develop heterogeneity with

respect to their invasive and metastatic characteristics,

antigenic specificity, state of differentiation, and response

to drugs and hormones (128). It is thought that some major

selection process occurs to favor the growth of one cell

over another, thus a dominant clonal population of cells may

emerge. This may be a result of competition for nutrients,

ability to evade the immune system, and resistance to

chemotherapeutic drugs.

The concepts of initiation and promotion support the

notion that cancer is not a "one-hit" event. Evidence

obtained from studies done with oncogenes and antioncogenes

further supports this concept. Weinberg (102) showed that

when rat endothelial fibroblasts were transfected with the

c-Ha-ras and c-myc oncogenes alone, no transforming effect

was observed. However, when c-myc and c-Ha-ras were














transfected together, multiple foci of transformed cells were

obtained. These cells had the capabilities to grow very

rapidly in culture and seed tumors in nude mice. Acting

together, c-myc and c-Ha-ras could do what neither gene could

do on its own.

Additional evidence for multistep carcinogenesis is seen

with the retinoblastoma (rb) gene. Retinoblastoma is a

childhood ocular tumor which requires both alleles of the rb

gene to be mutated in order for the disease to occur.

Knudson (97) has proposed that the rb gene behaves as an

"anti-oncogene", in that one normal allele is sufficient to

protect against the disease. His "two hit" model for this

disease suggests that two mutagenic events occur at 13q14

of chromosome 13. These two events can be in the form of 2

germline events, 1 germline and 1 somatic, or two somatic.

The rb gene is now thought to have an involvement in other

human malignancies including osteosarcoma (79) and mammary

carcinoma (56,80), and its activation provides an example of

multistep cancer in humans.



The Neoplastic Phenotype and Steps of Tumor Progression



Much effort has gone into comparing phenotypic

characteristics of in vitro transformed cells with those of















cancer in vivo. These types of studies have greatly

increased understanding of cancer cell biochemistry.

Unfortunately, many biochemical characteristics of cultured

cells are dissociable from their abilities to produce tumors

in animals (142). Furthermore, individual cells of

malignant tumors from animals and humans exhibit extensive

biochemical differences. These differences are reflected in

cell surface composition, enzyme levels, immunogenicity,

and response to cancer drugs.

Some general characteristics of transformed malignant

cells growing in culture include the following (142):

1. Histiologic characteristics of malignant cells in

vivo. The nuclei are increased in both size and number,

and there is a great deal of variation in the sizes and

and shapes of the cells. Also, there are increased

nuclear:cytoplasmic ratios, and the formation of

clusters of cells may be observed.

2. Differences in growth characteristics are common:

a. Transformed cells in culture are immortalized.

Malignant transformed cells can be passage in

culture for an indefinite period of time.

b. Transformed cells tend to pile up in culture and are

not subject to contact inhibition seen with















normal cells. As a result, malignant cells in

culture may grow to a much greater density.

c. Transformed cells seem to have much lower require-

ments for serum and/or growth factors to survive

in culture than normal cells do.

d. There also seems to be a loss of anchorage

dependence with transformed cells. They may

no longer need to grow attached to solid surfaces,

and can grow in soft agar.

e. It has been observed that when transformed cells in

culture are subjected to biochemical restrictions,

they do not stop growing. An example of this is a

lack of response to serum starvation.

3. In vitro transformed cells may also change their surface

properties. Changes of this nature include; alteration

in structure of surface glycolipids and glycoproteins,

loss of surface fibronectins, increased agglutination

by lectins, changes in surface antigens which may

be tumor specific and involved in immune responses,

and increases in the degree of amino acid uptake.

4. Cultured malignant cells produce increased levels of the

enzymes involved in DNA synthesis. They also















produce higher levels of other enzymes such as proteases

and collagenases.

5. Different transformed cells have varying levels of

nucleotides. Some may have higher cAMP levels or

increased cGMP:cAMP ratios than their normal cell

counterparts.

6. Transformed cells in culture have been shown to

produce growth factors involved in tumor growth.

These include angiogenesis factors, and transforming

growth factors (TGF). These may be produced to favor

their own growth (autocrine function).

7. Fetal antigens, placental hormones, and fetal

enzymes have been shown to be produced in increased

amounts in cultured tumor cells. This is

characteristic of tumor cells in vivo.

8. Ability to produce tumors in experimental animals is

a characteristic of malignant cells.

In addition to biological and physiological changes in

transformed cells, changes at the molecular level occur

as well. Genetic instability during tumor progression is

characterized by a variety of aberrations in the genome

including point mutations, deletions, rearrangements,

amplifications, chromosome translocations and abnormal














chromosome number (aneuploidy). It is thought that

aberrations such as point mutations, deletions, and

rearrangements are events associated with initiation

processes, whereas gross chromosomal changes occur as the

tumor progresses in malignancy (142). There are certain

chromosomal deletions, translocations and trisomies which are

characteristically associated with specific cancers. These

are called non-random chromosomal alterations. Changes in

ploidy are associated with many tumor types in advanced

stages, and are somewhat random in that no definitive pattern

of chromosome number is associated with a particular tumor

type. In more advanced cancers both random and non-random

chromosomal changes can be found. Continuous chromosomal

changes can bring about tumor heterogeneity and the selection

of more highly invasive and metastatic cancers. Thus, tumor

progression has been called a highly accelerated evolutionary

process.

Malignant tumors have several important in vivo

characteristics. At the cellular level, they have a greater

fraction of cells in S-phase, and are less differentiated

than their normal counterpart tissues. In order for tumor

cells to grow, divide, and metastasize, cell growth must

outnumber cell death. Therefore, angiogenesis factors are














important to the growth of malignant tumors, as rapidly

growing tumors often outgrow their blood supplies. It is

thought that malignant tumor cells may produce their own

growth factors, angiogenesis factors, and collagenases,

enabling them to compete with other cells for nutrients,

and eventually invade surrounding tissues.



Specific Questions Addressed During the Course of This
Project


Unrestrained cell growth is a common component of

neoplastic phenotypes. Proto-oncogenes are genes which have

been shown to be involved in regulation of cellular growth

and differentiation. They are found in all normal nucleated

animal cells. Their conversion to transforming genes or

oncogenes by one or more of several possible mechanisms may

allow the transformation of cells in vitro and generate

neoplasms in vivo. Exploration of how these potential

regulators of growth control interact with one another and

with other genomic components may enlighten our understanding

of how normal cellular replication or differentiation events

change with transformation.

It is possible that several proposed mechanisms of

proto-oncogene activation will lead to increased production














of transcript. Examination of gene copy numbers and gene

expression will offer clues to possible mechanisms involved

in activation of proto-oncogenes. The following was a

general basis for this project:



Normal cellular genes, when mutated by several suggested
mechanisms, may contribute to the tumorigenesis and
biologic behavior of chondrosarcoma and malignant
fibrous histiocytoma.


From this, the following hypotheses were derived:


1) Increases in proto-oncogene transcript levels may
be due to gene amplification.
2) There are differences in chromatin structure
between amplified and single copy proto-oncogenes.

To test the first hypothesis, tumor RNA and DNA samples

were evaluated for proto-oncogene transcript levels and gene

copy numbers of c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and

v-src. These genes were studied because of previous

associations with sarcomas in humans and other animals.

It was desirable to study potential regulatory changes

which accompanied proto-oncogene amplification and increased

transcript production. Therefore, the second hypothesis was

tested by studying the locations of DNAse I hypersensitive

sites. These sites represent areas where regulatory inter-

actions are thought to occur. Changes in locations of these










19



sites may offer clues to regulatory mechanisms involved in

proto-oncogene transcript production.


















CHAPTER 2
REVIEW OF THE LITERATURE



Differentiation of Mesenchyme



The precise pathways taken by mesenchymal cells

undergoing differentiation have been somewhat of a con-

troversial issue. Therefore, two proposed models of mesen-

chymal differentiation will be presented here. The first of

these models, the radial model of mesenchymal

differentiation, is a currently accepted model proposed by

Hadju (76). Each soft tissue and hematopoietic phenotype

directly originates from a primitive undifferentiated

mesenchymal cell. In this scheme, there are no precursor

cells and no branching of cell types. Rather, the

differentiated phenotypes are separated from each other

only by a primitive mesenchymal cell. Because of this,

close relationships exist between phenotypes.

More recently, another model has been proposed by Brooks

(23) and is shown in figure 1. The two novel features of

this model are the insertion of an intermediate precursor

20



























Primitive
Uncommited
Mesenchymal
Cell


Endothelio- ---> Endothelial
blast Cell

Myofibroblast-->Pericyte
/ i'*Smooth Muscle

Primitive /Chondro- __-----Chondrocyte
)rohistioblast" Osteoblast ->Osteocyte

Fibroblast--- Fibrocyte

-Lipoblast > Lipocyte

Schwannoblast -4Schwann Cell

*? ----- ^ Rhabdomyoblast->Rhabdomyocyte


Figure 1. Hypothetical model of mesenchymal differentiation as
proposed by Brooks. *

* Taken from Brooks, J.J. 1986. The Significance of Double
Phenotypic Patterns and Markers in Human Sarcomas. A New
Model of Mesenchymal Differentiation. Am. J. Pathol. 125:
113-123.














between the primitive cell and some differentiated

phenotypes, and a branching system reflecting close

relationships between some phenotypes and not others. This

model also recognizes the myofibroblast and the

chondroosteoblast.



Chondrosarcoma and Malignant Fibrous Histiocytoma



Chondrosarcoma is a malignant tumor of cartilage (figure

2). It has been well established that the basic

proliferating tissue is cartilagenous (39). Primary

chondrosarcomas are tumors which can arise de novo in

extraskeletal tissues or in mixed tumors such as teratomas.

The majority of chondrosarcomas are "myxoid". Those composed

of hyaline cartilage are more uncommon. Secondary

chondrosarcomas arise most commonly in osteosarcomas, and can

sometimes develop in patients with multiple exostoses. Rarely

do they develop from an enchondroma, a benign cartilagenous

tumor. In addition to primary and secondary chondrosarcomas

there are dedifferentiated chondrosarcomas which give rise to

more malignant tumors such as osteosarcomas, fibrosarcomas,

or malignant fibrous histiocytomas (MFH) (39).
























































Figure 2. Histiologic appearance of chondrosarcoma. This
section was taken from a patient with areas of grade I (less
dense cellularity) and grade III tumor. The appearance of
the grade III area closely resembles that of an MFH (shown
below).














Chondrosarcoma is primarily a tumor of adulthood (39).

The incidence of bone tumors in general is highest

during adolescence with a rate of 3 per 100,000 (61). The

incidence falls to 0.2 per 100,000 at ages 30-35 and rises

slowly thereafter to an incidence rate equal to that of

adolescence (30, 39). Chondrosarcoma is the third most

common type of bone tumor and makes up approximately 13

percent of all malignant bone tumors (85). More than 75

percent of chondrosarcomas occur in the trunk and the upper

ends of the femora and humeri. It is much less common for

these tumors to be located in the distal extremeties such as

the elbows and ankles (39).

Many chondrosarcomas are palpable, but many of those

affecting the trunk or long bones of the extremeties which

have not broken the cortex may cause pain alone to indicate

the presence of the lesion. Roentgenograms provide a very

helpful means for diagnosis. Osseous destruction in the area

of the lesion combined with irregular densities from calci-

fication and ossification are commonly observed. Central

chondrosarcomas of long bones commonly produce fusiform

expansion of the shaft associated with thickening of the

cortex (39).














Chondrosarcomas usually have a slow clinical evolution.

Metastasis is relatively rare and occurs late. The basic

therapeutic goal is to control the lesion locally and to

prevent local recurrence. Therefore, radical early

surgical treatment is desirable (39). A long followup after

treatment is necessary because recurrence may develop many

years later. The overall survival is approximately 50

percent at 5 years (180).

With respect to prognosis, the correlation between poor

differentiation, rapid growth rate, and metastasis is high.

Clinical study results suggest that a high cure rate is

expected for patients with more differentiated tumors (135).

A grading system exists for chondrosarcoma and is important

in terms of predicting survival and establishing the most

effective treatment protocol (39).

Criteria for grading chondrosarcomas are those of Evans

et al. (57), and include the following: Grade I tumors have

the presence of or domination of cells with small densely

staining nuclei, an inter-cellular background of a chondroid

or myxoid nature, frequent calcification patches, and

multiple nuclei present within a single lacuna. Grade II

tumor characteristics include; areas where a significant

fraction of the nuclei are of a moderate size, a mitotic














index of 0-2 per 10 high power fields (40X), dense

cellularity, paler staining nuclei, a background which is

more more myxoid than chondroid, and a greater cellularity

/increased nuclear size limited to isolated areas.

The criteria for the classification of a chondrosarcoma

as grade III are a mitotic index greater than 2 mitoses per

10 high power fields (40X), increased nuclear size compared

to those of grade II tumors, very dense cellularity which

may appear MFH like, and the absence of a chondroid or

myxoid background.

Malignant fibrous histiocytomas (MFH) (figure 3) are

soft tissue tumors whose cell of origin has been disputed,

but current evidence indicates that these are are immature

mesenchymal cells (15, 90). Malignant fibrous histiocytomas

usually occur in deeper structures such as deep fascia and

skeletal muscle. They also have been seen in soft tissues of

the extremities, mediastinum, and retroperitoneum, and may

occur within bones in areas of infarction or prior radiation.

As a group, these tumors comprise only about 0.8 percent of

all bone tumors (39), but are somewhat more common in soft

tissues. There seems to be a slightly higher percentage of

males with this disease than females, and nearly any age may

be affected (190). As with other varieties of bone tumors,























































Figure 3. Histiologic appearance of MFH.














pain and swelling are the most frequent symptoms. As with

chondrosarcomas, roentgenographic analysis is helpful in

preliminary diagnosis of the tumor.

Histiologic features of these tumors usually include a

high degree of variation, multinucleated tumor cells,

nuclear hyperchromasia and a high mitotic activity. A

typical pattern of growth can be described as the arrangement

of tumor cells around a central point, producing radiating

spokes, grouped at right angles to each other (storifiorm

pattern).

With respect to prognosis, subcutaneous tumors generally

have a better prognosis than the more deep seated lesions.

The recurrence of MFHs is said to be 44 percent with a two

year survival of 60 percent (190). There is no widely

accepted grading system for deep seated MFHs as for

chondrosarcomas. Other important prognostic factors include

mitotic index, degree of cellular polymorphism, tumor size

and tumor stage (180).

Staging criteria for both chondrosarcomas and MFHs are

those described by Enneking et al. (52) for sarcomas

originating from mesenchymal tissue of the musculoskeletal

system. This system takes into account surgical grade,

local extent, and presence or absence of regional














metastasis. Grade is further classified as low and high,

local extent as intracompartmental and extracompartmental,

and the extent of regional or distant metastasis is defined

as either present or absent.



Proto-Oncogenes



During the early 1970s it was discovered that a single

gene carried by a retrovirus could cause cancer in animals.

Soon thereafter, it was thought that the oncogenes acquired

by retroviruses might be derived from normal cellular genes

present in the host. It was later shown by Stehlin et al.

(164) that cDNA specific for the v-src region of Rous

Sarcoma Virus could detect closely related sequences in the

genome of normal chicken cells. This gene, now called c-src

has been found in all other vertibrate species including man

(162).

Since the discovery of cellular sequences homologous to

v-src, cellular counterparts (c-onc) for the other viral (v-

onc) oncogenes have also been found (16). These cellular

sequences are known as cellular oncogenes or proto-oncognes.

There are now more than forty known proto-oncogenes which are

expressed in most normal mammalian cells (77). The c-ras and














c-myc genes are transcribed in almost all mammalian cells

(levels may be low at about 5-20 molecules of RNA per cell)

whereas most other proto-oncogenes seem to be more tissue

specific (77). For example, c-myb is expressed in

hematopoietic cells but not elsewhere (191). C-sis RNA has

been detected in very few normal cell types, including

rapidly dividing cells of the human placenta and endothelial

cells (8). Since proto-oncogenes are so conserved between

species, it seems likely that their gene products play an

essential role in normal cellular growth and development.

There are several possible mechanisms by which proto-

oncogenes may be activated to oncogenes. The first of these

mechanisms involves insertional mutagenesis. The over

expression of a proto-oncogene may occur after the

integration of a new promoter. For instance, the c-mos

proto-oncogene of mice which is biologically inactive after

molecular cloning, can be experimentally converted into a

potent oncogene by addition of a strong transcriptional

promoter (18). Another example of this mechanism comes from

similar activation of the c-Ha-ras proto-oncogene of

rats (40). These oncogenes are created by ligation of cloned

DNA segments, and acquire transforming capabilities because

their transcripts are produced at much higher levels than














those afforded by native promoters of the normal proto-

oncogenes. In vivo, the c-myc and c-erb-B-1 proto-oncogenes

present in several avian hematopoietic neoplasias have become

activated after adjacent integration of an avian leukosis

proviral DNA segment. This viral segment provides a strong

transcriptional promoter which replaces indigenous promoters

of these genes (83, 131).

A second mechanism of activation involves overexpression

due to amplification of the proto-oncogene (gene dosage

effects). The c-myc proto-oncogene is amplified 30-50 times

in HL-60 promyelocytic leukemia cells (32), and in a

neuroendocrinal tumor the the colon (1). A c-Ki-ras gene is

amplified 3-5 times in a human colon carcinoma cell line

(115), and 60 fold in an adrenocortical tumor of mice

(149). Human neuroblastomas were found to contain 30-100

copies of the N-myc gene (150). This was later confirmed,

and shown to be associated with patient survival (152). A

human chronic myelogenous leukemia cell line was discovered

to have multiple copies of the c-abl gene (33). In each of

these cases, gene dosage effects are thought to be

responsible for increases in transcript levels and gene

product.














A third mechanism involves enhancer/promoter activity.

Enhancer sequences may increase utilization of

transcriptional promoters to which they become linked. The

affected promoter may be several kilobases away in either 5'

or 3' directions (74). One example of this is the presence

of retrovirus genome fragments downstream from the c-myc gene

in some avian lymphomas (131). Here, the retrovirus elements

appear to act by contributing an enhancer sequence rather

than a promoter. It is entirely possible that point

mutations at key regulatory sites such as promoter regions

rather than coding regions may result in in proto-oncogene

activation. This could facilitate the deregulation of a

proto-oncogene, i.e. one with abnormal transcriptional

control, or one which is inappropriately expressed.

A fourth mechanism involves the c-myc gene in

particular. Work with Burkitt lymphomas has demonstrated the

juxtaposition of the c-myc gene and immunoglobulin genes

following a translocation event. As a result of this

translocation, the c-myc gene loses all or part of its own

regulatory exon and acquires normally unlinked sequences

involved in immunoglobulin production (104). Rearranged c-

myb sequences have been found in certain mouse plasmacytomas














(122) but their detailed structure and mechanism of

activiation remain to be elucidated.

The fifth mechanism centers around structural

alterations in the proto-oncogene and protein product. This

mechanism is well documented in the case of the oncogenic

proteins encoded by the ras genes. It was discovered that in

the case of the oncogene in the T24/EJ human bladder

carcinoma cell line, a point mutation at position 12

converted the c-Ha-ras proto-oncogene to an oncogene. This G

to T transversion causes glycine which is normally the 12th

residue of the encoded 21,000 dalton protein to be replaced

by a valine (170). Another activated version of this gene

encodes an aspartate residue at this position (144).

Studies done with genes of the Ki-ras group also showed

that when the 12th residue was altered in this manner,

oncogenic activation of the c-Ki-ras gene was observed (24).

A slight variation of these results was obtained through the

study of a human lung carcinoma c-Ha-ras oncogene found to

have a mutation at amino acid 61 of the p21 protein (198).

These changes do not seem to affect the levels of expression

of these genes, only the activities of encoded proteins. It

is therefore suggested that the codons specifying residues

12 and 61 represent critical sites which, when mutated, will














often generate oncogenic alleles. It seems that point

mutations elsewhere in the ras proto-oncogenes merely serve

to inactivate the genes instead of converting them to

oncogenes (102).

Finally, the possibility that unknown mechanisms of

activation may be at work must not be overlooked. There may

be mechanisms of activation which have not been determined.

It is possible that new mechanisms may eventually be

implicated in proto-oncogene activation.



Biochemistry of Oncogene Products



Cytoplasmic Kinases



One of the first oncogene proteins of this class to

be recognized and studied was the 60,000 dalton protein of

the v-src gene (pp60 v-src) (89). Other oncogene products

with tyrosine-specific protein kinase activity include yes,

abl, fps, fgr, and ros (6, 77). These proteins are all

located at the inner surface of the cytoplasmic membrane and

a comparison of their amino acid sequences has shown that

they are related to each other (35). A region of

approximately 250 amino acids in pp60 src is responsible for














the kinase activity, and a corresponding domain is found in

other tyrosine kinases with a high degree of amino acid

conservation between them. This kinase domain is also found

in the cytoplasmic cyclic AMP dependent serine protein

kinases in mos and raf, and in serine specific kinases

located in the cytosol (118). A similar sequence domain has

been found in the membrane-bound receptor-like products erb-

B-l, fins, and neu, all of which have tyrosine kinase

activity, indicating a distant evolutionary relationship

between all protein kinases (77).

Originally, it was thought that this activity was

exclusive to oncogenes. However, a protein derived from the

c-src gene was isolated from normal cells and shown to have

tyrosine specific kinase activity (31). Since then, other

membrane-bound cellular proteins with similar activities have

been identified. These have offered clues as to what the

oncogene kinases may be doing. It has been shown that the

receptors for platelet derived growth factor (PDGF) and

insulin-like growth factor (IGF) have a tyrosine-specific

kinase activity (77). It has also been proposed that

tyrosine phosphorylation is an early event in the

transduction of mitogenic signals through the membrane.

Although pp6Osrc resides at the inner surface of the














membrane, and does not posess receptor activity, it probably

does play some role in the early signalling process (35).

Therefore, the presence of a tyrosine kinase encoded by a

viral oncogene might result in a continuous, deregulated

mitogenic signal for cell division.

Just how the cell responds to these signals is presently

unknown. Many attempts have been made to find the cellular

targets for phosphorylation by pp60src and by growth factor

receptors. One effect of pp60src which is thought to be of

importance is that it leads to increased protein

phosphorylation on serine residues (38). The phosphorylation

of the S6 ribosomal protein on a serine residue is thought to

be a critical event in the mitogenic stimulation of normal

quiescent cells. This may occur via a serine kinase

intermediate which might be activated directly or indirectly

by the pp60src tyrosine kinase (38).

Two different biochemical pathways have been shown to be

important in the mitogenic stimulation of cells and a

possible involvement with both has been shown for pp60src.

Both of these pathways involve the generation of second

messengers. The first involves the generation of cyclic AMP

by membrane bound adenylate cyclase, leading to increased














levels of intracellular cyclic AMP. This can lead to the

activation of cytoplasmic cyclic AMP-dependent serine

specific protein kinases. Activation of other serine

specific kinases, particularly protein kinase C may then

occur (59). It is protein kinase C which is thought to play

a central role in the various responses to mitogenic

stimulation. Graziani (66) has shown that tyrosine

phosphorylation of the cyclic AMP dependent protein kinases

by pp60 c-src occurs in transformed cells. Therefore, it is

possible that pp60src interacts with the pathway which

regulates cell proliferation through cyclic AMP and protein

kinase C.

Another pathway in which src may be involved also leads

to the activation of protein kinase C. Full activity of this

protein requires two cofactors; calcium, and diacylglycerol

(127). Both of these can be generated in response to

extracellualr signals such as acetylcholine or PDGF. The

result of the interaction of these molecules with their

receptors is a breakdown of inositol phospholipids located in

the membrane to yield diacylglycerol. This activates protein

kinase C, and inositoltriphosphate which can affect calcium

levels within the cell. Sugimoto et al. (167) showed that

pp60src could phosphorylate inositol phospholipids in vitro














and that in RSV transformed cells there is a buildup of

intermediates in the inositol lipid breakdown pathway. They

postulated that the primary target of pp60src might be lipid

and not protein.

Much remains unknown about the biochemical action of

pp60src and the rest of the tyrosine kinase family.

Phosphorylation of tyrosine seems to be a general phenomenon

for initiating cell division and inappropriate tyrosine

kinase activity could explain the loss of growth control

associated with transformed cells. The phosphorylation of

inositol lipids by at least two of the tyrosine kinases src

and ros is interesting, but the significance in transformed

cells remains to be determined.



Ras Proteins



The 21,000 dalton (p21) proteins of three human cellular

ras genes; Harvey (Ha), Kirsten (Ki), and N-ras are very

closely related in sequence. In the first 150 amino acids

there are a maximum of 14 amino acid differences between the

three proteins. The ras proteins therefore have been highly

conserved throughout evolution, and are thought to play an

essential role in cell growth (77). The ras p21s are














located at the inner surface of the plasma membrane and

although the viral proteins are phosphorylated at amino acid

residue 59 which is a threonine, the human p21s do not have a

threonine at 59, nor are they phosphorylated. The ras genes,

which are cell cycle dependent (94), are activated by point

mutations and therefore the modes of action of normal and

transforming p21s are of interest.

Both transforming and normal cellular p21s bind GTP and

GDP equally and have a GTPase activity (116). However, the

transforming version of p21 hydrolyzes GTP about 10 times

more slowly than the normal proteins (77). The normal ras

roteins are thought to interact with a receptor in response

to an external signal, bind GTP and interact with an as yet

unknown molecule to generate a second messenger (77).

Adenylate cyclase is unlikely to be directly involved

because the G proteins associated with it have different

molecular weights from ras p21 (77). Since transforming p21

has reduced GTPase activity, this could result in abnormally

high levels of the second messenger.

Ras encoded proteins are also regulators of inositol

triphosphate. Some of the proteins involved in the inositol

lipid breakdown pathway are GTP binding proteins and it is

possible ras may be one of these. Calcium has long been














implicated in cell proliferation. The increase in

intracellular calcium which occurs when cells are fertilized

or stimulated by growth factors may depend on the formation

of inositol triphosphate. This can act as a second

messenger to release intracellular stores of calcium. It has

been postulated, and some evidence exists that the activated

ras gene protein which binds but cannot hydrolyze GTP, can

initiate the formation of inositol triphosphate in an

uncontrolled fashion, independent of cellular growth factors

(12).

In order to expand the current understanding of the

functions of ras proteins, it will be necessary to identify

which protein(s) they interact with in the cell. Attempts

which have been made to copreciptiate ras associated proteins

with anti-ras antibodies have been unsuccessful, indicating

either that associations are weak, or they depend on intact

membrane structure (77).



Growth Factors and Their Receptors



Certain oncogene products are known to be transforming

versions of a growth factor and several growth factor

receptors. The erb-B-1 oncogene is a truncated version of the














epidermal growth factor (EGF) receptor gene (46, 82). The

neu (erb-B-2) oncogene, first detected by transfection assays

has homology with erb-B-1 and also encodes a receptor-like

molecule (145). The sis oncogene codes for one subunit of

PDGF (45). Recently, it has been shown that v-fms is derived

from the normal cellular gene encoding the receptor for

colony stimulating factor 1 (CSF-1) (111, 146).

The erb-B-1 oncogene protein is different from the

normal EGF receptor in that the extracellular EGF binding

domain is absent (17). It is possible that this truncated

receptor is in an activated configuration even in the absence

of EGF stimulation. More about the erb-B-1 oncogene product

will be discussed below. It has been predicted that other

known growth factor receptors in addition to those for EGF

and CSF-1 such as those for PDGF could be altered or

inappropriately expressed to yield oncogenic proteins. So

far no spontaneous examples of this have been reported.

Oncogenic changes in a growth factor protein are well

exemplified in the case of the sis oncogene product. The c-

sis protein sequences are homologous to one of the chains of

PDGF, and are normally produced in only a restricted number

of cell types; including bone marrow megakaryocytes (77),

human placental cells, and endothelial cells (8). Receptors














for PDGF have been found mainly on mesenchymal and glial

cells (165). In the case of virally transformed fibroblasts,

the v-sis sequences are fused to the env sequences of the

virus and this allows export of the abnormal PDGF-like

molecule to the membrane.

Abnormal expression of any mitogenic factor such as sis

may make it a possible candidate for a role in oncogenesis,

providing the cells which produce it have the appropriate

receptors. It is thought that high levels of sis expression

cause transformation, presumably by autocrine stimulation via

the PDGF receptor. The sis oncogene protein will be

discussed in further detail below.

Many tumor cells release transforming growth factors

(TGF). One class of these, TGF alpha is closely related in

sequence to EGF and interacts with the EGF receptor (43).

Other evidence suggests that TGF molecules function normally

as necessary mitogens for embryonic development (163, 179).

Inappropriate expression in adult cells could be a step in

transformation.


Nuclear Proteins














The products of five oncogenes; myc, myb, fos, ski (77),

and B-lym (65) are known to be located in the nucleus. The

expression of c-myc, c-fos, and c-myb appears to be dependent

on the proliferative state of the cell (2, 100, 120 175).

Quiescent 3T3 cells for example, have undetectable levels of

c-fos mRNA but within 30 minutes of stimulation by PDGF

(100), the levels are dramatically increased. This is only

transient, and after about 2 hours the high levels disappear

(120). Thus the interaction of PDGF with its receptor not

only facilitates activated intracellular phosphorylation

events and the breakdown of inositol lipids, but also leads

to the generation of a nuclear signal to switch on c-fos

expression. Since phosphorylation of intracellular proteins

occurs within a few minutes of mitogenic stimulation it is

likely that c-fos expression is a direct result of these

events. Like c-fos, the c-myc gene is expressed at very low

levels in quiescent cells, and its transcript levels

increase transiently after stimulation with PDGF, insulin,

and serum (19, 22, 67, 95, 126, 175).

The roles of c-fos and c-myc gene products will be

discussed in more detail below. It will be mentioned for

now that since c-myc and c-fos gene expression follows a

direct relationship to cell cycle, it is generally believed














that their protein products are involved in the regulation of

cell division. Inappropriate expression of these nuclear

proteins could keep the cell cycling even under conditions

which would normally be sufficient to switch off further

growth.



Molecular. Biological, and Physiological Characteristics
of Proto-Oncogenes Examined in This Study


Growth Factor Related



Erb-B-1. As mentioned previously, the product of the

erb-B-1 oncogene is a truncated version of the receptor for

EGF (181). It is a glycoprotein with protein kinase activity

and has the capability to transform cells, while the normal

growth factor and receptor do not (17). The erb-B-1

oncogene protein product represents the EGF receptor short

of both its large extracellular domain which binds the

ligand and either 32 or 71 amino acids from its carboxy

terminus (181). The transforming protein is 71 amino acids

in length and includes a hydrophobic region which resides at

the cell surface, a hydrophobic domain that spans the plasma

membrane, and a shortened cytoplasmic domain which possesses

the protein-tyrosine kinase activity (197). This














truncation could have several possible consequences, any of

which may contribute to neoplastic transformation. For

instance, only a small fraction of the erb-B-1 oncogene

product reaches the plasma membrane (17, 148). The remainder

never leaves the golgi apparatus and retains an immature

mannose-rich form. This is in contrast to the normal

behavior of intact transmembrane receptors.

The EGF receptor is returned to the interior of the

cell after binding ligand, a regulatory mechanism seemingly

designed to protect the cell from an over abundance of

stimuli. By contrast, the product of the erb-B-1 oncogene

cannot bind ligand and may be located permanently to the

surface of the cell (17, 148, 181).

The EGF receptor displays the full force of its protein

kinase activity only after binding ligand. The erb-B-1

oncogene product is presumably released from this dependence

and is constitutively active (17). The kinase activities

associated with the erb-B-1 oncogene product are

constitutive, and the appearance of this protein on the

plasma membrane seems to be a prerequisite for transformation

(14).

With respect to tumor activity, it has been reported

that abnormally high levels of the erb-B-1 oncogene were














found in 40 percent of primary brain tumors of glial origin

(108). Abnormally high copy numbers of the HER-2/neu

(c-erb-B-2) gene have been found in mammary carcinomas.

This in turn has been associated with patient survival and

time to relapse in diseased individuals (158).

Sis. The sis oncogene encodes one of the two subunits

(PDGF 2-B) of platelet derived growth factor (45). Following

synthesis, the 28kd product (p28sis) of the sis oncogene

assembles into a homodimer and is trimmed to a smaller

polypeptide (140).

The product of the sis oncogene may transform cells by

an autocrine function. Evidence exists that some cells

release a homodimer of p28sis, whose structure and activity

resemble those of PDGF (63). Application of antibodies

against PDGF to these cells arrests their growth (87). There

is also reason to suspect that the sis oncogene product, or

that of its cellular progenitor c-sis need not leave the

cell in order to invoke neoplastic growth (13). Instead, the

transforming protein may combine with a receptor while still

inside the cell.

There is also the question of why the sis oncogene

protein can transform cells, while the c-sis protein cannot.

It is not known if there are mutations in oncogenic sis that














alter the capabilities of its product. Also unclear is

whether or not the homodimer produced from it has abnormal

activity compared to the related but different subunits of

PDGF. Whether the formation of homodimer causes PDGF 2-B to

be processed abnormally, or if the sis oncogene product acts

at an anomalous site inside the cell are questions yet to be

answered. It is possible that cells produce factors which

cooperate with sis in neoplastic transformation (17). All of

these issues only obviate the fact that much more needs to be

done before a full understanding of the sis gene and its

product can be obtained.

The presence of the c-sis gene has been demonstrated in

several tumor types. Eva et al. (54) reported that cell

lines from both human sarcomas and gliomas were analyzed for

the presence of sis message. It was found to be at elevated

levels in 5/6 of sarcoma cell lines and 3/5 of glioma cell

lines studied. Sis message has also been found to be at

elevated levels in the metastases of two stomach carcinomas

(172).


Protein Kinases














C-Ha-ras. C-Ha-ras is a member of the ras oncogene

family, and is cell cycle dependant (94). As described

previously, activation of c-ras to an oncogene is

accomplished by point mutations at specific sites which

render its protein product oncogenic to the cell (34, 170).

Mutations of this nature have been found in approximately 15

percent of sarcomas (77). Oncogenes of the ras family may

be active in human carcinoma cell lines, as well as primary

human tumor specimens of several sites such as colon, lung,

gall bladder, urinary bladder, pancreas rhabdomyosarcoma

(42, 136,137), and in prostate cancer (186). These genes are

also present in human hematopoietic neoplasias; including

primary acute myelogenous leukemias, and cell lines derived

from acute lymphocytic leukemias, T cell leukemias and

chronic myelogenous leukemias (55, 161).



Src. The src oncogene as described above codes for a

protein, which like its normal proto-oncogene counterpart,

is a protein-tyrosine kinase. In order to study its

mechanism of activation, c-src has been molecularly cloned

from both chicken and human DNA. Nucleotide sequencing has

revealed the similarities between the protein coding regions

and those of v-src (171). Unlike v-src, c-src is very














complex and contains 11 introns. The exact mechanism by

which RSV acquired genomic c-src information is unclear

(196). It has been proposed that during a round of

infection, a non-oncogenic RSV progenitor transduced genomic

DNA after viral integration and excision, and then the

introns were removed by processing. Also, it may have

somehow incorporated c-src messenger RNA (77).

The differences between c-src and v-src have been

addressed by making use of in vitro recombinants of viral

and cellular genes. Results have shown that some of the

amino acid changes in v-src are biologically important for

transformation. This was also demonstrated by the fact that

high levels of c-src expression alone did not transform

cells (130). It has also been shown that if v-src is

expressed in cells at levels comparable with those of c-src

in normal cells, then transformation is observed (92).

It may be that both qualitative and quantitative changes in

src expression are required for transformation.

Src gene protein (p60src) activity is present in

normal tissues where organ specific levels have been found.

Jacobs et al. (91) have reported that levels of pp60c-src

were highest in brain followed by kidney, lung, muscle, and

connective tissue. It was also determined that a 4-20 fold














increase of pp60c-src kinase activity was present in human

skin tumors compared to normal skin (7).



Nuclear Related Proto-oncoqenes



Fos. The fos gene was first discovered as the oncogene

of two related murine viruses that cause osteogenic sarcoma.

The name fos refers to its origins in the FBJ and FBR

osteogenic sarcoma viruses. The fos oncogene, like other

oncogenes, causes the transformation of cells and is

derived from a normal cellular gene. The cellular and viral

fos genes have an interesting relationship to each other.

The first 332 amino acids of v-fos and murine c-fos differ in

only five positions but the remaining 49 amino acids are

completely different. The 104 bases at the C-terminus of c-

fos are deleted in v-fos, and although this changes the

reading frame and alters subsequent amino acids, the

mobilities of the proteins are similar (v-fos 55 kd, c-fos,

62 kd) (184).

The fos gene seems to serve as a kind of master switch

for turning on other various genes in response to a wide

range of stimuli including growth factors. Fos may act as a

sensor which detects incoming signals at the cellular














membrane and converts them to lasting responses such as cell

division and possibly memory formation (113).

The c-fos gene was recognized as a cell cycle dependent

gene early after studies with it began. C-fos can be rapidly

activated by the treatment of quiescent cells with PDGF, EGF,

nerve cell growth factor, and serum containing growth factors

(69). This led to speculation that c-fos had something to

do with cellular growth control.

Studies with nerve cells revealed additional information

about the activities of the c-fos gene. It was found that

c-fos expression is controlled by factors which differentiate

and trigger nerve cell activity. In vitro experiments have

indicated that c-fos induction depends on the ability of

neuroactive agents to open calcium channels (119). Calcium

entry is a normal component of neuronal responses to

stimulation. It was found that a dramatic increase in c-fos

gene activity occurs in the brains of mice treated with

metrazole, a drug which causes epilepsy like seizures (119).

The synthesis of c-fos proteins was found to occur primarily

in the nerve tracts stimulated by metrazole. The results

suggested that the c-fos protein mediates the long term

adaptation of nerve cells to metrazole stimulation.














There is now evidence for genes which seem to be

directly controlled by c-fos. For example, a set of genes

which code for fat cell proteins which become active when fat

cells differentiate has been identified. One of these genes

adipocyte P2 (aP2), was found to have a regulatory site 125

base pairs upstream from its promoter. This regulatory site

binds proteins which undergo undefined changes during

maturation. It is hypothesized that changes in the binding

proteins mediate activation of the aP2 gene (44).

Experiments were then performed to determine whether

the c-fos protein was one of these regulatory proteins. Data

from immunoprecipitation analyses showed that the binding

complex contains the c-fos protein itself, or at least a very

related protein (44). Further studies need to be done to

clarify this issue.

Site directed mutagenesis studies have been done in the

c-fos promoter region. Various deletions were studied for

effects on the c-fos gene's responses to various stimulatory

agents. It was found that a 22 base pair region located 300

base pairs 5' to the promoter is necessary for enhanced

expression of c-fos in response to serum stimulation (68,

177, 178). This region is called serum response element

(SRE). These same investigators have isolated a protein














which binds specifically to the SRE. The protein appears to

be necessary for c-fos response to serum stimulation,

however proof that the protein directly activates c-fos

remains to be obtained. SRE variants have been constructed,

each with altered protein binding capabilities. It was found

that the ability to stimulate transcription correllates with

avidity of protein binding (68,81).

The SRE is not the only region thought to be important

in c-fos gene regulation. C-fos is activated by several

different stimulatory agents. It is thought that these

stimulatory agents do not all act in the same manner to

affect c-fos expression. The current idea is that there are

multiple regulatory elements for the gene. Epidermal growth

factor and phorbol esters seem to work through the SRE, but

there is evidence that c-fos activation by PDGF may be

mediated through a different site 25 base pairs 5' of the SRE

(64, 177, 178).

Some c-fos stimulating agents use cyclic AMP as a

messenger. Mutations in the SRE do not seem to affect c-fos

activation by cyclic AMP (68, 177). Therefore, gene

activation by cyclic AMP uses other unidentified regulatory

sequences. Calcium ions which mediate c-fos during nerve

cell activation may use yet another site (64, 68, 177, 178).














The different stimulatory agents which appear to use

different regulatory proteins to enhance c-fos expression,

induce different nuclear proteins (64, 177, 178), and these

are called c-fos related antigens (FRA).

C-fos may be subject to negative regulation as well.

Verma et al. (184) have shown evidence to suggest that cells

may have factors which repress fos transcription, but more

needs to be done before this can be fully characterized and

understood.

Both the products of the v-fos (p55/v-fos) and c-fos

(P62/c-fos) genes may be part of nuclear complexes (60,

184). For example, the c-fos protein complex and several

FRAs bind specifically to a sequence element referred to as

the HeLa cell activator protein 1 (AP-1) binding site (60).

Structural studies and immunoprecipitation analyses were

performed with this complex. One of the Fos-associated

proteins, (p39) was found to be the protein product of c-jun

(138).

The p39/jun protein is one of the major polypeptides

identified in AP-1 oligonucleotide affinity chromatography

extracts of cellular proteins. The preparations of AP-1

were found to contain c-fos and several FRAs (20). Some of

these proteins seem to bind to the AP-1 site directly, while

c-fos appears to bind indirectly through protein/protein














interactions (20). Cell surface stimulation results in an

increase in c-fos and c-jun products. The products of the

two genes along with several other related proteins form a

complex which associates with transcriptional control

elements containing AP-1 sites (20, 60, 138). This

potentially can then mediate long term responses which

regulate growth control and development (113).

With respect to tumor activity, there seems to be some

controversy in the literature with regard to what types of

neoplasms are associated with c-fos expression. It has been

reported that c-fos has not been found consistently in any

type of neoplasm (77). However, Slamon et al. have reported

that c-fos is expressed in all tumor types including

carcinomas, sarcomas, and hematopoietic malignancies (159).

Myc. The myc family of cellular proto-oncogenes

contains three well defined members, c-myc, N-myc, and L-myc.

The first defined and most thoroughly studied member of this

family, c-myc, was identified as the cellular homolog to the

transforming gene of avian transforming virus MC29 (17). The

two other well characterized myc family genes N-myc and L-myc

were isolated on the basis of their homology to c-myc and

their frequent amplification in certain classes of human

tumors. The N-myc gene was originally isolated from human














neuroblastomas, a pediatric tumor of embryonal origin that

arises in the peripheral nervous system.

The N-myc and c-myc genes have a very similar overall

structure, exhibit extensive homology in their coding

regions, and encode similar sized nuclear proteins (41, 99).

It has been confirmed that N-myc has transforming activity

equivalent to that of c-myc in the rat embryo fibroblast

assay (150). The N-myc gene has been found to be amplified

in all human neuroblastomas having cytogenetic

characteristics of gene amplification such as homogenously

staining regions or double minutes (147). Patterns of N-myc

amplification in neuroblastomas have been associated with

tumor progression. A greater copy number of the N-myc gene

is associated with a more advanced stage of the tumor.

(152). N-myc activation has thusfar been found to occur only

by amplification and only in a restricted set of tumors.

In addition to neuroblastomas, N-myc amplification has

been observed in a subset of small cell lung carcinomas

(SCLC) and in a few retinoblastomas (98, 106, 124). Like

neuroblastomas, these tumors have neural characteristics.

Considering the oncogenic potentials and similarities of

c-myc and N-myc, the reason for relatively restricted

activation of the N-myc gene as opposed to the c-myc gene is














unknown. The N-myc gene may play a special role in certain

types of neural tumors. Also, N-myc amplification events may

be specially targeted in the precursor cells of these tumors

(2).

The L-myc gene was isolated by two independent methods.

The gene was first isolated on the basis of its amplification

in a subset of SCLCs (123). The gene was independently

isolated from unamplified genomes on the basis of its

homology to c-myc and N-myc (2). So far, activation of the

L-myc gene has only been observed in some SCLCs. Details

with respect to the structure and transforming potential of

L-myc call for further study, and will not be discussed here.

The expression of the c-myc gene has been shown to

follow a fixed relation to cell cycle. Growth arrested

fibroblasts (serum deprivation) in GO show a burst of c-myc

transcription during the GO/G1 transition when stimulated to

divide by either serum addition or insulin. The c-myc

transcript levels then decrease slowly as the cells proceed

through the cell cycle, and are present at basal levels

during S phase (19, 175). Nuclear run on assays with serum

released GO fibroblasts suggest that c-myc expression is

primarily regulated post-transcriptionally, at the level of

message degradation (19).














The c-myc protein product is a double stranded DNA

binding protein thought to interact with other genes, perhaps

those involved in cellular growth control. It is thought

that the myc protein can bind to the regulatory regions of

genes it controls, regulating transcription either by direct

activation or by inhibition of suppression (17).

The precise function the of c-myc protein has not been

elucidated. It is generally thought that its primary

function is to mediate a signals) associated with cell

division and thus, regulation of its expression is required

for normal cell growth (2). Experiments with c-myc antisense

RNA have shown that the ability of cells to divide can be

blocked (192).

The c-myc gene has been found to be present in many

types of sarcomas, carcinomas, and hematopoietic neoplasias

(32, 54, 77, 172). The two most widely studied mechanisms

of oncogenic activation of this gene are translocation

(seen in Burkitt lymphoma), and gene amplification. In the

case of Burkitt lymphoma the c-myc gene is translocated from

chromosome 8 to chromosome 14 or from chromosome 8 to 22. As

a result, the c-myc gene loses all or a portion of its first

exon, and acquires normally unlinked sequences involved in

immunoglobulin gene production (104, 121). C-myc gene















amplification has been found in many tumor types, and cell

lines, including HL-60 (promyelocytic leukemia), and COLO 320

(colon carcinoma) cell lines (2, 32).

The amplified region of the c-myc gene has been closely

studied in HL-60 cells. It has been shown that the amplified

region is very large and contains multiple copies of the

entire c-myc gene. Sequencing data indicates that

amplified c-myc gene units or "amplicons" appear to be

structurally normal (2). High levels of c-myc transcript

have been observed in HL-60 cell lines as well as the COLO

320 line and this has been attributed to gene dosage effects.

As a result of these investigations, it is commonly assumed

that when high levels of c-myc transcript are accompanied by

multiple copies, gene amplification is the cause of increased

expression.



Chromatin Structure Analysis of the C-myc Gene



The chromosomes of eukaryotes replicate, undergo

meiosis and mitosis, recombine, segregate, and are

transcribed. The occurence of these processes is

mediated through the interaction of chromosomal DNA and

proteins (72). In order for these proteins to act, specific














regions of the DNA must be accessible to binding.

Nuclease hypersensitive sites in chromatin are thought to be

regions that are open, and will allow DNA interaction

with proteins (70, 72). Therefore, it is thought that these

regions are specific for regulation of genes by cis and trans

acting factors. These protein accessible regions are

identified by their susceptibility to cleavage with

nuclease, and have been described to be twice as sensitive

as other areas of chromatin (72). DNAse I

hypersensitive sites are thought to represent approximately

1 percent of the entire genome (72). They were first

identified by Varshavsky (182) and by Scott and Wigmore

(151) who did studies with SV 40 chromatin. The presence of

these sites in chromatin of mammalian cells was discovered by

Wu and Elgin (195). These sites have been found in the

chromatin of plants, animals fungi, and in viral genomes (72,

189). Therefore they are considered to be very important in

the field of biology, and to the understanding of how

genetic regulation occurs among various species of

eukaryotes.

The indirect end labeling technique is most commonly

used in mapping locations of DNAse I hypersensitive sites.

This follows isolation of nuclei, treatment with DNAse I














and purification of the genomic DNA. Its most useful feature

is that it allows mapping of DNAse I sensitive sites in a

single direction (72). Regions of DNAse I sensitivity are

usually the size of a nucleosomal repeat which is

approximately 150-100 base pairs (72). This can make precise

mapping difficult, but resolution can be improved by fine

mapping techniques.

DNAse I hypersensitive sites have been associated with a

wide variety of functions (72). In Saccharomyces cerevisiae,

hypersensitive sites have been seen near centromeres,

silencers, recombination sites, origins of replication,

activation sequences, promoters, and potential sites of

transcription termination (72). Therefore, these sites are

probably associated with cis acting factors (72).

Topoisomerases I and II, RNA polymerase II, and some

transcription factors have been associated with DNAse I

hypersensitive sites (72). The proteins associated with most

sites in genes which have been studied have yet to be

identified.

The mechanisms which are involved in the formation and

maintainance of DNAse I hypersensitive sites are not clear.

Because the functions of these sites are so diverse, several

mechanisms are likely to be involved (72). It is thought















that interaction with trans-acting factors may be one

of these mechanisms (72). The base composition of the DNA,

methylation, looping, conformation, and torsional stress may

also have an involvement in this process (72).

Fundamental knowledge of these principles should provide

insight into molecular bases of regulation. Thus, it is a

well accepted fact that DNase I hypersensitive sites

represent regions where potential regulatory interactions are

thought to occur. Specific DNA sequences of this nature have

been shown to be located in promoter regions for such genes

as globin (51), immunoglobulin (53), c-myc (48, 73, 155,

156), heatshock (129, 193, 194), SV 40 early region (47),

and dihydrofolate reductase (154). The remainder of this

review will focus on those involving the c-myc gene.

As previously mentioned, amplification and translocation

are well known and widely studied potential c-myc activation

mechanisms. As a result of translocation to chromosome 14 in

Burkitt lymphoma, c-myc loses all or part of exon 1. This

exon is thought to serve primarily as a regulatory region, as

it is transcribed but not translated (77). Therefore, c-myc

may be deregulated by its loss, and may be influenced by

promoters of other genes proximal to its translocated site.

In the case of HL-60 cells (30-50 copies of the c-myc gene),














it was presumed that gene dosage effects are responsible for

observed increases in c-myc transcript. In both these

instances, gross structural mutations appear to be

responsible for observed changes in transcript levels. It

has only been recently that we have begun to understand the

effects these aberrations have on c-myc regulation or

deregulation as the cases may be.

During the past few years, data obtained from chromatin

structure analyses have demonstrated that changes in c-myc

gene regulatory sites accompany gross structural

abnormalities in both transocated and amplified states of

this gene. DNAse I sensitive sites in the 5' region of

exon 1 in HL-60 cells and Burkitt lymphoma cells have been

investigated in 2 separate studies by Siebenlist et al. (155,

156). DNAse I hypersensitive sites in HL-60 cells were

studied before and after differentiation with DMSO. Results

showed that 4 DNAse I sensitive sites were present in

untreated HL-60 cells (sites A, B, C, and D, figure 4). When

differentiation was induced with DMSO, site B was lost.

Further studies showed that the loss of this site accompanied

a timely decline in c-myc transcript production.

In a separate study, DNase I hypersensitive sites were

studied in both normal and rearranged c-myc alleles in a














HL-60 (DNASE 1) A B C D V= + HL--60
HL-60 (S 1) G
BL-31 (DNASE I) ABCD
= TRANSCRIPT ELONGATION BLOCK (BL)







A B C D E F G
I I I I V
V


PO PI P2

C7MYC EXON 1 EXON 2



1KB




Figure 4. Summary of chromatin structure analyses previously
described for the c-myc gene. Sites A, B, C, and D are DNAse
I hypersensitive sites found in both HL-60 cells (Siebenlist
et al (155)), and Burkitt lymphoma (BL-31) cells (Siebenlist
et al. (156)). Site B (indicated by open arrow) has been
described by Siebenlist et al. (155) to be involved in the
maintainance of c-myc transcript production in HL-60 cells,
and is therefore marked with a (+) symbol. Sites E and F
(solid arows) represent transcription attenuation sites found
in Burkitt lymphoma biopsies and cell lines and are marked by
a (-) symbol (25, 199). Site G is an S-1 nuclease sensitive
site described by Grosso and Pitot (73) in HL-60 cells.














Burkitt lymphoma cell line (BL-31) and a normal B cell line.

Three different hypersensitive patterns which differed in

relative band intensities were observed. These correlated

with the three different transcriptional states of the c-myc

gene examined in this study (normal B cell myc, unrearranged

BL-31 c-myc, and translocated BL-31 c-myc). The locations of

hypersensitive sites which were observed were identical to

those for HL-60 cells (sites A,B,C,D) and are also shown in

figure 4.

Other groups working with Burkitt lymphoma have mapped

sites in the first intron and 3' region of exon 1. These

were later found to be transcription attenuation sites.

The loss of transcription elongation blocks at these sites is

now thought to be a possible candidate for deregulation of

myc in Burkitt lymphomas. Cesarman et al. (25) mapped a

previously found DNAse I hypersensitive site (9, 10, 49) to a

region near a Pvu II site in exon one. They reported that

23/26 Burkitt lymphoma cell lines and biopsies had point

mutations at various sites in a specific region extending 34

bases 5' and 38 bases 3' to the Pvu II site in exon 1 (site E

figure 4). These point mutations accompanied changes in

transcription, namely the removal of a block to transcription

mapped to the same region.














Zajac-Kaye et al. (199) noted similar findings in 5/7

Burkitt lymphoma cell lines. Their data indicated that a 20

base pair region in the first exon (site F, figure 4) was

susceptible to sporadic point mutations. Mutations in this

region abolished binding of a regulatory protein known to

down regulate c-myc transcription.



Relevance to This Project



The c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-

src proto-oncogenes have been studied in other human tumor

systems. The object of this study is to determine whether or

not these genes play a significant role in the biology of

chondrosarcoma and MFH. Studying transcript levels and copy

numbers of these genes will offer clues to possible

involvements in the pathogenesis and progression of these

tumors. Furthermore, chromatin structure analysis will

enhance understanding of mechanisms involved in transcript

regulation.



















CHAPTER 3
MATERIALS AND METHODS


Slot-Blottinq of RNA and DNA



Preparation of Total Cellular RNA



Total cellular RNA was prepared from surgically obtained

tumor, normal muscle, and bone marrow tissue specimens from

patients treated at Shands Hospital, University of Florida.

These specimens included; 20 chondrosarcomas, 23 malignant

fibrous histiocytomas (MFH), 9 normal muscle, and 6 bone

marrow tissue specimens. Approximately 1-3 hr after surgical

removal the tissues were frozen at -70 C until use. Total

cellular RNA was prepared as described by Chirgwin et al.

(26). Before use, all glassware and centrifuge tubes were

rendered nuclease-free with 0.1 percent diethylpyrocarbonate

(DEPC) in deionized water and thouroghly dried. All stock

solutions were freed of RNAse by adding several drops of 0.2

percent DEPC and subsequent autoclaving.

Tumor and normal tissues weighing 1.0-1.2 gr, or

approximately 10 E6 cultured cells were placed into 10 ml of

67














a solution containing 4M guanidine isothiocyanate (Ultra-

pure, BRL, Gaithersburg, MD), 25mM sodium citrate, pH 7.0,

and 0.1 M 2-mercaptoethanol. The mixture was then

homogenized using a tissuemizer (Brinkman Instruments), and

0.75 ml of 1 M acetic acid was added. The suspensions were

layered into SW 50 ultra-centrifuge tubes (Beckman

instruments) containing a 1.5 ml pad of 5.7 M cesium

chloride. The samples were centrifuged for 16 hr at 20 C and

35,000 rpm.

Following centrifugation, the pellets were resuspended

in 1.0 ml DEPC treated, sterile H20, and extracted once with

25:25:1 phenol:chloroform: isoamyl alcohol. The RNA was

then precipitated by adding 100 ul of 4 M potassium acetate,

and 2.5 ul ethanol. Recovery of the RNA was accomplished by

centrifugation at 10,000 rpm for 20 min at 4 C. The pellets

were resuspended in 0.2 mM EDTA, visualized on formaldehyde

agarose gels (as described for northern blotting below),

quantitated by absorbance at 280 nm, and stored in aliquots

at -70 C.


Preparation of Genomic DNA














Genomic DNA was prepared from the same tumor, muscle, and

bone marrow tissue specimens as described above. Tissues

were frozen in liquid nitrogen and ground to a fine powder

with a morter and postal. This powder, or approximately 10

E6 cells was suspended in 9.2 ml of STE (100mM NaCI, 20mM

Tris, pH 8.0, 10mM EDTA). Two hundred ul of 0.5M EDTA and

200ul of proteinase K (10mg/ml) were then added, and the

mixture was incubated overnight at 65 C.

Following incubation, the mixture was extracted once

with an equal volume of phenol, once with an equal volume of

phenol/chloroform-isoamyl alcohol (24:1), and finally once

again with an equal volume of chloroform-isoamyl alcohol.

The DNA was recovered by spooling after the addition of an

equal volume of isopropanol, and resuspended in 10mM Tris,

1mM EDTA.



RNA Slot-Blotting



Quantites of 10, 5, and 2.5 ug of total cellular RNA were

denatured in a solution containing 100 ul of water and 300ul

of an RNA denaturant solution containing 6.15 M formaldehyde

and 10X SSC (sodium chloride, sodium citrate). The samples

were incubated at 65 C for 15 min and loaded onto a mini-














fold II slot-blotter (Schleicher & Schuell, Keene, NH) using

procedures described by Wahl (187) The slot-blotter

contained a Nitro-Plus 2000 filter (Micron Separation

Sciences) onto which the RNA was blotted.



DNA Slot-Blotting



Quantities of 20, 10, and 5 ug of DNA were suspended in

400 ul of 10mM Tris, ImM EDTA, pH 7.0, and 40 ul 3M NaOH,

then incubated at 65 C for 45 min. After incubation, the

samples were cooled on ice, 400 ul of 2 M ammonium acetate

were added, and the samples were loaded onto a minifold II

slot-blotter as described above.

After slot-blotting, the filters were air dried to

completion, then baked in a vacuum oven at 80 C for 2 hr.

The blots were then incubated at 42 C overnight in a pre-

hybridization solution (5 ml/ 100 square cm) containing 5X

SSC, 10X Denhardt's solution (0.2 percent ficoll, 0.2 percent

polyvinylpyrrolidone (PVP), 0.2 percent bovine serum albumin

(BSA)), 0.05M sodium phosphate pH 6.7, 500ug/ul sonicated,

denatured salmon DNA, 5 percent dextran sulfate (Pharmacia

Chemical Co., Piscataway,NJ), and 50 percent formamide (112,

160).














Preparation of Radiolabeled Probes



Descriptions, sources, and methods of labeling for all

probes used in slot-blot, Southern blot, northern blot, and

chromatin structure analysis are summarized in Table 1.

Figure 5 shows locations of the different c-myc probes as

well as other probes located on chromosome 8 used in mapping

and dilutional analysis of c-myc amplicons in MFHs.

Restriction maps of all other probes in Table 1 are shown in

figure 6.



Nick Translated Probes



Probes were nick translated by adding 250 ng of DNA

to a reaction mixture which contained 80 uCi 32P (dATP), 5.0

ul of 10X (dCTP, dGTP, dTTP), 1.25 ul of lmg/ml bovine serum

albumin (BSA), 5 ul of nick translation buffer (0.5 M

Tris HCL, pH 7.8, 0.1 M 2-mercaptoethanol, and 0.05 M MgCl2),

1.5 ul DNAse I/Polymerase I (BRL, Gaithersburg, MD), and

deionized H20 to a final volume of 25 ul. The reaction was

run at 15 C for 45 min. Labeled DNA was separated from

unincorporated nucleotides using a Biogel A-15m (Biorad,

Rockville Center, NY) column.















Table 1. Summary of probes used in slot-blotting, Southern
blotting, northern blotting, and chromatin structure
analyses.



Probe Source* Use** Reference Method of Description
Name Labeling+


1,4


N.T


9.0 kb Eco R i/
Hind III human
genomic fragment
cloned into PBR
322


C-Ha-ras


C-fos





C-sis


137




36





71




108


V-erb-B-1 1


N.T. 6.4 kb Bam HI
human genomic
fragment cloned
into PBR 322

N.T. 6.4 kb Xho I/
Nco I human
genomic fragment
cloned into PBR
322

N.T. 1.0 kb human
genomic fragment
cloned into
pSP 6


N.T


1.7 kb Pvu II/
Sst I genomic
fragment from
avian erythro-
blastosis virus


N.T. 800 bp Pvu II
genomic fragment
from avian
sarcoma virus
prague A strain


C-myc


V-src















Table 1. Continued.


Probe Source* Use** Reference Method of Description
Name Labeling+


Beta-
actin


TK
(pTK 11)


pGEM-H MYC 4


1,2





2,3,4


p380-8A


Carbonic
Anhydrase
(H 25-3.8) 6


21





188




78







183


N.T. 800 bp Nco I/
Taq I genomic
fragment from
chicken beta-
actin gene


N.T/ 1.25 kb Sma I
P.E. Bam H I human
genomic fragment


N.T


1020 bp Pst I
human cDNA
fragment cloned
into pGEM 1


N.T. 1.8 kb Sal I/SstI
human genomic
fragment cloned
into puc 19



N.T. 3.8 kb Eco R I
human genomic
fragment cloned
into PBR 325


Thyro-
globulin
(HT .96)


N.T. 960 bp Pst I
human cDNA cloned
into puc 8
















Continued.


Probe Source* Use** Reference Method of Description
Name Labeling+


Beta-
actin



PMC 41


P.E.



N.T.


C-myc
Sca/Xho I 9


P.E.


2.0 kb Bam HI
human cDNA
fragment

1.6 kb Cla I/
Eco RI human
genomic fragment
cloned into PBR
322


355 bp Sca I/
Xho I human
genomic fragment


* Sources: 1) Oncor, Inc, Gaitersburg, MD, 2) Oncogene
Sciences, Mineola, NY, 3) Dr. Harvey Bradshaw, 4)
Dr. Ken Soprano, 5) Dr. Carlo Croce, 6) American Type
Culture Collection, Rockville, MD, 7) Dr. Larry Kedes,
8) Dr. Robert Gallo, and 9) Made from c-myc plasmid
obtained from Oncor, Inc. (above)

** Uses: 1) Slot-blot hybridization, 2) northern blot
hybridization, 3) titration of c-myc gene copy number
in MFHs, 4) Mapping of c-myc amplicons in MFHs,
5) mapping DNAse I hypersensitive sites in cell lines
from the 3' direction, and 6) mapping DNAse I
hypersensitive sites in cell lines from the 5'
direction (fine mapping analysis).

+ Method of labeling: N.T. = nick translation, P.E. =
random primer extension.


Table 1.







































p0P1 P2
E
PO Pl P2

SCA VXHO I


I- I-
0(0
&&a


0
0
o 0
* __ U


PMO-41

------PGEM H MYC
POEM H MYC


C-MYC


Figure 5. Locations of the different c-myc probes as
described in table 1. Also shown are other probes located on
chromosome 8 which were used in mapping of c-myc amplicons in
MFHs.


I I


CA-2


MYC
TG


0


- =- I, ~


H25-3.8 P380--A


U

HT.96


BeDlam


*^



























C-HA RAS 6.4KB



--- E -_-
II I I I ,


C--FOS 6.4 KB





I '" '* -- --- I
TII



C-SIS 1.0KB



LTR LTR

V- ERB-A V-ERB-B


LTR .. LTR
"- I I
V-SRC

0.8KB

- !-


,I I 'IIi




BETA ACTIN 0.8 KB







PTK 11 1.25 KB



-I_ I-


1.7 KB BETA-ACIN 2.0KB












Figure 6. Restriction maps of non-c-myc/chromosome 8 probes
as described in table 1.














Probes Labeled by Random Primer Extension



Two hundred ng of DNA were denatured at 90 C for 2 min.

After denaturation, five ul of 5X primer extension buffer (1M

hepes, pH 6.6, 25mM MgC12, 50 mM 2-mercaptoethanol, 0.25 M

Tris HCL, pH 8.0, 0.1 mM dCTP/dGTP/dTTP, 2mg/ml BSA, 15 mg/ml

primer), 5 units Klenow (BRL, Gaithersburg, MD), 100 uCi 32P

(dATP) and H20 were added to a final volume of 25 ul. This

reaction mixture was allowed to sit at room temperature for

16 hr. Unincorporated nucleotides were separated from

labeled DNA as described for nick translations.



Hybridization of Slot-Blots



Slot- blots were hybridized at 42 C with 3.0 X 10 E6

cpm (1.0 X 10 E8 cpm/ug) of probe for at least 20 hr in 15 ml

of hybridization solution containing 5X SSC, IX Denhardt's

solution, 0.02 M sodium phosphate, pH 6.7, 100ug/ml

sonicated, denatured salmon DNA, 10 percent dextran sulfate,

50 percent formamide, and 6 percent water (112).

Post hybridization washes were carried out by washing

the filters twice for 15 min at room temperature with 2X

sodium chloride, sodium phosphate, EDTA (SSPE), 0.1 percent














sodium dodecyl sulfate (SDS). The blots were then washed

twice again at 50 C with 0.1X SSC, 0.1 percent SDS for 30

min each and exposed to preflashed X-ray film for 36-48 hr at

70 C with intensifying screens.

Slot-blots were rehybridized after treatment of the

membrane to remove bound probe. This was accomplished by

pouring 1 liter of 0.1 X SSPE, 0.1 percent SDS heated to 90 C

over the blots. The solution was then cooled to 70 C and

removed. (187)



Southern Blot Analysis



Genomic DNA was prepared as described above from tissue

samples and cell lines. Aliquots of DNA were restricted with

appropriate restriction endonucleases and electrophoresed

through 0.8 percent agarose gels (65 volts, 16 hr).

Digested DNA was then transferred to Zetabind (AMF Cuno,

Meriden, CT), pre-washed in 0.1 X SSC, 0.1 percent SDS at 65

C for 1 hr, and hybridized (2 X 10 E6 cpm/ml/10 E8 cpm/ug)

using pre-hybridization and hybridization conditions

previously described for slot-blotting (112, 160). Post

hybridization washes were performed by washing the membrane

at room temperature for 15 min, once with 2 X SSC,














0.1 percent SDS, and once again with 0.1 X SSC, 0.1 percent

SDS. The blots were then washed twice for 30 min at 60 C,

with 0.1 X SSC, 0.1 percent SDS and exposed to X-ray film

at -70 C with intensifying screens. Rehybridization of the

blots was accomplished after removal of bound probe. This

removal process consisted of washing the blots in 0.1 X SSC,

0.5 percent SDS at 80 C for 15-20 min.



Northern Blot Analysis



Total cellular RNA was prepared from cell lines as

described for slot-blotting. RNA was denatured at 55 C for

15 min in an RNA denaturant containing 5ul 10X MOPS, 8.75 ul

37 percent formaldehyde, 25 ul formamide (ultra-pure BRL,

Gaithersburg, MD), and water to a final volume of 50 ul.

After denaturation, 10 ul of RNA formaldehyde loading dye was

added (500 ul formamide, 162 ul 37 % formaldehyde, 350 ul

glycerol, 100 ul 10X MOPS, bromophenol blue). The samples

were electrophoresed through 1.2 percent formaldehyde

agarose gels (139). The gels were prepared by melting 4.2 gr

agarose in 304.5 ml water. After cooling, 35 ml MOPS and

10.5 ml formaldehyde were added. Samples were














electrophoresed in running buffer which consisted of iX MOPS

and 10 percent formaldehyde (volume/37%) at 120 volts for 3-

3.5 hr.

After electrophoresis, the gels were rinsed several

times in deionized water, then soaked in 10X SSC for 45 min.

Blotting stacks were assembled as for Southern blotting.

Overnight transfers to Zetabind membranes were completed in

20X SSC. Blots were pre-washed in 0.1 X SSC, 0.5 percent SDS

at 65 C for 1 hr. Pre-hybridization and hybridization

conditions (2.0 X 10 E6 cpm/ml/ 10 E8 cpm/ug), as well as

post-hybridization washes and rehybridization procedures were

identical to those described for Southern analysis.



Chromatin Structure Analysis



Cell Lines Used in Chromatin Structure Analysis



UR-HCL-1. The UR-HCL-1 cell line is a human MFH tumor

cell line obtained from ATCC.



P3C. The P3C cell line is an MFH tumor cell line

obtained from Dr. Byron Croker, Department of Pathology,

University of Florida. The cell line was made by culturing














an MFH from a patient treated at Shands Hospital, University

of Florida.



ST 486. The ST 486 cell line is a Burkitt lymphoma

cell line obtained from ATCC. This cell line was used as a

positive control for chromatin structure analyses, as DNAse

I hypersensitive patterns for Burkitt lymphoma c-myc have

been described (156).



HFF. The HFF normal human fibroblast cell line was

obtained from Dr. Kenneth Rand, Department of Pathology,

University of Florida.



Preparation of Nuclei



Cells were grown in Dulbeccos MEM (minimal essential

medium) (Gibco, Gaithersburg, MD), supplemented with 10

percent fetal bovine serum (Gibco, Gaithersburg, MD).

Nuclei were isolated from dividing cells (approximately 2.0 X

10 E8). The cells were washed in 100 ml IX phosphate

buffered saline (PBS), and centrifuged at 2,000 rpm, for 3

min at 4 C. The pellet was resuspended in 10 ml IX

RSB (10mM Tris, pH 7.4, 10mM NaCl, 3mM Mg C12), 0.5 percent














nonidet P40 (NP 40), 10 ul 0.1M phenyl methyl sulfate (PMSF),

then incubated on ice (0 C) for 5 min. The nuclei were

recovered by centrifugation at 2,000 rpm at 4 C for 3 min.

The pellet was then washed 3 times with 100, 50 and 20 ml of

IX RSB followed by centrifugation at 2,000 rpm at 4C for 3

min.

The nuclei were then resuspended in IX RSB and digested

with varying concentrations of DNAse I (Boerhinger Mannheim)

for 10 min at 37 C. Controls were 0 ug/ml DNAse I incubated

at both 0 and 37 C.



Isolation of Genomic DNA From DNAseI Treated Nuclei



After digestion with DNAse I, the samples were placed on

ice and 1/10 volume 0.25M EDTA was added along with 1/20

volume 10 percent SDS and 1/20 volume proteinase K. After

incubation overnight at 37 C, the samples were extracted

with an equal volume phenol, an equal volume phenol/24:l

chloroform-isoamyl alcohol, then a third time with an equal

volume of 24:1 chloroform isoamyl alcohol. DNA was

precipitated by adding 4M potassium acetate to a final

concentration of 0.3M, and the addition of 2-3 volumes cold

95 percent ethanol. After precipitation at 20 C overnight,














DNA was recovered by centrifugation at 10,000 rpm at 4 C for

15 min. The pellet was resuspended in 300 ul 50mM Tris,

pH 8.0, 10 mM EDTA. RNAse A (1mg/mi) was added to a final

concentration of 50 ug/ml and incubated overnight at 37 C.

The mixture was then phenol/chloroform extracted, and the DNA

precipitated as described above. The final DNA pellet was

resuspended in 10mM Tris, ImM EDTA.

Aliquots of DNA from each of the cell lines were

restricted with either Eco Rl (mapping of DNAse I

hypersensitive sites from a 3' direction (pmc 41 probe)), or

Sca I (mapping of these sites from a 5' direction (Sca I/Xho

I fragment probe)), and analyzed using Southern blotting and

hybridization methods described above. The mapping of DNAse

I hypersensitive site locations was accomplished through the

use of the indirect end labeling technique. This technique

allows mapping of the DNAse I hypersensitive sites in one

direction, and is described in figure 7 using restriction

with Eco R1 and hybridization with pmc 41 as an example.



Chromatin Structure/ Fibroblast Cell Synchrony Experiment



HFF cells were grown to 70-80 percent confluency in

Dulbeccos MEM supplemented with 10 percent fetal bovine















5-

5-_


& v 3
se ,

._ __.___ ,vv A 3'


PMC41




ISOLATE DNA, DIGEST WITH ECO RI



SOUTHERN TRANSFER, PROBE WITH PMC 41


9.6


Figure 7. Illustration of the indirect end labeling
technique. This technique allows 5' or 3' orientation of
the locations of DNAse I hypersensitive sites. It always
yields pieces of DNA which have a restriction site on one
side, therefore allowing analysis of DNA segments in one
direction. Various concentrations of DNAse I are used to
allow partial digestion. If DNAse I cut at sites 1 and 2,
analysis by this method would yield three bands on a Southern
blot. A main band of 9.6 kb which corresponds to the Eco
Rl/Eco Rl fragment would be present along with bands of sizes
corresponding to the lengths of the DNAseI cleavage sites to
the 3' ECO R I site.














serum. At this confluency level, cells were actively cycling

(confirmed by Northern blot hybridization with the TK probe

data shown below). The cells were then made quiescent by

the addition of MEM containing 0.1 percent serum, and

subsequent incubation at 37 C for 3 days.

MEM supplemented with 10 percent fetal bovine serum was

then added to release the cells. Nuclei isolation/DNAse I

treatment (as described above) and RNA isolation procedures

(as described above) were conducted at 0 hr (GO), 0.5 hr, 1

hr, 2 hr, 3 hr after serum release, and during log phase

growth. DNAse I hypersensitive sites were evaluated by

Southern blot hybridization procedures using the pmc 41 probe

as described above. RNA samples were analyzed for c-myc,

TK, and actin transcript levels using Northern blot

procedures as previously described.



Strategy for Fine Mapping DNAse I Hypersensitive Sites 3' to
C-Myc Exon 1


An overall scheme for fine mapping of DNAse I

hypersensitive sites from a 5' direction is shown in figure

8. Selected restriction enzyme sites in the region of a

DNAse I hypersensitive site 3' to c-myc exon 1 (discussed





















C-MYC EXON 1


'V
AI

PO P1 P2

PROBE


SCA /)01 I
SCA I/PVU II
SCA I/BSM I
SCA I/VMAE III


1 KB


355BP
805 BP
920BP
97OBP


S= TRANSCRIPT ELONGATION BLOCK (BL)


Figure 8. Location of the Sca I/Xho I probe and other
restriction sites in and around c-myc exon 1 used in fine
mapping of DNAse I hypersensitive sites from the 5' direction
in P3C and HFF cell lines. These sites were used to generate
P3C DNA fragments of known sizes, which were internal size
markers on the mapping blot (see materials and methods).
Also shown are the c-myc promoters (P0, P1, and P2), and
known c-myc transcript elongation block sites which are
indicated by solid arrows.


EXON 2














below) were chosen to generate fragments used as internal gel

markers to map DNAse I hypersensitive sites in this region.

First, P3C genomic DNA was restricted to completion with

Sca I (Boerhinger Mannheim). Five ug aliquots of this DNA

were then restricted a second time with either Pvu II (BRL,

Gaithersburg, MD), Bsm I (New England Biolabs), or Mae III

(Boehinger Mannheim).

Bsm I cut a single time within the c-myc gene, therefore

the restriction reaction was allowed to go to completion

overnight at 65 C. Pvu II and Mae III cut at multiple

sites within the c-myc gene. Reactions were therefore

controlled to prevent complete digestion of the DNA.

Partial digestion of DNA with an enzyme which cuts at

multiple sites between a desired site and Sca I will yield

band sizes corresponding to distances between the desired

site and the Sca I site.

Five ug aliquots of Sca I restricted P3C DNA were

restricted with various concentrations of Pvu II (37 C) and

Mae III (55 C) for 30 min reaction times. Concentrations

which yielded optimum visualization of the desired marker

band sizes were used to restrict P3C DNAs for use as markers

in mapping analyses.














DNAse I treated DNA samples (samples with optimum

visualization of DNAse I generated bands from previous

analysis) from P3C (0.2 and 0.5 ug/ml) and HFF (0.5 ug/ml)

cell lines were restricted with Sca I. Five ug aliquots of

these samples along with marker DNAs generated as described

above were electrophoresed in 1.5 percent agarose gels (65

volts, 20 hr), and blotted onto Zetabind. Lambda DNA

digested with Eco R 1 and Hind III was run on either side of

the gel to assure that the gel ran evenly. Prehybridization,

hybridization (2.0 X 10 E 8 cpm/ml/ 10 E8 cpm/ug), and

washing conditions were identical to those described for

Southern blotting.



Polyacrylamide Gel Electrophoresis (PAGE)



The discontinuous system for PAGE as described by

Laemmli (101) was used in this analysis. Stacking gels were

4 percent acrylamide (total) in 0.125 M Tris-HCl, pH 6.8, and

0.1 percent SDS. Separating gels were 8.5 percent total

acrylamide in 0.375 M Tris-HCL, pH 8.8, and 0.1 percent SDS.

Both gels were cross-linked with 2.7 percent bis acrylamide,

and polymerization was catalyzed with 0.005 percent TEMED,

and 0.05 percent ammonium persulfate.














Twenty ug of protein were combined with an equal volume

of treatment buffer (0.125 M Tris-HCL, pH6.8, 4 percent SDS,

20 percent glycerol, and 10 percent 2-mercaptoethanol),

incubated at 90 C for 1.5 min, ice-quenched, then loaded onto

the gels. Molecular weight markers ranging from 31,000 to

200,000 daltons (Biorad) were loaded as well.

A tank buffer which consisted of 25 mM Tris-HCL, pH 8.3,

0.192 M glycine, and 0.1 percent SDS was used as a running

buffer. Gels were electrophoresed in a Hoefer SE 600

vertical slab unit at 30 ma/l.5 mm gel thickness.



Western Blotting and Immunoperoxidase Assay



Western blotting of proteins was carried out at 0.6 amps

for 45 min at 4 C. Proteins were blotted onto 0.2 um pore

size nitrocellulose (Schleicher & Schuell, Keene, NH) using

methods described by Towbin (176). Transfer was carried out

using a Hoeffer TE 52 Transphor unit. Following transfer,

the blots were air-dried, then incubated for 3 hr in PBST (IX

phosphate buffered saline (PBS), 0.05 percent Tween 20) and 2

percent BSA. An anti- human c-myc monoclonal antibody (HL-

40) (IGG 1, ascites purified by protein A column) obtained

from Dr. Henry Neiman was then diluted (0.1 mg/ml) in PBST, 2














percent BSA, added to one blot, and allowed to incubate at

room temperature for 1 hr with light agitation. As a control

for non-specific binding, an identical blot was incubated

with an anti-met 72 monoclonal antibody (K 88. 151. G 127)

(IGG 1, ascites purified by protein A column), (0.01 mg/ml in

PBST, 2 percent BSA) obtained from Dr. Arthur Kimura. The

blots were then washed 3 times for 5 min each with PBST.

This was followed by incubation with a 1 ug/ml solution of a

horseradish peroxidase conjugated goat-anti-mouse Ig

(Southern Biotechnology Associates) diluted in PBST, 2

percent BSA for 1 hr at room temperature. Blots were then

washed 3 times with PBST as before, and incubated with a 180

ug/ml solution of the substrate, diaminobenzoate (DAB), in

PBST, and 0.01 percent H202 for 2-3 minutes. The reaction

was stopped with excess H20. Quantification of c-myc protein

bands was carried out by reflectance densitometry.

As a control for quantification, a third identical blot

was stained with a 0.1 percent solution of india ink in PBST

for 1 hr at room temperature, then destined with PBST until

the desired resolution was achieved.

















CHAPTER 4
RESULTS



Quantitation of Moderately Degraded RNA Using the Slot-
Blotting Technique



The relationship between RNA degradation and accuracy of

quantitation was evaluated because in many instances tumor

tissues were not immediately (1-3 hr) available for

processing after surgical removal, and message degradation

occurs rapidly. Intact total cellular RNA from HL-60 cells

was degraded in 0.2 N NaOH at 0, 0.5, 1,2,5, and 30 minute

intervals. After evaluation by formaldehyde gel

electrophoresis using procedures previously described

(northern blotting section), (figure 9), the sample at 0

minutes showed completely intact RNA, the samples at 0.5, 1,

2, and 5 minutes, moderately degraded RNA, and at 30 minutes

the RNA was extensively degraded. Analysis of these samples

by slot-blotting (figure 10) demonstrated that moderately

degraded RNA as shown in lanes B, C, D and E, is

as sensitive to quantitative changes as intact total

cellular RNA (lane A). Extensively degraded RNA shown in

91




Full Text
143
C-mvc Protein Levels in the P3C, UR HCL 1, HFF, and ST 486
Cell Lines
A comparison of c-myc protein levels was made between
cells with amplified (P3C) and single copy (UR HCL 1) c-myc
genes, cells in which c-myc is thought to be an oncogene (ST
486), and normal fibroblasts during peak levels of c-myc
transcript production. This was done to determine if the
increased levels of c-myc transcript seen in P3C cells were
translated. Twenty ug of protein from P3C, UR HCL 1, ST 486
and HFF cells (GO, 0.5, 1, and 2 hours after serum release,
and during log phase growth), were evaluated for relative c-
myc protein levels using PAGE and Western blotting techniques
as previously described.
The c-myc monoclonal antibody (HL-40) bound to a 65 kd
protein band (132) as determined by molecular weight markers
run on the gel. The control panel, incubated with anti-met
72 (72/K 88.151.G127), showed no non-specific binding, and
staining with india ink indicated that protein quantitation
was consistent between samples (figure 26). Relative levels
of c-myc protein were determined by reflectance densitomety,
and values were normalized to those of fibroblasts in GO
(figure 26). Fibroblasts 0.5 and 2 hours after serum


177
160. Southern, E.M. 1975. Detection of Specific Sequences
Among DNA Fragments Separated by Gel Electrophoresis.
J. Mol. Biol. 98: 503.
161. Souyri, M., and E. Fleissner. 1983. Identification By
Transfection of Transforming Sequences in DNA of Human
T Cell Leukemias. Proc. Natl. Acad. Sci. U.S.A. 80:
6676.
162. Spector, D.H., H.E. Varmus, and J.M. Bishop. 1978.
Nucleotide Sequences Related to the Transforming Gene
of Avian Sarcoma Virus are Present in DNA of Infected
Vertebrates. Proc. Natl. Acad. Sci. U.S.A. 75: 4102.
163. Sporn, M.B. and G.J. Todaro. 1980. Autocrine
Secretion and Malignant Transformation of Cells. N.
Eng. J. Med. 303: 878.
164. Stehelin, D. H.E. Varmus, J.M. Bishop, and P.K. Vogt.
1976. DNA Related to the Transforming Gene(s) of Avian
Sarcoma Viruses is Present in Normal Avian DNA.
Nature 260: 170.
165. Stiles, C.D. 1983. The Molecular Biology of Platelet
Derived Growth Factor. Cell 33: 653.
166. Strandberg, J.C. 1986. The Expression of Mvc and Ras
Oncogenes in Chondrosarcomas and Malignant Fibrous
Histiocytomas. Masters Thesis, University of Florida.
167. Sugimoto, Y., M. Whitman, L.C. Cantley, and R.L.
Erikson. 1984. Evidence That the Rous Sarcoma Virus
Transforming Gene Product Phosphorylates
Phosphatidylinositol and Diacylglycerol. Proc. Natl.
Acad. Sci. U.S.A. 81: 2117.
168. Swift, M., and C. Chase. 1979. Cancer in Families With
Xeroderma Pigmentosum. J. Natl. Cancer Inst. 62:
1415.
169. Swift, M., L. Sholman, M. Perry, and C.Chase. 1976.
Malignant Neoplasms in the Families of Patients With
Ataxia Telangiectasia. Can. Res. 36: 209.
170. Tabin, C.J., S.M. Bradley, C.I. Bargmann, and R.A.
Weinberg. 1982. Mechanism of Activation of a Human
Oncogene. Nature 300: 143.


REFERENCES
1. Alitalo, K., M. Schwab, C.C. Lin, H.E. Varmus, and
J.M. Bishop. 1983. Homogenously Staining Chromosomal
Regions Contain Amplified Copies of an Abundantly
Expressed Cellular Oncogene C-myc in Malignant
Neuroendocrine Cells From a Human Colon Carcinoma.
Proc. Natl. Acad. Sci. U.S.A. 80: 1707.
2. Alt, F.W., R. DePinho, K. Zimmerman, E. Legouy, K.
Halton, P. Ferrier, A. Tesfaye, G. Yancopoulos, and
P. Nisen, 1986. The C-myc Oncogene Family. Cold
Spring Harbour Symposium on Quantitative Biology. LI:
931-941.
3. Baas, F., H Bikker, A. Geurts, R. Melsert, P.L.
Pearson, J.J. De Vijlder, and G.J. VanOmmen. 1985.
The Human Thyroglobulin Gene: A Polymorphic Marker
Localized Distal to C-myc on Chromosome 8, Band q24.
Human Genetics 69: 138.
4. Baltimore, D. 1970. Viral RNA-Dependent DNA
Polymerase. Nature 226: 1209.
5. Baltimore, D. 1976. Viruses, Polymerases, and
Cancer. Science 192: 632.
6. Barbacid, M., and A.V. Lauver. 1981. The Gene
Products of McDonough Feline Sarcoma Virus Have an In
Vitro Associated Protein Kinase That Phosphorylates
Tyrosine Residues. Lack of Detection of This
Enzymatic Activity In Vivo. J. Virol. 40: 812.
7. Barnekow, A., E. Paul, and M. Schartl. 1987.
Expression of the C-src Proto-oncogene in Human Skin
Tumors. Can. Res. 47: 235.
160


are due to some unknown mechanism other than gene
amplification. Additionally, differences in chromatin
structure between amplified and single copy c-myc in
MFH cells may represent a compensatory response to increased
c-myc transcript production. Increased levels of c-myc
protein provide further evidence that c-myc may be an
oncogene in these cells.
IX


48
C-Ha-ras. C-Ha-ras is a member of the ras oncogene
family, and is cell cycle dependant (94). As described
previously, activation of c-ras to an oncogene is
accomplished by point mutations at specific sites which
render its protein product oncogenic to the cell (34, 170).
Mutations of this nature have been found in approximately 15
percent of sarcomas (77) Oncogenes of the ras family may
be active in human carcinoma cell lines, as well as primary
human tumor specimens of several sites such as colon, lung,
gall bladder, urinary bladder, pancreas rhabdomyosarcoma
(42, 136,137), and in prostate cancer (186). These genes are
also present in human hematopoietic neoplasias; including
primary acute myelogenous leukemias, and cell lines derived
from acute lymphocytic leukemias, T cell leukemias and
chronic myelogenous leukemias (55, 161) .
Src. The src oncogene as described above codes for a
protein, which like its normal proto-oncogene counterpart,
is a protein-tyrosine kinase. In order to study its
mechanism of activation, c-src has been molecularly cloned
from both chicken and human DNA. Nucleotide seguencing has
revealed the similarities between the protein coding regions
and those of v-src (171). Unlike v-src, c-src is very


82
nonidet P40 (NP 40), 10 ul 0.1M phenyl methyl sulfate (PMSF),
then incubated on ice (0 C) for 5 min. The nuclei were
recovered by centrifugation at 2,000 rpm at 4 C for 3 min.
The pellet was then washed 3 times with 100, 50 and 20 ml of
IX RSB followed by centrifugation at 2,000 rpm at 4C for 3
min.
The nuclei were then resuspended in IX RSB and digested
with varying concentrations of DNAse I (Boerhinger Mannheim)
for 10 min at 37 C. Controls were 0 ug/ml DNAse I incubated
at both 0 and 37 C.
Isolation of Genomic DNA From DNAsel Treated Nuclei
After digestion with DNAse I, the samples were placed on
ice and 1/10 volume 0.25M EDTA was added along with 1/20
volume 10 percent SDS and 1/20 volume proteinase K. After
incubation overnight at 37 C, the samples were extracted
with an equal volume phenol, an equal volume phenol/24:1
chloroform-isoamyl alcohol, then a third time with an equal
volume of 24:1 chloroform isoamyl alcohol. DNA was
precipitated by adding 4M potassium acetate to a final
concentration of 0.3M, and the addition of 2-3 volumes cold
95 percent ethanol. After precipitation at 20 C overnight,


150
Increases in transcript levels of c-sis were observed in
MFHs; however, only single copies of the c-sis gene were
seen. This suggests that increases in sis transcript levels
are due to some undetermined mechanism other than gene
amplification.
The myc gene product is postulated to be a double
stranded DNA binding protein capable of participating in the
regulation of cell division (17, 94, 95, 96). In vitro
experiments suggest that c-myc genes are of a cell-cycle
dependent nature in that levels of c-myc transcript like
those of c-jun (143) and c-fos (120) increase during the
G0/G1 transition and decrease to GO levels during S-phase.
These are unlike more "traditional" cell-cycle dependent
genes such as histone H-2b and TK whose transcript levels
peak during S phase (175).
Beta-actin is commonly used as a standard to normalize
the expression of other genes because it is single copy, and
constitutively expressed in most tissues. In these studies,
it was appropriate to normalize to TK instead of actin for
transcript levels of cell cycle dependent proto-oncogenes; c-
myc, c-Ha-ras, and c-fos. Normalization to actin would not
correct for the different frequencies of cell division seen
in different tumors. Although the c-myc gene is not cell
cycle dependent in the "traditional" sense, normal tissues or


ACKNOWLEDGEMENTS
I would like to thank the members of my committee, Dr.
Byron Croker, Dr. Warren Ross, Dr. Lindsey Hutt-Fletcher, Dr.
Linda Smith, and Dr. Harry Ostrer, for their assistance and
advice. I would especially like to thank Dr. Croker for his
faith in my abilities, his guidance, encouragement, and
friendship.
I would also like to thank Dr. Susan Chrysogelos for all
her help during the past 18 months. I am most appreciative
of all she has taught me. Special thanks go to Dr. Cheryl
Zack and Herb Houck for all their help and encouragement, to
Jerry Phipps for his assistance in obtaining surgical
specimens, and to my fellow graduate students for their help
and encouragement. In addition, I am most grateful to my
friends who have been very supportive, particularly Gail
Waldman, Patty Leginus, and Patty DeHaan.
Lastly, I would like to thank my husband Ron, my
grandparents, Karen, and Ken for all their much needed love,
support and encouragement. I have no words which could
adequately express my gratitude to my Mom and Dad. Without
them none of this would have ever been possible. They have
taught me so many things, but I think the most important
lesson I have learned from them is that family is one of
life's greatest treasures. I will never forget that. I will
never forget everything they have done for me.
IV


52
There is now evidence for genes which seem to be
directly controlled by c-fos. For example, a set of genes
which code for fat cell proteins which become active when fat
cells differentiate has been identified. One of these genes
adipocyte P2 (aP2), was found to have a regulatory site 125
base pairs upstream from its promoter. This regulatory site
binds proteins which undergo undefined changes during
maturation. It is hypothesized that changes in the binding
proteins mediate activation of the aP2 gene (44).
Experiments were then performed to determine whether
the c-fos protein was one of these regulatory proteins. Data
from immunoprecipitation analyses showed that the binding
complex contains the c-fos protein itself, or at least a very
related protein (44) Further studies need to be done to
clarify this issue.
Site directed mutagenesis studies have been done in the
c-fos promoter region. Various deletions were studied for
effects on the c-fos gene's responses to various stimulatory
agents. It was found that a 22 base pair region located 300
base pairs 5' to the promoter is necessary for enhanced
expression of c-fos in response to serum stimulation (68,
177, 178). This region is called serum response element
(SRE). These same investigators have isolated a protein


117
Table 17. DNA quantitation of non-cell-cycle dependent genes
as determined by slot-blot analysis. *
MUSCLE
BONE
MARROW
CS
MFH
C-SIS:ACTIN
0.9+0.1
1.0+0.1
1.2+0.2
1.2+0.2
V-ERB-B-1:
1.0+0.1
1.0+0.2
1.0+0.2
1.1+0.2
ACTIN
V-SRC:ACTIN
0.9+0.3
0.9+0.1
1.2+0.2
1.1+0.2
TOTAL CASES
9
6
20
23
Values shown are mean generactin ratios


MFHs show no significant differences in expression of c-Ha-
ras, c-fos, v-erb-B-1, and v-src. C-sis RNA levels are 2-to
3- fold greater in MFHs. DNA analysis shows c-myc to be a
single copy gene in all tissues except 6 MFHs which have
between 2 and 11 copies. C-myc amplicons were found to be
large, extending at least 50 kb 5' to the c-myc promoter and
slightly 3' of exon 3. These same tumors have increased
levels of c-myc transcript as determined from RNA analysis.
C-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-src are single copy
genes in all tissues.
Chromatin structure studies show that amplified c-myc
in P3C cells does not contain a DNAse I hypersensitive site
near the P0 promoter region 5' to exon 1, known to be
involved in maintaining c-myc transcript production in HL-
60 cells. Additionally, a new site is present in a region
known to contain a block to transcription elongation in
Burkitt lymphomas. These changes are not seen during normal
upregulation of c-myc in GO serum released fibroblasts (G0/G1
transition).
These data suggest that increased levels of c-myc
expression are due to gene dosage, while those of c-sis
viii


26
index of 0-2 per 10 high power fields (40X), dense
cellularity, paler staining nuclei, a background which is
more more myxoid than chondroid, and a greater cellularity
/increased nuclear size limited to isolated areas.
The criteria for the classification of a chondrosarcoma
as grade III are a mitotic index greater than 2 mitoses per
10 high power fields (40X), increased nuclear size compared
to those of grade II tumors, very dense cellularity which
may appear MFH like, and the absence of a chondroid or
myxoid background.
Malignant fibrous histiocytomas (MFH) (figure 3) are
soft tissue tumors whose cell of origin has been disputed,
but current evidence indicates that these are are immature
mesenchymal cells (15, 90). Malignant fibrous histiocytomas
usually occur in deeper structures such as deep fascia and
skeletal muscle. They also have been seen in soft tissues of
the extremities, mediastinum, and retroperitoneum, and may
occur within bones in areas of infarction or prior radiation.
As a group, these tumors comprise only about 0.8 percent of
all bone tumors (39), but are somewhat more common in soft
tissues. There seems to be a slightly higher percentage of
males with this disease than females, and nearly any age may
be affected (190). As with other varieties of bone tumors,


109
(table 14). Mean TK:actin ratios are 1.1 in bone marrow,
1.1 in chondrosarcomas, and 1.5 in MFHs (figures 12 and 13).
C-myc:TK ratios in bone marrows, chondrosarcomas, and MFHs
were 0.9, 1.2, and 1.3 respectively. There are no
significant differences between these groups for levels of c-
myc transcript (p>0.05). C-Ha-ras and c-fos transcript
levels among bone marrows, chondrosarcomas, and MFHs range
from 0.8 to 1.1 and are not significantly different
(p>0.05).
C-sis, v-erb-B-1, and v-src gene transcript levels are
detectable in muscle, giving mean generactin values ranging
from 0.9 to 1.0 (table 15). C-sis:actin ratios in
bone marrows, chondrosarcomas and MFHs are 1.1, 1.6 and 3.1
respectively. There are significantly higher transcript
levels of c-sis in MFHs compared to the other 3 groups
(p<0.05) (figure 14). V-erb-B-1 and v-src transcript levels
in bone marrows, chondrosarcomas and MFHs range from 0.9 to
1.2 and show no significant differences between any of the
groups for transcript levels of these two genes (p >0.05).
DNA Slot-Blot Results and Determination of C-mvc
Gene Copy Number


179
181. Ulrich, A., L. Coussens, J.S. Hayflick, T.J. Dull, A.
Gray, A.W. Tam, J. Lee, Y. Yarden, T.A. Libermann, J.
Schlessinger, J. Downward, E. Mayes, N. Whittle, M.D.
Waterfield, and P.H. Seeburg. 1984. Human Epidermal
Growth Factor Receptor cDNA Sequence and Aberrant
Expression of the Amplified Gene in A 431 Epidermoid
Carcinoma Cells. Nature 309; 418.
182. Varshavsky, A.J., O.H. Sundin, and M.J. Bohn. 1978.
SV 40 Viral Minichromosomes: Preferential Exposure of
the Origin of Replication as Probed by Restriction
Endonucleases. NAR 5: 3469.
183. Vent, P.J., T.B. Shows, P.J. Curtis, and R. Tashian.
1983. Polymorphic Gene for Human Carbonic Anhydrase II:
A Molecular Disease Marker Located on Chromosome 8.
Proc.Natl. Acad. Sci. U.S.A. 80: 4437.
184. Verma, I.M., J. Deschamps, C. Van Beueren, and P.
Sassone- Corsi. 1986. Human fos Gene. Cold Spring
Harbor Symposium on Quantitative Biology. 51: 949.
185. Vijayolaxmi, H., J. Evans, J.H. Ray, and J. German.
1983. Blooms Syndrome: Evidence for an Increased
Mutation Frequency In Vivo. Science 221: 851.
186. Viola, M., F. Fromowitz, S. Oravez, S. Deb, G. Finkel,
J. Lundy, P. Hand, A. Thor, and J. Schlom. 1986.
Expression of Ras Oncogene p21 in Prostate Cancer. The
New. Eng. J. Med. 314: 133.
187. Wahl, G. 1985. Rapid Detection of DNA and RNA Using
Slot-Blotting. (Application Update No. 371).
Schleicher & Schuell, Inc., Keene, NH.
188. Watt, R., L.W. Stanton, K.B. Marcy, R.C. Gallo, C.M.
Croch, and G. Rovera. 1983. Nucleotide Sequence of
Cloned cDNA of Human C-myc Oncogene. Nature 303:
725.
189. Weiss, R.A. 1973. Possible Episomes in
Eukaryotes. L.G. Silvestri, ed. Amsterdam, North-
Holland.
Weiss, S.W., and F.M. Enzinger. 1978. Malignant
Fibrous Histiocytoma: An Analysis of 200 Cases.
Cancer 41: 2250.
190.


77
Probes Labeled by Random Primer Extension
Two hundred ng of DNA were denatured at 90 C for 2 min.
After denaturation, five ul of 5X primer extension buffer (1M
hepes, pH 6.6, 25mM MgC12, 50 mM 2-mercaptoethanol, 0.25 M
Tris HCL, pH 8.0, 0.1 mM dCTP/dGTP/dTTP, 2mg/ml BSA, 15 mg/ml
primer), 5 units Klenow (BRL, Gaithersburg, MD), 100 uCi 32P
(dATP) and H20 were added to a final volume of 25 ul. This
reaction mixture was allowed to sit at room temperature for
16 hr. Unincorporated nucleotides were separated from
labeled DNA as described for nick translations.
Hybridization of Slot-Blots
Slot- blots were hybridized at 42 C with 3.0 X 10 E6
cpm (1.0 X 10 E8 cpm/ug) of probe for at least 20 hr in 15 ml
of hybridization solution containing 5X SSC, IX Denhardt1s
solution, 0.02 M sodium phosphate, pH 6.7, 100ug/ml
sonicated, denatured salmon DNA, 10 percent dextran sulfate,
50 percent formamide, and 6 percent water (112).
Post hybridization washes were carried out by washing
the filters twice for 15 min at room temperature with 2X
sodium chloride, sodium phosphate, EDTA (SSPE), 0.1 percent


15
produce higher levels of other enzymes such as proteases
and collagenases.
5. Different transformed cells have varying levels of
nucleotides. Some may have higher cAMP levels or
increased cGMP:cAMP ratios than their normal cell
counterparts.
6. Transformed cells in culture have been shown to
produce growth factors involved in tumor growth.
These include angiogenesis factors, and transforming
growth factors (TGF). These may be produced to favor
their own growth (autocrine function).
7. Fetal antigens, placental hormones, and fetal
enzymes have been shown to be produced in increased
amounts in cultured tumor cells. This is
characteristic of tumor cells in vivo.
8. Ability to produce tumors in experimental animals is
a characteristic of malignant cells.
In addition to biological and physiological changes in
transformed cells, changes at the molecular level occur
as well. Genetic instability during tumor progression is
characterized by a variety of aberrations in the genome
including point mutations, deletions, rearrangements,
amplifications, chromosome translocations and abnormal


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PROTO-ONCOGENE EXPRESSION IN HUMAN
CHONDROSARCOMA AND MALIGNANT FIBROUS HISTIOCYTOMA
By
Jane Carolyn Strandberg Gibson
May, 1989
Chairman: Dr. Byron P. Croker
Major Department: Pathology and Laboratory Medicine
Total cellular RNA and genomic DNA were extracted from
20 chondrosarcomas, 23 malignant fibrous histiocytomas (MFH),
9 muscle, and 6 bone marrow specimens. Levels of RNA and
gene copy numbers of c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-
1, v-src, thymidine kinase (TK) and actin were quantified
densitometrically from slot-blot analysis. C-myc, c-Ha-ras,
and c-fos transcript levels are undetectable in muscle. Mean
c-myc:TK ratios do not differ significantly among groups of
bone marrows, chondrosarcomas and 17 MFHs (p > 0.05). Six
MFHs have a mean c-myc:TK ratio of 2.0 which is significantly
higher than the other groups (p > 0.05). Intergroup
comparisons between bone marrows, chondrosarcomas, and
vii


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS V
ABSTRACT vii
CHAPTERS
1 INTRODUCTION 1
Mechanisms of Tumor Development 1
Multistep Carcinogenesis 7
The Neoplastic Phenotype and Steps of Tumor
Progression 12
Specific Questions Addressed During the Course
of This Project 17
2 REVIEW OF THE LITERATURE 2 0
Differentiation of Mesenchyme 20
Chondrosarcoma and Malignant Fibrous Histio
cytoma 22
Proto-Oncogenes 29
Biochemistry of Oncogene Products 3 4
Cytoplasmic Kinases 34
Ras Proteins 38
Growth Factors and Their Receptors 40
Nuclear Proteins 42
Molecular, Biological, and Physiological Char
acteristics of Proto-Oncogenes Examined in
This Study 44
Growth Factor Related 44
Protein Kinases 47
Nuclear Related Proto-Oncogenes 50
Chromatin Structure Analysis of the C-myc
Gene 59
Relevance To This Project 66
3 MATERIALS AND METHODS 67
Slot-Blotting of RNA and DNA 67
Preparation of Total Cellular RNA 67
Preparation of Genomic DNA 68
RNA Slot-Blotting 69
DNA Slot-Blotting 70
v


19
sites may offer clues to regulatory mechanisms involved in
proto-oncogene transcript production.


58
The c-myc protein product is a double stranded DNA
binding protein thought to interact with other genes, perhaps
those involved in cellular growth control. It is thought
that the myc protein can bind to the regulatory regions of
genes it controls, regulating transcription either by direct
activation or by inhibition of suppression (17).
The precise function the of c-myc protein has not been
elucidated. It is generally thought that its primary
function is to mediate a signal(s) associated with cell
division and thus, regulation of its expression is required
for normal cell growth (2). Experiments with c-myc antisense
RNA have shown that the ability of cells to divide can be
blocked (192).
The c-myc gene has been found to be present in many
types of sarcomas, carcinomas, and hematopoietic neoplasias
(32, 54, 77, 172). The two most widely studied mechanisms
of oncogenic activation of this gene are translocation
(seen in Burkitt lymphoma), and gene amplification. In the
case of Burkitt lymphoma the c-myc gene is translocated from
chromosome 8 to chromosome 14 or from chromosome 8 to 22. As
a result, the c-myc gene loses all or a portion of its first
exon, and acquires normally unlinked sequences involved in
immunoglobulin gene production (104, 121). C-myc gene


155
The DNAse I sensitivity assay can provide general
locations of DNAse I hypersensitive sites. Considering the
potentially large areas of these "sites", resolution of this
technique may be low. This may explain why site A in
lymphocytes and site 1 in UR HCL 1, HFF, and P3C cells
mapped to different locations (figure 27). Another
possibility is that some DNAse I sites for a particular gene
are cell-type specific, while others are shared between cell
types.
Although a transcript attenuation site in the same area
as sites E and F (figure 27 ) has been proposed for amplified
c-myc in HL-60 cells, a precise location has not yet been
described. The importance of fine mapping P3C c-myc DNAse I
hypersensitive sites in this region can therefore be
appreciated. Sites in the exon 1/intron 1 region were fine
mapped from a 5' direction, and it was found that site 5
mapped to the same location as site F, which was one of the
transcript attenuation sites previously described (figure
27) .
Based on the changes in chromatin structure seen with
the c-myc gene in P3C cells, one would expect to see
decreased levels of myc transcript. This conflicts with what
was observed with northern blot analysis. Therefore


104
Table 9. C-sis, v-erb-B-1, and v-src:actin ratios from slot-
blot analyses of genomic DNA from chondrosarcomas.
CHONDROSARCOMA SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
CS-1
1.25
0.933
0.749
CS-2
0.925
0.995
0.766
CS-3
1.28
0.846
1.28
CS-4
1.14
0.997
0.803
CS-5
1.36
1.38
1.35
CS-6
1.31
1.34
1.37
CS-7
1.24
1.24
1.22
CS-8
1.12
0.976
1.00
CS-9
1.17
1.06
1.24
CS-10
1.04
0.842
1.39
CS-11
1.22
0.784
1.25
CS-12
1.18
1.26
1.30
CS-13
1.29
1.29
1.32
CS-14
1.14
0.944
1.16
CS-15
1.23
0.898
1.19
CS-16
1.31
0.910
1.31
CS-17
1.39
0.805
1.20
CS-18
1.14
0.817
1.20
CS-19
0.699
1.12
0.682
CS-2 0
1.28
1.34
1.08


152
required to produce a cell with an amplified cmyc gene are
unknown. It has been postulated by Alt (2) and others, that
if c-myc genes could regulate expression of other genes, then
maybe amplification is selecting for regulation of various
growth regulatory genes.
The data reported here are consistent with the
hypothesis that increases in c-myc transcript production are
due to gene amplification. A hypothesis that changes in
chromatin structure exist between amplified and single copy
c-myc in MFH cell lines was tested as well. Studies with
DNAse I demonstrated differences in chromatin structure
between amplified and single copy c-myc genes in MFH cell
lines. Changes which accompanied c-myc gene amplification
include the disappearance of a DNAse I hypersensitive site
5' of exon 1, and the appearance of a new site in the first
intron. The meaning of these data can be more fully realized
when compared to those of Siebenlist and Leder (156), and
Siebenlist and Kelly (155) (figure 27). These two studies
reported that changes in chromatin structure accompanied c-
myc structural mutations in Burkitt lymphoma (translocation)
and HL-60 cells (amplification) (figure 27).
DNAse I hypersensitive sites 2 and 3 for HFF and UR HCL
1 cell lines, and site 3 in P3C cells were located in the


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Linda J. Smith
Assistant Professor of Pathology
and Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Har ry LQdtrer
Assistant Professor of
Biochemistry and Molecular Biology
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
May 1989
Dean, College of
Medicine
Dean, Graduate School


180
191. Westin, E.H., F. Wong-Staal, E.P. Gelman, R. Dalla-
Favera, T.S. Papas, J.A. Lautenberger, A. Eva, E.P.
Reddy, S.R. Tronick, S.A. Aaronson, and R.C. Gallo.
1982. Expression of Cellular Homologues of Retroviral
Oncogenes in Human Hematopoietic Cells. Proc. Natl.
Acad. Sci. U.S.A. 79: 2490.
192. Wickstrom, E., T. Bacon, A. Gonzalez, D. Freeman, G.
Lyman, and E. Wickstrom. 1988. Human Promyelocytic
Leukemia HL-60 Cell Proliferation and C-myc Protein
Expression are Inhibited by an Antisense
Pentadecadeoxynucleotide Targeted Against C-myc mRNA.
Proc. Natl. Acad. Sci. U.S.A. 85: 1028.
193. Wu, C. 1984. Two Protein Binding Sites in Chromatin
Implicated in the Activation of Heat-Shock Genes.
Nature 309: 229.
194. Wu, C. 1984. Activating Protein Factor Binds In Vitro
to Upstream Control Sequences in Heat Shock Gene
Chromatin. Nature 311: 81.
195. Wu, C., P.M. Bingham, K.J. Livak, R. Holmgren, and S.C.
Elgin. 1979. The Chromatin Structure of Specific
Genes 1. Evidence For Higher Order Domains of Defined
DNA Sequences. Cell 16: 797.
196. Wyke, J. 1983. Evoloution of Oncogenes From C-src to
V-src. Nature 304: 491.
197. Yamamoto, T., T. Nishida, N. Miyajima, S.Kawai, T. Ooi,
and K. Toyoshima. 1983. The Erb-B Gene of Avian
Erythroblastosis Virus is a Member of the Src Gene
Family. Cell 35: 71.
198. Yuasa, Y., S.K. Srivastava, C.Y. Dunn, J.S. Rhim, E.P.
Reddy, and S.A. Aaronson. 1983. Acquisition of
Transforming Properties by Alternative Point Mutations
Within C-bas/has Human Proto-Oncogene. Nature 303:
775.
199. Zajac-Kaye, M., E. Gelman, and D. Levens. 1988. A
Point Mutation in the C-myc Locus of a Burkitt Lymphoma
Abolishes Binding of a Nuclear Protein. Science 240:
1776.


30
c-myc genes are transcribed in almost all mammalian cells
(levels may be low at about 5-20 molecules of RNA per cell)
whereas most other proto-oncogenes seem to be more tissue
specific (77). For example, c-myb is expressed in
hematopoietic cells but not elsewhere (191). C-sis RNA has
been detected in very few normal cell types, including
rapidly dividing cells of the human placenta and endothelial
cells (8). Since proto-oncogenes are so conserved between
species, it seems likely that their gene products play an
essential role in normal cellular growth and development.
There are several possible mechanisms by which proto
oncogenes may be activated to oncogenes. The first of these
mechanisms involves insertional mutagenesis. The over
expression of a proto-oncogene may occur after the
integration of a new promoter. For instance, the c-mos
proto-oncogene of mice which is biologically inactive after
molecular cloning, can be experimentally converted into a
potent oncogene by additon of a strong transcriptional
promoter (18). Another example of this mechanism comes from
similar activation of the c-Ha-ras proto-oncogene of
rats (40). These oncogenes are created by ligation of cloned
DNA segments, and acquire transforming capabilities because
their transcripts are produced at much higher levels than


2
more common in people who frequently used snuff (117). In
1775, Pott reported a high incidence of scrotal cancer in
men who were chimney sweeps (117). More discoveries of this
nature were made in subsequent years, leading to attempts to
induce cancer in animals with chemicals. One of the first
succesful attempts was made in 1915 by Yamagiwa and Ichikawa
who induced skin carcinomas by the repeated application of
coal tar to the ears of rabbits (117). Subsequent studies
focused on identifying the actual carcinogenic chemicals in
the compounds which could induce cancer. Today, the list of
known carcinogenic chemicals is quite extensive and includes
a wide variety of different chemicals. Examples of
carcinogenic substances include industrial chemicals such as
aromatic hydrocarbons, halogenated hydrocarbons,
nitrosamines, intercalating agents, alkylating agents, nickel
and chromium compounds, asbestos, vinyl chloride,
diethylstilbesterol, and certain naturally occurring
substances such as aflatoxins and radon gas.
Chemical carcinogens are capable of interacting with a
wide variety of cellular macromolecules. This usually
involves the alkylation of nucleophilic groups on nucleic
acids or the reaction of electrophilic groups of the
carcinogen with proteins (142). Some chemical carcinogens


113
Table 15. RNA quantitation of non cell-cycle dependent genes
as determined by slot-blot analysis. *
MUSCLE
BONE
MARROW
CS
MFH
C-SIS:ACTIN
1.0+0.1
1.1+0.1
1.6+1.5
3.1+6.8
V-ERB-B-1:
0.9+0.1
1.2+0.2
1.1+0.7
1.0+0.4
ACTIN
V-SRC:ACTIN
1.0+0.1
1.1+0.1
0.9+0.2
1.2+0.7
TOTAL CASES
9
6
20
23
Values shown are mean generactin ratios


54
The different stimulatory agents which appear to use
different regulatory proteins to enhance c-fos expression,
induce different nuclear proteins (64, 177, 178), and these
are called c-fos related antigens (FRA).
C-fos may be subject to negative regulation as well.
Verma et al. (184) have shown evidence to suggest that cells
may have factors which repress fos transcription, but more
needs to be done before this can be fully characterized and
understood.
Both the products of the v-fos (p55/v-fos) and c-fos
(P62/c-fos) genes may be part of nuclear complexes (60,
184). For example, the c-fos protein complex and several
FRAs bind specifically to a sequence element referred to as
the HeLa cell activator protein 1 (AP-1) binding site (60).
Structural studies and immunoprecipitation analyses were
performed with this complex. One of the Fos-associated
proteins, (p39) was found to be the protein product of c-jun
(138) .
The p39/jun protein is one of the major polypeptides
identified in AP-1 oligonucleotide affinity chromatography
extracts of cellular proteins. The preparations of AP-1
were found to contain c-fos and several FRAs (20). Some of
these proteins seem to bind to the AP-1 site directly, while
c-fos appears to bind indirectly through protein/protein


PROTO-ONCOGENE EXPRESSION IN HUMAN
CHONDROSARCOMA AND MALIGNANT FIBROUS HISTIOCYTOMA
By
JANE CAROLYN STRANDBERG GIBSON
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSTIY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989


174
130. Parker, R.C., H.E. Varmus, and J.M. Bishop. 1984.
Expression of V-src and Chicken C-src in Rat Cells
Demonstrates Qualitative Differences Between pp60 V-src
and pp60 C-src. Cell 37: 131.
131. Payne, G.S., J.M. Bishop, and H.E. Varmus. 1982.
Multiple Arrangements of Viral DNA and an Activated
Host Oncogene in Bursal Lymphomas. Nature 295; 209.
132. Persson, H., L. Hennighausen, R. Taub, W. DeGrado, and
P. Leder. 1984. Antibodies to Human C-myc Oncogene
Product: Evidence of an Evolutionarily Conserved
Protein Induced During Cell Proliferation. Science
225: 687.
133. Poiesz, B.J., F.W. Ruscett, A.F. Gazdar, P.A. Buhn,
J.D. Minna, and R.C. Gallo. 1980. Detection and
Isolation of Type C Retrovirus Particles From Fresh and
Cultured Lymphocytes of a Patient With Cutaneous T-Cell
Lymphoma. Proc. Natl. Acad. Sci. U.S.A. 77: 7415.
134. Pollard, T.D., and R.R. Weihuing. 1974. Actin and
Myosin and Cell Movement. Crit. Rev. Biochem. 2: 1.
135. Pritchard, D.J., R.J. Lunke, W.F. Taylor, D.C. Dahlin,
and B.E. Medley. 1980. Chondrosarcoma: A
Clinicopathologic and Statistical Analysis. Cancer
45: 149.
136. Pulciani, S., E. Santos, A.V. Lauver, L.K. Long, S.A.
Aaronson, and M. Barbacid. 1982. Oncogenes in Solid
Human Tumors. Nature 300: 539.
137. Pulciani, S., E. Santos, A.V. Lauver, L.K. Long, and M.
Barbacid. 1982. Transforming Genes in Human Tumors.
1982. J.C.B. 51: 61.
138. Rauscher, F.J., D.R. Cohen, T.Curran, T.J. Bos, P.K.
Vogt, D. Bohman, R. Tijan, and B. Franza. 1988.
Fos-Associated Protein p39 is the Product of the
jun Proto-Oncogene. Science 240: 1010.
139. Rave, N., R. Crkvenjakov, and H. Boedtker, 1979.
Identification of Procollagen mRNAs Transferred to
Diazobenzyloxymethyl Paper From Formaldehyde Agarose
Gels. NAR 6: 3559.


101
Table 6. C-myc, c-Ha-ras, and c-fos:TK ratios from slot-blot
analyses of total cellular RNA from chondrosarcomas. Also
shown are TK:actin ratios which were used as molecular
measures of cell cycle.
CHONDROSARCOMA SAMPLE
c-mvc
TK
c-Ha-ras
TK
c-fos
TK
TK
actin
CS-1
0.693
0.522
1.02
1.16
CS-2
1.02
0.971
1.06
0.752
CS-3
0.911
1.64
0.934
0.596
CS-4
0.531
0.218
0.292
1.16
CS-5
0.452
0.594
2.49
1.19
CS-6
0.468
0.195
1.60
2.19
CS-7
1.41
1.02
1.35
0.608
CS-8
1.03
0.575
1.16
0.861
CS-9
1.19
0.887
1.43
0.653
CS-10
0.804
0.666
1.06
1.50
CS-11
2.49
1.62
0.982
0.370
CS-12
0.726
0.383
1.14
0.698
CS-13
2.17
0.502
0.894
0.839
CS-14
1.80
1.90
0.976
0.301
CS-15
0.812
0.353
1.20
1.52
CS-16
**
0.466
0.927
2.47
CS-17
0.416
0.468
1.00
2.56
CS-18
1.10
1.46
0.760
0.996
CS-19
2.63
0.233
0.934
0.331
CS-2 0
1.23
1.06
1.54
0.536
**
C-myc was not detectable


88
DNAse I treated DNA samples (samples with optimum
visualization of DNAse I generated bands from previous
analysis) from P3C (0.2 and 0.5 ug/ml) and HFF (0.5 ug/ml)
cell lines were restricted with Sea I. Five ug aliguots of
these samples along with marker DNAs generated as described
above were electrophoresed in 1.5 percent agarose gels (65
volts, 20 hr), and blotted onto Zetabind. Lambda DNA
digested with Eco R 1 and Hind III was run on either side of
the gel to assure that the gel ran evenly. Prehybridization,
hybridization (2.0 X 10 E 8 cpm/ml/ 10 E8 cpm/ug), and
washing conditions were identical to those described for
Southern blotting.
Polyacrylamide Gel Electrophoresis (PAGE)
The discontinuous system for PAGE as described by
Laemmli (101) was used in this analysis. Stacking gels were
4 percent acrylamide (total) in 0.125 M Tris-HCl, pH 6.8, and
0.1 percent SDS. Separating gels were 8.5 percent total
acrylamide in 0.375 M Tris-HCL, pH 8.8, and 0.1 percent SDS.
Both gels were cross-linked with 2.7 percent bis acrylamide,
and polymerization was catalyzed with 0.005 percent TEMED,
and 0.05 percent ammonium persulfate.


86
C-MYC EXON 1 EXON 2
SCA l/XHO 1
355 BP
PROBE
1 1
SCAI/PVUII
805 BP
1KB
SCAI/BSMI
SCA l/MAE III
920 BP
970 BP
y = TRANSCRIPT ELONGATION BLOCK (BL)
Figure 8. Location of the Sea I/Xho I probe and other
restriction sites in and around c-myc exon 1 used in fine
mapping of DNAse I hypersensitive sites from the 5' direction
in P3C and HFF cell lines. These sites were used to generate
P3C DNA fragments of known sizes, which were internal size
markers on the mapping blot (see materials and methods).
Also shown are the c-myc promoters (PO, PI, and P2), and
known c-myc transcript elongation block sites which are
indicated by solid arrows.


Ill
10
2.5
MT-1
MT2
MT-3
MT4
MT5
MT6
MT7
MT8
MT0
MT10
MT11
MT12
MT-13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 12. Slot-blot of total cellular RNA from MFHs
hybridized with the TK probe. Quantities of 10, 5, and 2.5
ug of RNA were slot-blotted onto nitro-plus 2000, and
hybridized with the TK probe (3.0 x 10 E6 cpm/ 10 E8
cpm/ug).


181
200. Zimmerman, K., G. Yancopoulos, R. Collum, R. Smith, N.
Kohl, K. Denis, M. Nau, O. Witte, D. Toran-Allerand, C.
Gee, J. Minna, and F. Alt. 1986. Differential
Expression of Myc Family Genes During Murine
Development. Nature 319; 780.


92
A B C D E F
Figure 9. Formaldehyde gel electrophoresis of 10 ug of HL-
60 RNA following alkaline degredation with 0.2 N NaOH and
neutralization with 0.1 M Tris, Ph 7.5 after (a) 0 min (b)
0.5 min (c) 1 min (d) 2 min (e) 5 min and (f) 30 min. Lane a
shows totally intact RNA, lanes b, c, d, and e intermediate
degredation, and lane f, extensively degraded RNA.


45
truncation could have several possible consequences, any of
which may contribute to neoplastic transformation. For
instance, only a small fraction of the erb-B-1 oncogene
product reaches the plasma membrane (17, 148). The remainder
never leaves the golgi apparatus and retains an immature
mannose-rich form. This is in contrast to the normal
behavior of intact transmembrane receptors.
The EGF receptor is returned to the interior of the
cell after binding ligand, a regulatory mechanism seemingly
designed to protect the cell from an over abundance of
stimuli. By contrast, the product of the erb-B-1 oncogene
cannot bind ligand and may be located permanantly to the
surface of the cell (17, 148, 181).
The EGF receptor displays the full force of its protein
kinase activity only after binding ligand. The erb-B-1
oncogene product is presumably released from this dependence
and is constitutively active (17). The kinase activities
associated with the erb-B-1 oncogene product are
constitutive, and the appearance of this protein on the
plasma membrane seems to be a prerequisite for transformation
(14) .
With respect to tumor activity, it has been reported
that abnormally high levels of the erb-B-1 oncogene were


57
unknown. The N-myc gene may play a special role in certain
types of neural tumors. Also, N-myc amplification events may
be specially targeted in the precursor cells of these tumors
(2) .
The L-myc gene was isolated by two independent methods.
The gene was first isolated on the basis of its amplification
in a subset of SCLCs (123). The gene was independently
isolated from unamplified genomes on the basis of its
homology to c-myc and N-myc (2). So far, activation of the
L-myc gene has only been observed in some SCLCs. Details
with respect to the structure and transforming potential of
L-myc call for further study, and will not be discussed here.
The expression of the c-myc gene has been shown to
follow a fixed relation to cell cycle. Growth arrested
fibroblasts (serum deprivation) in GO show a burst of c-myc
transcription during the G0/G1 transition when stimulated to
divide by either serum addition or insulin. The c-myc
transcript levels then decrease slowly as the cells proceed
through the cell cycle, and are present at basal levels
during S phase (19, 175). Nuclear run on assays with serum
released GO fibroblasts suggest that c-myc expression is
primarily regulated post-transcriptionally, at the level of
message degradation (19).


115
Tables 16 and 17 indicate the gene copy numbers for TK,
actin, and the six genes evaluated in this study as
determined by DNA slot-blot guantitation. C-myc:actin ratios
for all 9 muscle, 6 bone marrow and 20 chondrosarcoma tissues
show c-myc to be a single copy gene. The same is true for
17 of the MFHs, while 6 of these tumors have 2 or more copies
of c-myc. C-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-src:actin
values indicate that these genes are single copy in all
normal and neoplastic tissue groups.
The 6 MFHs with 2 or greater copies of c-myc are
samples MT-8, MT-16, MT-17, MT-18, MT-20,and MT-22. When RNA
values for this subset of MFHs were examined, it was found
that these 6 MFHs have increased c-myc transcript levels as
well (figure 15). Therefore, 17 of the MFHs have a mean c-
myc:TK ratio of 1.0 which is not significantly different
than that of chondrosarcomas (1.2), or bone marrow tissues
(0.9). The 6 MFHs with 2 or greater copies of myc have a
mean myc:TK ratio of 2.0 which is significantly higher than
the other groups.
These 6 MFHs also have higher freguencies of cell
division as measured by TK:actin ratios. Statistical
evaluation of the relationship between c-myc gene copy number
and cell division was performed using chi sguare analysis.


127
9.6 KB
P3C URHCL1
2010
a*
(UGDNA)
5 2 2010
4.'. .-I-'. \ ''ll ..
*>-* ?*; 4 ..
t: >.- .. '-<*
. /* r '
J * rv *
.^¡vZ : il* i'
Figure 17. Southern blot analysis of c-myc DNA copy number
in UR HCL 1 and P3C cell lines. Twenty, 10, 5, and 2 ug of
DNA from each cell line were restricted with Hind III,
electrophoresed through 0.8 percent agarose gels and
hybridized with the pGEM H MYC probe (See materials and
methods). Band size was determined by lambda DNA marker
bands produced by restriction with Hind III.


120
Table 18. RNA TKractin ratios and c-myc gene copy numbers
for MFHs. Chi square analysis showed a positive correlation
between c-myc gene copy number and cell division as measured
by TKractin ratios (p<0.05). The cut off point was 2 for c-
myc gene copy number and for TKractin values.
11
1
10
1
9
8
7
6
C-myc
Gene 5
Copy
Number 4
3
2
1
1
1
1
6
4 5 2
1
12 3 4
TK
Act in


60
regions of the DNA must be accesible to binding.
Nuclease hypersensitive sites in chromatin are thought to be
regions that are open, and will allow DNA interaction
with proteins (70, 72). Therefore, it is thought that these
regions are specific for regulation of genes by cis and trans
acting factors. These protein accessible regions are
identified by their susceptibility to cleavage with
nuclease, and have been described to be twice as sensitive
as other areas of chromatin (72). DNAse I
hypersensitive sites are thought to represent approximately
1 percent of the entire genome (72) They were first
identified by Varshavsky (182) and by Scott and Wigmore
(151) who did studies with SV 40 chromatin. The presence of
these sites in chromatin of mammalian cells was discovered by
Wu and Elgin (195). These sites have been found in the
chromatin of plants, animals fungi, and in viral genomes (72,
189). Therefore they are considered to be very important in
the field of biology, and to the understanding of how
genetic regulation occurs among various species of
eukaryotes.
The indirect end labeling technique is most commonly
used in mapping locations of DNAse I hypersensitive sites.
This follows isolation of nuclei, treatment with DNAse I


102
Table 7. C-sis, v-erb-B-1, and v-src:actin ratios from slot-
blot analyses of total cellular RNA from chondrosarcomas.
CHONDROSARCOMA SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
CT-1
2.42
0.821
0.993
CT-2
1.87
1.37
1.20
CT-3
0.637
0.060
0.841
CT-4
2.60
2.70
0.798
CT-5
1.22
2.57
0.888
CT-6
2.29
0.928
0.783
CT-7
0.180
2.31
1.01
CT-8
0.604
0.714
0.863
CT-9
6.08
1.86
0.697
CT-10
2.32
1.26
1.29
CT-11
0.130
1.28
0.900
CT-12
2.14
1.15
0.700
CT-13
0.457
0.979
0.801
CT-14
0.135
0.561
0.777
CT-15
3.38
0.822
1.33
CT-16
0.247
0.549
1.11
CT-17
1.79
0.511
0.890
CT-18
1.97
0.448
0.697
CT-19
0.637
0.688
0.770
CT-2 0
0.148
0.605
0.838


121
and DNA slot-blots. This serves as a control for a single
copy, constitutively expressed gene for RNA and DNA
analyses. It also demonstrates consistency of quantity and
quality of RNA.
Since quantitative signal intensity limitations of the
slot-blotting assay do not allow determination of gene copy
numbers greater than two, c-myc copy numbers for the 6 MFHs
with 2 or greater copies of the gene were further evaluated
using Southern blot/ DNA dilutional analysis.
Tumor DNA samples from 6 MFHs (mt-8, mt-16, mt-17, mt-
18, mt-20, and mt-22) and from normal muscle tissues were
prepared as described above. Aliquots of 10, 5, 2, and 1 ug
of tumor DNA and 10 ug of normal muscle DNA were analyzed
using Southern blot hybridization with the p GEM H MYC probe
(table 1). This probe hybridizes to a 9.6 kb fragment of
Hind III restricted genomic DNA. Band sizes were determined
by comparison to lambda DNA marker bands produced by
restriction with Hind III (figure 16). C-myc gene copy
numbers were determined by laser densitometry
(transmittance). Values for tumor DNA are normalized to
those of muscle DNA which are controls for single copy c-myc.
These 6 MFHs were found to have c-myc gene copy numbers of
(A-F) 8.7, 8.4, 9.6, 9.9, 10.6, and 2.2, which corresponded


175
140. Robbins, K.C., H.N. Antoniades, S.G. Davare, M.W.
Hynkapiller, and S.A. Aaronson. 1983. Structural and
Immunological Similarities Between Simian Sarcoma Virus
Gene Products and Human Platelet Derived Growth Factor.
Nature 305: 605.
141. Rodriguez-Pena, A. and E. Rozengurt. 1985. Serum Like
Phorbol Esters Rapidly Activate protein Kinase C in
Intact Quiescent Fibroblasts. EMBO J. 4: 71.
142. Ruddon, R.W. 1987. Cancer Biology Second Edition.
New York, Oxford University Press.
143. Rysek, R.P., S.I. Hirai, M. Yaniv, and R. Bravo. 1988.
Transcriptional Activation of C-jun During the G0/G1
Transition in Mouse Fibroblasts. Nature 334; 535.
144. Santos, E., E.P. Reddy, S. Pulciani, R.J. Feldmen, and
M. Barbacid. 1983. Spontaneous Activation of a Human
Proto-oncogene. Proc. Natl. Acad. Sci. U.S.A. 80:
4679.
145. Schecter, A.L., F.F. Stern, L. Vaidyanathan, S.J.
Decker, J.A. Drebin, M.I. Greene, and R.A. Weinberg.
1984. The Neu Oncogene: An Erb-B Related Gene
Encoding 185,000 Mr Tumour Antigen. Nature 312: 513.
146. Scherr, C.J., C.W. Rettenmier, R. Sacca, M.F. Roussel,
A.T. Look, and E.R. Stanley. 1985. The C-fms Proto
-Oncogene Product is Related to the Receptor for the
Mononuclear Phagocyte Growth Factor, CSF-1. Cell 41:
665.
147. Schimke, R. 1984. Gene Amplification in Cultured
Animal Cells. Cell 37: 705.
148. Schmidt, J.A., H. Beug, and M.J. Hayman. 1985.
Effects of Inhibitors of Glycoprotein Processing on
the Synthesis and Biological Activity of the Erb-B
Oncogene. EMBO J. 4: 105.
149. Schwab, M., K. Alitalo, H.E. Varmus, J.M. Bishop, and
A.D. George. 1983. A Cellular Oncogene (C-Ki-ras) is
Amplified, Overexpressed, and Located Within
Karyotypic Abnormalities in Mouse Adrenocortical tumor
Cells. Nature 303: 497.


78
sodium dodecyl sulfate (SDS). The blots were then washed
twice again at 50 C with 0.IX SSC, 0.1 percent SDS for 30
min each and exposed to preflashed X-ray film for 36-48 hr at
70 C with intensifying screens.
Slot-blots were rehybridized after treatment of the
membrane to remove bound probe. This was accomplished by
pouring 1 liter of 0.1 X SSPE, 0.1 percent SDS heated to 90 C
over the blots. The solution was then cooled to 70 C and
removed. (187)
Southern Blot Analysis
Genomic DNA was prepared as described above from tissue
samples and cell lines. Aliquots of DNA were restricted with
appropriate restriction endonucleases and electrophoresed
through 0.8 percent agarose gels (65 volts, 16 hr).
Digested DNA was then transferred to Zetabind (AMF Cuno,
Meriden, CT), pre-washed in 0.1 X SSC, 0.1 percent SDS at 65
C for 1 hr, and hybridized (2 X 10 E6 cpm/ml/10 E8 cpm/ug)
using pre-hybridization and hybridization conditions
previously described for slot-blotting (112, 160). Post
hybridization washes were performed by washing the membrane
at room temperature for 15 min, once with 2 X SSC,


65
Burkitt lymphoma cell line (BL-31) and a normal B cell line.
Three different hypersensitive patterns which differed in
relative band intensities were observed. These correlated
with the three different transcriptional states of the c-myc
gene examined in this study (normal B cell myc, unrearranged
BL-31 c-myc, and translocated BL-31 c-myc). The locations of
hypersensitive sites which were observed were identical to
those for HL-60 cells (sites A,B,C,D) and are also shown in
figure 4.
Other groups working with Burkitt lymphoma have mapped
sites in the first intron and 3' region of exon 1. These
were later found to be transcription attenuation sites.
The loss of transcription elongation blocks at these sites is
now thought to be a possible candidate for deregulation of
myc in Burkitt lymphomas. Cesarman et al. (25) mapped a
previously found DNAse I hypersensitive site (9, 10, 49) to a
region near a Pvu II site in exon one. They reported that
23/26 Burkitt lymphoma cell lines and biopsies had point
mutations at various sites in a specific region extending 34
bases 5' and 38 bases 3' to the Pvu II site in exon 1 (site E
figure 4). These point mutations accompanied changes in
transcription, namely the removal of a block to transcription
mapped to the same region.


63
it was presumed that gene dosage effects are responsible for
observed increases in c-myc transcript. In both these
instances, gross structural mutations appear to be
responsible for observed changes in transcript levels. It
has only been recently that we have begun to understand the
effects these aberrations have on c-myc regulation or
deregulation as the cases may be.
During the past few years, data obtained from chromatin
structure analyses have demonstrated that changes in c-myc
gene regulatory sites accompany gross structural
abnormalities in both transocated and amplified states of
this gene. DNAse I sensitive sites in the 5' region of
exon 1 in HL-60 cells and Burkitt lymphoma cells have been
investigated in 2 separate studies by Siebenlist et al. (155,
156). DNAse I hypersensitive sites in HL-60 cells were
studied before and after differentiation with DMSO. Results
showed that 4 DNAse I sensitive sites were present in
untreated HL-60 cells (sites A, B, C, and D, figure 4). When
differentiation was induced with DMSO, site B was lost.
Further studies showed that the loss of this site accompanied
a timely decline in c-myc transcript production.
In a separate study, DNase I hypersensitive sites were
studied in both normal and rearranged c-myc alleles in a


38
and that in RSV transformed cells there is a buildup of
intermediates in the inositol lipid breakdown pathway. They
postulated that the primary target of pp60src might be lipid
and not protein.
Much remains unknown about the biochemical action of
pp60src and the rest of the tyrosine kinase family.
Phosphorylation of tyrosine seems to be a general phenomenon
for initiating cell division and inappropriate tyrosine
kinase activity could explain the loss of growth control
associated with transformed cells. The phosphorylation of
inositol lipids by at least two of the tyrosine kinases src
and ros is interesting, but the significance in transformed
cells remains to be determined.
Ras Proteins
The 21,000 dalton (p21) proteins of three human cellular
ras genes; Harvey (Ha), Kirsten (Ki), and N-ras are very
closely related in seguence. In the first 150 amino acids
there are a maximum of 14 amino acid differences between the
three proteins. The ras proteins therefore have been highly
conserved throughout evolution, and are thought to play an
essential role in cell growth (77). The ras p21s are


Copyright 1989
by
Jane Carolyn Strandberg Gibson


22
between the primitive cell and some differentiated
phenotypes, and a branching system reflecting close
relationships between some phenotypes and not others. This
model also recognizes the myofibroblast and the
chondroosteoblast.
Chondrosarcoma and Malignant Fibrous Histiocytoma
Chondrosarcoma is a malignant tumor of cartilage (figure
2). It has been well established that the basic
proliferating tissue is cartilagenous (39). Primary
chondrosarcomas are tumors which can arise de novo in
extraskeletal tissues or in mixed tumors such as teratomas.
The majority of chondrosarcomas are "myxoid". Those composed
of hyaline cartilage are more uncommon. Secondary
chondrosarcomas arise most commonly in osteosarcomas, and can
sometimes develop in patients with multiple exostoses. Rarely
do they develop from an enchondroma, a benign cartilagenous
tumor. In addition to primary and secondary chondrosarcomas
there are dedifferentiated chondrosarcomas which give rise to
more malignant tumors such as osteosarcomas, fibrosarcomas,
or malignant fibrous histiocytomas (MFH) (39).


28
pain and swelling are the most frequent symptoms. As with
chondrosarcomas, roentgenographic analysis is helpful in
preliminary diagnosis of the tumor.
Histiologic features of these tumors usually include a
high degree of variation, multinucleated tumor cells,
nuclear hyperchromasia and a high mitotic activity. A
typical pattern of growth can be described as the arrangement
of tumor cells around a central point, producing radiating
spokes, grouped at right angles to each other (storifiorm
pattern).
With respect to prognosis, subcutaneous tumors generally
have a better prognosis than the more deep seated lesions.
The recurrence of MFHs is said to be 44 percent with a two
year survival of 60 percent (190). There is no widely
accepted grading system for deep seated MFHs as for
chondrosarcomas. Other important prognostic factors include
mitotic index, degree of cellular polymorphism, tumor size
and tumor stage (180).
Staging criteria for both chondrosarcomas and MFHs are
those described by Enneking et al. (52) for sarcomas
originating from mesenchymal tissue of the musculoskeletal
system. This system takes into account surgical grade,
local extent, and presence or absence of regional


35
the kinase activity, and a corresponding domain is found in
other tyrosine kinases with a high degree of amino acid
conservation between them. This kinase domain is also found
in the cytoplasmic cyclic AMP dependent serine protein
kinases in mos and raf, and in serine specific kinases
located in the cytosol (118). A similar sequence domain has
been found in the membrane-bound receptor-like products erb-
B1, fms, and neu, all of which have tyrosine kinase
activity, indicating a distant evoloutionary relationship
between all protein kinases (77).
Originally, it was thought that this activity was
exclusive to oncogenes. However, a protein derived from the
c-src gene was isolated from normal cells and shown to have
tyrosine specific kinase activity (31). Since then, other
membrane-bound cellular proteins with similar activities have
been identified. These have offered clues as to what the
oncogene kinases may be doing. It has been shown that the
receptors for platelet derived growth factor (PDGF) and
insulin-like growth factor (IGF) have a tyrosine-specific
kinase activity (77). It has also been proposed that
tyrosine phosphorylation is an early event in the
transduction of mitogenic signals through the membrane.
Although pp60src resides at the inner surface of the


110
Table 14. RNA quantitation of cell-cycle dependent genes as
determined by slot-blot analysis. *
MUSCLE
BONE
MARROW
CS
MFH
TK:ACTIN
ND
1.1+0.1
1.1+0.7
1.5+0.8
C-MYC:TK
ND
0.9+0.2
1.2+0.7
1.3+0.4 **
C-HA-RAS:TK
ND
1.0+0.2
0.8+0.5
0.9+0.9
C-FOS:TK
ND
1.0+0.1
1.1+0.4
1.00.4
TOTAL CASES
9
6
20
23
* Values shown
are mean gene
:actin and
gene:TK
ratios
** 17 MFHs with single copy c-myc had a mean myc:TK ratio of
1.0+0.2. Six MFHs with 2 or greater copies of myc had a
mean myc:TK ratio of 2.0+0.1.
ND Not detectable


CHAPTER 2
REVIEW OF THE LITERATURE
Differentiation of Mesenchyme
The precise pathways taken by mesenchymal cells
undergoing differentiation have been somewhat of a con
troversial issue. Therefore, two proposed models of mesen
chymal differentiation will be presented here. The first of
these models, the radial model of mesenchymal
differentiation, is a currently accepted model proposed by
Hadju (76). Each soft tissue and hematopoietic phenotype
directly originates from a primitive undifferentiated
mesenchymal cell. In this scheme, there are no precursor
cells and no branching of cell types. Rather, the
differentiated phenotypes are separated from each other
only by a primitive mesenchymal cell. Because of this,
close relationships exist between phenotypes.
More recently, another model has been proposed by Brooks
(23) and is shown in figure 1. The two novel features of
this model are the insertion of an intermediate precursor
20


PROTO-ONCOGENE EXPRESSION IN HUMAN
CHONDROSARCOMA AND MALIGNANT FIBROUS HISTIOCYTOMA
By
JANE CAROLYN STRANDBERG GIBSON
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSTIY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989

Copyright 1989
by
Jane Carolyn Strandberg Gibson

To my husband Ron, to my grandparents, to Karen and
Ken, and to Mom and Dad; whose love, support, and belief
me will always be my inspiration.
in

ACKNOWLEDGEMENTS
I would like to thank the members of my committee, Dr.
Byron Croker, Dr. Warren Ross, Dr. Lindsey Hutt-Fletcher, Dr.
Linda Smith, and Dr. Harry Ostrer, for their assistance and
advice. I would especially like to thank Dr. Croker for his
faith in my abilities, his guidance, encouragement, and
friendship.
I would also like to thank Dr. Susan Chrysogelos for all
her help during the past 18 months. I am most appreciative
of all she has taught me. Special thanks go to Dr. Cheryl
Zack and Herb Houck for all their help and encouragement, to
Jerry Phipps for his assistance in obtaining surgical
specimens, and to my fellow graduate students for their help
and encouragement. In addition, I am most grateful to my
friends who have been very supportive, particularly Gail
Waldman, Patty Leginus, and Patty DeHaan.
Lastly, I would like to thank my husband Ron, my
grandparents, Karen, and Ken for all their much needed love,
support and encouragement. I have no words which could
adequately express my gratitude to my Mom and Dad. Without
them none of this would have ever been possible. They have
taught me so many things, but I think the most important
lesson I have learned from them is that family is one of
life's greatest treasures. I will never forget that. I will
never forget everything they have done for me.
IV

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS V
ABSTRACT vii
CHAPTERS
1 INTRODUCTION 1
Mechanisms of Tumor Development 1
Multistep Carcinogenesis 7
The Neoplastic Phenotype and Steps of Tumor
Progression 12
Specific Questions Addressed During the Course
of This Project 17
2 REVIEW OF THE LITERATURE 2 0
Differentiation of Mesenchyme 20
Chondrosarcoma and Malignant Fibrous Histio
cytoma 22
Proto-Oncogenes 29
Biochemistry of Oncogene Products 3 4
Cytoplasmic Kinases 34
Ras Proteins 38
Growth Factors and Their Receptors 40
Nuclear Proteins 42
Molecular, Biological, and Physiological Char
acteristics of Proto-Oncogenes Examined in
This Study 44
Growth Factor Related 44
Protein Kinases 47
Nuclear Related Proto-Oncogenes 50
Chromatin Structure Analysis of the C-myc
Gene 59
Relevance To This Project 66
3 MATERIALS AND METHODS 67
Slot-Blotting of RNA and DNA 67
Preparation of Total Cellular RNA 67
Preparation of Genomic DNA 68
RNA Slot-Blotting 69
DNA Slot-Blotting 70
v

Preparation of Radiolabeled Probes 71
Nick Translated Probes 71
Probes Labeled by Random Primer Extension 77
Hybridization of Slot-Blots 77
Southern Blot Analysis 78
Northern Blot Analysis 79
Chromatin Structure Analysis 80
Cell Lines Used in Chromatin Structure
Analysis 80
Preparation of Nuclei 81
Isolation of Genomic DNA From DNAse I Treated
Nuclei 82
Chromatin Structure/Fibroblast Cell Synchrony
Experiment 83
Strategy for Fine Mapping DNAse I Hyper
sensitive Sites 3' to C-myc Exon 1 85
Polyacrylamide Gel Electrophoresis (PAGE) 88
Western Blotting and Immunoperoxidase Assay 89
4 RESULTS 91
Quantitation of Moderately Degraded RNA Using
the Slot-Blotting Technique 91
RNA Slot-Blot Results 96
DNA Slot-Blot Results and Determination of C-myc
Gene Copy Number 109
Regions Contained in the C-myc Amplicon 123
Chromatin Structure Analyses 125
C-myc Gene Copy Number and Transcript Levels
in P3C, UR HCL 1, HFF and ST 486 Cell
Lines 125
Locations of DNAse I Hypersensitve Sites in
P3C, UR HCL 1, HFF, and ST 486 Cell Lines... 126
Chromatin Structure of the C-myc Gene During
the G0/G1 Transition in the HFF Normal Human
Fibroblast Cell Line 134
Fine Mapping Analysis of DNAse I Hyper
sensitive Sites in P3C Cells From a 5'
Direction 139
C-myc Protein Levels in the P3C, UR HCL 1, HFF
and ST 486 Cell Lines 143
5 DISCUSSION 146
REFERENCES 160
BIOGRAPHICAL SKETCH 182
VI

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PROTO-ONCOGENE EXPRESSION IN HUMAN
CHONDROSARCOMA AND MALIGNANT FIBROUS HISTIOCYTOMA
By
Jane Carolyn Strandberg Gibson
May, 1989
Chairman: Dr. Byron P. Croker
Major Department: Pathology and Laboratory Medicine
Total cellular RNA and genomic DNA were extracted from
20 chondrosarcomas, 23 malignant fibrous histiocytomas (MFH),
9 muscle, and 6 bone marrow specimens. Levels of RNA and
gene copy numbers of c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-
1, v-src, thymidine kinase (TK) and actin were quantified
densitometrically from slot-blot analysis. C-myc, c-Ha-ras,
and c-fos transcript levels are undetectable in muscle. Mean
c-myc:TK ratios do not differ significantly among groups of
bone marrows, chondrosarcomas and 17 MFHs (p > 0.05). Six
MFHs have a mean c-myc:TK ratio of 2.0 which is significantly
higher than the other groups (p > 0.05). Intergroup
comparisons between bone marrows, chondrosarcomas, and
vii

MFHs show no significant differences in expression of c-Ha-
ras, c-fos, v-erb-B-1, and v-src. C-sis RNA levels are 2-to
3- fold greater in MFHs. DNA analysis shows c-myc to be a
single copy gene in all tissues except 6 MFHs which have
between 2 and 11 copies. C-myc amplicons were found to be
large, extending at least 50 kb 5' to the c-myc promoter and
slightly 3' of exon 3. These same tumors have increased
levels of c-myc transcript as determined from RNA analysis.
C-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-src are single copy
genes in all tissues.
Chromatin structure studies show that amplified c-myc
in P3C cells does not contain a DNAse I hypersensitive site
near the P0 promoter region 5' to exon 1, known to be
involved in maintaining c-myc transcript production in HL-
60 cells. Additionally, a new site is present in a region
known to contain a block to transcription elongation in
Burkitt lymphomas. These changes are not seen during normal
upregulation of c-myc in GO serum released fibroblasts (G0/G1
transition).
These data suggest that increased levels of c-myc
expression are due to gene dosage, while those of c-sis
viii

are due to some unknown mechanism other than gene
amplification. Additionally, differences in chromatin
structure between amplified and single copy c-myc in
MFH cells may represent a compensatory response to increased
c-myc transcript production. Increased levels of c-myc
protein provide further evidence that c-myc may be an
oncogene in these cells.
IX

CHAPTER 1
INTRODUCTION
Mechanisms of Tumor Development
Fundamental requirements for successful prevention and
sometimes treatment of cancer are knowledge and understanding
of its causitive factors. This task is not an easy one by
any means. Agents having abilities to contribute to or
cause cancer are called carcinogens. Studies to determine
the roles these agents play in neoplastic processes have
focused on three general classes of carcinogens: chemical,
physical, and biological.
Carcinogenic chemicals and ionizing radiation are known
to affect DNA at a structural level and to be mutagenic
under certain conditions. Therefore, one of the long
standing theories of carcinogenesis has been that cancer is
caused by genetic mutations.
Evidence that chemicals can induce cancer has been
reported for more than two centuries. The first observations
of chemically induced cancer were made in humans. The first
of these was in 1761 when Hill noticed that nasal cancer was
1

2
more common in people who frequently used snuff (117). In
1775, Pott reported a high incidence of scrotal cancer in
men who were chimney sweeps (117). More discoveries of this
nature were made in subsequent years, leading to attempts to
induce cancer in animals with chemicals. One of the first
succesful attempts was made in 1915 by Yamagiwa and Ichikawa
who induced skin carcinomas by the repeated application of
coal tar to the ears of rabbits (117). Subsequent studies
focused on identifying the actual carcinogenic chemicals in
the compounds which could induce cancer. Today, the list of
known carcinogenic chemicals is quite extensive and includes
a wide variety of different chemicals. Examples of
carcinogenic substances include industrial chemicals such as
aromatic hydrocarbons, halogenated hydrocarbons,
nitrosamines, intercalating agents, alkylating agents, nickel
and chromium compounds, asbestos, vinyl chloride,
diethylstilbesterol, and certain naturally occurring
substances such as aflatoxins and radon gas.
Chemical carcinogens are capable of interacting with a
wide variety of cellular macromolecules. This usually
involves the alkylation of nucleophilic groups on nucleic
acids or the reaction of electrophilic groups of the
carcinogen with proteins (142). Some chemical carcinogens

3
are known to react with cellular RNA as in the case of
dimethylnitrosamine (105), but most react with DNA (142).
Reaction of chemical carcinogens with DNA can facilitate the
induction of heritable changes in cells and may lead to
malignant transformation. Thus, it is generally believed
that this is the most likely mechanism for chemical
initiation of carcinogenesis. Representative agents from
virtually all classes of chemical carcinogens have been shown
to affect DNA in some way. The actions of many of these have
been found to result in the formation of base-adducts. The
potential biological conseguences of these are are several:
Base adducts may stabilize intercalation reactions. For
example, if the flat planar rings of a polycyclic hydrocarbon
were stably integrated between the stacked bases of double
helical DNA, the helix would be distorted. This could lead
to a frame-shift mutation which would occur during DNA
replication past the point of intercalation (86).
Many of the base adducts formed by carcinogens involve
modification of N-3 or N-7 positions on purines. This
induces an instability in the glycosidic bond between the
purine base and deoxyribose. The destabilized structure can
then undergo cleavage by DNA glycosylase, resulting in loss
of the base, and creation of an apurinic site in the DNA.

4
This open space can then be filled by any base, resulting in
a base transition (purine-pyrimidine base change) (110).
Interaction with some carcinogens has been shown to
favor a conformational transition of DNA from its usual
double-helical B form to a Z DNA form (125). This could
alter the ability of certain genes to be transcribed, since
B-Z conformational transitions are thought to be involved in
regulating chromatin structure (142).
Both X-rays and ultraviolet radiation also produce
damage to DNA. As with chemical carcinogens, this damage
induces DNA repair processes, some of which are error prone
and lead to mutations. Studies have shown that the
development of malignant transformations in cultured cells
after irradiation requires fixation of the initial damamge
into a heritable change. This is experimentally accomplished
by allowing clonal proliferation and expression of the
transformed phenotypes (109).
In addition to chemical and physical carcinogens,
biological carcinogens exist as well. It was long suspected
that various forms of cancer, particularly certain lymphomas
and leukemias, were caused or at least cocaused by
transmissable viruses. The known carcinogenic effects of
certain chemicals, irradiation, chronic irritation and

5
hormones did not fit with the notion of an infectious origin
of cancer. Early studies attempted to transmit malignant
disease by inoculation of filtered extracts prepared from
diseased tissues. It was later demonstrated by Ellerman and
Bang in 1908 that chicken leukemia could be transmitted by
cell-free filtered extracts (50). They were among the first
to demonstrate the viral etiology of this disease. An example
of a virus thought to cause cancer in humans is seen with
Human T-cell leukemia virus 1 (HTLV-1), a transmissible
virus thought to cause leukemia (133).
Other oncogenic RNA viruses are capable of participating
in transformation. Understanding of the molecular mechanisms
involved in cellular transformation by these viruses is based
on the Nobel prize winning work of Baltimore and Temin (5,
173). In the early 1960s, Temin demonstrated that mutations
in the Rous sarcoma virus (RSV) genome of RSV-infected
chicken cells could be induced at a high rate. It was also
shown that mutation of an RSV gene present in an infected
cell often changed the morphology of the cell, and the virus
genome was stably inherited by progeny cells (173). This led
to the notion that virus genetic information was contained in
a regularly inherited structure of the host cell as a
"provirus" integrated into the host cell's genome. The

6
problem with the provirus hypothesis was that there was no
known way for the tumor virus RNA to be converted into DNA
and integrated. Temin and Baltimore independently
demonstrated the presence of a virus coded, RNA directed, DNA
polymerase activity now known as reverse transcriptase (4,
174). As a result of this work, Temin proposed the
"protovirus" theory in which he postulated that genomes of
oncogenic viruses arose during evolution from normal
cellular DNA altered by some exogeneous carcinogen (173).
The normal cellular homologues of viral oncogenes (v-
onc) are known as proto-oncogenes (c-onc). These are thought
to have been evolutionarily conserved in the genomes of most
animal cells over a long period of time. They seem to be
involved in control of cellular growth and proliferation. It
is likely that their activations to oncogenic states occur
from one or more rare events such as translocations,
amplifications, point mutations or other aberrations of key
nucleotide sequences (102). Highly oncogenic viruses
presumably arose from genetic recombination events between
viruses of low oncogenicity and proto-oncogenes. The
combination of these two elements seems to have produced
highly transforming viral genomes. Many of these viruses are
replication defective, and do not form complete viruses

7
unless coinfected with a "helper" virus. Recombination
between replication-competent helper viruses and cellular
genes also may have produced highly oncogenic virus strains.
The "oncogene" hypothesis of Huebner and Todaro (88)
postulates that the cells of most or all vertebrates contain
"virogenes". These genes include sequences responsible for
transformation and are transmitted vertically form parent to
offspring. In this hypothesis, the occurrence of cancer may
be determined by the derepression of endogenous viral
oncogenes. Activation of repressed genes could result from
exposure of cells to chemical carcinogens, irradiation,
normal aging, or a combination thereof. This theory provides
an explanation for the known vertical transmission of certain
animal viruses. It also explains the observed necessity of
synergistic interactions between chemical carcinogens and
irradiation for transformation by some oncogenic viruses.
Multistep Carcinogenesis
The idea that development of cancer is a multistage
process arose from early studies of virus induced tumors, and
from discovery of cocarcinogenic effects of croton oil.
Rous discovered that certain virus induced skin papillomas in

8
rabbits regressed after a period of time. The papillomas
could be made to reappear if the skin was stressed by
punching holes in it or treating it with irritants such as
turpentine or chloroform (142). These experiments led to
conclusions that tumor cells could exist in a latent or
dormant state, and tumor induction processes and subseguent
growth of the tumor involved different mechanisms. These
mechanisms are known as initiation and promotion (62).
Studies of events involved in the initiation and
promotion phases of carcinogenesis were greatly aided by
isolation and identification of initiating agents such as
urethane, and the purification of the components of croton
oil which had promoting activities. The promoting substances
were found to be diesters of the ditepene alcohol; phorbol
(84). Of these, 12-0-tetradecanoylphorbol-13-acetate (TPA)
is the most potent promoter (11).
Initiation of transformation in normal cells by a
carcinogenic agent involves a permanent, heritable change in
gene expression. This could occur by direct genotoxic or
mutational events, where the carcinogenic agent reacts with
DNA directly. It may also occur via indirect "epigenetic"
events which regulate gene expression without direct
interaction with DNA seguences. Many feel initiating events

9
have a direct impact on DNA itself. According to Ruddon
(142), this theory depends on three kinds of evidence.
1. Agents which damage DNA are freguently carcinogenic.
It has been shown that chemical carcinogens are usually
activated to generate electrophilic agents. These form
specific reaction products with DNA. In some cases, as with
alkyl 0-6-guanine, the extent of product formation has been
shown to correlate with mutagenicity and carcinogenicity of
the agent (142).
2. Most carcinogenic agents are mutagens. A number of
in vitro test systems using mutational events in
microorganisms have been developed to rapidly screen the
mutagenic potential of various chemical agents. One of the
best known is the Ames test. Ames and his collegues have
shown that about 90 percent of all carcinogens are also
mutagenic (114). Very few noncarcinogens showed significant
mutagenicity in this test system.
3. The incidence of cancer in patients with DNA-repair
deficiencies is increased. In individuals with certain
recessively inherited disorders, the prevalence of cancer is
significantly higher than in the general population (103,
153). The common characteristic shared between these
disorders is the inability to repair some kinds of physical

10
or chemical damage to DNA. Such examples include, xeroderma
pigmentosum (deficiency in excision repair), ataxia
telangiectasia (greater sensitivity to X-irradiation, more
prone to leukemia and other cancers), Fanconi1s syndrome
(deficiency in repair of cross-linked bases, repair of X-ray
or UV induced damage), and Bloom's syndrome (increased
propensity to develop cancer, high genetic instability of
chromosomes). The high incidence of cancer in patients with
these diseases constitutes the best available evidence for a
causal relationship between mutagenicity and carcinogenicity
in humans (168, 169, 185).
Tumor initiating agents most likely interact with DNA to
induce mutations, rearrangements or amplifications, producing
a genotypically altered cell. The initiated cell then
undergoes clonal expansion influenced by promoting agents
which act as mitogens for the transformed cell (142). It has
been suggested that promoting activities may be mediated by
cellular membrane events. Direct action of promoters on DNA
has also been proposed (142). As a result, multiple clones
of cells are likely to be initiated by a DNA damaging agent.
Then, through a rare second event, one or a small number of
these clones progresses to malignant cancer.

11
Tumor promotion is itself a multistage process
sometimes labeled collectively as "tumor progression". Tumor
promotion is thought to be a stage of cell proliferation and
clonal expansion induced by mitogenic stimuli. The
progression phase is the evolution of genotypically and
phenotypically altered cells resulting from genetic
instability (128). During tumor progression which can take
years in humans, individual tumors develop heterogeneity with
respect to their invasive and metastatic characteristics,
antigenic specificity, state of differentiation, and response
to drugs and hormones (128). It is thought that some major
selection process occurs to favor the growth of one cell
over another, thus a dominant clonal population of cells may
emerge. This may be a result of competition for nutrients,
ability to evade the immune system, and resistance to
chemotherapeutic drugs.
The concepts of initiation and promotion support the
notion that cancer is not a "one-hit" event. Evidence
obtained from studies done with oncogenes and antioncogenes
further supports this concept. Weinberg (102) showed that
when rat endothelial fibroblasts were transfected with the
c-Ha-ras and c-myc oncogenes alone, no transforming effect
was observed. However, when c-myc and c-Ha-ras were

12
transfected together, multiple foci of transformed cells were
obtained. These cells had the capabilities to grow very
rapidly in culture and seed tumors in nude mice. Acting
together, c-myc and c-Ha-ras could do what neither gene could
do on its own.
Additional evidence for multistep carcinogenesis is seen
with the retinoblastoma (rb) gene. Retinoblastoma is a
childhood ocular tumor which requires both alleles of the rb
gene to be mutated in order for the disease to occur.
Knudson (97) has proposed that the rb gene behaves as an
"anti-oncogene", in that one normal allele is sufficient to
protect against the disease. His "two hit" model for this
disease suggests that two mutagenic events occur at 13ql4
of chromosome 13. These two events can be in the form of 2
germline events, 1 germline and 1 somatic, or two somatic.
The rb gene is now thought to have an involvement in other
human malignancies including osteosarcoma (79) and mammary
carcinoma (56,80), and its activation provides an example of
multistep cancer in humans.
The Neoplastic Phenotype and Steps of Tumor Progression
Much effort has gone into comparing phenotypic
characteristics of in vitro transformed cells with those of

13
cancer in vivo. These types of studies have greatly
increased understanding of cancer cell biochemistry.
Unfortunately, many biochemical characteristics of cultured
cells are dissociable from their abilities to produce tumors
in animals (142). Furthermore, individual cells of
malignant tumors from animals and humans exhibit extensive
biochemical differences. These differences are reflected in
cell surface composition, enzyme levels, immunogenicity,
and response to cancer drugs.
Some general characteristics of transformed malignant
cells growing in culture include the following (142):
1. Histiologic characteristics of malignant cells in
vivo. The nuclei are increased in both size and number,
and there is a great deal of variation in the sizes and
and shapes of the cells. Also, there are increased
nuclear:cytoplasmic ratios, and the formation of
clusters of cells may be observed.
2. Differences in growth characteristics are common:
a. Transformed cells in culture are immortalized.
Malignant transformed cells can be passaged in
culture for an indefinite period of time,
b. Transformed cells tend to pile up in culture and are
not subject to contact inhibition seen with

normal cells. As a result, malignant cells in
culture may grow to a much greater density.
14
c.Transformed cells seem to have much lower require
ments for serum and/or growth factors to survive
in culture than normal cells do.
d. There also seems to be a loss of anchorage
dependence with transformed cells. They may
no longer need to grow attached to solid surfaces,
and can grow in soft agar.
e. It has been observed that when transformed cells in
culture are subjected to biochemical restrictions,
they do not stop growing. An example of this is a
lack of response to serum starvation.
3. In vitro transformed cells may also change their surface
properties. Changes of this nature include; alteration
in structure of surface glycolipids and glycoproteins,
loss of surface fibronectins, increased agglutination
by lectins, changes in surface antigens which may
be tumor specifc and involved in immune responses,
and increases in the degree of amino acid uptake.
4. Cultured malignant cells produce increased levels of the
enzymes involved in DNA synthesis. They also

15
produce higher levels of other enzymes such as proteases
and collagenases.
5. Different transformed cells have varying levels of
nucleotides. Some may have higher cAMP levels or
increased cGMP:cAMP ratios than their normal cell
counterparts.
6. Transformed cells in culture have been shown to
produce growth factors involved in tumor growth.
These include angiogenesis factors, and transforming
growth factors (TGF). These may be produced to favor
their own growth (autocrine function).
7. Fetal antigens, placental hormones, and fetal
enzymes have been shown to be produced in increased
amounts in cultured tumor cells. This is
characteristic of tumor cells in vivo.
8. Ability to produce tumors in experimental animals is
a characteristic of malignant cells.
In addition to biological and physiological changes in
transformed cells, changes at the molecular level occur
as well. Genetic instability during tumor progression is
characterized by a variety of aberrations in the genome
including point mutations, deletions, rearrangements,
amplifications, chromosome translocations and abnormal

16
chromosome number (aneuploidy). It is thought that
aberrations such as point mutations, deletions, and
rearrangements are events associated with initiation
processes, whereas gross chromosomal changes occur as the
tumor progresses in malignancy (142). There are certain
chromosomal deletions, translocations and trisomies which are
characteristically associated with specific cancers. These
are called non-random chromosomal alterations. Changes in
ploidy are associated with many tumor types in advanced
stages, and are somewhat random in that no definitive pattern
of chromosome number is associated with a particular tumor
type. In more advanced cancers both random and non-random
chromosomal changes can be found. Continuous chromosomal
changes can bring about tumor heterogeneity and the selection
of more highly invasive and metastatic cancers. Thus, tumor
progression has been called a highly accelerated evolutionary
process.
Malignant tumors have several important in vivo
characteristics. At the cellular level, they have a greater
fraction of cells in S-phase, and are less differentiated
than their normal counterpart tissues. In order for tumor
cells to grow, divide, and metastasize, cell growth must
outnumber cell death. Therefore, angiogenesis factors are

17
important to the growth of malignant tumors, as rapidly
growing tumors often outgrow their blood supplies. It is
thought that malignant tumor cells may produce their own
growth factors, angiogenesis factors, and collagenases,
enableing them to compete with other cells for nutrients,
and eventually invade surrounding tissues.
Specific Questions Addressed During the Course of This
Proiect
Unrestrained cell growth is a common component of
neoplastic phenotypes. Proto-oncogenes are genes which have
been shown to be involved in regulation of cellular growth
and differentiation. They are found in all normal nucleated
animal cells. Their conversion to transforming genes or
oncogenes by one or more of several possible mechanisms may
allow the transformation of cells in vitro and generate
neoplasms in vivo. Exploration of how these potential
regulators of growth control interact with one another and
with other genomic components may enlighten our understanding
of how normal cellular replication or differentiation events
change with transformation.
It is possible that several proposed mechanisms of
proto-oncogene activation will lead to increased production

18
of transcript. Examination of gene copy numbers and gene
expression will offer clues to possible mechanisms involved
in activation of proto-oncogenes. The following was a
general basis for this project:
Normal cellular genes, when mutated by several suggested
mechanisms, may contribute to the tumorigenesis and
biologic behavior of chondrosarcoma and malignant
fibrous histiocytoma.
From this, the following hypotheses were derived:
1) Increases in proto-oncogene transcript levels may
be due to gene amplification.
2) There are differences in chromatin structure
between amplified and single copy proto-oncogenes.
To test the first hypothesis, tumor RNA and DNA samples
were evaluated for proto-oncogene transcript levels and gene
copy numbers of c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and
v-src. These genes were studied because of previous
associations with sarcomas in humans and other animals.
It was desirable to study potential regulatory changes
which accompanied proto-oncogene amplification and increased
transcript production. Therefore, the second hypothesis was
tested by studying the locations of DNAse I hypersensitive
sites. These sites represent areas where regulatory inter
actions are thought to occur. Changes in locations of these

19
sites may offer clues to regulatory mechanisms involved in
proto-oncogene transcript production.

CHAPTER 2
REVIEW OF THE LITERATURE
Differentiation of Mesenchyme
The precise pathways taken by mesenchymal cells
undergoing differentiation have been somewhat of a con
troversial issue. Therefore, two proposed models of mesen
chymal differentiation will be presented here. The first of
these models, the radial model of mesenchymal
differentiation, is a currently accepted model proposed by
Hadju (76). Each soft tissue and hematopoietic phenotype
directly originates from a primitive undifferentiated
mesenchymal cell. In this scheme, there are no precursor
cells and no branching of cell types. Rather, the
differentiated phenotypes are separated from each other
only by a primitive mesenchymal cell. Because of this,
close relationships exist between phenotypes.
More recently, another model has been proposed by Brooks
(23) and is shown in figure 1. The two novel features of
this model are the insertion of an intermediate precursor
20

21
Primitive
Uncommited
Mesenchymal
Cell
->
Endothelio-
blast
yofibroblast
Chondro-
Osteoblast
-> Endothelial
Cell
Pericyte
Smooth Muscle
>Chondrocyte
'Fibroblast
Lipoblast
Schwannoblast
Fibrocyte
> Lipocyte
Schwann Cell
Rhabdomyoblast^Rhabdomyocyte
Figure 1. Hypothetical model of mesenchymal differentiation as
proposed by Brooks. *
* Taken from Brooks, J.J. 1986. The Significance of Double
Phenotypic Patterns and Markers in Human Sarcomas. A New
Model of Mesenchymal Differentiation. Am. J. Pathol. 125:
113-123.

22
between the primitive cell and some differentiated
phenotypes, and a branching system reflecting close
relationships between some phenotypes and not others. This
model also recognizes the myofibroblast and the
chondroosteoblast.
Chondrosarcoma and Malignant Fibrous Histiocytoma
Chondrosarcoma is a malignant tumor of cartilage (figure
2). It has been well established that the basic
proliferating tissue is cartilagenous (39). Primary
chondrosarcomas are tumors which can arise de novo in
extraskeletal tissues or in mixed tumors such as teratomas.
The majority of chondrosarcomas are "myxoid". Those composed
of hyaline cartilage are more uncommon. Secondary
chondrosarcomas arise most commonly in osteosarcomas, and can
sometimes develop in patients with multiple exostoses. Rarely
do they develop from an enchondroma, a benign cartilagenous
tumor. In addition to primary and secondary chondrosarcomas
there are dedifferentiated chondrosarcomas which give rise to
more malignant tumors such as osteosarcomas, fibrosarcomas,
or malignant fibrous histiocytomas (MFH) (39).

23
Figure 2. Histiologic appearance of chondrosarcoma. This
section was taken from a patient with areas of grade I (less
dense cellularity) and grade III tumor. The appearance of
the grade III area closely resembles that of an MFH (shown
below).

24
Chondrosarcoma is primarily a tumor of adulthood (39).
The incidence of bone tumors in general is highest
during adolescence with a rate of 3 per 100,000 (61). The
incidence falls to 0.2 per 100,000 at ages 30-35 and rises
slowly thereafter to an incidence rate equal to that of
adolescence (30, 39). Chondrosarcoma is the third most
common type of bone tumor and makes up approximately 13
percent of all malignant bone tumors (85). More than 75
percent of chondrosarcomas occur in the trunk and the upper
ends of the femora and humeri. It is much less common for
these tumors to be located in the distal extremeties such as
the elbows and ankles (39).
Many chondrosarcomas are palpable, but many of those
affecting the trunk or long bones of the extremeties which
have not broken the cortex may cause pain alone to indicate
the presence of the lesion. Roentgenograms provide a very
helpful means for diagnosis. Osseous destruction in the area
of the lesion combined with irregular densities from calci
fication and ossification are commonly observed. Central
chondrosarcomas of long bones commonly produce fusiform
expansion of the shaft associated with thickening of the
cortex (39).

25
Chondrosarcomas usually have a slow clinical evolution.
Metastasis is relatively rare and occurs late. The basic
therapeutic goal is to control the lesion locally and to
prevent local recurrence. Therefore, radical early
surgical treatment is desirable (39). A long followup after
treatment is necessary because recurrence may develop many
years later. The overall survival is approximately 50
percent at 5 years (180).
With respect to prognosis, the correlation between poor
differentiation, rapid growth rate, and metastasis is high.
Clinical study results suggest that a high cure rate is
expected for patients with more differentiated tumors (135).
A grading system exists for chondrosarcoma and is important
in terms of predicting survival and establishing the most
effective treatment protocol (39).
Criteria for grading chondrosarcomas are those of Evans
et al. (57), and include the following: Grade I tumors have
the presence of or domination of cells with small densely
staining nuclei, an inter-cellular background of a chondroid
or myxoid nature, freguent calcification patches, and
multiple nuclei present within a single lacuna. Grade II
tumor characteristics include; areas where a significant
fraction of the nuclei are of a moderate size, a mitotic

26
index of 0-2 per 10 high power fields (40X), dense
cellularity, paler staining nuclei, a background which is
more more myxoid than chondroid, and a greater cellularity
/increased nuclear size limited to isolated areas.
The criteria for the classification of a chondrosarcoma
as grade III are a mitotic index greater than 2 mitoses per
10 high power fields (40X), increased nuclear size compared
to those of grade II tumors, very dense cellularity which
may appear MFH like, and the absence of a chondroid or
myxoid background.
Malignant fibrous histiocytomas (MFH) (figure 3) are
soft tissue tumors whose cell of origin has been disputed,
but current evidence indicates that these are are immature
mesenchymal cells (15, 90). Malignant fibrous histiocytomas
usually occur in deeper structures such as deep fascia and
skeletal muscle. They also have been seen in soft tissues of
the extremities, mediastinum, and retroperitoneum, and may
occur within bones in areas of infarction or prior radiation.
As a group, these tumors comprise only about 0.8 percent of
all bone tumors (39), but are somewhat more common in soft
tissues. There seems to be a slightly higher percentage of
males with this disease than females, and nearly any age may
be affected (190). As with other varieties of bone tumors,

27
Figure 3.
Histiologic appearance
of MFH.

28
pain and swelling are the most frequent symptoms. As with
chondrosarcomas, roentgenographic analysis is helpful in
preliminary diagnosis of the tumor.
Histiologic features of these tumors usually include a
high degree of variation, multinucleated tumor cells,
nuclear hyperchromasia and a high mitotic activity. A
typical pattern of growth can be described as the arrangement
of tumor cells around a central point, producing radiating
spokes, grouped at right angles to each other (storifiorm
pattern).
With respect to prognosis, subcutaneous tumors generally
have a better prognosis than the more deep seated lesions.
The recurrence of MFHs is said to be 44 percent with a two
year survival of 60 percent (190). There is no widely
accepted grading system for deep seated MFHs as for
chondrosarcomas. Other important prognostic factors include
mitotic index, degree of cellular polymorphism, tumor size
and tumor stage (180).
Staging criteria for both chondrosarcomas and MFHs are
those described by Enneking et al. (52) for sarcomas
originating from mesenchymal tissue of the musculoskeletal
system. This system takes into account surgical grade,
local extent, and presence or absence of regional

29
metastasis. Grade is further classified as low and high,
local extent as intracompartmental and extracompartmental,
and the extent of regional or distant metastasis is defined
as either present or absent.
Proto-Oncogenes
During the early 1970s it was discovered that a single
gene carried by a retrovirus could cause cancer in animals.
Soon thereafter, it was thought that the oncogenes acquired
by retroviruses might be derived from normal cellular genes
present in the host. It was later shown by Stehlin et al.
(164) that cDNA specific for the v-src region of Rous
Sarcoma Virus could detect closely related sequences in the
genome of normal chicken cells. This gene, now called c-src
has been found in all other vertibrate species including man
(162) .
Since the discovery of cellular sequences homologous to
v-src, cellular counterparts (c-onc) for the other viral (v-
onc) oncogenes have also been found (16). These cellular
sequences are known as cellular oncogenes or proto-oncognes.
There are now more than forty known proto-oncogenes which are
expressed in most normal mammalian cells (77). The c-ras and

30
c-myc genes are transcribed in almost all mammalian cells
(levels may be low at about 5-20 molecules of RNA per cell)
whereas most other proto-oncogenes seem to be more tissue
specific (77). For example, c-myb is expressed in
hematopoietic cells but not elsewhere (191). C-sis RNA has
been detected in very few normal cell types, including
rapidly dividing cells of the human placenta and endothelial
cells (8). Since proto-oncogenes are so conserved between
species, it seems likely that their gene products play an
essential role in normal cellular growth and development.
There are several possible mechanisms by which proto
oncogenes may be activated to oncogenes. The first of these
mechanisms involves insertional mutagenesis. The over
expression of a proto-oncogene may occur after the
integration of a new promoter. For instance, the c-mos
proto-oncogene of mice which is biologically inactive after
molecular cloning, can be experimentally converted into a
potent oncogene by additon of a strong transcriptional
promoter (18). Another example of this mechanism comes from
similar activation of the c-Ha-ras proto-oncogene of
rats (40). These oncogenes are created by ligation of cloned
DNA segments, and acquire transforming capabilities because
their transcripts are produced at much higher levels than

31
those afforded by native promoters of the normal proto
oncogenes. In vivo, the c-myc and c-erb-B-1 proto-oncogenes
present in several avian hematopoietic neoplasias have become
activated after adjacent integration of an avian leukosis
proviral DNA segment. This viral segment provides a strong
transcriptional promoter which replaces indigenous promoters
of these genes (83, 131).
A second mechanism of activation involves overexpression
due to amplification of the proto-oncogene (gene dosage
effects). The c-myc proto-oncogene is amplified 30-50 times
in HL-60 promyelocytic leukemia cells (32), and in a
neuroendocrinal tumor the the colon (1). A c-Ki-ras gene is
amplified 3-5 times in a human colon carcinoma cell line
(115), and 60 fold in an adrenocortical tumor of mice
(149). Human neuroblastomas were found to contain 30-100
copies of the N-myc gene (150). This was later confirmed,
and shown to be associated with patient survival (152). A
human chronic myelogenous leukemia cell line was discovered
to have multiple copies of the c-abl gene (33). In each of
these cases, gene dosage effects are thought to be
responsible for increases in transcript levels and gene
product.

32
A third mechanism involves enhancer/promoter activity.
Enhancer sequences may increase utilization of
transcriptional promoters to which they become linked. The
affected promoter may be several kilobases away in either 5'
or 3' directions (74). One example of this is the presence
of retrovirus genome fragments downstream from the c-myc gene
in some avian lymphomas (131). Here, the retrovirus elements
appear to act by contributing an enhancer sequence rather
than a promoter. It is entirely possible that point
mutations at key regulatory sites such as promoter regions
rather than coding regions may result in in proto-oncogene
activation. This could facilitate the deregulation of a
proto-oncogene, i.e. one with abnormal transcriptional
control, or one which is inappropriately expressed.
A fourth mechanism involves the c-myc gene in
particular. Work with Burkitt lymphomas has demonstrated the
juxtaposition of the c-myc gene and immunoglobulin genes
following a translocation event. As a result of this
translocation, the c-myc gene loses all or part of its own
regulatory exon and acquires normally unlinked sequences
involved in immunoglobulin production (104). Rearranged c-
myb sequences have been found in certain mouse plasmacytomas

33
(122) but their detailed structure and mechanism of
activiation remain to be elucidated.
The fifth mechanism centers around structural
alterations in the proto-oncogene and protein product. This
mechanism is well documented in the case of the oncogenic
proteins encoded by the ras genes. It was discovered that in
the case of the oncogene in the T24/EJ human bladder
carcinoma cell line, a point mutation at position 12
converted the c-Ha-ras proto-oncogene to an oncogene. This G
to T transversion causes glycine which is normally the 12th
residue of the encoded 21,000 dalton protein to be replaced
by a valine (170). Another activated version of this gene
encodes an aspartate residue at this position (144).
Studies done with genes of the Ki-ras group also showed
that when the 12th residue was altered in this manner,
oncogenic activation of the c-Ki-ras gene was observed (24).
A slight variation of these results was obtained through the
study of a human lung carcinoma c-Ha-ras oncogene found to
have a mutation at amino acid 61 of the p21 protein (198).
These changes do not seem to affect the levels of expression
of these genes, only the activities of encoded proteins. It
is therefore suggested that the codons specifying residues
12 and 61 represent critical sites which, when mutated, will

34
often generate oncogenic alleles. It seems that point
mutations elsewhere in the ras proto-oncogenes merely serve
to inactivate the genes instead of converting them to
oncogenes (102).
Finally, the possibility that unknown mechanisms of
activation may be at work must not be overlooked. There may
be mechanisms of activation which have not been determined.
It is possible that new mechanisms may eventually be
implicated in proto-oncogene activation.
Biochemistry of Oncogene Products
Cytoplasmic Kinases
One of the first oncogene proteins of this class to
be recognized and studied was the 60,000 dalton protein of
the v-src gene (pp60 v-src) (89). Other oncogene products
with tyrosine-specific protein kinase activity include yes,
abl, fps, fgr, and ros (6, 77). These proteins are all
located at the inner surface of the cytoplasmic membrane and
a comparison of their amino acid sequences has shown that
they are related to each other (35). A region of
approximately 250 amino acids in pp60 src is responsible for

35
the kinase activity, and a corresponding domain is found in
other tyrosine kinases with a high degree of amino acid
conservation between them. This kinase domain is also found
in the cytoplasmic cyclic AMP dependent serine protein
kinases in mos and raf, and in serine specific kinases
located in the cytosol (118). A similar sequence domain has
been found in the membrane-bound receptor-like products erb-
B1, fms, and neu, all of which have tyrosine kinase
activity, indicating a distant evoloutionary relationship
between all protein kinases (77).
Originally, it was thought that this activity was
exclusive to oncogenes. However, a protein derived from the
c-src gene was isolated from normal cells and shown to have
tyrosine specific kinase activity (31). Since then, other
membrane-bound cellular proteins with similar activities have
been identified. These have offered clues as to what the
oncogene kinases may be doing. It has been shown that the
receptors for platelet derived growth factor (PDGF) and
insulin-like growth factor (IGF) have a tyrosine-specific
kinase activity (77). It has also been proposed that
tyrosine phosphorylation is an early event in the
transduction of mitogenic signals through the membrane.
Although pp60src resides at the inner surface of the

36
membrane, and does not posess receptor activity, it probably
does play some role in the early signalling process (35).
Therefore, the presence of a tyrosine kinase encoded by a
viral oncogene might result in a continuous, deregulated
mitogenic signal for cell division.
Just how the cell responds to these signals is presently
unknown. Many attempts have been made to find the cellular
targets for phosphorylation by pp60src and by growth factor
receptors. One effect of pp60src which is thought to be of
importance is that it leads to increased protein
phosphorylation on serine residues (38). The phosphorylation
of the S6 ribosomal protein on a serine residue is thought to
be a critical event in the mitogenic stimulation of normal
quiescent cells. This may occur via a serine kinase
intermediate which might be activated directly or indirectly
by the pp60src tyrosine kinase (38).
Two different biochemical pathways have been shown to be
important in the mitogenic stimulation of cells and a
possible involvement with both has been shown for pp60src.
Both of these pathways involve the generation of second
messengers. The first involves the generation of cyclic AMP
by membrane bound adenylate cyclase, leading to increased

37
levels of intracellular cyclic AMP. This can lead to the
activation of cytoplasmic cyclic AMP-dependent serine
specific protein kinases. Activation of other serine
specific kinases, particularly protein kinase C may then
occur (59). It is protein kinase C which is thought to play
a central role in the various responses to mitogenic
stimulation. Graziani (66) has shown that tyrosine
phosphorylation of the cyclic AMP dependent protein kinases
by pp60 c-src occurs in transformed cells. Therefore, it is
possible that pp60src interacts with the pathway which
regulates cell proliferation through cyclic AMP and protein
kinase C.
Another pathway in which src may be involved also leads
to the activation of protein kinase C. Full activity of this
protein requires two cofactors; calcium, and diacylglycerol
(127). Both of these can be generated in response to
extracellualr signals such as acetylcholine or PDGF. The
result of the interaction of these molecules with their
receptors is a breakdown of inositol phospholipids located in
the membrane to yield diacylglycerol. This activates protein
kinase C, and inositoltriphosphate which can affect calcium
levels within the cell. Sugimoto et al. (167) showed that
pp60src could phosphorylate inositol phospholipids in vitro

38
and that in RSV transformed cells there is a buildup of
intermediates in the inositol lipid breakdown pathway. They
postulated that the primary target of pp60src might be lipid
and not protein.
Much remains unknown about the biochemical action of
pp60src and the rest of the tyrosine kinase family.
Phosphorylation of tyrosine seems to be a general phenomenon
for initiating cell division and inappropriate tyrosine
kinase activity could explain the loss of growth control
associated with transformed cells. The phosphorylation of
inositol lipids by at least two of the tyrosine kinases src
and ros is interesting, but the significance in transformed
cells remains to be determined.
Ras Proteins
The 21,000 dalton (p21) proteins of three human cellular
ras genes; Harvey (Ha), Kirsten (Ki), and N-ras are very
closely related in seguence. In the first 150 amino acids
there are a maximum of 14 amino acid differences between the
three proteins. The ras proteins therefore have been highly
conserved throughout evolution, and are thought to play an
essential role in cell growth (77). The ras p21s are

39
located at the inner surface of the plasma membrane and
although the viral proteins are phosphorylated at amino acid
residue 59 which is a threonine, the human p21s do not have a
threonine at 59, nor are they phosphorylated. The ras genes,
which are cell cycle dependent (94), are activated by point
mutations and therefore the modes of action of normal and
transforming p21s are of interest.
Both transforming and normal cellular p21s bind GTP and
GDP egually and have a GTPase activity (116). However, the
transforming version of p21 hydrolyzes GTP about 10 times
more slowly than the normal proteins (77) The normal ras
roteins are thought to interact with a receptor in response
to an external signal, bind GTP and interact with an as yet
unknown molecule to generate a second messenger (77).
Adenylate cyclase is unlikely to be directly involved
because the G proteins associated with it have different
molecular weights from ras p21 (77). Since transforming p21
has reduced GTPase activity, this could result in abnormally
high levels of the second messenger.
Ras encoded proteins are also regulators of inositol
triphosphate. Some of the proteins involved in the inositol
lipid breakdown pathway are GTP binding proteins and it is
possible ras may be one of these. Calcium has long been

40
implicated in cell proliferation. The increase in
intracellular calcium which occurs when cells are fertilized
or stimulated by growth factors may depend on the formation
of inositol triphosphate. This can act as a second
messenger to release intracellular stores of calcium. It has
been postulated, and some evidence exists that the activated
ras gene protein which binds but cannot hydrolyze GTP, can
initiate the formation of inositol triphosphate in an
uncontrolled fashion, independant of cellular growth factors
(12) .
In order to expand the current understanding of the
functions of ras proteins, it will be necessary to identify
which protein(s) they interact with in the cell. Attempts
which have been made to copreciptiate ras associated proteins
with anti-ras antibodies have been unsuccessful, indicating
either that associations are weak, or they depend on intact
membrane structure (77).
Growth Factors and Their Receptors
Certain oncogene products are known to be transforming
versions of a growth factor and several growth factor
receptors. The erb-B-1 oncogene is a truncated version of the

41
epidermal growth factor (EGF) receptor gene (46, 82). The
neu (erb-B-2) oncogene, first detected by transfection assays
has homology with erb-B-1 and also encodes a receptor-like
molecule (145). The sis oncogene codes for one subunit of
PDGF (45). Recently, it has been shown that v-fms is derived
from the normal cellular gene encoding the receptor for
colony stimulating factor 1 (CSF-1) (111, 146).
The erb-B-1 oncogene protein is different from the
normal EGF receptor in that the extracellular EGF binding
domain is absent (17). It is possible that this truncated
receptor is in an activated configuration even in the absence
of EGF stimulation. More about the erb-B-1 oncogene product
will be discussed below. It has been predicted that other
known growth factor receptors in addition to those for EGF
and CSF-1 such as those for PDGF could be altered or
inappropriately expressed to yield oncogenic proteins. So
far no spontaneous examples of this have been reported.
Oncogenic changes in a growth factor protein are well
exemplified in the case of the sis oncogene product. The c-
sis protein sequences are homologous to one of the chains of
PDGF, and are normally produced in only a restricted number
of cell types; including bone marrow megakaryocytes (77),
human placental cells, and endothelial cells (8). Receptors

42
for PDGF have been found mainly on mesenchymal and glial
cells (165). In the case of virally transformed fibroblasts,
the v-sis sequences are fused to the env sequences of the
virus and this allows export of the abnormal PDGF-like
molecule to the membrane.
Abnormal expression of any mitogenic factor such as sis
may make it a possible candidate for a role in oncogenesis,
providing the cells which produce it have the appropriate
receptors. It is thought that high levels of sis expression
cause transformation, presumably by autocrine stimulation via
the PDGF receptor. The sis oncogene protein will be
discussed in further detail below.
Many tumor cells release transforming growth factors
(TGF). One class of these, TGF alpha is closely related in
sequence to EGF and interacts with the EGF receptor (43).
Other evidence suggests that TGF molecules function normally
as necessary mitogens for embryonic development (163, 179).
Inappropriate expression in adult cells could be a step in
transformation.
Nuclear Proteins

43
The products of five oncogenes; myc, myb, fos, ski (77),
and B-lym (65) are known to be located in the nucleus. The
expression of c-myc, c-fos, and c-myb appears to be dependent
on the proliferative state of the cell (2, 100, 120 175).
Quiescent 3T3 cells for example, have undetectable levels of
c-fos mRNA but within 30 minutes of stimulation by PDGF
(100), the levels are dramatically increased. This is only
transient, and after about 2 hours the high levels disappear
(120). Thus the interaction of PDGF with its receptor not
only facilitates activated intracellular phosphorylation
events and the breakdown of inositol lipids, but also leads
to the generation of a nuclear signal to switch on c-fos
expression. Since phosphorylation of intracellular proteins
occurs within a few minutes of mitogenic stimulation it is
likely that c-fos expression is a direct result of these
events. Like c-fos, the c-myc gene is expressed at very low
levels in quiescent cells, and its transcript levels
increase transiently after stimulation with PDGF, insulin,
and serum (19, 22, 67, 95, 126, 175).
The roles of c-fos and c-myc gene products will be
discussed in more detail below. It will be mentioned for
now that since c-myc and c-fos gene expression follows a
direct relationship to cell cycle, it is generally believed

44
that their protein products are involved in the regulation of
cell division. Inappropriate expression of these nuclear
proteins could keep the cell cycling even under conditions
which would normally be sufficient to switch off further
growth.
Molecular. Biological, and Physiological Characteristics
of Proto-Oncogenes Examined in This Study
Growth Factor Related
Erb-B-1. As mentioned previously, the product of the
erb-B-1 oncogene is a truncated version of the receptor for
EGF (181) It is a glycoprotein with protein kinase activity
and has the capability to transform cells, while the normal
growth factor and receptor do not (17). The erb-B-1
oncogene protein product represents the EGF receptor short
of both its large extracellular domain which binds the
ligand and either 32 or 71 amino acids from its carboxy
terminus (181). The transforming protein is 71 amino acids
in length and includes a hydrophobic region which resides at
the cell surface, a hydrophobic domain that spans the plasma
membrane, and a shortened cytoplasmic domain which possesses
the protein-tyrosine kinase activity (197). This

45
truncation could have several possible consequences, any of
which may contribute to neoplastic transformation. For
instance, only a small fraction of the erb-B-1 oncogene
product reaches the plasma membrane (17, 148). The remainder
never leaves the golgi apparatus and retains an immature
mannose-rich form. This is in contrast to the normal
behavior of intact transmembrane receptors.
The EGF receptor is returned to the interior of the
cell after binding ligand, a regulatory mechanism seemingly
designed to protect the cell from an over abundance of
stimuli. By contrast, the product of the erb-B-1 oncogene
cannot bind ligand and may be located permanantly to the
surface of the cell (17, 148, 181).
The EGF receptor displays the full force of its protein
kinase activity only after binding ligand. The erb-B-1
oncogene product is presumably released from this dependence
and is constitutively active (17). The kinase activities
associated with the erb-B-1 oncogene product are
constitutive, and the appearance of this protein on the
plasma membrane seems to be a prerequisite for transformation
(14) .
With respect to tumor activity, it has been reported
that abnormally high levels of the erb-B-1 oncogene were

46
found in 40 percent of primary brain tumors of glial origin
(108). Abnormally high copy numbers of the HER-2/neu
(c-erb-B-2) gene have been found in mammary carcinomas.
This in turn has been associated with patient survival and
time to relapse in diseased individuals (158).
Sis. The sis oncogene encodes one of the two subunits
(PDGF 2-B) of platelet derived growth factor (45). Following
synthesis, the 28kd product (p28sis) of the sis oncogene
assembles into a homodimer and is trimmed to a smaller
polypeptide (140).
The product of the sis oncogene may transform cells by
an autocrine function. Evidence exists that some cells
release a homodimer of p28sis, whose structure and activity
resemble those of PDGF (63). Application of antibodies
against PDGF to these cells arrests their growth (87). There
is also reason to suspect that the sis oncogene product, or
that of its cellular progenitor c-sis need not leave the
cell in order to invoke neoplastic growth (13). Instead, the
transforming protein may combine with a receptor while still
inside the cell.
There is also the guestion of why the sis oncogene
protein can transform cells, while the c-sis protein cannot.
It is not known if there are mutations in oncogenic sis that

47
alter the capabilities of its product. Also unclear is
whether or not the homodimer produced from it has abnormal
activity compared to the related but different subunits of
PDGF. Whether the formation of homodimer causes PDGF 2-B to
be processed abnormally, or if the sis oncogene product acts
at an anomalous site inside the cell are questions yet to be
answered. It is possible that cells produce factors which
cooperate with sis in neoplastic transformation (17). All of
these issues only obviate the fact that much more needs to be
done before a full understanding of the sis gene and its
product can be obtained.
The presence of the c-sis gene has been demonstrated in
several tumor types. Eva et al. (54) reported that cell
lines from both human sarcomas and gliomas were analyzed for
the presence of sis message. It was found to be at elevated
levels in 5/6 of sarcoma cell lines and 3/5 of glioma cell
lines studied. Sis message has also been found to be at
elevated levels in the metastases of two stomach carcinomas
(172) .
Protein Kinases

48
C-Ha-ras. C-Ha-ras is a member of the ras oncogene
family, and is cell cycle dependant (94). As described
previously, activation of c-ras to an oncogene is
accomplished by point mutations at specific sites which
render its protein product oncogenic to the cell (34, 170).
Mutations of this nature have been found in approximately 15
percent of sarcomas (77) Oncogenes of the ras family may
be active in human carcinoma cell lines, as well as primary
human tumor specimens of several sites such as colon, lung,
gall bladder, urinary bladder, pancreas rhabdomyosarcoma
(42, 136,137), and in prostate cancer (186). These genes are
also present in human hematopoietic neoplasias; including
primary acute myelogenous leukemias, and cell lines derived
from acute lymphocytic leukemias, T cell leukemias and
chronic myelogenous leukemias (55, 161) .
Src. The src oncogene as described above codes for a
protein, which like its normal proto-oncogene counterpart,
is a protein-tyrosine kinase. In order to study its
mechanism of activation, c-src has been molecularly cloned
from both chicken and human DNA. Nucleotide seguencing has
revealed the similarities between the protein coding regions
and those of v-src (171). Unlike v-src, c-src is very

49
complex and contains 11 introns. The exact mechanism by
which RSV acquired genomic c-src information is unclear
(196). It has been proposed that during a round of
infection, a non-oncogenic RSV progenitor transduced genomic
DNA after viral integration and excision, and then the
introns were removed by processing. Also, it may have
somehow incorporated c-src messenger RNA (77).
The differences between c-src and v-src have been
addressed by making use of in vitro recombinants of viral
and cellular genes. Results have shown that some of the
amino acid changes in v-src are biologically important for
transformation. This was also demonstrated by the fact that
high levels of c-src expression alone did not transform
cells (130). It has also been shown that if v-src is
expressed in cells at levels comparable with those of c-src
in normal cells, then transformation is observed (92).
It may be that both qualitative and quantitative changes in
src expression are required for transformation.
Src gene protein (p60src) activity is present in
normal tissues where organ specific levels have been found.
Jacobs et al. (91) have reported that levels of pp60c-src
were highest in brain followed by kidney, lung, muscle, and
connective tissue. It was also determined that a 4-20 fold

50
increase of pp60c-src kinase activity was present in human
skin tumors compared to normal skin (7).
Nuclear Related Proto-oncogenes
Fos. The fos gene was first discovered as the oncogene
of two related murine viruses that cause osteogenic sarcoma.
The name fos refers to its origins in the FBJ and FBR
osteogenic sarcoma viruses. The fos oncogene, like other
oncogenes, causes the transformation of cells and is
derived from a normal cellular gene. The cellular and viral
fos genes have an interesting relationship to each other.
The first 332 amino acids of v-fos and murine c-fos differ in
only five positions but the remaining 49 amino acids are
completely different. The 104 bases at the C-terminus of c-
fos are deleted in v-fos, and although this changes the
reading frame and alters subseguent amino acids, the
mobilities of the proteins are similar (v-fos 55 kd, c-fos,
62 kd) (184) .
The fos gene seems to serve as a kind of master switch
for turning on other various genes in response to a wide
range of stimuli including growth factors. Fos may act as a
sensor which detects incoming signals at the cellular

51
membrane and converts them to lasting responses such as cell
division and possibly memory formation (113).
The c-fos gene was recognized as a cell cycle dependent
gene early after studies with it began. C-fos can be rapidly
activated by the treatment of quiescent cells with PDGF, EGF,
nerve cell growth factor, and serum containing growth factors
(69). This led to speculation that c-fos had something to
do with cellular growth control.
Studies with nerve cells revealed additional information
about the activities of the c-fos gene. It was found that
c-fos expression is controlled by factors which differentiate
and trigger nerve cell activity. In vitro experiments have
indicated that c-fos induction depends on the ability of
neuroactive agents to open calcium channels (119). Calcium
entry is a normal component of neuronal responses to
stimulation. It was found that a dramatic increase in c-fos
gene activity occurs in the brains of mice treated with
metrazole, a drug which causes epilepsy like seizures (119).
The synthesis of c-fos proteins was found to occur primarily
in the nerve tracts stimulated by metrazole. The results
suggested that the c-fos protein mediates the long term
adaptation of nerve cells to metrazole stimulation.

52
There is now evidence for genes which seem to be
directly controlled by c-fos. For example, a set of genes
which code for fat cell proteins which become active when fat
cells differentiate has been identified. One of these genes
adipocyte P2 (aP2), was found to have a regulatory site 125
base pairs upstream from its promoter. This regulatory site
binds proteins which undergo undefined changes during
maturation. It is hypothesized that changes in the binding
proteins mediate activation of the aP2 gene (44).
Experiments were then performed to determine whether
the c-fos protein was one of these regulatory proteins. Data
from immunoprecipitation analyses showed that the binding
complex contains the c-fos protein itself, or at least a very
related protein (44) Further studies need to be done to
clarify this issue.
Site directed mutagenesis studies have been done in the
c-fos promoter region. Various deletions were studied for
effects on the c-fos gene's responses to various stimulatory
agents. It was found that a 22 base pair region located 300
base pairs 5' to the promoter is necessary for enhanced
expression of c-fos in response to serum stimulation (68,
177, 178). This region is called serum response element
(SRE). These same investigators have isolated a protein

53
which binds specifically to the SRE. The protein appears to
be necessary for c-fos response to serum stimulation,
however proof that the protein directly activates c-fos
remains to be obtained. SRE variants have been constructed,
each with altered protein binding capabilities. It was found
that the ability to stimulate transcription correllates with
avidity of protein binding (68,81).
The SRE is not the only region thought to be important
in c-fos gene regulation. C-fos is activated by several
different stimulatory agents. It is thought that these
stimulatory agents do not all act in the same manner to
affect c-fos expression. The current idea is that there are
multiple regulatory elements for the gene. Epidermal growth
factor and phorbol esters seem to work through the SRE, but
there is evidence that c-fos activation by PDGF may be
mediated through a different site 25 base pairs 5' of the SRE
(64, 177, 178).
Some c-fos stimulating agents use cyclic AMP as a
messenger. Mutations in the SRE do not seem to affect c-fos
activation by cyclic AMP (68, 177). Therefore, gene
activation by cyclic AMP uses other unidentified regulatory
seguences. Calcium ions which mediate c-fos during nerve
cell activation may use yet another site (64, 68, 177, 178).

54
The different stimulatory agents which appear to use
different regulatory proteins to enhance c-fos expression,
induce different nuclear proteins (64, 177, 178), and these
are called c-fos related antigens (FRA).
C-fos may be subject to negative regulation as well.
Verma et al. (184) have shown evidence to suggest that cells
may have factors which repress fos transcription, but more
needs to be done before this can be fully characterized and
understood.
Both the products of the v-fos (p55/v-fos) and c-fos
(P62/c-fos) genes may be part of nuclear complexes (60,
184). For example, the c-fos protein complex and several
FRAs bind specifically to a sequence element referred to as
the HeLa cell activator protein 1 (AP-1) binding site (60).
Structural studies and immunoprecipitation analyses were
performed with this complex. One of the Fos-associated
proteins, (p39) was found to be the protein product of c-jun
(138) .
The p39/jun protein is one of the major polypeptides
identified in AP-1 oligonucleotide affinity chromatography
extracts of cellular proteins. The preparations of AP-1
were found to contain c-fos and several FRAs (20). Some of
these proteins seem to bind to the AP-1 site directly, while
c-fos appears to bind indirectly through protein/protein

55
interactions (20). Cell surface stimulation results in an
increase in c-fos and c-jun products. The products of the
two genes along with several other related proteins form a
complex which associates with transcriptional control
elements containing AP-1 sites (20, 60, 138). This
potentially can then mediate long term responses which
regulate growth control and development (113).
With respect to tumor activity, there seems to be some
controversy in the literature with regard to what types of
neoplasms are associated with c-fos expression. It has been
reported that c-fos has not been found consistently in any
type of neoplasm (77). However, Slamon et al. have reported
that c-fos is expressed in all tumor types including
carcinomas, sarcomas, and hematopoietic malignancies (159).
Myc. The myc family of cellular proto-oncogenes
contains three well defined members, c-myc, N-myc, and L-myc.
The first defined and most thoroughly studied member of this
family, c-myc, was identified as the cellular homolog to the
transforming gene of avian transforming virus MC29 (17). The
two other well characterized myc family genes N-myc and L-myc
were isolated on the basis of their homology to c-myc and
their freguent amplification in certain classes of human
tumors. The N-myc gene was originally isolated from human

56
neuroblastomas, a pediatric tumor of embryonal origin that
arises in the peripheral nervous system.
The N-myc and c-myc genes have a very similar overall
structure, exhibit extensive homology in their coding
regions, and encode similar sized nuclear proteins (41, 99).
It has been confirmed that N-myc has transforming activity
equivalent to that of c-myc in the rat embryo fibroblast
assay (150). The N-myc gene has been found to be amplified
in all human neuroblastomas having cytogenetic
characteristics of gene amplification such as homogenously
staining regions or double minutes (147). Patterns of N-myc
amplification in neuroblastomas have been associated with
tumor progression. A greater copy number of the N-myc gene
is associated with a more advanced stage of the tumor.
(152). N-myc activation has thusfar been found to occur only
by amplification and only in a restricted set of tumors.
In addition to neuroblastomas, N-myc amplification has
been observed in a subset of small cell lung carcinomas
(SCLC) and in a few retinoblastomas (98, 106, 124). Like
neuroblastomas, these tumors have neural characteristics.
Considering the oncogenic potentials and similarities of
c-myc and N-myc, the reason for relatively restricted
activation of the N-myc gene as opposed to the c-myc gene is

57
unknown. The N-myc gene may play a special role in certain
types of neural tumors. Also, N-myc amplification events may
be specially targeted in the precursor cells of these tumors
(2) .
The L-myc gene was isolated by two independent methods.
The gene was first isolated on the basis of its amplification
in a subset of SCLCs (123). The gene was independently
isolated from unamplified genomes on the basis of its
homology to c-myc and N-myc (2). So far, activation of the
L-myc gene has only been observed in some SCLCs. Details
with respect to the structure and transforming potential of
L-myc call for further study, and will not be discussed here.
The expression of the c-myc gene has been shown to
follow a fixed relation to cell cycle. Growth arrested
fibroblasts (serum deprivation) in GO show a burst of c-myc
transcription during the G0/G1 transition when stimulated to
divide by either serum addition or insulin. The c-myc
transcript levels then decrease slowly as the cells proceed
through the cell cycle, and are present at basal levels
during S phase (19, 175). Nuclear run on assays with serum
released GO fibroblasts suggest that c-myc expression is
primarily regulated post-transcriptionally, at the level of
message degradation (19).

58
The c-myc protein product is a double stranded DNA
binding protein thought to interact with other genes, perhaps
those involved in cellular growth control. It is thought
that the myc protein can bind to the regulatory regions of
genes it controls, regulating transcription either by direct
activation or by inhibition of suppression (17).
The precise function the of c-myc protein has not been
elucidated. It is generally thought that its primary
function is to mediate a signal(s) associated with cell
division and thus, regulation of its expression is required
for normal cell growth (2). Experiments with c-myc antisense
RNA have shown that the ability of cells to divide can be
blocked (192).
The c-myc gene has been found to be present in many
types of sarcomas, carcinomas, and hematopoietic neoplasias
(32, 54, 77, 172). The two most widely studied mechanisms
of oncogenic activation of this gene are translocation
(seen in Burkitt lymphoma), and gene amplification. In the
case of Burkitt lymphoma the c-myc gene is translocated from
chromosome 8 to chromosome 14 or from chromosome 8 to 22. As
a result, the c-myc gene loses all or a portion of its first
exon, and acquires normally unlinked sequences involved in
immunoglobulin gene production (104, 121). C-myc gene

59
amplification has been found in many tumor types, and cell
lines, including HL-60 (promyelocytic leukemia), and COLO 320
(colon carcinoma) cell lines (2, 32).
The amplified region of the c-myc gene has been closely
studied in HL-60 cells. It has been shown that the amplified
region is very large and contains multiple copies of the
entire c-myc gene. Seguencing data indicates that
amplified c-myc gene units or "amplicons" appear to be
structurally normal (2). High levels of c-myc transcript
have been observed in HL-60 cell lines as well as the COLO
320 line and this has been attributed to gene dosage effects.
As a result of these investigations, it is commonly assumed
that when high levels of c-myc transcript are accompanied by
multiple copies, gene amplification is the cause of increased
expression.
Chromatin Structure Analysis of the C-myc Gene
The chromosomes of eukaryotes replicate, undergo
meiosis and mitosis, recombine, segregate, and are
transcribed. The occurence of these processes is
mediated through the interaction of chromosomal DNA and
proteins (72). In order for these proteins to act, specific

60
regions of the DNA must be accesible to binding.
Nuclease hypersensitive sites in chromatin are thought to be
regions that are open, and will allow DNA interaction
with proteins (70, 72). Therefore, it is thought that these
regions are specific for regulation of genes by cis and trans
acting factors. These protein accessible regions are
identified by their susceptibility to cleavage with
nuclease, and have been described to be twice as sensitive
as other areas of chromatin (72). DNAse I
hypersensitive sites are thought to represent approximately
1 percent of the entire genome (72) They were first
identified by Varshavsky (182) and by Scott and Wigmore
(151) who did studies with SV 40 chromatin. The presence of
these sites in chromatin of mammalian cells was discovered by
Wu and Elgin (195). These sites have been found in the
chromatin of plants, animals fungi, and in viral genomes (72,
189). Therefore they are considered to be very important in
the field of biology, and to the understanding of how
genetic regulation occurs among various species of
eukaryotes.
The indirect end labeling technique is most commonly
used in mapping locations of DNAse I hypersensitive sites.
This follows isolation of nuclei, treatment with DNAse I

61
and purification of the genomic DNA. Its most useful feature
is that it allows mapping of DNAse I sensitive sites in a
single direction (72). Regions of DNAse I sensitivity are
usually the size of a nucleosomal repeat which is
approximately 150-100 base pairs (72) This can make precise
mapping difficult, but resolution can be improved by fine
mapping technigues.
DNAse I hypersensitive sites have been associated with a
wide variety of functions (72). In Saccharomvces cerevisiae.
hypersensitive sites have been seen near centromeres,
silencers, recombination sites, origins of replication,
activation sequences, promoters, and potential sites of
transcription termination (72). Therefore, these sites are
probably associated with cis acting factors (72).
Topoisomerases I and II, RNA polymerase II, and some
transcription factors have been associated with DNAse I
hypersensitive sites (72). The proteins associated with most
sites in genes which have been studied have yet to be
identified.
The mechanisms which are involved in the formation and
maintainance of DNAse I hypersensitive sites are not clear.
Because the functions of these sites are so diverse, several
mechanisms are likely to be involved (72). It is thought

62
that interaction with trans-acting factors may be one
of these mechanisms (72). The base composition of the DNA,
methylation, looping, conformation, and torsional stress may
also have an involvement in this process (72).
Fundamental knowledge of these principles should provide
insight into molecular bases of regulation. Thus, it is a
well accepted fact that DNase I hypersensitive sites
represent regions where potential regulatory interactions are
thought to occur. Specific DNA seguences of this nature have
been shown to be located in promoter regions for such genes
as globin (51), immunoglobulin (53), c-myc (48, 73, 155,
156), heatshock (129, 193, 194), SV 40 early region (47),
and dihydrofolate reductase (154). The remainder of this
review will focus on those involving the c-myc gene.
As previously mentioned, amplification and translocation
are well known and widely studied potential c-myc activation
mechanisms. As a result of translocation to chromosome 14 in
Burkitt lymphoma, c-myc loses all or part of exon 1. This
exon is thought to serve primarily as a regulatory region, as
it is transcribed but not translated (77). Therefore, c-myc
may be deregulated by its loss, and may be influenced by
promoters of other genes proximal to its translocated site.
In the case of HL-60 cells (30-50 copies of the c-myc gene),

63
it was presumed that gene dosage effects are responsible for
observed increases in c-myc transcript. In both these
instances, gross structural mutations appear to be
responsible for observed changes in transcript levels. It
has only been recently that we have begun to understand the
effects these aberrations have on c-myc regulation or
deregulation as the cases may be.
During the past few years, data obtained from chromatin
structure analyses have demonstrated that changes in c-myc
gene regulatory sites accompany gross structural
abnormalities in both transocated and amplified states of
this gene. DNAse I sensitive sites in the 5' region of
exon 1 in HL-60 cells and Burkitt lymphoma cells have been
investigated in 2 separate studies by Siebenlist et al. (155,
156). DNAse I hypersensitive sites in HL-60 cells were
studied before and after differentiation with DMSO. Results
showed that 4 DNAse I sensitive sites were present in
untreated HL-60 cells (sites A, B, C, and D, figure 4). When
differentiation was induced with DMSO, site B was lost.
Further studies showed that the loss of this site accompanied
a timely decline in c-myc transcript production.
In a separate study, DNase I hypersensitive sites were
studied in both normal and rearranged c-myc alleles in a

64
\
A
I
HL-60 (DNASE I)
HL-60 (S 1)
BL-31 (DNASE I)
f = TRANSCRIPT
A B C D V = + HL-60
G
ABCD
ELONGATION BLOCK (BL)
+
B
I
V
TV
PO
CD E F
I I TT
P1 P2
G
C-MYC EXON 1 EXON 2
/
I 1
1 KB
Figure 4. Summary of chromatin structure analyses previously
described for the c-myc gene. Sites A, B, C, and D are DNAse
I hypersensitive sites found in both HL-60 cells (Siebenlist
et al (155)), and Burkitt lymphoma (BL-31) cells (Siebenlist
et al. (156)). Site B (indicated by open arrow) has been
described by Siebenlist et al. (155) to be involved in the
maintainance of c-myc transcript production in HL-60 cells,
and is therefore marked with a (+) symbol. Sites E and F
(solid arows) represent transcription attenuation sites found
in Burkitt lymphoma biopsies and cell lines and are marked by
a (-) symbol (25, 199). Site G is an S-l nuclease sensitive
site described by Grosso and Pitot (73) in HL-60 cells.

65
Burkitt lymphoma cell line (BL-31) and a normal B cell line.
Three different hypersensitive patterns which differed in
relative band intensities were observed. These correlated
with the three different transcriptional states of the c-myc
gene examined in this study (normal B cell myc, unrearranged
BL-31 c-myc, and translocated BL-31 c-myc). The locations of
hypersensitive sites which were observed were identical to
those for HL-60 cells (sites A,B,C,D) and are also shown in
figure 4.
Other groups working with Burkitt lymphoma have mapped
sites in the first intron and 3' region of exon 1. These
were later found to be transcription attenuation sites.
The loss of transcription elongation blocks at these sites is
now thought to be a possible candidate for deregulation of
myc in Burkitt lymphomas. Cesarman et al. (25) mapped a
previously found DNAse I hypersensitive site (9, 10, 49) to a
region near a Pvu II site in exon one. They reported that
23/26 Burkitt lymphoma cell lines and biopsies had point
mutations at various sites in a specific region extending 34
bases 5' and 38 bases 3' to the Pvu II site in exon 1 (site E
figure 4). These point mutations accompanied changes in
transcription, namely the removal of a block to transcription
mapped to the same region.

66
Zajac-Kaye et al. (199) noted similar findings in 5/7
Burkitt lymphoma cell lines. Their data indicated that a 20
base pair region in the first exon (site F, figure 4) was
susceptible to sporadic point mutations. Mutations in this
region abolished binding of a regulatory protein known to
down regulate c-myc transcription.
Relevance to This Project
The c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-
src proto-oncogenes have been studied in other human tumor
systems. The object of this study is to determine whether or
not these genes play a significant role in the biology of
chondrosarcoma and MFH. Studying transcript levels and copy
numbers of these genes will offer clues to possible
involvements in the pathogenesis and progression of these
tumors. Furthermore, chromatin structure analysis will
enhance understanding of mechanisms involved in transcript
regulation.

CHAPTER 3
MATERIALS AND METHODS
Slot-Blotting of RNA and DNA
Preparation of Total Cellular RNA
Total cellular RNA was prepared from surgically obtained
tumor, normal muscle, and bone marrow tissue specimens from
patients treated at Shands Hospital, University of Florida.
These specimens included; 20 chondrosarcomas, 23 malignant
fibrous histiocytomas (MFH), 9 normal muscle, and 6 bone
marrow tissue specimens. Approximately 1-3 hr after surgical
removal the tissues were frozen at -70 C until use. Total
cellular RNA was prepared as described by Chirgwin et al.
(26). Before use, all glassware and centrifuge tubes were
rendered nuclease-free with 0.1 percent diethylpyrocarbonate
(DEPC) in deionized water and thouroghly dried. All stock
solutions were freed of RNAse by adding several drops of 0.2
percent DEPC and subseguent autoclaving.
Tumor and normal tissues weighing 1.0-1.2 gr, or
approximately 10 E6 cultured cells were placed into 10 ml of
67

68
a solution containing 4M guanidine isothiocyanate (Ultra-
pure, BRL, Gaithersburg, MD), 25mM sodium citrate, pH 7.0,
and 0.1 M 2-mercaptoethanol. The mixture was then
homogenized using a tissuemizer (Brinkman Instruments), and
0.75 ml of 1 M acetic acid was added. The suspensions were
layered into SW 50 ultra-centrifuge tubes (Beckman
instruments) containing a 1.5 ml pad of 5.7 M cesium
chloride. The samples were centrifuged for 16 hr at 20 C and
35,000 rpm.
Following centrifugation, the pellets were resuspended
in 1.0 ml DEPC treated, sterile H20, and extracted once with
25:25:1 phenol:chloroform: isoamyl alcohol. The RNA was
then precipitated by adding 100 ul of 4 M potassium acetate,
and 2.5 ul ethanol. Recovery of the RNA was accomplished by
centrifugation at 10,000 rpm for 20 min at 4 C. The pellets
were resuspended in 0.2 mM EDTA, visualized on formaldehyde
agarose gels (as described for northern blotting below),
quantitated by absorbance at 280 nm, and stored in aliquots
at -70 C.
Preparation of Genomic DNA

69
Genomic DNA was prepared from the same tumor, muscle, and
bone marrow tissue specimens as described above. Tissues
were frozen in liquid nitrogen and ground to a fine powder
with a morter and pestal. This powder, or approximately 10
E6 cells was suspended in 9.2 ml of STE (lOOmM NaCl, 20mM
Tris, pH 8.0, lOmM EDTA). Two hundred ul of 0.5M EDTA and
200ul of proteinase K (10mg/ml) were then added, and the
mixture was incubated overnight at 65 C.
Following incubation, the mixture was extracted once
with an equal volume of phenol, once with an equal volume of
phenol/chloroform-isoamyl alcohol (24:1), and finally once
again with an equal volume of chloroform-isoamyl alcohol.
The DNA was recovered by spooling after the addition of an
equal volume of isopropanol, and resuspended in lOmM Tris,
ImM EDTA.
RNA Slot-Blotting
Quantites of 10, 5, and 2.5 ug of total cellular RNA were
denatured in a solution containing 100 ul of water and 300ul
of an RNA denaturant solution containing 6.15 M formaldehyde
and 10X SSC (sodium chloride, sodium citrate). The samples
were incubated at 65 C for 15 min and loaded onto a mini-

70
fold II slot-blotter (Schleicher & Schuell, Keene, NH) using
procedures described by Wahl (187) The slot-blotter
contained a Nitro-Plus 2000 filter (Micron Separation
Sciences) onto which the RNA was blotted.
DNA Slot-Blotting
Quantities of 20, 10, and 5 ug of DNA were suspended in
400 ul of lOmM Tris, ImM EDTA, pH 7.0, and 40 ul 3M NaOH,
then incubated at 65 C for 45 min. After incubation, the
samples were cooled on ice, 400 ul of 2 M ammonium acetate
were added, and the samples were loaded onto a minifold II
slot-blotter as described above.
After slot-blotting, the filters were air dried to
completion, then baked in a vacuum oven at 80 C for 2 hr.
The blots were then incubated at 42 C overnight in a pre
hybridization solution (5 ml/ 100 square cm) containing 5X
SSC, 10X Denhardt's solution (0.2 percent ficoll, 0.2 percent
polyvinylpyrrolidone (PVP), 0.2 percent bovine serum albumin
(BSA)), 0.05M sodium phosphate pH 6.7, 500ug/ul sonicated,
denatured salmon DNA, 5 percent dextran sulfate (Pharmacia
Chemical Co., Piscataway,NJ), and 50 percent formamide (112,
160) .

71
Preparation of Radiolabeled Probes
Descriptions, sources, and methods of labeling for all
probes used in slot-blot, Southern blot, northern blot, and
chromatin structure analysis are summarized in Table 1.
Figure 5 shows locations of the different c-myc probes as
well as other probes located on chromosome 8 used in mapping
and dilutional analysis of c-myc amplicons in MFHs.
Restriction maps of all other probes in Table 1 are shown in
figure 6.
Nick Translated Probes
Probes were nick translated by adding 250 ng of DNA
to a reaction mixture which contained 80 uCi 32P (dATP), 5.0
ul of 10X (dCTP, dGTP, dTTP), 1.25 ul of lmg/ml bovine serum
albumin (BSA), 5 ul of nick translation buffer (0.5 M
Tris HCL, pH 7.8, 0.1 M 2-mercaptoethanol, and 0.05 M MgC12),
1.5 ul DNAse I/Polymerase I (BRL, Gaithersburg, MD), and
deionized H20 to a final volume of 25 ul. The reaction was
run at 15 C for 45 min. Labeled DNA was separated from
unincorporated nucleotides using a Biogel A-15m (Biorad,
Rockville Center, NY) column.

72
Table 1. Summary of probes used in slot-blotting, Southern
blotting, northern blotting, and chromatin structure
analyses.
Probe
Name
Source*
Use**
Reference
Method of
Labeling+
Description
C-myc
1
1,4
1
N.T
9.0 kb Eco R 1/
Hind III human
genomic fragment
cloned into PBR
322
C-Ha-ras
1
1
137
N.T.
6.4 kb Bam HI
human genomic
fragment cloned
into PBR 322
C-fos
1
1
36
N.T.
6.4 kb Xho 1/
Neo I human
genomic fragment
cloned into PBR
322
C-sis
2
1
71
N.T.
1.0 kb human
genomic fragment
cloned into
pSP 6
V-erb-B-1
1
1
108
N.T
1.7 kb Pvu 11/
Sst I genomic
fragment from
avian erythro
blastosis virus
V-src
1
1
37
N.T.
800 bp Pvu II
genomic fragment
from avian
sarcoma virus
prague A strain

73
Table 1. Continued.
Probe Source*
Name
Use**
Reference
Method of Description
Labelingt
Beta-
actin
1
1
28
N.T.
800 bp Nco 1/
Taq I genomic
fragment from
chicken beta-
actin gene
TK
(pTK 11)
3
1,2
21
N.T/
P.E.
1.25 kb Sma I
Bam H I human
genomic fragment
pGEM-H MYC
4
2,3,4
188
N.T
1020 bp Pst I
human cDNA
fragment cloned
into pGEM 1
p380-8A
5
4
78
N.T.
1.8 kb Sal I/SstI
human genomic
fragment cloned
into puc 19
Carbonic
Anhydrase
(H 25-3.8)
6
4
183
N.T.
3.8 kb Eco R I
human genomic
fragment cloned
into PBR 325
Thyro-
globulin
(HT .96)
6
4
3
N.T.
960 bp Pst I
human cDNA cloned
into puc 8

Table 1. Continued.
Probe
Name
Source*
Use**
Reference
Method of
Labeling+
Description
Beta-
actin
7
2
75
P.E.
2.0 kb Bam HI
human cDNA
fragment
PMC 41
8
5
73
N.T.
1.6 kb Cla 1/
Eco RI human
genomic fragment
cloned into PBR
322
C-myc
Sca/Xho
I 9
6
1
P.E.
355 bp Sea 1/
Xho I human
genomic fragment
* Sources: 1)
Oncor,
Inc, Gaitersburg, MD,
2) Oncogene
Sciences, Minela, NY, 3) Dr. Harvey Bradshaw, 4)
Dr. Ken Soprano, 5) Dr. Carlo Croce, 6) American Type
Culture Collection, Rockville, MD, 7) Dr. Larry Kedes,
8) Dr. Robert Gallo, and 9) Made from c-myc plasmid
obtained from Oncor, Inc. (above)
** Uses: 1) Slot-blot hybridization, 2) northern blot
hybridization, 3) titration of c-myc gene copy number
in MFHs, 4) Mapping of c-myc amplicons in MFHs,
5) mapping DNAse I hypersensitive sites in cell lines
from the 3' direction, and 6) mapping DNAse I
hypersensitive sites in cell lines from the 5'
direction (fine mapping analysis).
+ Method of labeling: N.T. = nick translation, P.E. =
random primer extension.

75
33:3
23.1
22
12
11.2
12
13
21.3
22.1
22.2
22.3
23
24.1
24.2
24.3
CA-2
<
o
Imyc
Itg
H25-3.8
ir
P0P1 P2
SCA l/XHO I
P380-8A
8
h h
(0 w
a a
PGEMH MYC
HT.96
CMYC
Figure 5. Locations of the different c-myc probes as
described in table 1. Also shown are other probes located on
chromosome 8 which were used in mapping of c-myc amplicons in
MFHs.

76
i i
a
HL
C-FOS 6.4 KB
5
o
£
s
C-HA RAS 6.4 KB
_ £

_
§ 8
o
G
Q
Q
9
Z
Z
Z
X UJ
I
X
I
ii
i
1
j
¡2 I x
\-¡-t L,
x x SE
C-SIS 1.0 KB
LTR
1
BETA ACTIN 0.8 KB
95
i£
LL
LTR
VSRC
0.8 KB
E
o ^
9 I
ECO
1i 1
1 1
X
S-
<:
PTK 11 1.25 KB
LTR
V- ERB-A V-ERB-B
fc
_x_
x_
(/>
LTR
£
as
B
jjj
ii
1.7 KB
BETAACTIN 2.0 KB
Figure 6. Restriction maps of non-c-myc/chromosome 8 probes
as described in table 1.

77
Probes Labeled by Random Primer Extension
Two hundred ng of DNA were denatured at 90 C for 2 min.
After denaturation, five ul of 5X primer extension buffer (1M
hepes, pH 6.6, 25mM MgC12, 50 mM 2-mercaptoethanol, 0.25 M
Tris HCL, pH 8.0, 0.1 mM dCTP/dGTP/dTTP, 2mg/ml BSA, 15 mg/ml
primer), 5 units Klenow (BRL, Gaithersburg, MD), 100 uCi 32P
(dATP) and H20 were added to a final volume of 25 ul. This
reaction mixture was allowed to sit at room temperature for
16 hr. Unincorporated nucleotides were separated from
labeled DNA as described for nick translations.
Hybridization of Slot-Blots
Slot- blots were hybridized at 42 C with 3.0 X 10 E6
cpm (1.0 X 10 E8 cpm/ug) of probe for at least 20 hr in 15 ml
of hybridization solution containing 5X SSC, IX Denhardt1s
solution, 0.02 M sodium phosphate, pH 6.7, 100ug/ml
sonicated, denatured salmon DNA, 10 percent dextran sulfate,
50 percent formamide, and 6 percent water (112).
Post hybridization washes were carried out by washing
the filters twice for 15 min at room temperature with 2X
sodium chloride, sodium phosphate, EDTA (SSPE), 0.1 percent

78
sodium dodecyl sulfate (SDS). The blots were then washed
twice again at 50 C with 0.IX SSC, 0.1 percent SDS for 30
min each and exposed to preflashed X-ray film for 36-48 hr at
70 C with intensifying screens.
Slot-blots were rehybridized after treatment of the
membrane to remove bound probe. This was accomplished by
pouring 1 liter of 0.1 X SSPE, 0.1 percent SDS heated to 90 C
over the blots. The solution was then cooled to 70 C and
removed. (187)
Southern Blot Analysis
Genomic DNA was prepared as described above from tissue
samples and cell lines. Aliquots of DNA were restricted with
appropriate restriction endonucleases and electrophoresed
through 0.8 percent agarose gels (65 volts, 16 hr).
Digested DNA was then transferred to Zetabind (AMF Cuno,
Meriden, CT), pre-washed in 0.1 X SSC, 0.1 percent SDS at 65
C for 1 hr, and hybridized (2 X 10 E6 cpm/ml/10 E8 cpm/ug)
using pre-hybridization and hybridization conditions
previously described for slot-blotting (112, 160). Post
hybridization washes were performed by washing the membrane
at room temperature for 15 min, once with 2 X SSC,

79
0.1 percent SDS, and once again with 0.1 X SSC, 0.1 percent
SDS. The blots were then washed twice for 30 min at 60 C,
with 0.1 X SSC, 0.1 percent SDS and exposed to X-ray film
at -70 C with intensifying screens. Rehybridization of the
blots was accomplished after removal of bound probe. This
removal process consisted of washing the blots in 0.1 X SSC,
0.5 percent SDS at 80 C for 15-20 min.
Northern Blot Analysis
Total cellular RNA was prepared from cell lines as
described for slot-blotting. RNA was denatured at 55 C for
15 min in an RNA denaturant containing 5ul 10X MOPS, 8.75 ul
37 percent formaldehyde, 25 ul formamide (ultra-pure BRL,
Gaithersburg, MD), and water to a final volume of 50 ul.
After denaturation, 10 ul of RNA formaldehyde loading dye was
added (500 ul formamide, 162 ul 37 % formaldehyde, 350 ul
glycerol, 100 ul 10X MOPS, bromophenol blue). The samples
were electrophoresed through 1.2 percent formaldehyde
agarose gels (139). The gels were prepared by melting 4.2 gr
agarose in 304.5 ml water. After cooling, 35 ml MOPS and
10.5 ml formaldehyde were added. Samples were

80
electrophoresed in running buffer which consisted of IX MOPS
and 10 percent formaldehyde (volume/37%) at 120 volts for 3-
3.5 hr.
After electrophoresis, the gels were rinsed several
times in deionized water, then soaked in 10X SSC for 45 min.
Blotting stacks were assembled as for Southern blotting.
Overnight transfers to Zetabind membranes were completed in
2OX SSC. Blots were pre-washed in 0.1 X SSC, 0.5 percent SDS
at 65 C for 1 hr. Pre-hybridization and hybridization
conditions (2.0 X 10 E6 cpm/ml/ 10 E8 cpm/ug), as well as
post-hybridization washes and rehybridization procedures were
identical to those described for Southern analysis.
Chromatin Structure Analysis
Cell Lines Used in Chromatin Structure Analysis
UR-HCL-1. The UR-HCL-1 cell line is a human MFH tumor
cell line obtained from ATCC.
P3C. The P3C cell line is an MFH tumor cell line
obtained from Dr. Byron Croker, Department of Pathology,
University of Florida. The cell line was made by culturing

81
an MFH from a patient treated at Shands Hospital, University
of Florida.
ST 486. The ST 486 cell line is a Burkitt lymphoma
cell line obtained from ATCC. This cell line was used as a
positive control for chromatin structure analyses, as DNAse
I hypersensitive patterns for Burkitt lymphoma c-myc have
been described (156).
HFF. The HFF normal human fibroblast cell line was
obtained from Dr. Kenneth Rand, Department of Pathology,
University of Florida.
Preparation of Nuclei
Cells were grown in Dulbeccos MEM (minimal essential
medium) (Gibco, Gaithersburg, MD), supplemented with 10
percent fetal bovine serum (Gibco, Gaithersburg, MD).
Nuclei were isolated from dividing cells (approximately 2.0 X
10 E8). The cells were washed in 100 ml IX phosphate
buffered saline (PBS), and centrifuged at 2,000 rpm, for 3
min at 4 C. The pellet was resuspended in 10 ml IX
RSB (lOmM Tris, pH 7.4, lOmM NaCl, 3mM Mg C12), 0.5 percent

82
nonidet P40 (NP 40), 10 ul 0.1M phenyl methyl sulfate (PMSF),
then incubated on ice (0 C) for 5 min. The nuclei were
recovered by centrifugation at 2,000 rpm at 4 C for 3 min.
The pellet was then washed 3 times with 100, 50 and 20 ml of
IX RSB followed by centrifugation at 2,000 rpm at 4C for 3
min.
The nuclei were then resuspended in IX RSB and digested
with varying concentrations of DNAse I (Boerhinger Mannheim)
for 10 min at 37 C. Controls were 0 ug/ml DNAse I incubated
at both 0 and 37 C.
Isolation of Genomic DNA From DNAsel Treated Nuclei
After digestion with DNAse I, the samples were placed on
ice and 1/10 volume 0.25M EDTA was added along with 1/20
volume 10 percent SDS and 1/20 volume proteinase K. After
incubation overnight at 37 C, the samples were extracted
with an equal volume phenol, an equal volume phenol/24:1
chloroform-isoamyl alcohol, then a third time with an equal
volume of 24:1 chloroform isoamyl alcohol. DNA was
precipitated by adding 4M potassium acetate to a final
concentration of 0.3M, and the addition of 2-3 volumes cold
95 percent ethanol. After precipitation at 20 C overnight,

83
DNA was recovered by centrifugation at 10,000 rpm at 4 C for
15 min. The pellet was resuspended in 300 ul 50mM Tris,
pH 8.0, 10 mM EDTA. RNAse A (lmg/ml) was added to a final
concentration of 50 ug/ml and incubated overnight at 37 C.
The mixture was then phenol/chloroform extracted, and the DNA
precipitated as described above. The final DNA pellet was
resuspended in lOmM Tris, ImM EDTA.
Aliquots of DNA from each of the cell lines were
restricted with either Eco R1 (mapping of DNAse I
hypersensitive sites from a 3' direction (pmc 41 probe)), or
Sea I (mapping of these sites from a 5' direction (Sea I/Xho
I fragment probe)), and analyzed using Southern blotting and
hybridization methods described above. The mapping of DNAse
I hypersensitive site locations was accomplished through the
use of the indirect end labeling technique. This technique
allows mapping of the DNAse I hypersensitive sites in one
direction, and is described in figure 7 using restriction
with Eco R1 and hybridization with pmc 41 as an example.
Chromatin Structure/ Fibroblast Cell Synchrony Experiment
HFF cells were grown to 70-80 percent confluency in
Dulbeccos MEM supplemented with 10 percent fetal bovine

84
1¡
S/WVY
PMC 41
i
ISOLATE DNA, DIGEST WITH ECO F
X
SOUTHERN TRANSFER, PROBE WITH PMC 41
Figure 7. Illustration of the indirect end labeling
technique. This technique allows 5' or 3' orientation of
the locations of DNAse I hypersensitive sites. It always
yields pieces of DNA which have a restriction site on one
side, therefore allowing analysis of DNA segments in one
direction. Various concentrations of DNAse I are used to
allow partial digestion. If DNAse I cut at sites 1 and 2,
analysis by this method would yield three bands on a Southern
blot. A main band of 9.6 kb which corresponds to the Eco
Rl/Eco R1 fragment would be present along with bands of sizes
corresponding to the lengths of the DNAsel cleavage sites to
the 3' ECO R I site.

85
serum. At this confluency level, cells were actively cycling
(confirmed by Northern blot hybridization with the TK probe
data shown below). The cells were then made quiescent by
the addition of MEM containing 0.1 percent serum, and
subsequent incubation at 37 C for 3 days.
MEM supplemented with 10 percent fetal bovine serum was
then added to release the cells. Nuclei isolation/DNAse I
treatment (as described above) and RNA isolation procedures
(as described above) were conducted at 0 hr (GO), 0.5 hr, 1
hr, 2 hr, 3 hr after serum release, and during log phase
growth. DNAse I hypersensitive sites were evaluated by
Southern blot hybridization procedures using the pmc 41 probe
as described above. RNA samples were analyzed for c-myc,
TK, and actin transcript levels using Northern blot
procedures as previously described.
Strategy for Fine Mapping DNAse I Hypersensitive Sites 31 to
C-Mvc Exon 1
An overall scheme for fine mapping of DNAse I
hypersensitive sites from a 5' direction is shown in figure
8. Selected restriction enzyme sites in the region of a
DNAse I hypersensitive site 3' to c-myc exon 1 (discussed

86
C-MYC EXON 1 EXON 2
SCA l/XHO 1
355 BP
PROBE
1 1
SCAI/PVUII
805 BP
1KB
SCAI/BSMI
SCA l/MAE III
920 BP
970 BP
y = TRANSCRIPT ELONGATION BLOCK (BL)
Figure 8. Location of the Sea I/Xho I probe and other
restriction sites in and around c-myc exon 1 used in fine
mapping of DNAse I hypersensitive sites from the 5' direction
in P3C and HFF cell lines. These sites were used to generate
P3C DNA fragments of known sizes, which were internal size
markers on the mapping blot (see materials and methods).
Also shown are the c-myc promoters (PO, PI, and P2), and
known c-myc transcript elongation block sites which are
indicated by solid arrows.

87
below) were chosen to generate fragments used as internal gel
markers to map DNAse I hypersensitive sites in this region.
First, P3C genomic DNA was restricted to completion with
Sea I (Boerhinger Mannheim). Five ug aliquots of this DNA
were then restricted a second time with either Pvu II (BRL,
Gaithersburg, MD), Bsm I (New England Biolabs), or Mae III
(Boehinger Mannheim).
Bsm I cut a single time within the c-myc gene, therefore
the restriction reaction was allowed to go to completion
overnight at 65 C. Pvu II and Mae III cut at multiple
sites within the c-myc gene. Reactions were therefore
controlled to prevent complete digestion of the DNA.
Partial digestion of DNA with an enzyme which cuts at
multiple sites between a desired site and Sea I will yield
band sizes corresponding to distances between the desired
site and the Sea I site.
Five ug aliquots of Sea I restricted P3C DNA were
restricted with various concentrations of Pvu II (37 C) and
Mae III (55 C) for 30 min reaction times. Concentrations
which yielded optimum visualization of the desired marker
band sizes were used to restrict P3C DNAs for use as markers
in mapping analyses.

88
DNAse I treated DNA samples (samples with optimum
visualization of DNAse I generated bands from previous
analysis) from P3C (0.2 and 0.5 ug/ml) and HFF (0.5 ug/ml)
cell lines were restricted with Sea I. Five ug aliguots of
these samples along with marker DNAs generated as described
above were electrophoresed in 1.5 percent agarose gels (65
volts, 20 hr), and blotted onto Zetabind. Lambda DNA
digested with Eco R 1 and Hind III was run on either side of
the gel to assure that the gel ran evenly. Prehybridization,
hybridization (2.0 X 10 E 8 cpm/ml/ 10 E8 cpm/ug), and
washing conditions were identical to those described for
Southern blotting.
Polyacrylamide Gel Electrophoresis (PAGE)
The discontinuous system for PAGE as described by
Laemmli (101) was used in this analysis. Stacking gels were
4 percent acrylamide (total) in 0.125 M Tris-HCl, pH 6.8, and
0.1 percent SDS. Separating gels were 8.5 percent total
acrylamide in 0.375 M Tris-HCL, pH 8.8, and 0.1 percent SDS.
Both gels were cross-linked with 2.7 percent bis acrylamide,
and polymerization was catalyzed with 0.005 percent TEMED,
and 0.05 percent ammonium persulfate.

89
Twenty ug of protein were combined with an equal volume
of treatment buffer (0.125 M Tris-HCL, pH6.8, 4 percent SDS,
20 percent glycerol, and 10 percent 2-mercaptoethanol),
incubated at 90 C for 1.5 min, ice-quenched, then loaded onto
the gels. Molecular weight markers ranging from 31,000 to
200,000 daltons (Biorad) were loaded as well.
A tank buffer which consisted of 25 mM Tris-HCL, pH 8.3,
0.192 M glycine, and 0.1 percent SDS was used as a running
buffer. Gels were electrophoresed in a Hoefer SE 600
vertical slab unit at 30 ma/1.5 mm gel thickness.
Western Blotting and Immunoperoxidase Assay
Western blotting of proteins was carried out at 0.6 amps
for 45 min at 4 C. Proteins were blotted onto 0.2 um pore
size nitrocellulose (Schleicher & Schuell, Keene, NH) using
methods described by Towbin (176). Transfer was carried out
using a Hoeffer TE 52 Transphor unit. Following transfer,
the blots were air-dried, then incubated for 3 hr in PBST (IX
phosphate buffered saline (PBS), 0.05 percent Tween 20) and 2
percent BSA. An anti- human c-myc monoclonal antibody (HL-
40) (IGG 1, ascites purified by protein A column) obtained
from Dr. Henry Neiman was then diluted (0.1 mg/ml) in PBST, 2

90
percent BSA, added to one blot, and allowed to incubate at
room temperature for 1 hr with light agitation. As a control
for non-specific binding, an identical blot was incubated
with an anti-met 72 monoclonal antibody (K 88. 151. G 127)
(IGG 1, ascites purified by protein A column), (0.01 mg/ml in
PBST, 2 percent BSA) obtained from Dr. Arthur Kimura. The
blots were then washed 3 times for 5 min each with PBST.
This was followed by incubation with a 1 ug/ml solution of a
horseradish peroxidase conjugated goat-anti-mouse Ig
(Southern Biotechnology Associates) diluted in PBST, 2
percent BSA for 1 hr at room temperature. Blots were then
washed 3 times with PBST as before, and incubated with a 180
ug/ml solution of the substrate, diaminobenzoate (DAB), in
PBST, and 0.01 percent H202 for 2-3 minutes. The reaction
was stopped with excess H20. Quantification of c-myc protein
bands was carried out by reflectance densitometry.
As a control for guantification, a third identical blot
was stained with a 0.1 percent solution of india ink in PBST
for 1 hr at room temperature, then destained with PBST until
the desired resolution was achieved.

CHAPTER 4
RESULTS
Quantitation of Moderately Degraded RNA Using the Slot-
Blotting Technique
The relationship between RNA degradation and accuracy of
quantitation was evaluated because in many instances tumor
tissues were not immediately (1-3 hr) available for
processing after surgical removal, and message degradation
occurs rapidly. Intact total cellular RNA from HL-60 cells
was degraded in 0.2 N NaOH at 0, 0.5, 1,2,5, and 30 minute
intervals. After evaluation by formaldehyde gel
electrophoresis using procedures previously described
(northern blotting section), (figure 9), the sample at 0
minutes showed completely intact RNA, the samples at 0.5, 1,
2, and 5 minutes, moderately degraded RNA, and at 30 minutes
the RNA was extensively degraded. Analysis of these samples
by slot-blotting (figure 10) demonstrated that moderately
degraded RNA as shown in lanes B, C, D and E, is
as sensitive to quantitative changes as intact total
cellular RNA (lane A). Extensively degraded RNA shown in
91

92
A B C D E F
Figure 9. Formaldehyde gel electrophoresis of 10 ug of HL-
60 RNA following alkaline degredation with 0.2 N NaOH and
neutralization with 0.1 M Tris, Ph 7.5 after (a) 0 min (b)
0.5 min (c) 1 min (d) 2 min (e) 5 min and (f) 30 min. Lane a
shows totally intact RNA, lanes b, c, d, and e intermediate
degredation, and lane f, extensively degraded RNA.

93
A
B
C
D
E
F
Densitometer readings for slot-blot of alkaline
degraded total cellular RNA from HL-60 cells
as shown above.
A 8.71 E5
E 8.64 E5
C 9.03 E5
D 8.41 E5
E 8.66 E5
F 1.09 E5
Figure 10. Slot-blot of alkaline degraded HL-60 total
cellular RNA hybridized with the c-myc probe (3.0 X 10 E6
cpm / 1 X 10 E 8 cpm/ug). Densitometric values (peak areas
sq um/ug) correspond to hybridization signals for 2.5 ug of
RNA neutralized after (a) 0 min (b) 0.5 min (c) 1 min (d) 2
min (e) 5 min and (f) 30 min. Densitometric values (shown
below blot) for lanes A-E indicated that moderately degraded
RNA was as reliably quantitated by slot-blotting as totally
intact RNA. Extensively degraded RNA (lane F) was not, and
no samples showing this level of degredation were used in
these analyses.

94
lane F was unacceptable for analysis because it could not be
evaluated for gene expression as quantitatively as total
cellular RNA. Also, the hybridization signal intensity of
the extensively degraded RNA was just below the linear range
for this system which was determined to be 1.10 X 10 E5-2.2 X
10E 6 square um/ug RNA (166). No patient samples
showing this level of degradation were used. All RNA samples
used in this study conformed to the criteria for moderate
degredation after visualization by formaldehyde gel
electrophoresis (figure 11).
RNA from 20 chondrosarcomas, 23 malignant fibrous
histiocytomas, 9 normal muscle (normal non-proliferating
mesenchymal tissue), and 6 bone marrow (normally
proliferating mesenchymal tissue) tissues were screened for
proto-oncogene transcript levels and gene copy numbers of c-
myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-src. These
genes were studied because of previous associations with
sarcomas in humans and other animals. The v-erb-B-1 and v-src
gene probes were used because c-erb-B-1 and c-src were not
available.
Hybridization signal intensities from these blots were
normalized to those of beta-actin and thymidine kinase (TK).
Beta-actin is a constitutively expressed non-cell-cycle

95
z z s z z z
11* 11 11
u A to ^ -* -*>
Figure 11. RNA formaldehyde gel electrophoresis of total
cellular RNA from 6 MFHs. These samples illustrate typical
moderate degredation seen with RNA samples extracted from
tumor and normal tissues.

96
dependent gene (75, 134), and was used to normalize
hybridization signal intensities for c-sis, v-erb-B-1, and v-
src, which also are non-cell-cycle-dependent. Hybridization
signal intensities of cell-cycle dependent genes c-myc, c-Ha-
ras, and c-fos were normalized to TK, also a cell-cycle-
dependent gene (93) Ratios of TK/actin were taken as a
molecular measurement of cell division.
The extent of hybridization for slot-blots was estimated
by densitometric scanning of the x-ray film (Bio-rad video
densitometer). Results were reported as the logarithm of the
peak areas for 10, 5, and 2.5 ug for RNA, and 20, 10, and 5
ug for DNA. The mean values for the 3 aliquots of each RNA
and DNA sample were calculated and used to determine a
gene:actin and gene:TK ratio (tables 2-13). All DNA values
are normalized to actin as are RNA values for c-sis, v-erb-
1, and v-src. C-myc, c-Ha-ras, and c-fos RNA levels are
normalized to TK. Significant differences between any two
groups were determined with Students T test.
RNA Slot-Blot Results
Thymidine kinase, c-myc, c-Ha-ras, and c-fos are
undetectable in muscle using this method of detection

97
Table 2. C-myc, c-Ha-ras, and c-fos:TK ratios from slot-
blot analyses of total cellular RNA from normal muscle and
bone marrow tissues. Also shown are TK:actin ratios which
were used as molecular measures of cell cycle.
SAMPLE
c-mvc
TK
c-Ha-ras
TK
c-fos
TK
TK
actin
MUSCLE
MFH patients
MM-1
*
*
*
*
MM-2
*
*
*
k
MM-3
*
*
*
*
MM-4
*
*
:k
*
MM-5
*
*
*
*
CHONDROSARCOMA
patients
MC-1
*
*
*
*
MC-2
*
*
*
*
MC-3
*
k
*
*
MC-4

*
k
*
BONE MARROWS
BM-1
1.18
1.28
1.05
0.909
BM-2
0.945
1.15
0.935
1.01
BM-3
0.848
0.831
0.834
1.02
BM-4
0.804
0.972
0.935
1.13
BM-5
0.551
1.05
1.08
1.28
BM-6
0.957
0.813
0.968
1.15
* C-myc, c-Ha-ras, c-fos, and TK were undetectable in normal
muscle tissues by these analyses.

98
Table 3. C-sis, v-erb-B-1, and v-src:actin ratios from
slot-blot analyses of total cellular RNA from normal muscle
and bone marrow tissues.
SAMPLE
c-sis v-erb-B-1 v-src
actin actin actin
MFH patients
MM-1
0.970
0.770
0.926
MM-2
0.989
0.882
1.02
MM-3
0.963
0.842
0.973
MM-4
0.904
0.835
1.00
MM5
0.911
1.02
1.07
CHONDROSARCOMA
patients
MC-1
0.933
0.897
0.949
MC-2
0.947
0.765
0.763
MC-3
0.897
0.981
1.06
MC-4
1.14
0.949
0.893
Bone marrows
BM-1
1.15
1.28
1.18
BM-2
1.10
1.18
0.989
BM-3
1.09
0.863
0.876
BM-4
0.925
1.10
1.26
BM-5
1.16
1.22
0.998
BM-6
1.02
1.27
1.07

99
Table 4. C-myc, c-Ha-ras, and c-fos:actin ratios from slot-
blot analyses of genomic DNA from normal muscle and bone
marrow tissues. Also shown are TK:actin ratios.
SAMPLE
c-mvc c-Ha-ras c-fos TK
actin actin actin actin
MFH patients
MM-1
1.06
1.01
0.964
0.810
MM-2
1.03
1.01
0.898
0.951
MM-3
1.27
1.03
0.876
0.920
MM-4
0.948
0.958
0.979
0.944
MM-5
0.964
0.964
0.918
0.996
CHONDROSARCOMA
patients
MC-1
1.21
1.17
0.952
0.955
MC-2
1.02
1.06
0.842
0.747
MC-3
1.22
1.05
0.868
1.10
MC-4
1.03
1.21
0.952
0.924
Bone marrows
BM-1
0.746
0.918
0.858
0.903
BM-2
1.16
0.990
1.21
1.21
BM-3
0.853
1.08
0.975
1.02
BM-4
0.743
0.792
0.743
0.847
BM-5
1.09
0.903
1.13
1.27
BM-6
0.786
0.731
0.786
0.897

100
Table 5. C-sis, v-erb-B-1, and v-src:actin ratios from
slot-blot analyses of genomic DNA from normal muscle and bone
marrow tissues.
SAMPLE
c-sis v-erb-B-1 v-src
actin actin actin
MFH patients
MM-1
0.979
0.907
0.927
MM-2
0.962
1.00
1.04
MM-3
1.01
1.03
1.01
MM-4
0.876
0.856
0.825
MM-5
0.933
0.989
0.893
CHONDROSARCOMA
patients
MC-1
0.856
1.06
0.968
MC-2
0.852
0.974
0.888
MC-3
0.953
1.05
0.108
MC-4
0.925
0.973
0.957
Bone marrows
BM-1
1.06
1.00
0.940
BM-2
1.05
1.14
1.13
BM-3
0.829
0.846
0.919
BM-4
0.861
0.757
0.822
BM-5
1.18
1.19
0.894
BM-6
0.882
1.09
0.844

101
Table 6. C-myc, c-Ha-ras, and c-fos:TK ratios from slot-blot
analyses of total cellular RNA from chondrosarcomas. Also
shown are TK:actin ratios which were used as molecular
measures of cell cycle.
CHONDROSARCOMA SAMPLE
c-mvc
TK
c-Ha-ras
TK
c-fos
TK
TK
actin
CS-1
0.693
0.522
1.02
1.16
CS-2
1.02
0.971
1.06
0.752
CS-3
0.911
1.64
0.934
0.596
CS-4
0.531
0.218
0.292
1.16
CS-5
0.452
0.594
2.49
1.19
CS-6
0.468
0.195
1.60
2.19
CS-7
1.41
1.02
1.35
0.608
CS-8
1.03
0.575
1.16
0.861
CS-9
1.19
0.887
1.43
0.653
CS-10
0.804
0.666
1.06
1.50
CS-11
2.49
1.62
0.982
0.370
CS-12
0.726
0.383
1.14
0.698
CS-13
2.17
0.502
0.894
0.839
CS-14
1.80
1.90
0.976
0.301
CS-15
0.812
0.353
1.20
1.52
CS-16
**
0.466
0.927
2.47
CS-17
0.416
0.468
1.00
2.56
CS-18
1.10
1.46
0.760
0.996
CS-19
2.63
0.233
0.934
0.331
CS-2 0
1.23
1.06
1.54
0.536
**
C-myc was not detectable

102
Table 7. C-sis, v-erb-B-1, and v-src:actin ratios from slot-
blot analyses of total cellular RNA from chondrosarcomas.
CHONDROSARCOMA SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
CT-1
2.42
0.821
0.993
CT-2
1.87
1.37
1.20
CT-3
0.637
0.060
0.841
CT-4
2.60
2.70
0.798
CT-5
1.22
2.57
0.888
CT-6
2.29
0.928
0.783
CT-7
0.180
2.31
1.01
CT-8
0.604
0.714
0.863
CT-9
6.08
1.86
0.697
CT-10
2.32
1.26
1.29
CT-11
0.130
1.28
0.900
CT-12
2.14
1.15
0.700
CT-13
0.457
0.979
0.801
CT-14
0.135
0.561
0.777
CT-15
3.38
0.822
1.33
CT-16
0.247
0.549
1.11
CT-17
1.79
0.511
0.890
CT-18
1.97
0.448
0.697
CT-19
0.637
0.688
0.770
CT-2 0
0.148
0.605
0.838

103
Table 8. C-myc, c-Ha-ras, and c-fos:actin ratios from slot-
blot analyses of genomic DNA from chondrosarcomas. Also shown
are TK:actin ratios.
CHONDROSARCOMA SAMPLE
c-mvc
actin
c-Ha-ras
actin
c-fos
actin
TK
actin
CS-1
0.980
0.825
0.948
0.951
CS-2
0.757
0.933
1.20
1.06
CS-3
0.878
0.929
0707
0.982
CS-4
0.955
0.700
0.642
1.06
CS-5
0.922
1.16
1.14
1.04
CS-6
0.775
0.769
0.656
0.973
CS-7
1.03
0.735
0.816
0.974
CS-8
0.933
0.698
0.872
0.903
CS-9
1.14
0.901
1.18
1.24
CS-10
1.23
1.35
0.956
1.23
CS-11
1.24
0.756
0.783
1.08
CS-12
0.993
0.868
1.03
0.833
CS-13
1.12
1.20
1.33
1.19
CS-14
1.08
0.800
1.24
1.04
CS-15
1.03
0.793
1.25
0.833
CS-16
0.885
1.23
0.799
1.19
CS-17
1.25
1.05
1.32
1.04
CS-18
1.09
1.04
0.774
0.879
CS-19
1.16
0.901
1.31
1.18
CS-2 0
1.05
1.14
1.09
1.17

104
Table 9. C-sis, v-erb-B-1, and v-src:actin ratios from slot-
blot analyses of genomic DNA from chondrosarcomas.
CHONDROSARCOMA SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
CS-1
1.25
0.933
0.749
CS-2
0.925
0.995
0.766
CS-3
1.28
0.846
1.28
CS-4
1.14
0.997
0.803
CS-5
1.36
1.38
1.35
CS-6
1.31
1.34
1.37
CS-7
1.24
1.24
1.22
CS-8
1.12
0.976
1.00
CS-9
1.17
1.06
1.24
CS-10
1.04
0.842
1.39
CS-11
1.22
0.784
1.25
CS-12
1.18
1.26
1.30
CS-13
1.29
1.29
1.32
CS-14
1.14
0.944
1.16
CS-15
1.23
0.898
1.19
CS-16
1.31
0.910
1.31
CS-17
1.39
0.805
1.20
CS-18
1.14
0.817
1.20
CS-19
0.699
1.12
0.682
CS-2 0
1.28
1.34
1.08

105
Table 10. C-myc, c-Ha-ras, and c-fos:TK ratios from slot-
blot analyses of total cellular RNA from MFHs. also shown
are TK:actin ratios which were used as molecular measures
of cell cycle.
MFH SAMPLE
c-myc
c-Ha-ras
c-fos
TK
TK
TK
TK
actin
MT-1
0.455
0.686
0.419
0.501
MT-2
1.24
0.412
1.27
0.560
MT-3
1.14
0.465
2.54
2.03
MT-4
1.00
0.184
1.15
1.01
MT-5
0.870
0.781
0.987
0.636
MT-6
1.31
0.474
0.862
2.19
MT-7
0.690
0.412
0.953
0.651
MT-8
1.68
1.05
0.520
2.35
MT-9
0.935
0.360
0.417
0.602
MT-10
1.00
1.25
0.458
0.393
MT-11
1.09
3.87
0.556
1.31
MT-12
1.21
0.346
0.968
1.29
MT-13
1.26
0.370
0.472
1.46
MT-14
1.04
3.40
0.855
1.10
MT-15
1.15
0.258
0.908
1.51
MT-16
1.92
0.198
0.899
1.63
MT-17
1.97
0.498
0.895
2.13
MT-18
1.96
0.215
0.955
3.69
MT-19
1.05
0.145
1.25
1.30
MT-20
1.93
1.27
1.04
1.33
MT-21
1.14
0.896
1.30
1.06
MT-22
1.96
1.20
1.04
1.71
MT-2 3
1.10
0.987
1.30
1.03

106
Table 11. C-sis, v-erb-B-1, and v-src: actin ratios from
slot-blot analyses of total cellular RNA from MFHs.
MFH SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
MT-1
1.25
0.113
0.283
MT-2
1.33
0.697
1.03
MT-3
1.65
1.27
0.616
MT-4
1.39
1.13
0.802
MT-5
1.30
1.38
1.94
MT-6
2.83
0.787
0.292
MT-7
1.54
0.680
1.70
MT-8
2.31
0.689
0.507
MT-9
1.14
1.35
0.992
MT-10
0.810
1.49
1.92
MT-11
2.29
1.02
2.92
MT-12
2.63
1.63
1.49
MT-13
2.19
1.50
1.71
MT-14
2.02
1.64
1.05
MT-15
1.56
1.17
0.790
MT-16
1.89
0.801
0.480
MT-17
34.0 **
0.832
2.32
MT-18
2.30
1.16
0.586
MT-19
1.87
0.558
1.04
MT-2 0
1.31
0.942
1.01
MT-21
1.29
1.09
1.01
MT-22
1.03
1.05
0.990
MT-2 3
1.43
1.05
1.01
**
Value was determined from titration of RNA, since signal
intensity was outside the linear range for this method.

107
Table 12. C-myc, c-Ha-ras, and c-fos: actin ratios from
slot-blot analyses of genomic DNA from MFHs. Also shown
are TK:actin ratios.
MFH SAMPLE
c-mvc
c-Ha-ras
c-fos
TK
actin
actin
actin
actin
MT-1
0.833
1.26
0.991
0.800
MT-2
1.06
0.638
0.938
0.964
MT-3
1.05
0.956
0.815
1.05
MT-4
1.21
0.897
1.25
1.06
MT-5
1.18
1.31
1.07
0.919
MT-6
1.96
1.24
0.812
0.988
MT-7
1.17
1.06
1.04
0.891
MT-8
1.13
0.952
1.15
0.980
MT-9
1.26
0.916
1.32
1.27
MT-10
1.08
1.39
0.732
1.07
MT-11
1.14
0.834
0.908
1.01
MT-12
1.39
1.27
1.22
1.24
MT-13
1.19
1.15
0.998
1.14
MT-14
1.11
1.09
1.19
1.28
MT-15
1.21
1.27
1.09
1.18
MT-16
1.81
1.21
1.30
1.16
MT-17
2.25
1.20
1.26
1.21
MT-18
2.41
1.03
1.17
1.19
MT-19
1.01
1.11
1.27
1.20
MT-2 0
2.72
1.19
1.02
1.09
MT-21
1.25
1.28
1.20
1.19
MT-2 2
2.69
1.33
1.13
1.14
MT-2 3
1.45
1.31
1.29
0.960

108
Table 13. C-sis, v-erb-B-1, and v-src: actin ratios from
slot-blot analyses of genomic DNA from MFHs.
MFH SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
MT-1
1.15
1.37
0.946
MT-2
1.22
0.851
0.784
MT-3
0.759
1.13
0.947
MT-4
1.27
0.943
0.860
MT-5
1.17
1.01
1.02
MT-6
1.40
0.946
1.07
MT-7
0.868
0.978
1.08
MT-8
1.02
0.987
0.674
MT-9
1.35
0.976
1.03
MT-10
0.830
1.41
1.36
MT-11
1.34
0.891
0.777
MT-12
0.789
1.29
1.28
MT-13
1.17
1.28
1.14
MT-14
1.30
1.23
1.24
MT-15
1.13
1.36
1.17
MT-16
1.20
0.881
1.03
MT-17
1.22
1.24
1.05
MT-18
1.10
1.11
1.08
MT-19
1.30
1.24
1.35
MT-2 0
1.25
1.16
1.27
MT-21
1.33
1.22
1.27
MT-2 2
1.28
1.28
1.24
MT-2 3
1.32
1.15
1.26

109
(table 14). Mean TK:actin ratios are 1.1 in bone marrow,
1.1 in chondrosarcomas, and 1.5 in MFHs (figures 12 and 13).
C-myc:TK ratios in bone marrows, chondrosarcomas, and MFHs
were 0.9, 1.2, and 1.3 respectively. There are no
significant differences between these groups for levels of c-
myc transcript (p>0.05). C-Ha-ras and c-fos transcript
levels among bone marrows, chondrosarcomas, and MFHs range
from 0.8 to 1.1 and are not significantly different
(p>0.05).
C-sis, v-erb-B-1, and v-src gene transcript levels are
detectable in muscle, giving mean generactin values ranging
from 0.9 to 1.0 (table 15). C-sis:actin ratios in
bone marrows, chondrosarcomas and MFHs are 1.1, 1.6 and 3.1
respectively. There are significantly higher transcript
levels of c-sis in MFHs compared to the other 3 groups
(p<0.05) (figure 14). V-erb-B-1 and v-src transcript levels
in bone marrows, chondrosarcomas and MFHs range from 0.9 to
1.2 and show no significant differences between any of the
groups for transcript levels of these two genes (p >0.05).
DNA Slot-Blot Results and Determination of C-mvc
Gene Copy Number

110
Table 14. RNA quantitation of cell-cycle dependent genes as
determined by slot-blot analysis. *
MUSCLE
BONE
MARROW
CS
MFH
TK:ACTIN
ND
1.1+0.1
1.1+0.7
1.5+0.8
C-MYC:TK
ND
0.9+0.2
1.2+0.7
1.3+0.4 **
C-HA-RAS:TK
ND
1.0+0.2
0.8+0.5
0.9+0.9
C-FOS:TK
ND
1.0+0.1
1.1+0.4
1.00.4
TOTAL CASES
9
6
20
23
* Values shown
are mean gene
:actin and
gene:TK
ratios
** 17 MFHs with single copy c-myc had a mean myc:TK ratio of
1.0+0.2. Six MFHs with 2 or greater copies of myc had a
mean myc:TK ratio of 2.0+0.1.
ND Not detectable

Ill
10
2.5
MT-1
MT2
MT-3
MT4
MT5
MT6
MT7
MT8
MT0
MT10
MT11
MT12
MT-13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 12. Slot-blot of total cellular RNA from MFHs
hybridized with the TK probe. Quantities of 10, 5, and 2.5
ug of RNA were slot-blotted onto nitro-plus 2000, and
hybridized with the TK probe (3.0 x 10 E6 cpm/ 10 E8
cpm/ug).

112
10
2.5
MT-1
MT-2
MT-3
MT4
MT-5
MT
MT7
MT8
MT9
MT10
MT11
MT12
MT13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 13. Slot-blot of total cellular RNA from MFHs
hybridized with the actin probe. Quantities of 10, 5, and
2.5 ug of RNA were slot-blotted onto nitro-plus 2000, and
hybridized with the actin probe (3.0 x 10 E6 cpm/ 10 E8
cpm/ug).

113
Table 15. RNA quantitation of non cell-cycle dependent genes
as determined by slot-blot analysis. *
MUSCLE
BONE
MARROW
CS
MFH
C-SIS:ACTIN
1.0+0.1
1.1+0.1
1.6+1.5
3.1+6.8
V-ERB-B-1:
0.9+0.1
1.2+0.2
1.1+0.7
1.0+0.4
ACTIN
V-SRC:ACTIN
1.0+0.1
1.1+0.1
0.9+0.2
1.2+0.7
TOTAL CASES
9
6
20
23
Values shown are mean generactin ratios

114
10 5 2.5
MT-1
MT-2
MT3
MT4
MT5
MT6
MT7
MT8
MT9
MT10
MT11
MT12
MT13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 14. Slot-blot of total cellular RNA from MFHs
hybridized with the c-sis probe. Quantities of 10, 5, and
2.5 ug of RNA were slot-blotted onto nitro-plus 2000, and
hybridized with the c-sis probe (3.0 x 10 E6 cpm/ 10 E8
cpm/ug).

115
Tables 16 and 17 indicate the gene copy numbers for TK,
actin, and the six genes evaluated in this study as
determined by DNA slot-blot guantitation. C-myc:actin ratios
for all 9 muscle, 6 bone marrow and 20 chondrosarcoma tissues
show c-myc to be a single copy gene. The same is true for
17 of the MFHs, while 6 of these tumors have 2 or more copies
of c-myc. C-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-src:actin
values indicate that these genes are single copy in all
normal and neoplastic tissue groups.
The 6 MFHs with 2 or greater copies of c-myc are
samples MT-8, MT-16, MT-17, MT-18, MT-20,and MT-22. When RNA
values for this subset of MFHs were examined, it was found
that these 6 MFHs have increased c-myc transcript levels as
well (figure 15). Therefore, 17 of the MFHs have a mean c-
myc:TK ratio of 1.0 which is not significantly different
than that of chondrosarcomas (1.2), or bone marrow tissues
(0.9). The 6 MFHs with 2 or greater copies of myc have a
mean myc:TK ratio of 2.0 which is significantly higher than
the other groups.
These 6 MFHs also have higher freguencies of cell
division as measured by TK:actin ratios. Statistical
evaluation of the relationship between c-myc gene copy number
and cell division was performed using chi sguare analysis.

116
Table 16. DNA
determined by
quantitation of cell-cycle dependent genes as
slot-blot analysis. *
BONE
MUSCLE
MARROW
CS
MFH
TK:ACTIN
0.9+0.1
1.0+0.2
1.0+0.1
1.10.1
C-MYCrACTIN
1.1+0.1
0.9+0.2
1.00.1
1.5+0.6**
C-HA-RAS:
1.1+0.1
0.90.1
0.9+0.2
1.1+0.2
ACTIN
C-FOSrACTIN
0.9+0.1
1.0+0.2
1.0+0.2
1.1+0.2
TOTAL CASES
9
6
20
23
* Values shown are mean gene:actin ratios
** 17 MFHs with single copy c-myc had a mean myc:actin
ratio of 1.2+0.1. Six MFHs with 2 or greater copies of
myc had a mean myciactin ratio of 2.3+0.4.

117
Table 17. DNA quantitation of non-cell-cycle dependent genes
as determined by slot-blot analysis. *
MUSCLE
BONE
MARROW
CS
MFH
C-SIS:ACTIN
0.9+0.1
1.0+0.1
1.2+0.2
1.2+0.2
V-ERB-B-1:
1.0+0.1
1.0+0.2
1.0+0.2
1.1+0.2
ACTIN
V-SRC:ACTIN
0.9+0.3
0.9+0.1
1.2+0.2
1.1+0.2
TOTAL CASES
9
6
20
23
Values shown are mean generactin ratios

118
C-MYC
10 5 2-5
MT-1
MT-2
MT-3
MT4
MT5
MT6
MT7
MT8
MT9
MT10
MT11
MT12
MT13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 15. Slot-blot of total cellular RNA and genomic DNA
from MFHs hybridized with the c-myc probe. Quantities of 10,
5, and 2.5 ug of RNA and 20, 10, and 5 ug of DNA were slot-
blotted onto nitro-plus 2000, and hybridized with the c-myc
probe (3.0 x 10 E6 cpm/ 10 E8 cpm/ug).

119
The results indicated a positive correlation between c-myc
gene copy number and cell division as represented by TKractin
ratios (p<0.05) (table 18).
The slot-blotting technique used in these studies was
primarily a technique for screening total cellular RNA and
genomic DNA from tumors and normal tissues for proto-oncogene
expression. A restricted range of linearity for
hybridization signal intensities was one technical
limitation of this system, therefore limiting its
reliability as a quantitative tool when signal intensities
exceeded or fell short of the linear range. For example, DNA
copy numbers greater than 2 could not be assessed in 6 MFHs.
Another technical limitation of the slot-blot is that it
cannot be ascertained what size transcript the probe
hybridized to. In these analyses, all probes were confirmed
for correct specificity by restriction digests and Southern
blot analysis prior to use in slot-blot hybridizations.
The beta-actin control was the most validating factor
for the slot-blotting assay used in this study.
Hybridization with this gene is an internal control for both
RNA and DNA slot-blots. As shown in these results, actin
values show little variability between samples on both RNA

120
Table 18. RNA TKractin ratios and c-myc gene copy numbers
for MFHs. Chi square analysis showed a positive correlation
between c-myc gene copy number and cell division as measured
by TKractin ratios (p<0.05). The cut off point was 2 for c-
myc gene copy number and for TKractin values.
11
1
10
1
9
8
7
6
C-myc
Gene 5
Copy
Number 4
3
2
1
1
1
1
6
4 5 2
1
12 3 4
TK
Act in

121
and DNA slot-blots. This serves as a control for a single
copy, constitutively expressed gene for RNA and DNA
analyses. It also demonstrates consistency of quantity and
quality of RNA.
Since quantitative signal intensity limitations of the
slot-blotting assay do not allow determination of gene copy
numbers greater than two, c-myc copy numbers for the 6 MFHs
with 2 or greater copies of the gene were further evaluated
using Southern blot/ DNA dilutional analysis.
Tumor DNA samples from 6 MFHs (mt-8, mt-16, mt-17, mt-
18, mt-20, and mt-22) and from normal muscle tissues were
prepared as described above. Aliquots of 10, 5, 2, and 1 ug
of tumor DNA and 10 ug of normal muscle DNA were analyzed
using Southern blot hybridization with the p GEM H MYC probe
(table 1). This probe hybridizes to a 9.6 kb fragment of
Hind III restricted genomic DNA. Band sizes were determined
by comparison to lambda DNA marker bands produced by
restriction with Hind III (figure 16). C-myc gene copy
numbers were determined by laser densitometry
(transmittance). Values for tumor DNA are normalized to
those of muscle DNA which are controls for single copy c-myc.
These 6 MFHs were found to have c-myc gene copy numbers of
(A-F) 8.7, 8.4, 9.6, 9.9, 10.6, and 2.2, which corresponded

122
MIO 5 2 1
B
M 10 5 2 1
C
M 10 5 2 1
Figure 16. Southern blot/DNA dilutional analysis of genomic
DNA from MFHs with 2 or greater copies of c-myc. Tumor DNA
samples of 10, 5, 2, and 1 ug (labeled 10, 5, 2 and 1) and
muscle DNA samples of 10 ug (labeled M) were digested with
Hind III, electrophoresed through 0.8 percent agarose gels,
blotted, and hybridized with p GEM H MYC. C-myc gene copy
numbers were calculated to be (A-F) 8.7, 8.4, 9.6, 9.9, 10.6
and 2.2 respectively. Samples A,B,C,D,E, and F correspond to
samples MT-16, MT-17, MT-18, MT-20, MT-22, and MT-8
respectively as shown in figure 15.

123
to MT-16, MT-17, MT-18, MT-20, MT-22, and MT-8 respectively.
Thus, five of the MFHs with increased transcript levels of c-
myc have between 8 and 11 copies of the c-myc gene, while a
sixth has 2 copies.
Regions Contained in the C-Mvc Amplicon
The Southern blots from the dilutional analysis of c-myc
copy number described above were rehybridized with the c-myc,
p 380-8A, H25-8A, and HT .96 probes to further examine the
copy number of the c-myc gene and surrounding regions in the
6 MFHs with c-myc amplification. This was done in order to
determine whether the entire c-myc gene was amplified, and to
determine if the promoter region was contained in the
amplicons. Copy numbers for the different areas of the c-myc
gene and surrounding areas were determined by laser
densitometry as described above for Southern blot dilutional
analysis of myc gene copy number.
Hybridization with the c-myc probe from Oncor which is
specific for all three myc exons, indicates that this region
is amplified approximately 10 times in mt-16, mt-17, mt-18,
mt-20, and mt-22, while mt-8 has 2 copies (table 19). The
same is true for the p GEM H MYC probe (discussed above)

124
Table 19. Copy numbers of 5' and 3' regions of the c-myc
gene and flanking regions in mt-8, mt-16, mt-17, mt-18, mt-
20, and mt-22 as determined by Southern blot analyses of
genomic DNA digested with Hind III and hybridized with the c-
myc, p GEM H MYC, p 380-8A, H25-3.8, and HT 0.96 probes. *
Probe
C-myc
pGEM H MYC
p380-8A
H25-3.8
HT 0.96
Size of
hybridized
fragment (kb)
9.6
9.6
6.8
9.4
3.4
Tumor
MT-8
2
2
2
1
1
MT-16
9
9
9
1
1
MT-17
9
8
9
1
1
MT-18
10
10
10
1
1
MT-2 0
10
10
10
1
1
MT-22
10
11
11
1
1
MUSCLE
1
1
1
1
1
* Copy numbers were determined by laser densitometry. Values
were normalized to that of muscle which has a c-myc gene
copy number of 1.

125
which was specific for the 3' end of the gene. These probes
were specific for overlapping regions, and both were used for
confirmatory purposes.
The p 380-8A probe which is specific for a region
approximately 50 kb upstream from the c-myc promoter
indicates an amplification of approximately 10 fold in mt-16,
mt-17, mt-18, mt-20, and mt-22, while mt-8 has 2 copies of
this region (table 19). Analyses with the thyroglobulin and
carbonic anhydrase probes, show single copy genes (table 19).
These results indicate that the amplfied regions of myc (8q
24) in MFHs are very large, and contain all three exons as
well as regulatory regions. The amplicons do not extend as
far 5' as the carbonic anhydrase gene (8q 22), or as far 3'
as the q terminal region of chromosome 8, where the
thyroglobulin gene is located.
Chromatin Structure Analysis
C-Mvc Gene Copy Number and Transcript Levels in P3C.
UR HCL 1, HFF and ST 486 Cell Lines
C-myc gene copy numbers were determined for the P3C and
UR HCL 1 cell lines. Twenty, 10, 5, and 2 ug of genomic DNA

126
from these cell lines were prepared as previously described,
and analyzed using the pGEM H MYC probe and conditions
established for Southern hybridizations. Gene copy number
was determined by laser densitometry and normalized to muscle
DNA. The P3C cell line was found to contain approximately 10
copies of the c-myc gene, while the UR HCL 1 cell line has a
single copy c-myc gene (figure 17).
Five and 10 ug aliquots of total cellular RNA from the
P3C, UR HCL 1, HFF, and ST 486 cell lines were evaluated for
relative c-myc transcript levels using northern blot
hybridization methods and the p GEM H MYC probe. The blot
was rehybridized with the human beta actin gene probe as a
control for RNA quantitation. P3C cells clearly showed
increased levels of c-myc transcript relative to the other
cell lines (figure 18).
Locations of DNAse I Sites in P3C. UR HCL 1, HFF, and St 486
Cell Lines
Data generated thusfar support the hypothesis that
increases in c-myc transcript are due to gene amplification.
The possibility that other regulatory changes may contribute
to this as well cannot be ruled out. Therefore, potential
differences in chromatin structure were evaluated between

127
9.6 KB
P3C URHCL1
2010
a*
(UGDNA)
5 2 2010
4.'. .-I-'. \ ''ll ..
*>-* ?*; 4 ..
t: >.- .. '-<*
. /* r '
J * rv *
.^¡vZ : il* i'
Figure 17. Southern blot analysis of c-myc DNA copy number
in UR HCL 1 and P3C cell lines. Twenty, 10, 5, and 2 ug of
DNA from each cell line were restricted with Hind III,
electrophoresed through 0.8 percent agarose gels and
hybridized with the pGEM H MYC probe (See materials and
methods). Band size was determined by lambda DNA marker
bands produced by restriction with Hind III.

128
510510510510
<4KB > PP* *
ACTIN
2.1KB
Figure 18. Northern blot analysis of total cellular RNA
from UR HCL 1, P3C, HFF, and ST 486 cell lines. Ten and 5 ug
aliquots of RNA from each cell line were electrophoresed
through 1.2 percent formaldehyde agarose gels, blotted, and
hybridized with the pGEM H MYC and beta actin probes (See
materials and methods). Message sizes were determined by
an RNA ladder.

129
amplified and single copy c-myc genes in MFH cell lines.
Locations of c-myc DNAse I hypersensitve sites were
determined for P3C, UR HCL 1, HFF, and ST 486 cell lines.
Initially, mapping of these sites was done from a 3'
direction using previously described methods. Various
concentrations of DNAse I were used for each cell line to
determine an optimal range of concentrations (i.e. ones
which yielded optimum visualization of DNAse I generated
bands). Figure 19 shows Southern blot analysis of DNAs from
DNAse I treated nuclei from the UR HCL 1 cell line. This
figure demonstrates the extent of digestion with varying
concentrations of DNAse I.
Genomic DNA was isolated from DNAse I treated nuclei as
previously described. Fifteen ug of DNA from the UR HCL 1,
HFF, and ST 486 cell lines and 7 ug of DNA from P3C DNAse I
treated nuclei were restricted with ECO Rl, and compared for
locations of DNAse I hypersensitive sites by Southern blot
hybridization with the pmc 41 probe (figure 20).
The optimum DNAse I concentrations for the P3C,
UR HCL-1, and HFF lines were 0.2, 0.5, and 1.0 ug/ml DNAse I,
while those for ST 486 were 0.1, 0.2, and 0.5 ug/ml DNAse I.
The controls shown for each cell line were 0 ug/ml DNAse I at
37 C degrees (lane marked 0). Locations of DNAse I
hypersensitive sites were determined by comparison to lambda

UR HCL 1
UG/ML DNASE I
130
Figure 19. Southern blot of UR HCL-1 genomic DNA from DNAse
I treated nuclei. After treatment of nuclei with various
concentrations of DNAse I (L-R; 0.1, 0.2, 0.5, 1, 2, 5,
ug/ml, controls consisted of 0 ug/ml DNAse I at both 0 and 37
C degrees), genomic DNA was isolated restricted with Eco R
I, electrophoresed through 0.8 percent agarose gels, and
hybridized with the pmc 41 probe. Samples 0.2, 0.5, and 1
ug/ml DNAse I gave optimum band visualization and were used
in a composite Southern blot shown in figure 20.

131
o
o
0>
ug/ml DNAse I
URtiCL 1
o in n
111
o H O o
HFF
o in n
O '-i o o
ST486
Irt CM H
I I I
O O O O
e
!c
<

44 *
Figure 20. Southern blot of P3C, UR HCL 1, HFF, and ST 486
genomic DNA from DNAse I treated nuclei. The concentrations
of DNAse I shown for each cell line were the ones which gave
optimal visualization of bands in the initial analyses. The
genomic DNA from DNAse I treated nuclei was restricted with
Eco R I, electrophoresed through 0.8 percent agarose gels,
blotted, and hybridized with the pmc 41 probe. Controls
shown for each cell line were 0 ug/ml DNAse at 37 C degrees.

132
DNA digested with either Hind III (right side of blot), or
both Eco R1 and Hind II together (left side of blot).
Controls at 0 ug/ml DNAse I at 0 and 37 C degrees
demonstrated that DNAse I generated bands are real, and
not due to endogenous nuclease activity. There is a band
seen in the control lane for the P3C cell line. Although
this band may be generated by endogenous nucleases, it ran
differently than the DNAse I band of similar size, and was
not relevant to these analyses.
Locations of DNAse I hypersensitive sites for the c-myc
gene in each of the cell lines studied are shown in figure
21. It was found that five DNAse I hypersensitive sites at
identical locations are present for UR HCL 1 and the normal
human fibroblast line HFF (sites 1,2,3,4,6, figure 21). One
site is located 5' to the first exon and 5' of promoter PO
(site 1). Two sites are located 5' of the first exon and 3'
of the PO promoter region (sites 2,3). Another site is
located in the 3' region of the first exon near a PVU II site
(site 4), and a fifth site was found to be 5' of exon 2 (site
6). The amplified c-myc gene in the P3C cell line also had
four of these sites (1,3,4,6), however a site near the PO
promoter region is not present (site 2), and a new site in
the 5' region of the first intron is seen (site 5). Each of

HIND III
133
C-MYC EXON 1
EXON 2
I
PO- P1 P2
I II II
I
6
I 1
1 KB
HFF 1 2 3 4 6
UR HCL 1 1 2 3 4 6
P3C 1 3 4 5 6
y = TRANSCRIPTION ELONGATION BLOCK (BL)
Figure 21. Locations of DNAse I hypersensitive sites in the
c-myc gene for each of the cell lines HFF, P3C, and UR HCL-1.
Also shown are promoter regions PO, PI, and P2.

134
the DNAse I bands for P3C cells were similar in intensity.
This suggests that most if not all of the copies of c-myc
have these changes in chromatin structure. Three DNAse I
hypersensitive sites were observed for the ST 486 cell line.
These were in identical regions as those previously reported
by Siebenlist et al. (156) in Burkitt lymphoma BL 31 cells.
Chromatin Structure of the C-mvc Gene During the G0/G1
Transition in the HFF Normal Human Fibroblast Cell Line
These data show that increases in transcript and
changes in DNAse I hypersensitive sites accompany c-myc gene
amplification in P3C cells. It was of interest to determine
if changes in DNAse I hypersensitive sites are seen in normal
cells during periods when peak levies of c-myc transcript are
produced. Increased levels of c-myc transcript production
have been observed during the G0/G1 transition in quiescent
fibroblasts after serum addition. Quiescent HFF cells
were evaluated at GO 0.5, 1, 2, and 3 hours after serum
release, and during log phase growth. Locations of DNAse I
hypersensitive sites in these cells were mapped from the 3'
direction using Southern hybridization with the pmc 41 probe.

135
Total cellular RNA from the cells at each of these time
points was evaluated by Northern blot analysis for transcript
levels of c-myc and TK to ascertain that the desired phases
of the cell cycle were represented (figure 22).
Hybridization with the c-myc probe demonstrates a profile of
transcript levels which starts out at a basal level during
GO, peaks 1 hr after serum release, then returns to levels
comparable to those of GO during log phase growth. Levels of
TK at the various time points indicate that transcript levels
are highest during log phase, and lowest during GO.
Hybridization with actin demonstrates consistent actin
transcript levels and RNA quantitation. These results are
expected for transcript levels of these genes during the
cell cycle.
Fifteen ug of genomic DNA isolated from DNAse I treated
nuclei at each time point discussed above were restricted
with ECO R1 and analyzed for Locations of DNAse I
hypersensitive sites as previously described (figure 23).
Mapping of sites was accomplished by band size comparisons to
those generated by digestion of lambda DNA with Eco R 1 and
Hind III. Locations of c-myc DNAse I hypersensitive sites
for HFF at each time point examined were found to be
identical to those previously described for HFF (sites

136
8 10
o M W -*
C-MYC
2.4 KB
TK
1.0 KB
ACT1N
2.0 KB
Figure 22. Northern blot analysis of total cellular RNA from
the HFF normal human fibroblast cell line. HFF cells were
made quiescent (GO) in MEM containing 0.1 percent fetal
bovine serum (37 C degrees, 3 days), then released by
addition of MEM supplemented with 10 percent fetal bovine
serum. RNA samples were evaluated for levels of c-myc, TK
and actin transcript at GO, 0.5, 1, 2, and 3 hours after
serum release, and during log phase growth (L) (see materials
and methods).

137
GO 0.5 HR 1 HR
UG/ML DNASE I
Figure 23. Southern blot analysis of genomic DNA from DNAse
I treated nuclei of HFF normal human fibroblast cells.
Nuclei isolated from HFF cells in GO, 0.5, 1, 2, and 3 hours
after serum release, and during log phase growth (L) were
treated with various concentrations of DNAse I (L-R; 0.1,
0.5, 1, 2, 5, and 0 ug/ml (37 C degrees)). Genomic DNA was
isolated, restricted with Eco R 1, electrophoresed through
0.8 percent agarose gels, blotted, and hybridized with the
pmc 41 c-myc probe (See materials and methods). Sizes of
bands were determined by comparison to lambda DNA digested
with Eco R 1 and Hind III.

138
2 HR
3 3 3-
3 HR
UG/ML DNASE I
3 3 3 m g
LOG PHASE
3 3 3 N §
M * ft*
*ft
Figure 23. contd.

139
1,2,3,4,6) in figure 21. Despite a peak of myc transcript
production 1 hr after serum release, no changes were observed
in the locations of these sites during the transition of HFF
fibroblasts from GO to Gl, or between any time points after
serum release.
Fine Mapping Analysis of DNAse I Hypersensitive Sites in P3C
Cells From a 51 Direction
Fine mapping of P3C c-myc DNAse I hypersensitive sites
(shown in figure 21) in the exon 1/ intron 1 region was done
from the 5' direction to more precisely determine their
locations (particularly site 5) relative to known
transcription elongation block sites. Five ug of P3C and 15
ug of HFF DNA from DNAse I treated nuclei were restricted
with Sea I. These, and P3C marker DNAs restricted with Sea
I/Mae III, Sea I/Bsm I, and Sea I/Pvu II were analyzed by
Southern blot hybridization using the Sea I/Xho I probe (see
materials and methods). The Southern blot from fine mapping
analysis of c-myc DNAse I hypersensitive sites in the 3'
region of exon 1, and in intron 1 is shown in figure 24. The
resolution of bands on this blot was found to be at least 25
base pairs. This is demonstrated by the easy resolution of
bands generated by digestion with Sea I/Mae III (970 bases)

140
2027 >
1904 >
1584 >
1375 >
947 >
831 >
564 i*
< 2027
< 1904
< 1584
< 1375
< 947
< 831
< 564
Figure 24. Southern blot of P3C and HFF DNAse I treated DNAs
used in fine mapping of DNAse I hypersensitive sites in the
exon 1/ intron 1 region. Also shown are P3C genomic DNA
marker fragments; Scal/Bsm I, Sea 1/ Mae III, and Scal/Pvu
II. P3C A and P3C B were treated with 0.2 and 0.5 ug/ml
DNAse I respectively (from figure 20). The HFF sample (from
figure 20) was treated with 0.5 ug/ml DNAse I. The blot was
hybridized with the 355 b.p. Sea 1/ Xho I fragment (2.0 X 10
E7 cpm/ml/ 10 E8 cpm/ug) (see materials and methods). Also
shown are lambda DNA markers produced by digestion with Eco R
I and Hind III.

141
and Sea 1/ Bsm I (920 bases) which differ by 50 base pairs
and are 0.5 cm apart on the blot. Lambda DNA digested with
Eco R I/Hind III was run on either side of the gel as size
markers, and to assure the gel ran evenly. Sizes of these
marker bands are also shown in figure 24. Two samples of
DNAse I treated DNAs from P3C cells (0.2 and 0.5 ug/ml, A
and B respectively) were run because both produced optimal
visualization of DNAse I generated bands, as did sample 0.5
ug/ml for HFF cells. Bands corresponding to sites 4 and 6
(figure 25) were observed in both the P3C and HFF cell lines,
and map to distances of 760 and 2025 base pairs 3' of the
Sea I site respectively. Site 5, which is present
exclusively in the P3C cell line, maps to a distance of 960
base pairs downstream of the Sea I site. As a result of
these analyses, site 4 can be placed approximately 45 base
pairs 5' of the Pvu II site in one region known to be a
transcription attenuation site. Site 5 can be placed in a
region 3' of exon 1, approximately 10 base pairs 5' of an Mae
III site. This site is located in a region also known to
contain a transcription elongation block, which extends 15
base pairs 5' and 5 base pairs 3' of the Mae III site. Site
6 was found to be approximately 465 bases 5' of exon 2 in the
same region as an SI nuclease sensitive site described by
Grosso and Pitot (73).

HIND III
142
C-MYC EXON I
EXON 2
\
I 1
1 KB
PROBE
4 5 6
= TRANSCRIPT ELONGATION BLOCK
Figure 25. Locations of c-myc DNAse I hypersensitive sites
4, 5, and 6 for P3C and HFF cell lines as determined from
fine mapping analysis from the 5' direction.

143
C-mvc Protein Levels in the P3C, UR HCL 1, HFF, and ST 486
Cell Lines
A comparison of c-myc protein levels was made between
cells with amplified (P3C) and single copy (UR HCL 1) c-myc
genes, cells in which c-myc is thought to be an oncogene (ST
486), and normal fibroblasts during peak levels of c-myc
transcript production. This was done to determine if the
increased levels of c-myc transcript seen in P3C cells were
translated. Twenty ug of protein from P3C, UR HCL 1, ST 486
and HFF cells (GO, 0.5, 1, and 2 hours after serum release,
and during log phase growth), were evaluated for relative c-
myc protein levels using PAGE and Western blotting techniques
as previously described.
The c-myc monoclonal antibody (HL-40) bound to a 65 kd
protein band (132) as determined by molecular weight markers
run on the gel. The control panel, incubated with anti-met
72 (72/K 88.151.G127), showed no non-specific binding, and
staining with india ink indicated that protein quantitation
was consistent between samples (figure 26). Relative levels
of c-myc protein were determined by reflectance densitomety,
and values were normalized to those of fibroblasts in GO
(figure 26). Fibroblasts 0.5 and 2 hours after serum

144
O-MYC
CONTROL
CONTROL
<0 r*
§
116 KD>
92 KD >
66 KD>
45 KD>
31 KD>
P3C ST 486 UR HCL 1 HFF/GO
HFF/0.5
HFF/1
HFF/2
HFF/L
6 5 2 1
1
2
1
1
Figure 26. Western blot showing relative levels of c-myc protein
in P3C, UR HCL 1, ST 486, and HFF (GO, 0.5, 1.0, 2.0 hr after
serum release, and during log phase growth (L)) cell lines.
Twenty ug of protein were separated by size using PAGE, and
electroblotted onto nitrocellulose. The panels were (L-R)
incubated with the HL-40 (anti c-myc) monoclonal antibody, the K
88.151.G127 (anti met 72) antibody (control for non-specific
binding), and the third panel was stained with 0.1 percent india
ink to control for protein quantitation. The c-myc monoclonal
antibody bound to a 65 kd protein band as determined by molecular
weight markers. Shown below the figure are the relative levels
of c-myc protein as determined by reflectance densitometry.
Values were normalized to myc protein levels in HFF cells at GO
which was given a value of 1.

145
release, and during log phase growth had the same amounts of
c-myc protein as in GO. Protein levels in UR HCL 1 cells and
fibroblasts 1 hr after serum release (presumably G0/G1
transition) were twice this level (2), while ST 486 and P3C
cells had 5 and 6 times as much protein respectively.

CHAPTER 5
DISCUSSION
An amplified c-myc gene and increased levels of c-myc
and c-sis transcript suggest an involvement of these genes
in the pathogenesis and progression of MFHs. No increased
transcript levels or amplified copy numbers of any of the
proto-oncogenes were found in chondrosarcomas. MFHs are more
malignant, have a higher fraction of dividing cells (39), and
are potentially more genetically unstable. It is therefore
not surprising that more proto-oncogene mutations would be
observed in these tumors.
Muscle and bone marrow specimens were compared as
examples of normal non-dividing and dividing mesenchymal
tissues. Results presented here show that transcript levels
of TK, c-myc, c-Ha-ras, and c-fos are undetectable in
skeletal muscle tissues while levels of c-sis, v-erb-B-1, v-
src and actin are present at detectable levels. These
results are expected since normal skeletal muscle is a non
dividing tissue and c-myc, c-Ha-ras, c-fos, and TK are cell
cycle dependent genes, while c-sis, v-erb-B-1, v-src and
146

147
actin are non-cell-cycle dependent. It has been reported
that actin transcript levels vary during stages of the cell
cycle (69). However, later studies with guiescent
fibrob"asts have shown that beta actin message levels were
consistent during all phases of the cell cycle after serum
release (175). These studies suggest that levels of actin
transcript production during phases of the cell cycle may
depend on cell type and culture conditions. Data reported
here indicates that actin levels are consistent during the
cell cycle in normal HFF fibroblasts. Therefore, actin was
used in normalization of results for slot-blot and northern
blot analyses.
These results for the v-erb-B-1 and v-src genes are
somewhat in agreement with those reported by Claycomb and
Lanson (27) and Leibovitch et al. (107) which show that c-
myc, c-Ha-ras, c-sis, c-src, and v-erb-B-1 transcripts are
present in skeletal muscle cells in culture while those of TK
and c-fos are not.
Transcript levels of cell cycle dependent proto
oncogenes and TK as well as non-cell-cycle dependent proto
oncogenes and actin were found to be detectable in all bone
marrow samples. It would be reasonable to suspect that all
of these proto-oncogenes including cell-cycle dependent genes

148
would be expressed because bone marrow is a normally dividing
tissue; this is what was observed. Detectable c-fos
transcript levels have also been observed in normal bone
marrow by Evinger-Hodges et al. (58).
The slot-blotting technique has advantages for an
analysis of this nature. Due to the moderately degraded
conditions of most RNA samples isolated from surgically
obtained tumor and normal tissue specimens (as described in
materials and methods), slot-blotting provides a workable
alternative to northern blotting which is not possible with
degraded RNA.
The most reliable information provided by the slot-blot
assay is a relative comparison of proto-oncogene transcript
levels between samples. This was accomplished by maintaining
constant pre-hybridization and hybridization conditions, as
well as film exposure times.
The slot-blot results presented here do not rule out a
tumorigenic involvement of proto-oncogenes which do not have
abnormal transcript levels or copy numbers. They do
however, offer some clues as to which potential proto
oncogene activation mechanisms may be at work in the cases of
the c-myc and c-sis genes in MFHs.
Several possible mechanisms have been described for the

149
activation of proto-oncogenes to oncogenes. It is possible
that any of these; insertional mutagenesis, enhancer/promoter
activity, amplification, gene rearrangements or point
mutations could result in the loss of normal transcriptional
constraints. It is reasonable to expect that increases in
transcriptional levels would result from all of these
mechanisms; including point mutations, if in regulatory
rather than coding regions.
Single copy genes and insignificant differences in
transcript levels between normal and tumor tissues were
observed for c-Ha-ras, c-fos, v-erb-B-1, and v-src. Although
no known mechanism of proto-oncogene activation is apparent
(above), this does not exclude an involvement of these genes
through point mutations at critical sites or some unknown
activation mechanism. In the case of the c-myc gene in 6
MFHs, evidence presented here supports the concept of gene
amplification as a mechanism of activiation of a proto
oncogene to an oncogene. Furthermore, TK:actin ratios
indicate that myc gene amplification may be driving cell
division in these tumors.
Increased transcript levels of c-myc, together with
multiple copies of the gene, suggest that abnormal amounts of
transcript are due at least in part to gene dosage effects.

150
Increases in transcript levels of c-sis were observed in
MFHs; however, only single copies of the c-sis gene were
seen. This suggests that increases in sis transcript levels
are due to some undetermined mechanism other than gene
amplification.
The myc gene product is postulated to be a double
stranded DNA binding protein capable of participating in the
regulation of cell division (17, 94, 95, 96). In vitro
experiments suggest that c-myc genes are of a cell-cycle
dependent nature in that levels of c-myc transcript like
those of c-jun (143) and c-fos (120) increase during the
G0/G1 transition and decrease to GO levels during S-phase.
These are unlike more "traditional" cell-cycle dependent
genes such as histone H-2b and TK whose transcript levels
peak during S phase (175).
Beta-actin is commonly used as a standard to normalize
the expression of other genes because it is single copy, and
constitutively expressed in most tissues. In these studies,
it was appropriate to normalize to TK instead of actin for
transcript levels of cell cycle dependent proto-oncogenes; c-
myc, c-Ha-ras, and c-fos. Normalization to actin would not
correct for the different frequencies of cell division seen
in different tumors. Although the c-myc gene is not cell
cycle dependent in the "traditional" sense, normal tissues or

151
tumors with a larger growth fraction would be expected to
have increased c-myc transcript levels regardless of the
factors driving cell division.
Southern blot analyses show that c-myc amplicons in MFHs
(which contain approximately 10 copies) are very large and
contain all three exons. Hybridization with the p380-8A
probe (specific for a region approximately 50 kb upstream
from the c-myc promoter) indicates that the c-myc promoter
region is amplified as well. Use of this probe in similar
analyses by Haluska and Croce (78) has shown this region to
be co-amplified with the c-myc gene in COLO 320 (colon
carcinoma cell line), but not with c-myc in HL-60 cells.
The measurements reported here for c-myc gene
amplification are in accordance with other studies which have
demonstrated c-myc gene amplification of large regions of DNA
in other tumor systems (2). The question of why c-myc
amplification occurs at all has been the focus of much
speculation. In the case of Wilm's tumor for example, high
levels of transcript have been observed from a single
gene (2). If c-myc is a nuclear regulatory protein found in
most normal cells then why would normal growth provide an
ever increasing pressure for selection of cells with elevated
levels of c-myc protein? The type of selective pressures

152
required to produce a cell with an amplified cmyc gene are
unknown. It has been postulated by Alt (2) and others, that
if c-myc genes could regulate expression of other genes, then
maybe amplification is selecting for regulation of various
growth regulatory genes.
The data reported here are consistent with the
hypothesis that increases in c-myc transcript production are
due to gene amplification. A hypothesis that changes in
chromatin structure exist between amplified and single copy
c-myc in MFH cell lines was tested as well. Studies with
DNAse I demonstrated differences in chromatin structure
between amplified and single copy c-myc genes in MFH cell
lines. Changes which accompanied c-myc gene amplification
include the disappearance of a DNAse I hypersensitive site
5' of exon 1, and the appearance of a new site in the first
intron. The meaning of these data can be more fully realized
when compared to those of Siebenlist and Leder (156), and
Siebenlist and Kelly (155) (figure 27). These two studies
reported that changes in chromatin structure accompanied c-
myc structural mutations in Burkitt lymphoma (translocation)
and HL-60 cells (amplification) (figure 27).
DNAse I hypersensitive sites 2 and 3 for HFF and UR HCL
1 cell lines, and site 3 in P3C cells were located in the

153
HL-60 (DNASE I) ABCD
HL60 (S1) G
BL-31 (DNASE I) ABCD
Y = TRANSCRIPTION ELONGATION
V = + HL-60
BLOCK (BL)
Q
Z
X
\
A
I
I
+ -
B C D E F
} I i TT
PO P1 P2
II II
2 3 4 5
G
I 1
1KB
C-MYC EXON 1
EXON 2
HFF 1 2 3 4 6
UR HCL 1 1 2 3 4 6
P3C 13 4 5 6
Figure 27. Summary of chromatin structure analyses
previously described for the c-myc gene (letters) and those
reported here (numerals). Sites A, B, C, and D are DNAse I
hypersensitive sites found in both HL-60 cells (Siebenlist et
al (155)), and Burkitt lymphoma (BL-31) cells (Siebenlist et
al. (156)). Site B (indicated by open arrow) has been
described by Siebenlist et al (155) to be involved in the
maintainance of c-myc transcript production in HL-60 cells,
and is therefore marked with a (+) symbol. Sites E and F
(solid arows) represent transcription attenuation sites found
in Burkitt lymphoma biopsies and cell lines and are marked by
a (-) symbol (25, 199). Site G is an S-l nuclease sensitive
site described by Grosso and Pitot (73) in HL-60 cells.

154
same regions as sites B and C (C for P3C cells) in HL-60 and
Burkitt lymphoma cells. Site 2, which is not seen in P3C
cells, was located in the same region as site B in HL-60
cells. The disappearance of this site has been shown to
accompany decreased c-myc transcript production in
differentiating HL-60 cells post treatment with DMSO. Even
though site 2 and site B map to different sides of the PO
promoter, they can be considered to be located in the same
region because DNAse I hypersensitive sites may include 150-
200 base pairs (about the size of a nucleosomal repeat).
Site 4 in HFF, UR HCL 1, and P3C cells was located in a
region which contains a PVU II site and a c-myc transcript
elongation block in Burkitt lymphoma biopsies and cell
lines (Site E, figure 27) (25). Site 5 was observed
exclusively in P3C cells. This site mapped to a region in
the first intron also known to contain a c-myc transcript
elongation block in Burkitt lymphoma cell lines
(Site F, figure 27) (199). Site 6 in the HFF, UR HCL 1, and
P3C cell lines has not yet been found by DNAse I
hypersensitive site analysis. However, an S-l nuclease
sensitive site in a similar region has been described by
Grosso and Pitot (73).

155
The DNAse I sensitivity assay can provide general
locations of DNAse I hypersensitive sites. Considering the
potentially large areas of these "sites", resolution of this
technique may be low. This may explain why site A in
lymphocytes and site 1 in UR HCL 1, HFF, and P3C cells
mapped to different locations (figure 27). Another
possibility is that some DNAse I sites for a particular gene
are cell-type specific, while others are shared between cell
types.
Although a transcript attenuation site in the same area
as sites E and F (figure 27 ) has been proposed for amplified
c-myc in HL-60 cells, a precise location has not yet been
described. The importance of fine mapping P3C c-myc DNAse I
hypersensitive sites in this region can therefore be
appreciated. Sites in the exon 1/intron 1 region were fine
mapped from a 5' direction, and it was found that site 5
mapped to the same location as site F, which was one of the
transcript attenuation sites previously described (figure
27) .
Based on the changes in chromatin structure seen with
the c-myc gene in P3C cells, one would expect to see
decreased levels of myc transcript. This conflicts with what
was observed with northern blot analysis. Therefore

156
potential changes in chromatin structure during c-myc
upregulation in normal cells were studied. It has been
shown that c-myc transcript levels peak during the G0/G1
transition when serum starved fibroblasts are released from
their quiescent states (175). Although transcript levels
peaked 1 hr after serum addition in HFF cells as shown by
northern blot analysis, no differences in DNAse I sensitive
sites were observed between fibroblasts in GO, those in log
phase, and after maximal physiologic stimulation (G0/G1). The
DNAse I sites found at each of the time points (GO, 0.5, 1,
2, 3 hours after serum release and during log phase growth)
are identical to those described previously for HFF (sites
1,2,3,4, and 6, figure 27). These data are consistent with
those reported by Blanchard et al. (19) which suggest that
cellular levels of c-myc transcript are primarily regulated
by post-transcriptional mechanisms at the level of message
degredation in normal cells.
C-myc chromatin structure analyses reported here, and
those previously reported for lymphocytes provide important
data as to the nature of regulatory interactions taking place
with amplified c-myc in MFHs. First, chromatin structure
data from quiescent and serum released HFF fibroblasts

157
indicate that in normal cells, post-transcriptional
regulation is adequate to control c-myc transcript levels.
When the c-myc gene is amplified in MFHs, which are
tumors of fibroblast origin, the normal post-transcriptional
regulatory mechanisms may not be sufficient to compensate for
the abundance of c-myc transcript produced. Changes in
chromatin structure suggest that regulatory changes take
place at the level of the gene as well. These changes in
chromatin structure are indicative of a cellular adjustment
to an abundance of myc expression. It is therefore concluded
that the differences in regulation between amplified and
single copy c-myc in MFHs may represent a compensatory
response to gene dosage effects.
Western blot analysis showed that P3C cells have
increased amounts of c-myc protein compared to UR HCL 1 cells
or normal HFF fibroblasts 1 hour after serum release from GO.
This suggests that c-myc gene amplification and increased
transcript levels have an impact on the P3C cells through
increased amounts of protein. These data further indicate
that despite any attempt by the P3C cells to compensate for
increased c-myc transcript, relatively high levels of c-myc
protein are produced. This is further evidence that c-myc
may be an oncogene in these cells.

158
Exactly what all of the consequences of c-myc gene
ampIfication in MFHs are, and whether multiple copies of the
c-myc gene are the only abnormal events influencing
increased levels of transcript production are questions yet
to be answered. C-myc gene amplification may be a mechanism
by tumor cells to obtain growth advantages over other
surrounding cells. It would be interesting to study this in
vivo. Individuals with neoplastic disease could be
evaluated for various parameters of tumor growth. These
include tumor stage, tumor size, angiogenesis factors, and
metastasis. In order for a study of this nature to be
meaningful, a large sample size and careful patient followups
would be required. Although this would take years to
complete, studies such as this would provide a more complete
understanding of how abnormal gene copy number may actually
effect tumor growth in individuals with cancer.
The following statements summarize the results from
this project.
1. Increased transcript levels of c-myc and c-sis were
observed in MFHs in vivo.
2. The c-sis gene is single copy in MFHs, therefore
transcript levels are increased by an unknown
mechanism other that gene amplification.

159
3. Increased c-myc transcript levels and TK:actin RNA
ratios correlated with c-myc gene amplification in
vivo.
4. Chromatin structure studies indicate that regulation
of c-myc in normal fibroblasts is post-
transcriptional .
5. When c-myc is amplified in MFH cells, changes in
chromatin structure may represent a compensatory
response to increased transcript levels.
6. Transcriptional and translational changes suggest
that amplification of c-myc in P3C cells represents
activation of a proto-oncogene to an oncogene.
7. C-myc gene amplification may be driving cell
division in MFHs.
8. In vitro studies support the notion that c-myc
gene amplfication may provide a selective growth
advantage to MFH cells.

REFERENCES
1. Alitalo, K., M. Schwab, C.C. Lin, H.E. Varmus, and
J.M. Bishop. 1983. Homogenously Staining Chromosomal
Regions Contain Amplified Copies of an Abundantly
Expressed Cellular Oncogene C-myc in Malignant
Neuroendocrine Cells From a Human Colon Carcinoma.
Proc. Natl. Acad. Sci. U.S.A. 80: 1707.
2. Alt, F.W., R. DePinho, K. Zimmerman, E. Legouy, K.
Halton, P. Ferrier, A. Tesfaye, G. Yancopoulos, and
P. Nisen, 1986. The C-myc Oncogene Family. Cold
Spring Harbour Symposium on Quantitative Biology. LI:
931-941.
3. Baas, F., H Bikker, A. Geurts, R. Melsert, P.L.
Pearson, J.J. De Vijlder, and G.J. VanOmmen. 1985.
The Human Thyroglobulin Gene: A Polymorphic Marker
Localized Distal to C-myc on Chromosome 8, Band q24.
Human Genetics 69: 138.
4. Baltimore, D. 1970. Viral RNA-Dependent DNA
Polymerase. Nature 226: 1209.
5. Baltimore, D. 1976. Viruses, Polymerases, and
Cancer. Science 192: 632.
6. Barbacid, M., and A.V. Lauver. 1981. The Gene
Products of McDonough Feline Sarcoma Virus Have an In
Vitro Associated Protein Kinase That Phosphorylates
Tyrosine Residues. Lack of Detection of This
Enzymatic Activity In Vivo. J. Virol. 40: 812.
7. Barnekow, A., E. Paul, and M. Schartl. 1987.
Expression of the C-src Proto-oncogene in Human Skin
Tumors. Can. Res. 47: 235.
160

161
8. Barrett, T.B., C.M. Gajdneck, C.M. Schwartz, S.M.
McDougall, and E.P. Benditt. 1984. Expression of the
Sis Gene by Endothelial Cells in Culture and In Vivo.
Proc. Natl. Acad. Sci. U.S.A. 81: 6772.
9. Bentley, D.L., and M. Groudine. 1986. Novel Promoter
Upstream of the Human C-myc and Regulation of C-myc
Expression in B-Cell Lymphomas. Molec. and Cell. Biol.
6: 3841.
10. Bentley, D.L, and M. Groudine. 1986. A Block to
Elongation is Largely Responsible fo Decreased
Transcript of C-myc in Differentiated HL-60 Cells.
Nature 321: 702.
11. Berenblum, I. 1975. Sequential Aspects of Chemical
Carcinogenesis: Skin. In Cancer: A Comprehensive
Treatise. F.F. Becker, ed. New York, Plenum Press,
pp. 323-344
12. Berridge, M.J., and R.F. Irvine 1984. Inositol
Triphosphate, a Novel Second Messenger in Cellular
Signal Transduction. Nature 312: 315-380.
13. Betsholtz, C., B. Wetermark, B. Ek, and C.H. Helden.
1984. Coexpression of a PDGF-Like Growth Factor and
PDGF Receptors in a Human Osteosarcoma Cell Line;
Implications For Autocrine Receptor Activation.
Cell 39: 447.
14. Beug, H. and M.J. Hayman. 1984. Temperature
Sensitive Mutants of Avian Erythroblastosis Virus:
Surface Expression of the Erb-B Product Correlates
With Transformation. Cell 36: 963.
15. Bevis, C.C., and B.P. Croker. 1985. Bone and Soft
Tissue Tumors; Contrast and Comparison by Leukocyte
Phenotyping. Laboratory Medicine 52: 1
16. Bishop, J.M. 1983. Cellular Oncogenes and
Retroviruses Ann. Rev. Biochenm. 52: 301.
17 .
23 .
Bishop, J.M.
1985. Viral Oncogenes.
Cell 42:

162
18. Blair, D.G., M. Oskarsson, T.G. Wook, W.L. McClements,
P.J. Fischinger, and G.G. Vande Woude. 1981.
Activation of the Transforming Potential of a Normal
Cell Sequence: A Molecular Model For Oncogenesis.
Science 212: 941.
19. Blanchard, J.M., M. Piechaczyk, C. Dani, J. Chambard,
A. Franchi, J. Pouyssegur, and P. Jeanteur. 1985.
C-myc Gene is Transcribed At High Rate In GO Arrested
Fibroblasts and is Post-Transcriptionally Regulated
in Response to Growth Factors. Nature 317: 443.
20. Bohmann, P., T. Bos, A. Admon, T. Nishimura, P. Vogt,
and R.J. Tjian. 1988. Human Proto-Oncogene C-jun
Encodes a DNA Binding Protein With Structural and
Functional Properties of Transcription Factor AP-1.
Science 238: 1386
21. Bradshaw, H.K., and P.L. Deininger. 1984. Human
Thymidine Kinase Gene: Molecular Cloning and
Nucleotide Sequence of a cDNA Expressible in Mammalian
Cells. Molec. Cell. Biol. 4: 2316.
22. Bravo, R., J. Burckhardt, T.Curran, and R. Muller.
1986. Expression of C-fos in NIH 3T3 Cells is Very
Low But Inducible Throughout the Cell Cycle. 1986.
EMBO J. 5: 695.
23. Brooks, J. 1986. The Significance of Double
Phenotypic Patterns and Markers in Human Sarcomas. A
New Model of Mesenchymal Differentiation. Am.J.
Path. 125: 113.
24. Capon, D.J., P.H. Seeburg, J.P. McGrath, H.S.
Hayflick, V. Edman, A.D. Levinson, and D.V. Goedell.
1983. Activation of Ki-ras 2 Gene in Human Colon and
Lung Carcinomas by Two Different Point Mutations.
1983. Nature 304: 507.
25. Cesarman, E., R. Dalla-Favera, D. Bentley, and M.
Groudine. 1988. Mutations in the First Exon Are
Associated With Altered Transcription of C-myc in
Burkitt Lymphoma. 1988. Science 238: 1272.

163
26. Chirgwin, J.M., A.E. Przybyla, and R.J. McDonald.
1979. Isolation of Biologically Active Ribonucleic
Acid From Sources Enriched in Ribonuclease.
Biochemistry 18: 5293.
27. Claycomb, W.C., and N.A. Lanson. 1987. Proto-Oncogene
Expression in Proliferating and Differentiating Cardiac
and Skeletal Muscle. Biochem J. 247: 701.
28. Cleveland, D.W., M.A. Lopata, R.J. MacDonald, N.J.
Conan W.J. Rutter, and M.W. Kirschner. 1980. Number
and Evoloutionary Conservation of Alpha and Beta
Tubulin and Cytoplasmic Beta and Gamma Actin Genes
Using Specific Cloned cDNA Probes. Cell 20: 95.
29. Cohen, S., G. Carpenter, and L.E. King. 1980.
Epidermal Growth Factor Receptor Protein Kinase
Interactions. Co-purification of Receptor and
Epidermal Growth factor Enhanced Phosphorylation
Activity. J. Biol. Chem. 255: 4834.
30. Coley, B.L. 1960. Neoplasms of Bone. Second
Edition. New York, Paul B. Hoebner, Inc.
31. Collett, M.S., E. Erikson, A.F. Purchio, J.S. Brugge,
and R.L. Erikson. 1979. A Normal Cellular Protein
Similar in Structure and Function to the Avian Sarcoma
Transforming Gene Product. Proc. Natl. Acad. Sci.
U.S.A. 76: 3159.
32. Collins, S.J., and M. Groudine. 1982. Amplification
of Endogenous Myc Related DNA Sequences in a Human
Myeloid Leukemia Cell Line. Nature 298: 679.
33. Collins, S.J., and M. Groudine. 1983. Rearrangement
and Amplification of C-abl Sequences in the Human
Chronic Myelogenous Leukemia Cell Line K-562. Proc.
Natl. Acad. Sci. U.S.A. 80: 4813.
34. Cooper, C.S., M. Park, D.G. Blair, M.A. Tainsky, K.
Huebner, C.M. Croch, and C.F. Vande Woude. 1984.
Molecular Cloning of a New Transforming Gene From a
Chemically Transformed Human Cell Line. Nature 311:
29.

164
35. Courtneidge, S.A., A.D. Levinson, and J.M. Bishop.
1980. The Protein Encoded by the Transforming Gene
of Avian Sarcoma Viurs and a Homologous Protein in
Normal Cells (pp 60 C-src) Are Associated With the
Plasma Membrane. Proc. Natl. Acad. Sci. U.S.A. 77:
3783 .
36. Curran, T., W.P. MacConnell, F. VanStraaten, and I.M.
Verma. 1983. Structure of the FBJ Murine Osteosarcoma
Virus Genome: Molecular Cloning of its Associated
Helper Virus and the Cellular Homolog of the V-fos Gene
From Mouse and Human Cells. Molec. Cell. Biol. 3:
914.
37. Dalla Favara, R., E.P. Gelman, R.C. Gallo, and F.
Wong-Staal. 1981. A Human One Gene Homologous to the
Transforming Gene (V-src) of Simian Sarcoma Virus.
Nature 292: 31.
38. Decker, S. 1981. Phosphorylation of Ribsomal Protein
S6 in Avian Sarcoma Virus-Transformed Chicken Embryo
Fibroblasts Proc. Natl. Acad. Sci., U.S.A. 78:
4112.
39. Dahlin, D.C., and K.K. Unni. 1986. Bone Tumors:
General Aspect- and Data on 8,452 Cases. Fourth
Edition. Springfield, Thomas, U.S.A.
40. DeFeo, D., M.A. Gonda, H.A. Young, E.H. Chang, D.R.
Lowy, E.M. Scolnick, and R.W. Ellis. 1981. Analysis
of Two Divergent Rat Genomic Clones Homologous to the
Transforming Gene of Harvey Murine Sarcoma Viurs.
Proc. Natl. Acad. Sci., U.S.A. 78: 3228.
41. DePinho, R., E Legouy, L. Feldman, N. Kohl, G.
Yancopoulos, and F. Alt. 1986. Structure and
Expression of the Murine N-myc Gene. Proc. Natl.
Acad. Sci. U.S.A. 83: 1827.
42. Der, C.J., T.G. Krontiris, and G.M. Cooper. 1982.
Transforming Genes of Human Bladder and Lung Carcinoma
Cell Lines are Homologous to the Ras Genes of Harvey
and Kirsten Sarcoma Viruses. Proc. Natl. Acad. Sci.
U.S.A. 79: 3637.

165
43. Derynck, R., A.B. Roberts, M.E. Winkler, E.Y. Chen,
and D.V. Goeddel. 1984. Human Transforming Growth
Factor Alpha: Precursor, Structure, and Expression
in E.Coli. Cell 38: 287.
44. Distel, R.J., R. Hyo-Sung, B. Rosen, D.L. Groves, and
B.M. Spiegelman. 1987. Nucleoprotein Complexes That
Regulate Gene Expression in Adipocyte Differentiation
Direct Participation of C-fos. Cell: 49: 835.
45. Doolittle, R.F., M.W. Hunkapiller, L.E. Hood, S.G.
DeVare, K.C. Robbins, S.A. Aaronson, and H.N.
Antoniades. 1983. Simian Sarcoma Virus One Gene
V-sis is Derived From the Gene Encoding a Platelet-
Derived Growth Factor. Science 221: 275.
46. Downward, J., Y. Yarden, E. Mayes, G.Scrace, N. Totty
P. Stockwell, A. Ullrich, J. Schlessinger, and M.D.
Waterfield. 1984. Close Similarity of Epidermal
Growth Factor Receptor and V-erb-B Oncogene Protein
Seguences. Nature 307: 521.
47. Dynan, W.S., and Tjian, R. 1983. The Promoter
Specific Transcription Factor SP 1 Binds to Upstream
Seguences in the SV 40 Early Promoter. Cell 35: 79
48. Dyson, P.J., T.D. Littlewood, A. Forster, and T.H.
Rabbits. 1985. Chromatin Structure of
Transcriptionally Active and Inactive Human C-myc
Alleles. EMBO J 4: 2885.
49. Eick, D., and G.W. Bornkamm 1986. Transcriptional
Arrest Within the First Exon is a Fast Control
Mechanism in C-myc Gene Expression. Nucleic
Acids Res. 14: 8331.
50. Ellerman, V., and O. Bang. 1908. Experimentelle
Leukamie Bei Huhnern. Centrallbl f. Bacteriol. 46:
595.
Emerson, B.M., and G. Felsenfeld. 1984. Specific
Factor Conferring Nuclease Hypersensitivity at the 5
End of the Chicken Adult Beta-Globin Gene. Proc.
Natl. Acad. Sci. U.S.A. 81: 95.
51.

166
52. Enneking, W.F., S.S. Spanier, and M.A. Goodman. 1980
A System for the Surgical Staging of Musculoskeletal
Sarcoma. Clin, and Ortho. Rel. Res. 153: 106.
53. Euphrussi, A., G. Church, S. Tonegawa, and W. Gilbert
1985. B Lineage Specific Interactions of an Ig
Enhancer With Cellular Factors In Vivo. Science
227: 134.
54. Eva, A., K.C. Robbins, P.R. Andersen, A. Srinivasan,
S. Tronick, E. Reddy, P. Elmore, N.W. Galen, A.T.
Lautenberger, J.A. Papas, E.H. Westin, F. Wong-Staal,
R.C. Gallo, and S.A. Aaronson. 1982. Cellular Genes
Analogous to Retroviurs One Genes are Transcribed in
Human Tumor Cells. Nature 295: 116.
55. Eva, A. S.R. Tronick, R.A. Gol, J. H. Pierce, and S.A
Aaronson. 1983. Transforming Genes of Human
Hematopoietic Tumors: Frequent Detection of Ras-
Related Oncogenes Whose Activation Appears to be
Independent of Tumor Phenotype. Proc. Natl. Acad.
Sci. U.S.A. 80: 4926.
56. Eva, Y., H.P. Lee, H. To, J. Shew, R. Bookstein,
P.Scully, and W. Lee. 1988. Inactivation of
Retinoblastoma Susceptibility Gene in Human Breast
Cancers. Science 241: 218.
57. Evans, H.L., A.G. Ayala, and M.M. Romsdahl. 1979.
Prognostic Factors in Chondrosarcoma of Bone. Cancer
40: 818.
58. Evinger-Hodges, M.J., K.A. Dicke, J.V. Gutterman, and
M. Blick. 1987. Proto-Oncogene Expression in Human
Normal Bone Marrow. Leukemia 1: 597.
59. Flockhart, D.A., and J.D. Corbin. 1982. Regulatory
Mechanisms in the Control of Protein Kinases. CRC
Crit. Rev. Biochem. 12: 133.
60. Franza, B.R., F.J. Rauscher, S.F. Josephs, and T.
Curran. 1988. The Fos Complex and Fos-Related
Antigens Recognize Elements That Contain AP-1 Binding
Sites. Science: 239: 1150.

167
61. Fraumeni, J.F. 1975. Bone Cancer: Epidemiologic and
Etiologic Considerations. In Frontiers of Radiation
Therapy and Oncology. Vol. 10. J.M. Vaeth, ed. Basel,
S. Karger, pp 17-27.
62. Friedewald, W.F. and P. Rous. 1944. The Initiating
and Promoting Elements in Tumor Production: An
Analysis of the Effects of Tar, Benzpyrene, and
Methylcholanthrene. J. Exp.Med. 80: 101.
63. Garrett, J.S., S.R. Couglin, H.L. Nilman, P.M.
Tremble, G.M. Geils, and L.T. Williams. 1984.
Blockade of Autocrine Stimulation in Simian Sarcoma
Viurs-Transformed Cells Reverses Down-Regulation of
Platelet Derived Growth Factor Receptors. Proc.
Natl. Acad. Sci. U.S.A. 81: 7466.
64. Gilman, M.Z., R.N. Wilson, and R.A. Weinberg. 1986.
Multiple Protein-Binding Sites in the 5' Flanking
Region Regulate C-fos Expression. Mol. Cell. Biol.
12: 4305.
65. Goubin, G., D.S. Goldman, J. Luce, P.E. Neiman, and
G.M. Cooper. 1983. Molecular Cloning and Nucleotide
Sequence of a Transforming Gene Detected by
Transfection of Chicken B-Cell Lymphoma DNA. Nature
302: 114.
66. Graziani, Y., J.L. Mailer, Y. Sugimoto, and R.L.
Erikson. 1984. In Cancer Cells. Vol 2. G.F. Vande
Woude, A.J. Levine, W.C. Topp, and J.D. Watson, eds.
Cold Spring Laboratory, Cold Spring Harbor, N.Y.
pp 27-35.
67. Greenberg, M.E., A.L. Hernamowski, and E.B. Ziff. 1986
Effect of Protein Synthesis Inhibitors on Growth Factor
Activation of C-fos, C-myc, and Actin Gene
Transcription. Mol. Cell. Biol. 4: 1050.
68. Greenberg, M.E., Z. Siegfried, and E.B. Ziff. 1987.
Mutation of the C-fos Symmetry Element Inhibits Serum
Inducibility of Transcription In Vivo and the Nuclear
Regulatory Factor Binding In Vitro. Mol. Cell. Biol.
3: 1217.

168
69. Greenberg, M.E., and E.B. Ziff. 1984. Stimulation of
3T3 Cells Induces Transcription of the C-fos Proto-
Oncogene. Nature: 311: 433.
70. Gridoni, D., W.S. Dynan, and R. Tjian. 1984. Multiple
Specific Contacts Between a Mammalian Transcription
Factor and its Cognate Promoters. Nature 312: 409.
71. Groffen, J., N. Heistekamp, J.R. Stephenson, A.G.
VanKessel, A. Deklein, G. Grosveld, and D. Bootsma.
1983. C-sis is Translocated From Chromosome 22 to
Chromosome 9 in Chronic Myelocytic Leukemia. J.
Exp. Med. 18 : 9 .
72. Gross, D.S., and W.T. Garrard. 1988. Nuclease
Hypersensitive Sites in Chromatin. Ann. Rev. Biochem.
57: 159.
73. Grosso, L., and H. Pitot. 1985. Chromatin Structure
of the C-myc Gene in HL-60 Cells During Alterations of
Transcriptional Activity. Can. Res. 45: 5035.
74. Gruss, P., R. Dahr, and G. Khoury. 1981. Simian Virus
40 Tandem Repeated Sequences an Element of the Early
Promoter. Proc. Natl. Acad. Sci. U.S.A. 78: 943.
75. Gunning, P., P. Ponte, H. Okayama, J. Engel, H. Blau,
and L. Kedes. 1983. Isolation and Characterization of
Full Length cDNA Clones for Human alpha, Beta, and
Gamma Actin mRNAs. Skeletal but not Cytoplasmic Actins
Have an Amino-Terminal Cysteine that is Subsequently
Removed. Molec. and Cell. Biol. 3: 787.
76. Hadju, S. 1979. Pathology of Soft Tissue Tumors.
Philadelphia, Lea and Febiger, p. 39.
77. Hall, A. 1986. Oncogenes. Genetic Engineering 5: 61.
78. Haluska, F.G., K Huebner, and C. Croch. 1987. P380
-8A 1.8 SaSs, A Single Copy Clone 5' of C-myc at 8q24
Which Recognizes an Sst I Polymorphism. NAR 15: 865.

169
79. Hansen, M.F., A. Koufos, B.L. Gallie, R.A. Phillips, 0.
Fodstad, A.Brogger, T. Gedde-Dahl, and W.K. Cavenee.
1985. Osteosarcoma and Retinoblastoma: A Shared
Chromosomal Mechanism Revealing Recessive
Predisposition. Proc. Natl. Acad. Sci. U.S.A. 82:
6216.
80. Harbour, J.W., S. Lai, J. Whang-Peng, A.F. Gazdar, J.D.
Minna, and F.J. Kaye. 1988. The Retinoblastoma Gene
in Human Breast Cancer. Science 241: 353.
81. Hayes, T.E., A.M. Kitchen, and B.H. Cochran. 1987.
Inducible Binding of a Factor to the C-fos Gene.
Proc. Natl. Acad. Sci. U.S.A. 5: 1272.
82. Hayman, M.J., and H. Beug. 1984. Identification of a
Form of the Avian Erythroblastosis Virus Erb-B Gene
Product at the Cell Surface. Nature 309: 460.
83. Hayward, W.S., B.G. Neel, and S.M. Astrin. 1981.
Activiation of a Cellular One Gene by Promoter
Insertion in ALV- Induced Lymphoid Leukosis. Nature
290: 475.
84. Hecker, E. 1971. Isolation and Characterization of
the Cocarcinogenic Principals From Croton Oil. Methods
Cancer Research 6: 439.
85. Henderson, E.D., and D.C. Dahlin. 1963.
Chondrosarcoma of Bone A Study of 288 Cases. J. Bone
Joint Surg. 45A: 1450.
86. Hogan, M.E., N. Datlagupta, and J.P. Whitlock. 1981.
Carcinogen Induced Alteration of DNA Structure.
J.Biol. Chem. 256: 4504.
87. Huang, J.S., S.S. Huang, and T.F. Devel. 1984.
Transforming Protein of Simian Sarcoma Virus Stimulates
Autocrine Cell Growth of SSV-Transformed Cells Through
PDGF Cell Surface Receptors. Cell 39: 79.
88. Huebner, R.J., and G.J. Todaro 1969. Oncogenes of RNA
Tumor Viruses as Determinants of Cancer. Proc. Natl.
Acad.Sci. U.S.A. 65: 1087.

170
89. Hunter, T., and B. Sefton. 1980. Transforming Gene
Product of Rous Sarcoma Virus Phosphorylates Tyrosine.
Proc. Natl. Acad. Sci. U.S.A. 77: 1311.
90. Iwasaki, H., T. Isayama, H.Johzaki, and M. Kikuchi.
1987. Malignant Fibrous Histiocytoma: Evidence of
Perivascular Mesenchymal Cell Origin Immunocyto-
chemical Studies With Monoclonal Anti-MFH Antibodies.
Am. J. Path. 128: 528.
91. Jacobs, C., and H. Rubsamen. 1983. Expression of pp60
C-src Protein Kinase in Adult and Fetal Human Tissue:
High Activities in Some Sarcomas and Mammary
Carcinomas. Can. Res. 43: 1696.
92. Jakobovits, E.B., J.E. Majors, and H.E. Varmus 1984.
Hormonal Regulation of the Rous Sarcoma Virus Src Gene
via a Heterologous Promoter Defines a Threshold Dose
For Cellular Transformation. Cell 38: 757.
93. Johnson, L.F., L. Gollakota, and A. Muench. 1982.
Regulation of Thymidine Kinase Enzyme Level in Serum
-Stimulated Mouse 3T6 Fibroblasts. Exp. Cell Res.
138: 79.
94. Kaczmarek, L. 1986. Proto-Oncogene Expression During
the Cell Cycle. Laboratory Investigation 54: 365.
95. Kelly, K., B.H. Cochran, C.D. Stiles, and P. Leder.
1983. Cell-Specific Regulation of the C-myc Gene by
Lymphocyte Mitogens and Platelet-Derived Growth
Factor. Cell 35: 603.
96. Kelly, K., and U. Siebenlist. 1985. The Role of C-myc
in the Proliferation of Normal and Neoplastic Cells.
Journal of Clinical Immunology 5: 65.
97. Knudson, A.G. 1985. Hereditary Cancer, Oncogenes and
Antioncogenes. Can. Res. 45: 1437.
98. Kohl, N.E., N. Kanda, R. Schreck, G. Bruns, S. Laft,
and F. Gilbert. 1983. Transpostions and Amplificaton
of Oncogene Related Seguence in Human Neuroblastomas.
Cell 35: 359.

171
99. Kohl, N., E. Legouy, R. DePinho, R. Smith, C.Gee, and
F. Alt. 1986. Human N-myc is Closely Related in
Organization and Nucleotide Sequence to C-myc. Nature
319: 73.
100. Kruijer, W., J.A. Cooper, T. Hunter, and I.M. Verma.
1984. Platelet-Derived Growth Factor Induces Rapid but
Transient Expression of the C-fos Gene and Protein.
1984. Nature 312: 711.
101. Laemmli, Y. 1970. Cleavage of Structural Protein
During the Assembly of the Head in Bacteriophage T4.
Nature 227: 280.
102. Land, H., L.F. Parada, and R.A. Weinberg. 1983.
Cellular Oncogenes and Multistep Carcinogenesis.
Science 222: 771.
103. LeBeau, M.M., and J.D. Rowley. Heritable Fragile Sites
in Cancer. Nature 308: 607.
104. Leder, P., J. Battey, G. Lenoir, C. Moulding, W.Murphy,
H. Potter, T. Stewart, and R. Taub. 1983.
Translocations Among Antibody Genes in Human Cancer.
Science 222: 765.
105. Lee, K.Y., W. Lijinsky, and P.N. Magee. 1964.
Methylation of Ribonucleic Acids of Liver and Other
Organs in Different Species Treated With C14 or H3
Dimethylnitrosamine In Vivo. J. Natl. Cancer Inst.
32: 65.
106. Lee, W., A. Murphree, and W. Benedict. 1984.
Expression and Amplification of the N-myc Gene in
Primary Retinoblastoma. Nature 309: 458.
107. Leibovitch, S.A., M.P. Leibovitch, M. Guiller, J.
Hillion, and J. Harel. 1986. Differentiation of
Proto-Oncogenes Related to Transformation and Cancer
Progression in Rat Myoblasts. Can. Res. 46: 4097.
108. Libermann, T.A., H.R. Nusbaum, N. Razn, R. Kris, I.
Lax, H. Soreq, N. Whittle, M.D. Waterfield, A. Ulrich,
and J. Schlessinger. 1985. Amplification, Enhanced
Expression and Possible Rearrangement of EGF Receptor
Gene in Primary Human Brain Tumors of Glial Origin.
Nature 313: 144.

172
109. Little, J.B. 1977. Radiation Carcinogenesis In Vitro:
Implications For Mechanisms. In Origins of Human
Cancer. H.H. Hiatt, J.D. Watson, and J.A.
Winsten, eds. Cold Spring Harbor Laboratory, Cold
Spring Harbor.
110. Loeb, L.A. 1985. Apurinic Sites As Mutagenic
Intermediates. Cell 40: 483.
111. Manger, R., L. Najita, E.J. Nichols, S. Hakomori, and
L. Rohrschneider 1984. Cell Surface Expression of
the McDonough Strain of Feline Sarcoma Virus Fms Gene
Product (GP 140 fms). Cell 39: 327.
112. Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982.
Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor Laboratory Publishers, Cold Spring Harbor,
New York.
113. Marx, J. 1987. The Fos Gene As a "Master Switch."
Science 237: 854.
114. McCann, J., E. Choe, E. Yamasaki, and B.N. Ames.
1975. Detection of Carcinogens as Mutagens in the
Salmonella Microsome Test: Assay of 300 Chemicals.
Proc. Natl. Acad. Sci. U.S.A. 72: 5135.
115. McCoy, M., J.T. Tode, J.M. Cunningham, E.H. Chang,
D.R. Lowy, and R.A. Weinberg. 1983. Characterization
of a Human Colon/Lung Carcinoma Gene. Nature 302:
79.
116. McGrath, J.P., D.J. Capon, D.V. Goeddel, and A.D.
Levinson. 1984. Comparative Biochemical Properties of
Normal and Activated Human Ras p21 Protein. Nature
310: 644.
117. Miller, E.C. 1978. Some Current Perspectives on
Chemical Carcinogenesis in Humans and Experimental
Animals. Can. Res. 38: 1479.
118. Moelling, K., B. Heimann, P. Beimling, U.R. Rapp, and
T. Sander. 1984. Serine and Threonine Specific
Protein Kinase Activities of Purified Gag-Mil and Gag
-Raf Proteins. Nature 312: 558.

173
119.Morgan, J.I., D.R. Cohen, J.L. Hempstead, and T.
Curran. 1986. Mapping Patterns of C-fos Expression
in the Central Nervous System After Seizure. Science
237: 192.
120. Muller, R., R. Bravo, J. Burckhardt, and T. Curran.
1984. Induction of C-fos Gene and Protein by Growth
Factors Preceedes Activation of C-myc. Nature 312:
716.
121. Murphy, W., H. Potter, T. Steward, and R. Taub. 1983.
Translocations Among Antibody Genes in Human Cancer.
Science 222: 765
122. Mushinski, J.F., M. Potter, S.R. Bauer, and E.P.
Reddy. 1983. DNA Rearrangement and Altered RNA
Expression of the C-myb Oncogene in Mouse Plasmacytoid
Lymphosarcomas. Science 220: 795.
123. Nau, M., B.Brooks, J. Batley, E. Sausville, A. Gazdar,
I. Kirsch, 0. McBride, V. Bertness, G. Hollis, and J.
Minna. 1985. L-myc, A New Myc Related Gene Amplified
and Expressed in Human Small Cell Lung Cancer. Nature
318: 69.
124. Nau, M., S. Brooks, D. Carney, A. Gazdar, J. Battey,
E. Sausville, and J. Minna. 1986. Human Small Cell
Lung Cancers Show Amplification and Expression of the
N-Myc Gene. Proc. Natl. Acad. Sci. U.S.A. 83: 1092.
125. Needle, S. 1981. New Twists To DNA and DNA-
Carcinogen Interactions. Nature 292: 292.
126. Nishikura, K., and J.M. Murray. 1987. Antisense RNA
of Proto-Oncogene C-fos Blocks Renewed Growth of
Quiescent 3T3 Cells. Molec. and Cell. Biol. 7: 639.
127. Nishizuka, Y. 1984. Turnover of Inositol Phospholipids
and Signal Transduction. Science 225:
128. Nowell, P.C. 1976. The Clonal Evolution of Tumor Cell
Populations. Science 194: 23.
129. Parker, C.S. and T. Topol. 1984. Drosophila RNA
Polymerase II Transcription Factor Binds to the
Regulatory Site of an HSP 70 Gene. Cell 37: 273.

174
130. Parker, R.C., H.E. Varmus, and J.M. Bishop. 1984.
Expression of V-src and Chicken C-src in Rat Cells
Demonstrates Qualitative Differences Between pp60 V-src
and pp60 C-src. Cell 37: 131.
131. Payne, G.S., J.M. Bishop, and H.E. Varmus. 1982.
Multiple Arrangements of Viral DNA and an Activated
Host Oncogene in Bursal Lymphomas. Nature 295; 209.
132. Persson, H., L. Hennighausen, R. Taub, W. DeGrado, and
P. Leder. 1984. Antibodies to Human C-myc Oncogene
Product: Evidence of an Evolutionarily Conserved
Protein Induced During Cell Proliferation. Science
225: 687.
133. Poiesz, B.J., F.W. Ruscett, A.F. Gazdar, P.A. Buhn,
J.D. Minna, and R.C. Gallo. 1980. Detection and
Isolation of Type C Retrovirus Particles From Fresh and
Cultured Lymphocytes of a Patient With Cutaneous T-Cell
Lymphoma. Proc. Natl. Acad. Sci. U.S.A. 77: 7415.
134. Pollard, T.D., and R.R. Weihuing. 1974. Actin and
Myosin and Cell Movement. Crit. Rev. Biochem. 2: 1.
135. Pritchard, D.J., R.J. Lunke, W.F. Taylor, D.C. Dahlin,
and B.E. Medley. 1980. Chondrosarcoma: A
Clinicopathologic and Statistical Analysis. Cancer
45: 149.
136. Pulciani, S., E. Santos, A.V. Lauver, L.K. Long, S.A.
Aaronson, and M. Barbacid. 1982. Oncogenes in Solid
Human Tumors. Nature 300: 539.
137. Pulciani, S., E. Santos, A.V. Lauver, L.K. Long, and M.
Barbacid. 1982. Transforming Genes in Human Tumors.
1982. J.C.B. 51: 61.
138. Rauscher, F.J., D.R. Cohen, T.Curran, T.J. Bos, P.K.
Vogt, D. Bohman, R. Tijan, and B. Franza. 1988.
Fos-Associated Protein p39 is the Product of the
jun Proto-Oncogene. Science 240: 1010.
139. Rave, N., R. Crkvenjakov, and H. Boedtker, 1979.
Identification of Procollagen mRNAs Transferred to
Diazobenzyloxymethyl Paper From Formaldehyde Agarose
Gels. NAR 6: 3559.

175
140. Robbins, K.C., H.N. Antoniades, S.G. Davare, M.W.
Hynkapiller, and S.A. Aaronson. 1983. Structural and
Immunological Similarities Between Simian Sarcoma Virus
Gene Products and Human Platelet Derived Growth Factor.
Nature 305: 605.
141. Rodriguez-Pena, A. and E. Rozengurt. 1985. Serum Like
Phorbol Esters Rapidly Activate protein Kinase C in
Intact Quiescent Fibroblasts. EMBO J. 4: 71.
142. Ruddon, R.W. 1987. Cancer Biology Second Edition.
New York, Oxford University Press.
143. Rysek, R.P., S.I. Hirai, M. Yaniv, and R. Bravo. 1988.
Transcriptional Activation of C-jun During the G0/G1
Transition in Mouse Fibroblasts. Nature 334; 535.
144. Santos, E., E.P. Reddy, S. Pulciani, R.J. Feldmen, and
M. Barbacid. 1983. Spontaneous Activation of a Human
Proto-oncogene. Proc. Natl. Acad. Sci. U.S.A. 80:
4679.
145. Schecter, A.L., F.F. Stern, L. Vaidyanathan, S.J.
Decker, J.A. Drebin, M.I. Greene, and R.A. Weinberg.
1984. The Neu Oncogene: An Erb-B Related Gene
Encoding 185,000 Mr Tumour Antigen. Nature 312: 513.
146. Scherr, C.J., C.W. Rettenmier, R. Sacca, M.F. Roussel,
A.T. Look, and E.R. Stanley. 1985. The C-fms Proto
-Oncogene Product is Related to the Receptor for the
Mononuclear Phagocyte Growth Factor, CSF-1. Cell 41:
665.
147. Schimke, R. 1984. Gene Amplification in Cultured
Animal Cells. Cell 37: 705.
148. Schmidt, J.A., H. Beug, and M.J. Hayman. 1985.
Effects of Inhibitors of Glycoprotein Processing on
the Synthesis and Biological Activity of the Erb-B
Oncogene. EMBO J. 4: 105.
149. Schwab, M., K. Alitalo, H.E. Varmus, J.M. Bishop, and
A.D. George. 1983. A Cellular Oncogene (C-Ki-ras) is
Amplified, Overexpressed, and Located Within
Karyotypic Abnormalities in Mouse Adrenocortical tumor
Cells. Nature 303: 497.

176
150. Schwab, M., H. Varmus, and J. Bishop. 1985. The
Human N-myc Gene Contributes to Tumorigenic Conversion
of Mammalian Cells in Culture. Nature 316: 160.
151. Scott, W.A., and D.J. Wigmore. 1978. Sites in Simian
Virus 40 Chromatin Which Are Preferentially Cleaved by
Endogenous Nucleases. Cell 15: 1511.
152. Seeger, R., G. Brodeur, H. Sather, A. Dalton, S.
Siegel, K. Wong, and O. Hammond. 1985. Association
of Multiple Copies of the N-myc Oncogene With Rapid
Progression of Neuroblastomas. N. Eng. J. Med. 313:
1111.
153. Setlow, R.B. 1978. Repairing Deficient Human
Disorders and Cancer. Nature 271: 713.
154. Shimada, T., K. Inokuchi, and A.W. Nienhuis. 1986.
Chromatin Structure of the Human Dihydrofolate
Reductase Gene Promoter. Multiple Protein Binding
Sites. J. Biol. Chem. 261: 1445.
155. Siebenlist, U., L. Henninghausen, J. Battey, and P.
Leder. 1984. Chromatin Structure and Protein Binding
in the Putative Regulatory Region of the C-myc Gene in
Burkitt Lymphoma. Cell 37: 381.
156. Siebenlist, U., P. Bressler, and K. Kelly. 1988. Two
Distinct Mechanisms of Transcriptional Control Operate
on C-myc During Differentiation of HL-60 Cells. Molec.
Cell. Biol. 8: 867.
157. Slamon, D., T. Boone, R. Seeger, D. Keith, V. Chazin,
H. Lee, and L. Souza. 1986. Identification and
Characterization of the Protein Encoded by the Human N
-myc Oncogene. Science 232: 768.
158. Slamon, D.J., G.M. Clark, S.G. Wong, W.J. Levin, A.
Ulrich, and W.L. Maguire. 1987. Human Breast Cancer:
Correlation of Relapse and Survival With Amplification
of the HER-2/neu Oncogene. Science 235: 177.
159. Slamon, D.J., J.B. DeKernion, I.M. Verma, and M.J.
Cline. 1984. Expression of Cellular Oncogenes in
Human Malignancies. Science: 224: 256.

177
160. Southern, E.M. 1975. Detection of Specific Sequences
Among DNA Fragments Separated by Gel Electrophoresis.
J. Mol. Biol. 98: 503.
161. Souyri, M., and E. Fleissner. 1983. Identification By
Transfection of Transforming Sequences in DNA of Human
T Cell Leukemias. Proc. Natl. Acad. Sci. U.S.A. 80:
6676.
162. Spector, D.H., H.E. Varmus, and J.M. Bishop. 1978.
Nucleotide Sequences Related to the Transforming Gene
of Avian Sarcoma Virus are Present in DNA of Infected
Vertebrates. Proc. Natl. Acad. Sci. U.S.A. 75: 4102.
163. Sporn, M.B. and G.J. Todaro. 1980. Autocrine
Secretion and Malignant Transformation of Cells. N.
Eng. J. Med. 303: 878.
164. Stehelin, D. H.E. Varmus, J.M. Bishop, and P.K. Vogt.
1976. DNA Related to the Transforming Gene(s) of Avian
Sarcoma Viruses is Present in Normal Avian DNA.
Nature 260: 170.
165. Stiles, C.D. 1983. The Molecular Biology of Platelet
Derived Growth Factor. Cell 33: 653.
166. Strandberg, J.C. 1986. The Expression of Mvc and Ras
Oncogenes in Chondrosarcomas and Malignant Fibrous
Histiocytomas. Masters Thesis, University of Florida.
167. Sugimoto, Y., M. Whitman, L.C. Cantley, and R.L.
Erikson. 1984. Evidence That the Rous Sarcoma Virus
Transforming Gene Product Phosphorylates
Phosphatidylinositol and Diacylglycerol. Proc. Natl.
Acad. Sci. U.S.A. 81: 2117.
168. Swift, M., and C. Chase. 1979. Cancer in Families With
Xeroderma Pigmentosum. J. Natl. Cancer Inst. 62:
1415.
169. Swift, M., L. Sholman, M. Perry, and C.Chase. 1976.
Malignant Neoplasms in the Families of Patients With
Ataxia Telangiectasia. Can. Res. 36: 209.
170. Tabin, C.J., S.M. Bradley, C.I. Bargmann, and R.A.
Weinberg. 1982. Mechanism of Activation of a Human
Oncogene. Nature 300: 143.

178
171. Takeya, T., and H. Hanafusa. 1982. DNA Sequence of the
Viral and Cellular Src Gene of Chickens. Complete
Nucleotide Sequence of an ECO R I Fragment of Recovered
Avian Src Virus Which Codes For GP 37 and pp60 Src.
Virol. 44: 12.
172. Tatosyan, A.G., S.A. Galetzk, F.L. Kisseljova, A.A.
Asanova, I.B. Zborovskaya, D.D. Spitovsky, E.S.
Revasova, P. Martin, and F.L. Kisseljov. 1985.
Oncogene Expression in Solid Human Tumors. Int. J.
Cancer 35; 731.
173. Temin, H.M. 1976. The DNA Provirus Hypothesis: The
Establishment and Implications of RNA Directed DNA
Synthesis. Science 192: 1075.
174. Temin, H.M., and S. Mizutani. 1970. RNA Dependent DNA
Polymerase in Virions of Rous Sarcoma Virus. Nature
226: 1211.
175. Thompson, C., P.B. Challoner, P. Neiman, and M.
Groudine. 1985. Levels of C-myc Oncogene mRNA are
Invariant Throughout the Cell Cycle. Nature 314:
363 .
176. Towbin, H., T. Stachelin, and T. Gordan. 1979.
Electrophoretic Transfer of Protein From
Polyacrylamide Gels to Nitrocellulose Sheets:
Procedure and Some Applications. Proc. Natl. Acad.
Sci. U.S.A. 9: 4350.
177. Treisman, R. 1986. Identification of a Protein-
Binding Site That Mediates Transcriptional Response
of the C-fos Gene to Serum Factors. Cell: .4: 567.
178. Treisman, R. 1986. Transient Accumulation of C-fos
RNA Following Serum Stimulation Requires a Conserved
5' Element and C-fos 3' Sequences. Cell 3: 889.
179. Twardzik, D.R., J.E. Ranchalis, and G.J. Todaro. 1982.
Mouse Embryonic Transforming Growth Factors Related to
Those Isolated From Tumor Cells. Can. Res. 42: 590.
Uhthoff, H., ed. 1983. Current Concepts of Diagnosis
and Treatment of Bone and Soft Tissue Tumors New
York, Springer Verlag Publishers.
180.

179
181. Ulrich, A., L. Coussens, J.S. Hayflick, T.J. Dull, A.
Gray, A.W. Tam, J. Lee, Y. Yarden, T.A. Libermann, J.
Schlessinger, J. Downward, E. Mayes, N. Whittle, M.D.
Waterfield, and P.H. Seeburg. 1984. Human Epidermal
Growth Factor Receptor cDNA Sequence and Aberrant
Expression of the Amplified Gene in A 431 Epidermoid
Carcinoma Cells. Nature 309; 418.
182. Varshavsky, A.J., O.H. Sundin, and M.J. Bohn. 1978.
SV 40 Viral Minichromosomes: Preferential Exposure of
the Origin of Replication as Probed by Restriction
Endonucleases. NAR 5: 3469.
183. Vent, P.J., T.B. Shows, P.J. Curtis, and R. Tashian.
1983. Polymorphic Gene for Human Carbonic Anhydrase II:
A Molecular Disease Marker Located on Chromosome 8.
Proc.Natl. Acad. Sci. U.S.A. 80: 4437.
184. Verma, I.M., J. Deschamps, C. Van Beueren, and P.
Sassone- Corsi. 1986. Human fos Gene. Cold Spring
Harbor Symposium on Quantitative Biology. 51: 949.
185. Vijayolaxmi, H., J. Evans, J.H. Ray, and J. German.
1983. Blooms Syndrome: Evidence for an Increased
Mutation Frequency In Vivo. Science 221: 851.
186. Viola, M., F. Fromowitz, S. Oravez, S. Deb, G. Finkel,
J. Lundy, P. Hand, A. Thor, and J. Schlom. 1986.
Expression of Ras Oncogene p21 in Prostate Cancer. The
New. Eng. J. Med. 314: 133.
187. Wahl, G. 1985. Rapid Detection of DNA and RNA Using
Slot-Blotting. (Application Update No. 371).
Schleicher & Schuell, Inc., Keene, NH.
188. Watt, R., L.W. Stanton, K.B. Marcy, R.C. Gallo, C.M.
Croch, and G. Rovera. 1983. Nucleotide Sequence of
Cloned cDNA of Human C-myc Oncogene. Nature 303:
725.
189. Weiss, R.A. 1973. Possible Episomes in
Eukaryotes. L.G. Silvestri, ed. Amsterdam, North-
Holland.
Weiss, S.W., and F.M. Enzinger. 1978. Malignant
Fibrous Histiocytoma: An Analysis of 200 Cases.
Cancer 41: 2250.
190.

180
191. Westin, E.H., F. Wong-Staal, E.P. Gelman, R. Dalla-
Favera, T.S. Papas, J.A. Lautenberger, A. Eva, E.P.
Reddy, S.R. Tronick, S.A. Aaronson, and R.C. Gallo.
1982. Expression of Cellular Homologues of Retroviral
Oncogenes in Human Hematopoietic Cells. Proc. Natl.
Acad. Sci. U.S.A. 79: 2490.
192. Wickstrom, E., T. Bacon, A. Gonzalez, D. Freeman, G.
Lyman, and E. Wickstrom. 1988. Human Promyelocytic
Leukemia HL-60 Cell Proliferation and C-myc Protein
Expression are Inhibited by an Antisense
Pentadecadeoxynucleotide Targeted Against C-myc mRNA.
Proc. Natl. Acad. Sci. U.S.A. 85: 1028.
193. Wu, C. 1984. Two Protein Binding Sites in Chromatin
Implicated in the Activation of Heat-Shock Genes.
Nature 309: 229.
194. Wu, C. 1984. Activating Protein Factor Binds In Vitro
to Upstream Control Sequences in Heat Shock Gene
Chromatin. Nature 311: 81.
195. Wu, C., P.M. Bingham, K.J. Livak, R. Holmgren, and S.C.
Elgin. 1979. The Chromatin Structure of Specific
Genes 1. Evidence For Higher Order Domains of Defined
DNA Sequences. Cell 16: 797.
196. Wyke, J. 1983. Evoloution of Oncogenes From C-src to
V-src. Nature 304: 491.
197. Yamamoto, T., T. Nishida, N. Miyajima, S.Kawai, T. Ooi,
and K. Toyoshima. 1983. The Erb-B Gene of Avian
Erythroblastosis Virus is a Member of the Src Gene
Family. Cell 35: 71.
198. Yuasa, Y., S.K. Srivastava, C.Y. Dunn, J.S. Rhim, E.P.
Reddy, and S.A. Aaronson. 1983. Acquisition of
Transforming Properties by Alternative Point Mutations
Within C-bas/has Human Proto-Oncogene. Nature 303:
775.
199. Zajac-Kaye, M., E. Gelman, and D. Levens. 1988. A
Point Mutation in the C-myc Locus of a Burkitt Lymphoma
Abolishes Binding of a Nuclear Protein. Science 240:
1776.

181
200. Zimmerman, K., G. Yancopoulos, R. Collum, R. Smith, N.
Kohl, K. Denis, M. Nau, O. Witte, D. Toran-Allerand, C.
Gee, J. Minna, and F. Alt. 1986. Differential
Expression of Myc Family Genes During Murine
Development. Nature 319; 780.

BIOGRAPHICAL SKETCH
Jane Carolyn Strandberg Gibson was born in Madison,
Wisconsin, on September 29, 1962, to James and Kathleen
Strandberg, and is the oldest of their 3 children.
Jane has lived in the Orlando, Florida, area since 1969,
where she graduated from Bishop Moore High School in 1980.
During that same year, she began her college career at the
University of Central Florida in Orlando. In 1982 she moved
to Gainesville and attended the University of Florida were
she received a Bachelor of Science degree in microbiology and
cell science in April,1984.
In the fall of 1984, she began Graduate School as a
student in the Department of Pathology at the University of
Florida. In August 1986, she received her Master of Science
degree in medical sciences-pathology. At that time she
decided to continue her work with proto-oncogene expression
in human sarcomas under the direction of Dr. Byron Croker.
Jane married Ronald Lee Gibson on May 7, 1988. Since that
time she has continued her work toward a Doctor of
Philosophy degree in the Department of Pathology and
Laboratory Medicine, University of Florida College of
Medicine.
182

I certify that I have read this study and that in ray
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Associate Professor of Pathology
and Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Warren E. Ross
Associate Professor of
Pharmacology and Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Hutt-Fletcher
Associate Professor of Pathology
and Laboratory Medicine
Linds e^M.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Linda J. Smith
Assistant Professor of Pathology
and Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Har ry LQdtrer
Assistant Professor of
Biochemistry and Molecular Biology
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
May 1989
Dean, College of
Medicine
Dean, Graduate School



Preparation of Radiolabeled Probes 71
Nick Translated Probes 71
Probes Labeled by Random Primer Extension 77
Hybridization of Slot-Blots 77
Southern Blot Analysis 78
Northern Blot Analysis 79
Chromatin Structure Analysis 80
Cell Lines Used in Chromatin Structure
Analysis 80
Preparation of Nuclei 81
Isolation of Genomic DNA From DNAse I Treated
Nuclei 82
Chromatin Structure/Fibroblast Cell Synchrony
Experiment 83
Strategy for Fine Mapping DNAse I Hyper
sensitive Sites 3' to C-myc Exon 1 85
Polyacrylamide Gel Electrophoresis (PAGE) 88
Western Blotting and Immunoperoxidase Assay 89
4 RESULTS 91
Quantitation of Moderately Degraded RNA Using
the Slot-Blotting Technique 91
RNA Slot-Blot Results 96
DNA Slot-Blot Results and Determination of C-myc
Gene Copy Number 109
Regions Contained in the C-myc Amplicon 123
Chromatin Structure Analyses 125
C-myc Gene Copy Number and Transcript Levels
in P3C, UR HCL 1, HFF and ST 486 Cell
Lines 125
Locations of DNAse I Hypersensitve Sites in
P3C, UR HCL 1, HFF, and ST 486 Cell Lines... 126
Chromatin Structure of the C-myc Gene During
the G0/G1 Transition in the HFF Normal Human
Fibroblast Cell Line 134
Fine Mapping Analysis of DNAse I Hyper
sensitive Sites in P3C Cells From a 5'
Direction 139
C-myc Protein Levels in the P3C, UR HCL 1, HFF
and ST 486 Cell Lines 143
5 DISCUSSION 146
REFERENCES 160
BIOGRAPHICAL SKETCH 182
VI


137
GO 0.5 HR 1 HR
UG/ML DNASE I
Figure 23. Southern blot analysis of genomic DNA from DNAse
I treated nuclei of HFF normal human fibroblast cells.
Nuclei isolated from HFF cells in GO, 0.5, 1, 2, and 3 hours
after serum release, and during log phase growth (L) were
treated with various concentrations of DNAse I (L-R; 0.1,
0.5, 1, 2, 5, and 0 ug/ml (37 C degrees)). Genomic DNA was
isolated, restricted with Eco R 1, electrophoresed through
0.8 percent agarose gels, blotted, and hybridized with the
pmc 41 c-myc probe (See materials and methods). Sizes of
bands were determined by comparison to lambda DNA digested
with Eco R 1 and Hind III.


34
often generate oncogenic alleles. It seems that point
mutations elsewhere in the ras proto-oncogenes merely serve
to inactivate the genes instead of converting them to
oncogenes (102).
Finally, the possibility that unknown mechanisms of
activation may be at work must not be overlooked. There may
be mechanisms of activation which have not been determined.
It is possible that new mechanisms may eventually be
implicated in proto-oncogene activation.
Biochemistry of Oncogene Products
Cytoplasmic Kinases
One of the first oncogene proteins of this class to
be recognized and studied was the 60,000 dalton protein of
the v-src gene (pp60 v-src) (89). Other oncogene products
with tyrosine-specific protein kinase activity include yes,
abl, fps, fgr, and ros (6, 77). These proteins are all
located at the inner surface of the cytoplasmic membrane and
a comparison of their amino acid sequences has shown that
they are related to each other (35). A region of
approximately 250 amino acids in pp60 src is responsible for


37
levels of intracellular cyclic AMP. This can lead to the
activation of cytoplasmic cyclic AMP-dependent serine
specific protein kinases. Activation of other serine
specific kinases, particularly protein kinase C may then
occur (59). It is protein kinase C which is thought to play
a central role in the various responses to mitogenic
stimulation. Graziani (66) has shown that tyrosine
phosphorylation of the cyclic AMP dependent protein kinases
by pp60 c-src occurs in transformed cells. Therefore, it is
possible that pp60src interacts with the pathway which
regulates cell proliferation through cyclic AMP and protein
kinase C.
Another pathway in which src may be involved also leads
to the activation of protein kinase C. Full activity of this
protein requires two cofactors; calcium, and diacylglycerol
(127). Both of these can be generated in response to
extracellualr signals such as acetylcholine or PDGF. The
result of the interaction of these molecules with their
receptors is a breakdown of inositol phospholipids located in
the membrane to yield diacylglycerol. This activates protein
kinase C, and inositoltriphosphate which can affect calcium
levels within the cell. Sugimoto et al. (167) showed that
pp60src could phosphorylate inositol phospholipids in vitro


161
8. Barrett, T.B., C.M. Gajdneck, C.M. Schwartz, S.M.
McDougall, and E.P. Benditt. 1984. Expression of the
Sis Gene by Endothelial Cells in Culture and In Vivo.
Proc. Natl. Acad. Sci. U.S.A. 81: 6772.
9. Bentley, D.L., and M. Groudine. 1986. Novel Promoter
Upstream of the Human C-myc and Regulation of C-myc
Expression in B-Cell Lymphomas. Molec. and Cell. Biol.
6: 3841.
10. Bentley, D.L, and M. Groudine. 1986. A Block to
Elongation is Largely Responsible fo Decreased
Transcript of C-myc in Differentiated HL-60 Cells.
Nature 321: 702.
11. Berenblum, I. 1975. Sequential Aspects of Chemical
Carcinogenesis: Skin. In Cancer: A Comprehensive
Treatise. F.F. Becker, ed. New York, Plenum Press,
pp. 323-344
12. Berridge, M.J., and R.F. Irvine 1984. Inositol
Triphosphate, a Novel Second Messenger in Cellular
Signal Transduction. Nature 312: 315-380.
13. Betsholtz, C., B. Wetermark, B. Ek, and C.H. Helden.
1984. Coexpression of a PDGF-Like Growth Factor and
PDGF Receptors in a Human Osteosarcoma Cell Line;
Implications For Autocrine Receptor Activation.
Cell 39: 447.
14. Beug, H. and M.J. Hayman. 1984. Temperature
Sensitive Mutants of Avian Erythroblastosis Virus:
Surface Expression of the Erb-B Product Correlates
With Transformation. Cell 36: 963.
15. Bevis, C.C., and B.P. Croker. 1985. Bone and Soft
Tissue Tumors; Contrast and Comparison by Leukocyte
Phenotyping. Laboratory Medicine 52: 1
16. Bishop, J.M. 1983. Cellular Oncogenes and
Retroviruses Ann. Rev. Biochenm. 52: 301.
17 .
23 .
Bishop, J.M.
1985. Viral Oncogenes.
Cell 42:


UR HCL 1
UG/ML DNASE I
130
Figure 19. Southern blot of UR HCL-1 genomic DNA from DNAse
I treated nuclei. After treatment of nuclei with various
concentrations of DNAse I (L-R; 0.1, 0.2, 0.5, 1, 2, 5,
ug/ml, controls consisted of 0 ug/ml DNAse I at both 0 and 37
C degrees), genomic DNA was isolated restricted with Eco R
I, electrophoresed through 0.8 percent agarose gels, and
hybridized with the pmc 41 probe. Samples 0.2, 0.5, and 1
ug/ml DNAse I gave optimum band visualization and were used
in a composite Southern blot shown in figure 20.


163
26. Chirgwin, J.M., A.E. Przybyla, and R.J. McDonald.
1979. Isolation of Biologically Active Ribonucleic
Acid From Sources Enriched in Ribonuclease.
Biochemistry 18: 5293.
27. Claycomb, W.C., and N.A. Lanson. 1987. Proto-Oncogene
Expression in Proliferating and Differentiating Cardiac
and Skeletal Muscle. Biochem J. 247: 701.
28. Cleveland, D.W., M.A. Lopata, R.J. MacDonald, N.J.
Conan W.J. Rutter, and M.W. Kirschner. 1980. Number
and Evoloutionary Conservation of Alpha and Beta
Tubulin and Cytoplasmic Beta and Gamma Actin Genes
Using Specific Cloned cDNA Probes. Cell 20: 95.
29. Cohen, S., G. Carpenter, and L.E. King. 1980.
Epidermal Growth Factor Receptor Protein Kinase
Interactions. Co-purification of Receptor and
Epidermal Growth factor Enhanced Phosphorylation
Activity. J. Biol. Chem. 255: 4834.
30. Coley, B.L. 1960. Neoplasms of Bone. Second
Edition. New York, Paul B. Hoebner, Inc.
31. Collett, M.S., E. Erikson, A.F. Purchio, J.S. Brugge,
and R.L. Erikson. 1979. A Normal Cellular Protein
Similar in Structure and Function to the Avian Sarcoma
Transforming Gene Product. Proc. Natl. Acad. Sci.
U.S.A. 76: 3159.
32. Collins, S.J., and M. Groudine. 1982. Amplification
of Endogenous Myc Related DNA Sequences in a Human
Myeloid Leukemia Cell Line. Nature 298: 679.
33. Collins, S.J., and M. Groudine. 1983. Rearrangement
and Amplification of C-abl Sequences in the Human
Chronic Myelogenous Leukemia Cell Line K-562. Proc.
Natl. Acad. Sci. U.S.A. 80: 4813.
34. Cooper, C.S., M. Park, D.G. Blair, M.A. Tainsky, K.
Huebner, C.M. Croch, and C.F. Vande Woude. 1984.
Molecular Cloning of a New Transforming Gene From a
Chemically Transformed Human Cell Line. Nature 311:
29.


116
Table 16. DNA
determined by
quantitation of cell-cycle dependent genes as
slot-blot analysis. *
BONE
MUSCLE
MARROW
CS
MFH
TK:ACTIN
0.9+0.1
1.0+0.2
1.0+0.1
1.10.1
C-MYCrACTIN
1.1+0.1
0.9+0.2
1.00.1
1.5+0.6**
C-HA-RAS:
1.1+0.1
0.90.1
0.9+0.2
1.1+0.2
ACTIN
C-FOSrACTIN
0.9+0.1
1.0+0.2
1.0+0.2
1.1+0.2
TOTAL CASES
9
6
20
23
* Values shown are mean gene:actin ratios
** 17 MFHs with single copy c-myc had a mean myc:actin
ratio of 1.2+0.1. Six MFHs with 2 or greater copies of
myc had a mean myciactin ratio of 2.3+0.4.


70
fold II slot-blotter (Schleicher & Schuell, Keene, NH) using
procedures described by Wahl (187) The slot-blotter
contained a Nitro-Plus 2000 filter (Micron Separation
Sciences) onto which the RNA was blotted.
DNA Slot-Blotting
Quantities of 20, 10, and 5 ug of DNA were suspended in
400 ul of lOmM Tris, ImM EDTA, pH 7.0, and 40 ul 3M NaOH,
then incubated at 65 C for 45 min. After incubation, the
samples were cooled on ice, 400 ul of 2 M ammonium acetate
were added, and the samples were loaded onto a minifold II
slot-blotter as described above.
After slot-blotting, the filters were air dried to
completion, then baked in a vacuum oven at 80 C for 2 hr.
The blots were then incubated at 42 C overnight in a pre
hybridization solution (5 ml/ 100 square cm) containing 5X
SSC, 10X Denhardt's solution (0.2 percent ficoll, 0.2 percent
polyvinylpyrrolidone (PVP), 0.2 percent bovine serum albumin
(BSA)), 0.05M sodium phosphate pH 6.7, 500ug/ul sonicated,
denatured salmon DNA, 5 percent dextran sulfate (Pharmacia
Chemical Co., Piscataway,NJ), and 50 percent formamide (112,
160) .


167
61. Fraumeni, J.F. 1975. Bone Cancer: Epidemiologic and
Etiologic Considerations. In Frontiers of Radiation
Therapy and Oncology. Vol. 10. J.M. Vaeth, ed. Basel,
S. Karger, pp 17-27.
62. Friedewald, W.F. and P. Rous. 1944. The Initiating
and Promoting Elements in Tumor Production: An
Analysis of the Effects of Tar, Benzpyrene, and
Methylcholanthrene. J. Exp.Med. 80: 101.
63. Garrett, J.S., S.R. Couglin, H.L. Nilman, P.M.
Tremble, G.M. Geils, and L.T. Williams. 1984.
Blockade of Autocrine Stimulation in Simian Sarcoma
Viurs-Transformed Cells Reverses Down-Regulation of
Platelet Derived Growth Factor Receptors. Proc.
Natl. Acad. Sci. U.S.A. 81: 7466.
64. Gilman, M.Z., R.N. Wilson, and R.A. Weinberg. 1986.
Multiple Protein-Binding Sites in the 5' Flanking
Region Regulate C-fos Expression. Mol. Cell. Biol.
12: 4305.
65. Goubin, G., D.S. Goldman, J. Luce, P.E. Neiman, and
G.M. Cooper. 1983. Molecular Cloning and Nucleotide
Sequence of a Transforming Gene Detected by
Transfection of Chicken B-Cell Lymphoma DNA. Nature
302: 114.
66. Graziani, Y., J.L. Mailer, Y. Sugimoto, and R.L.
Erikson. 1984. In Cancer Cells. Vol 2. G.F. Vande
Woude, A.J. Levine, W.C. Topp, and J.D. Watson, eds.
Cold Spring Laboratory, Cold Spring Harbor, N.Y.
pp 27-35.
67. Greenberg, M.E., A.L. Hernamowski, and E.B. Ziff. 1986
Effect of Protein Synthesis Inhibitors on Growth Factor
Activation of C-fos, C-myc, and Actin Gene
Transcription. Mol. Cell. Biol. 4: 1050.
68. Greenberg, M.E., Z. Siegfried, and E.B. Ziff. 1987.
Mutation of the C-fos Symmetry Element Inhibits Serum
Inducibility of Transcription In Vivo and the Nuclear
Regulatory Factor Binding In Vitro. Mol. Cell. Biol.
3: 1217.


169
79. Hansen, M.F., A. Koufos, B.L. Gallie, R.A. Phillips, 0.
Fodstad, A.Brogger, T. Gedde-Dahl, and W.K. Cavenee.
1985. Osteosarcoma and Retinoblastoma: A Shared
Chromosomal Mechanism Revealing Recessive
Predisposition. Proc. Natl. Acad. Sci. U.S.A. 82:
6216.
80. Harbour, J.W., S. Lai, J. Whang-Peng, A.F. Gazdar, J.D.
Minna, and F.J. Kaye. 1988. The Retinoblastoma Gene
in Human Breast Cancer. Science 241: 353.
81. Hayes, T.E., A.M. Kitchen, and B.H. Cochran. 1987.
Inducible Binding of a Factor to the C-fos Gene.
Proc. Natl. Acad. Sci. U.S.A. 5: 1272.
82. Hayman, M.J., and H. Beug. 1984. Identification of a
Form of the Avian Erythroblastosis Virus Erb-B Gene
Product at the Cell Surface. Nature 309: 460.
83. Hayward, W.S., B.G. Neel, and S.M. Astrin. 1981.
Activiation of a Cellular One Gene by Promoter
Insertion in ALV- Induced Lymphoid Leukosis. Nature
290: 475.
84. Hecker, E. 1971. Isolation and Characterization of
the Cocarcinogenic Principals From Croton Oil. Methods
Cancer Research 6: 439.
85. Henderson, E.D., and D.C. Dahlin. 1963.
Chondrosarcoma of Bone A Study of 288 Cases. J. Bone
Joint Surg. 45A: 1450.
86. Hogan, M.E., N. Datlagupta, and J.P. Whitlock. 1981.
Carcinogen Induced Alteration of DNA Structure.
J.Biol. Chem. 256: 4504.
87. Huang, J.S., S.S. Huang, and T.F. Devel. 1984.
Transforming Protein of Simian Sarcoma Virus Stimulates
Autocrine Cell Growth of SSV-Transformed Cells Through
PDGF Cell Surface Receptors. Cell 39: 79.
88. Huebner, R.J., and G.J. Todaro 1969. Oncogenes of RNA
Tumor Viruses as Determinants of Cancer. Proc. Natl.
Acad.Sci. U.S.A. 65: 1087.


46
found in 40 percent of primary brain tumors of glial origin
(108). Abnormally high copy numbers of the HER-2/neu
(c-erb-B-2) gene have been found in mammary carcinomas.
This in turn has been associated with patient survival and
time to relapse in diseased individuals (158).
Sis. The sis oncogene encodes one of the two subunits
(PDGF 2-B) of platelet derived growth factor (45). Following
synthesis, the 28kd product (p28sis) of the sis oncogene
assembles into a homodimer and is trimmed to a smaller
polypeptide (140).
The product of the sis oncogene may transform cells by
an autocrine function. Evidence exists that some cells
release a homodimer of p28sis, whose structure and activity
resemble those of PDGF (63). Application of antibodies
against PDGF to these cells arrests their growth (87). There
is also reason to suspect that the sis oncogene product, or
that of its cellular progenitor c-sis need not leave the
cell in order to invoke neoplastic growth (13). Instead, the
transforming protein may combine with a receptor while still
inside the cell.
There is also the guestion of why the sis oncogene
protein can transform cells, while the c-sis protein cannot.
It is not known if there are mutations in oncogenic sis that


69
Genomic DNA was prepared from the same tumor, muscle, and
bone marrow tissue specimens as described above. Tissues
were frozen in liquid nitrogen and ground to a fine powder
with a morter and pestal. This powder, or approximately 10
E6 cells was suspended in 9.2 ml of STE (lOOmM NaCl, 20mM
Tris, pH 8.0, lOmM EDTA). Two hundred ul of 0.5M EDTA and
200ul of proteinase K (10mg/ml) were then added, and the
mixture was incubated overnight at 65 C.
Following incubation, the mixture was extracted once
with an equal volume of phenol, once with an equal volume of
phenol/chloroform-isoamyl alcohol (24:1), and finally once
again with an equal volume of chloroform-isoamyl alcohol.
The DNA was recovered by spooling after the addition of an
equal volume of isopropanol, and resuspended in lOmM Tris,
ImM EDTA.
RNA Slot-Blotting
Quantites of 10, 5, and 2.5 ug of total cellular RNA were
denatured in a solution containing 100 ul of water and 300ul
of an RNA denaturant solution containing 6.15 M formaldehyde
and 10X SSC (sodium chloride, sodium citrate). The samples
were incubated at 65 C for 15 min and loaded onto a mini-


158
Exactly what all of the consequences of c-myc gene
ampIfication in MFHs are, and whether multiple copies of the
c-myc gene are the only abnormal events influencing
increased levels of transcript production are questions yet
to be answered. C-myc gene amplification may be a mechanism
by tumor cells to obtain growth advantages over other
surrounding cells. It would be interesting to study this in
vivo. Individuals with neoplastic disease could be
evaluated for various parameters of tumor growth. These
include tumor stage, tumor size, angiogenesis factors, and
metastasis. In order for a study of this nature to be
meaningful, a large sample size and careful patient followups
would be required. Although this would take years to
complete, studies such as this would provide a more complete
understanding of how abnormal gene copy number may actually
effect tumor growth in individuals with cancer.
The following statements summarize the results from
this project.
1. Increased transcript levels of c-myc and c-sis were
observed in MFHs in vivo.
2. The c-sis gene is single copy in MFHs, therefore
transcript levels are increased by an unknown
mechanism other that gene amplification.


76
i i
a
HL
C-FOS 6.4 KB
5
o
£
s
C-HA RAS 6.4 KB
_ £

_
§ 8
o
G
Q
Q
9
Z
Z
Z
X UJ
I
X
I
ii
i
1
j
¡2 I x
\-¡-t L,
x x SE
C-SIS 1.0 KB
LTR
1
BETA ACTIN 0.8 KB
95
i£
LL
LTR
VSRC
0.8 KB
E
o ^
9 I
ECO
1i 1
1 1
X
S-
<:
PTK 11 1.25 KB
LTR
V- ERB-A V-ERB-B
fc
_x_
x_
(/>
LTR
£
as
B
jjj
ii
1.7 KB
BETAACTIN 2.0 KB
Figure 6. Restriction maps of non-c-myc/chromosome 8 probes
as described in table 1.


79
0.1 percent SDS, and once again with 0.1 X SSC, 0.1 percent
SDS. The blots were then washed twice for 30 min at 60 C,
with 0.1 X SSC, 0.1 percent SDS and exposed to X-ray film
at -70 C with intensifying screens. Rehybridization of the
blots was accomplished after removal of bound probe. This
removal process consisted of washing the blots in 0.1 X SSC,
0.5 percent SDS at 80 C for 15-20 min.
Northern Blot Analysis
Total cellular RNA was prepared from cell lines as
described for slot-blotting. RNA was denatured at 55 C for
15 min in an RNA denaturant containing 5ul 10X MOPS, 8.75 ul
37 percent formaldehyde, 25 ul formamide (ultra-pure BRL,
Gaithersburg, MD), and water to a final volume of 50 ul.
After denaturation, 10 ul of RNA formaldehyde loading dye was
added (500 ul formamide, 162 ul 37 % formaldehyde, 350 ul
glycerol, 100 ul 10X MOPS, bromophenol blue). The samples
were electrophoresed through 1.2 percent formaldehyde
agarose gels (139). The gels were prepared by melting 4.2 gr
agarose in 304.5 ml water. After cooling, 35 ml MOPS and
10.5 ml formaldehyde were added. Samples were


85
serum. At this confluency level, cells were actively cycling
(confirmed by Northern blot hybridization with the TK probe
data shown below). The cells were then made quiescent by
the addition of MEM containing 0.1 percent serum, and
subsequent incubation at 37 C for 3 days.
MEM supplemented with 10 percent fetal bovine serum was
then added to release the cells. Nuclei isolation/DNAse I
treatment (as described above) and RNA isolation procedures
(as described above) were conducted at 0 hr (GO), 0.5 hr, 1
hr, 2 hr, 3 hr after serum release, and during log phase
growth. DNAse I hypersensitive sites were evaluated by
Southern blot hybridization procedures using the pmc 41 probe
as described above. RNA samples were analyzed for c-myc,
TK, and actin transcript levels using Northern blot
procedures as previously described.
Strategy for Fine Mapping DNAse I Hypersensitive Sites 31 to
C-Mvc Exon 1
An overall scheme for fine mapping of DNAse I
hypersensitive sites from a 5' direction is shown in figure
8. Selected restriction enzyme sites in the region of a
DNAse I hypersensitive site 3' to c-myc exon 1 (discussed


118
C-MYC
10 5 2-5
MT-1
MT-2
MT-3
MT4
MT5
MT6
MT7
MT8
MT9
MT10
MT11
MT12
MT13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 15. Slot-blot of total cellular RNA and genomic DNA
from MFHs hybridized with the c-myc probe. Quantities of 10,
5, and 2.5 ug of RNA and 20, 10, and 5 ug of DNA were slot-
blotted onto nitro-plus 2000, and hybridized with the c-myc
probe (3.0 x 10 E6 cpm/ 10 E8 cpm/ug).


CHAPTER 4
RESULTS
Quantitation of Moderately Degraded RNA Using the Slot-
Blotting Technique
The relationship between RNA degradation and accuracy of
quantitation was evaluated because in many instances tumor
tissues were not immediately (1-3 hr) available for
processing after surgical removal, and message degradation
occurs rapidly. Intact total cellular RNA from HL-60 cells
was degraded in 0.2 N NaOH at 0, 0.5, 1,2,5, and 30 minute
intervals. After evaluation by formaldehyde gel
electrophoresis using procedures previously described
(northern blotting section), (figure 9), the sample at 0
minutes showed completely intact RNA, the samples at 0.5, 1,
2, and 5 minutes, moderately degraded RNA, and at 30 minutes
the RNA was extensively degraded. Analysis of these samples
by slot-blotting (figure 10) demonstrated that moderately
degraded RNA as shown in lanes B, C, D and E, is
as sensitive to quantitative changes as intact total
cellular RNA (lane A). Extensively degraded RNA shown in
91


129
amplified and single copy c-myc genes in MFH cell lines.
Locations of c-myc DNAse I hypersensitve sites were
determined for P3C, UR HCL 1, HFF, and ST 486 cell lines.
Initially, mapping of these sites was done from a 3'
direction using previously described methods. Various
concentrations of DNAse I were used for each cell line to
determine an optimal range of concentrations (i.e. ones
which yielded optimum visualization of DNAse I generated
bands). Figure 19 shows Southern blot analysis of DNAs from
DNAse I treated nuclei from the UR HCL 1 cell line. This
figure demonstrates the extent of digestion with varying
concentrations of DNAse I.
Genomic DNA was isolated from DNAse I treated nuclei as
previously described. Fifteen ug of DNA from the UR HCL 1,
HFF, and ST 486 cell lines and 7 ug of DNA from P3C DNAse I
treated nuclei were restricted with ECO Rl, and compared for
locations of DNAse I hypersensitive sites by Southern blot
hybridization with the pmc 41 probe (figure 20).
The optimum DNAse I concentrations for the P3C,
UR HCL-1, and HFF lines were 0.2, 0.5, and 1.0 ug/ml DNAse I,
while those for ST 486 were 0.1, 0.2, and 0.5 ug/ml DNAse I.
The controls shown for each cell line were 0 ug/ml DNAse I at
37 C degrees (lane marked 0). Locations of DNAse I
hypersensitive sites were determined by comparison to lambda


168
69. Greenberg, M.E., and E.B. Ziff. 1984. Stimulation of
3T3 Cells Induces Transcription of the C-fos Proto-
Oncogene. Nature: 311: 433.
70. Gridoni, D., W.S. Dynan, and R. Tjian. 1984. Multiple
Specific Contacts Between a Mammalian Transcription
Factor and its Cognate Promoters. Nature 312: 409.
71. Groffen, J., N. Heistekamp, J.R. Stephenson, A.G.
VanKessel, A. Deklein, G. Grosveld, and D. Bootsma.
1983. C-sis is Translocated From Chromosome 22 to
Chromosome 9 in Chronic Myelocytic Leukemia. J.
Exp. Med. 18 : 9 .
72. Gross, D.S., and W.T. Garrard. 1988. Nuclease
Hypersensitive Sites in Chromatin. Ann. Rev. Biochem.
57: 159.
73. Grosso, L., and H. Pitot. 1985. Chromatin Structure
of the C-myc Gene in HL-60 Cells During Alterations of
Transcriptional Activity. Can. Res. 45: 5035.
74. Gruss, P., R. Dahr, and G. Khoury. 1981. Simian Virus
40 Tandem Repeated Sequences an Element of the Early
Promoter. Proc. Natl. Acad. Sci. U.S.A. 78: 943.
75. Gunning, P., P. Ponte, H. Okayama, J. Engel, H. Blau,
and L. Kedes. 1983. Isolation and Characterization of
Full Length cDNA Clones for Human alpha, Beta, and
Gamma Actin mRNAs. Skeletal but not Cytoplasmic Actins
Have an Amino-Terminal Cysteine that is Subsequently
Removed. Molec. and Cell. Biol. 3: 787.
76. Hadju, S. 1979. Pathology of Soft Tissue Tumors.
Philadelphia, Lea and Febiger, p. 39.
77. Hall, A. 1986. Oncogenes. Genetic Engineering 5: 61.
78. Haluska, F.G., K Huebner, and C. Croch. 1987. P380
-8A 1.8 SaSs, A Single Copy Clone 5' of C-myc at 8q24
Which Recognizes an Sst I Polymorphism. NAR 15: 865.


106
Table 11. C-sis, v-erb-B-1, and v-src: actin ratios from
slot-blot analyses of total cellular RNA from MFHs.
MFH SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
MT-1
1.25
0.113
0.283
MT-2
1.33
0.697
1.03
MT-3
1.65
1.27
0.616
MT-4
1.39
1.13
0.802
MT-5
1.30
1.38
1.94
MT-6
2.83
0.787
0.292
MT-7
1.54
0.680
1.70
MT-8
2.31
0.689
0.507
MT-9
1.14
1.35
0.992
MT-10
0.810
1.49
1.92
MT-11
2.29
1.02
2.92
MT-12
2.63
1.63
1.49
MT-13
2.19
1.50
1.71
MT-14
2.02
1.64
1.05
MT-15
1.56
1.17
0.790
MT-16
1.89
0.801
0.480
MT-17
34.0 **
0.832
2.32
MT-18
2.30
1.16
0.586
MT-19
1.87
0.558
1.04
MT-2 0
1.31
0.942
1.01
MT-21
1.29
1.09
1.01
MT-22
1.03
1.05
0.990
MT-2 3
1.43
1.05
1.01
**
Value was determined from titration of RNA, since signal
intensity was outside the linear range for this method.


64
\
A
I
HL-60 (DNASE I)
HL-60 (S 1)
BL-31 (DNASE I)
f = TRANSCRIPT
A B C D V = + HL-60
G
ABCD
ELONGATION BLOCK (BL)
+
B
I
V
TV
PO
CD E F
I I TT
P1 P2
G
C-MYC EXON 1 EXON 2
/
I 1
1 KB
Figure 4. Summary of chromatin structure analyses previously
described for the c-myc gene. Sites A, B, C, and D are DNAse
I hypersensitive sites found in both HL-60 cells (Siebenlist
et al (155)), and Burkitt lymphoma (BL-31) cells (Siebenlist
et al. (156)). Site B (indicated by open arrow) has been
described by Siebenlist et al. (155) to be involved in the
maintainance of c-myc transcript production in HL-60 cells,
and is therefore marked with a (+) symbol. Sites E and F
(solid arows) represent transcription attenuation sites found
in Burkitt lymphoma biopsies and cell lines and are marked by
a (-) symbol (25, 199). Site G is an S-l nuclease sensitive
site described by Grosso and Pitot (73) in HL-60 cells.


149
activation of proto-oncogenes to oncogenes. It is possible
that any of these; insertional mutagenesis, enhancer/promoter
activity, amplification, gene rearrangements or point
mutations could result in the loss of normal transcriptional
constraints. It is reasonable to expect that increases in
transcriptional levels would result from all of these
mechanisms; including point mutations, if in regulatory
rather than coding regions.
Single copy genes and insignificant differences in
transcript levels between normal and tumor tissues were
observed for c-Ha-ras, c-fos, v-erb-B-1, and v-src. Although
no known mechanism of proto-oncogene activation is apparent
(above), this does not exclude an involvement of these genes
through point mutations at critical sites or some unknown
activation mechanism. In the case of the c-myc gene in 6
MFHs, evidence presented here supports the concept of gene
amplification as a mechanism of activiation of a proto
oncogene to an oncogene. Furthermore, TK:actin ratios
indicate that myc gene amplification may be driving cell
division in these tumors.
Increased transcript levels of c-myc, together with
multiple copies of the gene, suggest that abnormal amounts of
transcript are due at least in part to gene dosage effects.


44
that their protein products are involved in the regulation of
cell division. Inappropriate expression of these nuclear
proteins could keep the cell cycling even under conditions
which would normally be sufficient to switch off further
growth.
Molecular. Biological, and Physiological Characteristics
of Proto-Oncogenes Examined in This Study
Growth Factor Related
Erb-B-1. As mentioned previously, the product of the
erb-B-1 oncogene is a truncated version of the receptor for
EGF (181) It is a glycoprotein with protein kinase activity
and has the capability to transform cells, while the normal
growth factor and receptor do not (17). The erb-B-1
oncogene protein product represents the EGF receptor short
of both its large extracellular domain which binds the
ligand and either 32 or 71 amino acids from its carboxy
terminus (181). The transforming protein is 71 amino acids
in length and includes a hydrophobic region which resides at
the cell surface, a hydrophobic domain that spans the plasma
membrane, and a shortened cytoplasmic domain which possesses
the protein-tyrosine kinase activity (197). This


153
HL-60 (DNASE I) ABCD
HL60 (S1) G
BL-31 (DNASE I) ABCD
Y = TRANSCRIPTION ELONGATION
V = + HL-60
BLOCK (BL)
Q
Z
X
\
A
I
I
+ -
B C D E F
} I i TT
PO P1 P2
II II
2 3 4 5
G
I 1
1KB
C-MYC EXON 1
EXON 2
HFF 1 2 3 4 6
UR HCL 1 1 2 3 4 6
P3C 13 4 5 6
Figure 27. Summary of chromatin structure analyses
previously described for the c-myc gene (letters) and those
reported here (numerals). Sites A, B, C, and D are DNAse I
hypersensitive sites found in both HL-60 cells (Siebenlist et
al (155)), and Burkitt lymphoma (BL-31) cells (Siebenlist et
al. (156)). Site B (indicated by open arrow) has been
described by Siebenlist et al (155) to be involved in the
maintainance of c-myc transcript production in HL-60 cells,
and is therefore marked with a (+) symbol. Sites E and F
(solid arows) represent transcription attenuation sites found
in Burkitt lymphoma biopsies and cell lines and are marked by
a (-) symbol (25, 199). Site G is an S-l nuclease sensitive
site described by Grosso and Pitot (73) in HL-60 cells.


83
DNA was recovered by centrifugation at 10,000 rpm at 4 C for
15 min. The pellet was resuspended in 300 ul 50mM Tris,
pH 8.0, 10 mM EDTA. RNAse A (lmg/ml) was added to a final
concentration of 50 ug/ml and incubated overnight at 37 C.
The mixture was then phenol/chloroform extracted, and the DNA
precipitated as described above. The final DNA pellet was
resuspended in lOmM Tris, ImM EDTA.
Aliquots of DNA from each of the cell lines were
restricted with either Eco R1 (mapping of DNAse I
hypersensitive sites from a 3' direction (pmc 41 probe)), or
Sea I (mapping of these sites from a 5' direction (Sea I/Xho
I fragment probe)), and analyzed using Southern blotting and
hybridization methods described above. The mapping of DNAse
I hypersensitive site locations was accomplished through the
use of the indirect end labeling technique. This technique
allows mapping of the DNAse I hypersensitive sites in one
direction, and is described in figure 7 using restriction
with Eco R1 and hybridization with pmc 41 as an example.
Chromatin Structure/ Fibroblast Cell Synchrony Experiment
HFF cells were grown to 70-80 percent confluency in
Dulbeccos MEM supplemented with 10 percent fetal bovine


159
3. Increased c-myc transcript levels and TK:actin RNA
ratios correlated with c-myc gene amplification in
vivo.
4. Chromatin structure studies indicate that regulation
of c-myc in normal fibroblasts is post-
transcriptional .
5. When c-myc is amplified in MFH cells, changes in
chromatin structure may represent a compensatory
response to increased transcript levels.
6. Transcriptional and translational changes suggest
that amplification of c-myc in P3C cells represents
activation of a proto-oncogene to an oncogene.
7. C-myc gene amplification may be driving cell
division in MFHs.
8. In vitro studies support the notion that c-myc
gene amplfication may provide a selective growth
advantage to MFH cells.


55
interactions (20). Cell surface stimulation results in an
increase in c-fos and c-jun products. The products of the
two genes along with several other related proteins form a
complex which associates with transcriptional control
elements containing AP-1 sites (20, 60, 138). This
potentially can then mediate long term responses which
regulate growth control and development (113).
With respect to tumor activity, there seems to be some
controversy in the literature with regard to what types of
neoplasms are associated with c-fos expression. It has been
reported that c-fos has not been found consistently in any
type of neoplasm (77). However, Slamon et al. have reported
that c-fos is expressed in all tumor types including
carcinomas, sarcomas, and hematopoietic malignancies (159).
Myc. The myc family of cellular proto-oncogenes
contains three well defined members, c-myc, N-myc, and L-myc.
The first defined and most thoroughly studied member of this
family, c-myc, was identified as the cellular homolog to the
transforming gene of avian transforming virus MC29 (17). The
two other well characterized myc family genes N-myc and L-myc
were isolated on the basis of their homology to c-myc and
their freguent amplification in certain classes of human
tumors. The N-myc gene was originally isolated from human


123
to MT-16, MT-17, MT-18, MT-20, MT-22, and MT-8 respectively.
Thus, five of the MFHs with increased transcript levels of c-
myc have between 8 and 11 copies of the c-myc gene, while a
sixth has 2 copies.
Regions Contained in the C-Mvc Amplicon
The Southern blots from the dilutional analysis of c-myc
copy number described above were rehybridized with the c-myc,
p 380-8A, H25-8A, and HT .96 probes to further examine the
copy number of the c-myc gene and surrounding regions in the
6 MFHs with c-myc amplification. This was done in order to
determine whether the entire c-myc gene was amplified, and to
determine if the promoter region was contained in the
amplicons. Copy numbers for the different areas of the c-myc
gene and surrounding areas were determined by laser
densitometry as described above for Southern blot dilutional
analysis of myc gene copy number.
Hybridization with the c-myc probe from Oncor which is
specific for all three myc exons, indicates that this region
is amplified approximately 10 times in mt-16, mt-17, mt-18,
mt-20, and mt-22, while mt-8 has 2 copies (table 19). The
same is true for the p GEM H MYC probe (discussed above)


17
important to the growth of malignant tumors, as rapidly
growing tumors often outgrow their blood supplies. It is
thought that malignant tumor cells may produce their own
growth factors, angiogenesis factors, and collagenases,
enableing them to compete with other cells for nutrients,
and eventually invade surrounding tissues.
Specific Questions Addressed During the Course of This
Proiect
Unrestrained cell growth is a common component of
neoplastic phenotypes. Proto-oncogenes are genes which have
been shown to be involved in regulation of cellular growth
and differentiation. They are found in all normal nucleated
animal cells. Their conversion to transforming genes or
oncogenes by one or more of several possible mechanisms may
allow the transformation of cells in vitro and generate
neoplasms in vivo. Exploration of how these potential
regulators of growth control interact with one another and
with other genomic components may enlighten our understanding
of how normal cellular replication or differentiation events
change with transformation.
It is possible that several proposed mechanisms of
proto-oncogene activation will lead to increased production


49
complex and contains 11 introns. The exact mechanism by
which RSV acquired genomic c-src information is unclear
(196). It has been proposed that during a round of
infection, a non-oncogenic RSV progenitor transduced genomic
DNA after viral integration and excision, and then the
introns were removed by processing. Also, it may have
somehow incorporated c-src messenger RNA (77).
The differences between c-src and v-src have been
addressed by making use of in vitro recombinants of viral
and cellular genes. Results have shown that some of the
amino acid changes in v-src are biologically important for
transformation. This was also demonstrated by the fact that
high levels of c-src expression alone did not transform
cells (130). It has also been shown that if v-src is
expressed in cells at levels comparable with those of c-src
in normal cells, then transformation is observed (92).
It may be that both qualitative and quantitative changes in
src expression are required for transformation.
Src gene protein (p60src) activity is present in
normal tissues where organ specific levels have been found.
Jacobs et al. (91) have reported that levels of pp60c-src
were highest in brain followed by kidney, lung, muscle, and
connective tissue. It was also determined that a 4-20 fold


40
implicated in cell proliferation. The increase in
intracellular calcium which occurs when cells are fertilized
or stimulated by growth factors may depend on the formation
of inositol triphosphate. This can act as a second
messenger to release intracellular stores of calcium. It has
been postulated, and some evidence exists that the activated
ras gene protein which binds but cannot hydrolyze GTP, can
initiate the formation of inositol triphosphate in an
uncontrolled fashion, independant of cellular growth factors
(12) .
In order to expand the current understanding of the
functions of ras proteins, it will be necessary to identify
which protein(s) they interact with in the cell. Attempts
which have been made to copreciptiate ras associated proteins
with anti-ras antibodies have been unsuccessful, indicating
either that associations are weak, or they depend on intact
membrane structure (77).
Growth Factors and Their Receptors
Certain oncogene products are known to be transforming
versions of a growth factor and several growth factor
receptors. The erb-B-1 oncogene is a truncated version of the


16
chromosome number (aneuploidy). It is thought that
aberrations such as point mutations, deletions, and
rearrangements are events associated with initiation
processes, whereas gross chromosomal changes occur as the
tumor progresses in malignancy (142). There are certain
chromosomal deletions, translocations and trisomies which are
characteristically associated with specific cancers. These
are called non-random chromosomal alterations. Changes in
ploidy are associated with many tumor types in advanced
stages, and are somewhat random in that no definitive pattern
of chromosome number is associated with a particular tumor
type. In more advanced cancers both random and non-random
chromosomal changes can be found. Continuous chromosomal
changes can bring about tumor heterogeneity and the selection
of more highly invasive and metastatic cancers. Thus, tumor
progression has been called a highly accelerated evolutionary
process.
Malignant tumors have several important in vivo
characteristics. At the cellular level, they have a greater
fraction of cells in S-phase, and are less differentiated
than their normal counterpart tissues. In order for tumor
cells to grow, divide, and metastasize, cell growth must
outnumber cell death. Therefore, angiogenesis factors are


53
which binds specifically to the SRE. The protein appears to
be necessary for c-fos response to serum stimulation,
however proof that the protein directly activates c-fos
remains to be obtained. SRE variants have been constructed,
each with altered protein binding capabilities. It was found
that the ability to stimulate transcription correllates with
avidity of protein binding (68,81).
The SRE is not the only region thought to be important
in c-fos gene regulation. C-fos is activated by several
different stimulatory agents. It is thought that these
stimulatory agents do not all act in the same manner to
affect c-fos expression. The current idea is that there are
multiple regulatory elements for the gene. Epidermal growth
factor and phorbol esters seem to work through the SRE, but
there is evidence that c-fos activation by PDGF may be
mediated through a different site 25 base pairs 5' of the SRE
(64, 177, 178).
Some c-fos stimulating agents use cyclic AMP as a
messenger. Mutations in the SRE do not seem to affect c-fos
activation by cyclic AMP (68, 177). Therefore, gene
activation by cyclic AMP uses other unidentified regulatory
seguences. Calcium ions which mediate c-fos during nerve
cell activation may use yet another site (64, 68, 177, 178).


72
Table 1. Summary of probes used in slot-blotting, Southern
blotting, northern blotting, and chromatin structure
analyses.
Probe
Name
Source*
Use**
Reference
Method of
Labeling+
Description
C-myc
1
1,4
1
N.T
9.0 kb Eco R 1/
Hind III human
genomic fragment
cloned into PBR
322
C-Ha-ras
1
1
137
N.T.
6.4 kb Bam HI
human genomic
fragment cloned
into PBR 322
C-fos
1
1
36
N.T.
6.4 kb Xho 1/
Neo I human
genomic fragment
cloned into PBR
322
C-sis
2
1
71
N.T.
1.0 kb human
genomic fragment
cloned into
pSP 6
V-erb-B-1
1
1
108
N.T
1.7 kb Pvu 11/
Sst I genomic
fragment from
avian erythro
blastosis virus
V-src
1
1
37
N.T.
800 bp Pvu II
genomic fragment
from avian
sarcoma virus
prague A strain


8
rabbits regressed after a period of time. The papillomas
could be made to reappear if the skin was stressed by
punching holes in it or treating it with irritants such as
turpentine or chloroform (142). These experiments led to
conclusions that tumor cells could exist in a latent or
dormant state, and tumor induction processes and subseguent
growth of the tumor involved different mechanisms. These
mechanisms are known as initiation and promotion (62).
Studies of events involved in the initiation and
promotion phases of carcinogenesis were greatly aided by
isolation and identification of initiating agents such as
urethane, and the purification of the components of croton
oil which had promoting activities. The promoting substances
were found to be diesters of the ditepene alcohol; phorbol
(84). Of these, 12-0-tetradecanoylphorbol-13-acetate (TPA)
is the most potent promoter (11).
Initiation of transformation in normal cells by a
carcinogenic agent involves a permanent, heritable change in
gene expression. This could occur by direct genotoxic or
mutational events, where the carcinogenic agent reacts with
DNA directly. It may also occur via indirect "epigenetic"
events which regulate gene expression without direct
interaction with DNA seguences. Many feel initiating events


178
171. Takeya, T., and H. Hanafusa. 1982. DNA Sequence of the
Viral and Cellular Src Gene of Chickens. Complete
Nucleotide Sequence of an ECO R I Fragment of Recovered
Avian Src Virus Which Codes For GP 37 and pp60 Src.
Virol. 44: 12.
172. Tatosyan, A.G., S.A. Galetzk, F.L. Kisseljova, A.A.
Asanova, I.B. Zborovskaya, D.D. Spitovsky, E.S.
Revasova, P. Martin, and F.L. Kisseljov. 1985.
Oncogene Expression in Solid Human Tumors. Int. J.
Cancer 35; 731.
173. Temin, H.M. 1976. The DNA Provirus Hypothesis: The
Establishment and Implications of RNA Directed DNA
Synthesis. Science 192: 1075.
174. Temin, H.M., and S. Mizutani. 1970. RNA Dependent DNA
Polymerase in Virions of Rous Sarcoma Virus. Nature
226: 1211.
175. Thompson, C., P.B. Challoner, P. Neiman, and M.
Groudine. 1985. Levels of C-myc Oncogene mRNA are
Invariant Throughout the Cell Cycle. Nature 314:
363 .
176. Towbin, H., T. Stachelin, and T. Gordan. 1979.
Electrophoretic Transfer of Protein From
Polyacrylamide Gels to Nitrocellulose Sheets:
Procedure and Some Applications. Proc. Natl. Acad.
Sci. U.S.A. 9: 4350.
177. Treisman, R. 1986. Identification of a Protein-
Binding Site That Mediates Transcriptional Response
of the C-fos Gene to Serum Factors. Cell: .4: 567.
178. Treisman, R. 1986. Transient Accumulation of C-fos
RNA Following Serum Stimulation Requires a Conserved
5' Element and C-fos 3' Sequences. Cell 3: 889.
179. Twardzik, D.R., J.E. Ranchalis, and G.J. Todaro. 1982.
Mouse Embryonic Transforming Growth Factors Related to
Those Isolated From Tumor Cells. Can. Res. 42: 590.
Uhthoff, H., ed. 1983. Current Concepts of Diagnosis
and Treatment of Bone and Soft Tissue Tumors New
York, Springer Verlag Publishers.
180.


157
indicate that in normal cells, post-transcriptional
regulation is adequate to control c-myc transcript levels.
When the c-myc gene is amplified in MFHs, which are
tumors of fibroblast origin, the normal post-transcriptional
regulatory mechanisms may not be sufficient to compensate for
the abundance of c-myc transcript produced. Changes in
chromatin structure suggest that regulatory changes take
place at the level of the gene as well. These changes in
chromatin structure are indicative of a cellular adjustment
to an abundance of myc expression. It is therefore concluded
that the differences in regulation between amplified and
single copy c-myc in MFHs may represent a compensatory
response to gene dosage effects.
Western blot analysis showed that P3C cells have
increased amounts of c-myc protein compared to UR HCL 1 cells
or normal HFF fibroblasts 1 hour after serum release from GO.
This suggests that c-myc gene amplification and increased
transcript levels have an impact on the P3C cells through
increased amounts of protein. These data further indicate
that despite any attempt by the P3C cells to compensate for
increased c-myc transcript, relatively high levels of c-myc
protein are produced. This is further evidence that c-myc
may be an oncogene in these cells.


94
lane F was unacceptable for analysis because it could not be
evaluated for gene expression as quantitatively as total
cellular RNA. Also, the hybridization signal intensity of
the extensively degraded RNA was just below the linear range
for this system which was determined to be 1.10 X 10 E5-2.2 X
10E 6 square um/ug RNA (166). No patient samples
showing this level of degradation were used. All RNA samples
used in this study conformed to the criteria for moderate
degredation after visualization by formaldehyde gel
electrophoresis (figure 11).
RNA from 20 chondrosarcomas, 23 malignant fibrous
histiocytomas, 9 normal muscle (normal non-proliferating
mesenchymal tissue), and 6 bone marrow (normally
proliferating mesenchymal tissue) tissues were screened for
proto-oncogene transcript levels and gene copy numbers of c-
myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-src. These
genes were studied because of previous associations with
sarcomas in humans and other animals. The v-erb-B-1 and v-src
gene probes were used because c-erb-B-1 and c-src were not
available.
Hybridization signal intensities from these blots were
normalized to those of beta-actin and thymidine kinase (TK).
Beta-actin is a constitutively expressed non-cell-cycle


98
Table 3. C-sis, v-erb-B-1, and v-src:actin ratios from
slot-blot analyses of total cellular RNA from normal muscle
and bone marrow tissues.
SAMPLE
c-sis v-erb-B-1 v-src
actin actin actin
MFH patients
MM-1
0.970
0.770
0.926
MM-2
0.989
0.882
1.02
MM-3
0.963
0.842
0.973
MM-4
0.904
0.835
1.00
MM5
0.911
1.02
1.07
CHONDROSARCOMA
patients
MC-1
0.933
0.897
0.949
MC-2
0.947
0.765
0.763
MC-3
0.897
0.981
1.06
MC-4
1.14
0.949
0.893
Bone marrows
BM-1
1.15
1.28
1.18
BM-2
1.10
1.18
0.989
BM-3
1.09
0.863
0.876
BM-4
0.925
1.10
1.26
BM-5
1.16
1.22
0.998
BM-6
1.02
1.27
1.07


81
an MFH from a patient treated at Shands Hospital, University
of Florida.
ST 486. The ST 486 cell line is a Burkitt lymphoma
cell line obtained from ATCC. This cell line was used as a
positive control for chromatin structure analyses, as DNAse
I hypersensitive patterns for Burkitt lymphoma c-myc have
been described (156).
HFF. The HFF normal human fibroblast cell line was
obtained from Dr. Kenneth Rand, Department of Pathology,
University of Florida.
Preparation of Nuclei
Cells were grown in Dulbeccos MEM (minimal essential
medium) (Gibco, Gaithersburg, MD), supplemented with 10
percent fetal bovine serum (Gibco, Gaithersburg, MD).
Nuclei were isolated from dividing cells (approximately 2.0 X
10 E8). The cells were washed in 100 ml IX phosphate
buffered saline (PBS), and centrifuged at 2,000 rpm, for 3
min at 4 C. The pellet was resuspended in 10 ml IX
RSB (lOmM Tris, pH 7.4, lOmM NaCl, 3mM Mg C12), 0.5 percent


33
(122) but their detailed structure and mechanism of
activiation remain to be elucidated.
The fifth mechanism centers around structural
alterations in the proto-oncogene and protein product. This
mechanism is well documented in the case of the oncogenic
proteins encoded by the ras genes. It was discovered that in
the case of the oncogene in the T24/EJ human bladder
carcinoma cell line, a point mutation at position 12
converted the c-Ha-ras proto-oncogene to an oncogene. This G
to T transversion causes glycine which is normally the 12th
residue of the encoded 21,000 dalton protein to be replaced
by a valine (170). Another activated version of this gene
encodes an aspartate residue at this position (144).
Studies done with genes of the Ki-ras group also showed
that when the 12th residue was altered in this manner,
oncogenic activation of the c-Ki-ras gene was observed (24).
A slight variation of these results was obtained through the
study of a human lung carcinoma c-Ha-ras oncogene found to
have a mutation at amino acid 61 of the p21 protein (198).
These changes do not seem to affect the levels of expression
of these genes, only the activities of encoded proteins. It
is therefore suggested that the codons specifying residues
12 and 61 represent critical sites which, when mutated, will


148
would be expressed because bone marrow is a normally dividing
tissue; this is what was observed. Detectable c-fos
transcript levels have also been observed in normal bone
marrow by Evinger-Hodges et al. (58).
The slot-blotting technique has advantages for an
analysis of this nature. Due to the moderately degraded
conditions of most RNA samples isolated from surgically
obtained tumor and normal tissue specimens (as described in
materials and methods), slot-blotting provides a workable
alternative to northern blotting which is not possible with
degraded RNA.
The most reliable information provided by the slot-blot
assay is a relative comparison of proto-oncogene transcript
levels between samples. This was accomplished by maintaining
constant pre-hybridization and hybridization conditions, as
well as film exposure times.
The slot-blot results presented here do not rule out a
tumorigenic involvement of proto-oncogenes which do not have
abnormal transcript levels or copy numbers. They do
however, offer some clues as to which potential proto
oncogene activation mechanisms may be at work in the cases of
the c-myc and c-sis genes in MFHs.
Several possible mechanisms have been described for the


HIND III
133
C-MYC EXON 1
EXON 2
I
PO- P1 P2
I II II
I
6
I 1
1 KB
HFF 1 2 3 4 6
UR HCL 1 1 2 3 4 6
P3C 1 3 4 5 6
y = TRANSCRIPTION ELONGATION BLOCK (BL)
Figure 21. Locations of DNAse I hypersensitive sites in the
c-myc gene for each of the cell lines HFF, P3C, and UR HCL-1.
Also shown are promoter regions PO, PI, and P2.


162
18. Blair, D.G., M. Oskarsson, T.G. Wook, W.L. McClements,
P.J. Fischinger, and G.G. Vande Woude. 1981.
Activation of the Transforming Potential of a Normal
Cell Sequence: A Molecular Model For Oncogenesis.
Science 212: 941.
19. Blanchard, J.M., M. Piechaczyk, C. Dani, J. Chambard,
A. Franchi, J. Pouyssegur, and P. Jeanteur. 1985.
C-myc Gene is Transcribed At High Rate In GO Arrested
Fibroblasts and is Post-Transcriptionally Regulated
in Response to Growth Factors. Nature 317: 443.
20. Bohmann, P., T. Bos, A. Admon, T. Nishimura, P. Vogt,
and R.J. Tjian. 1988. Human Proto-Oncogene C-jun
Encodes a DNA Binding Protein With Structural and
Functional Properties of Transcription Factor AP-1.
Science 238: 1386
21. Bradshaw, H.K., and P.L. Deininger. 1984. Human
Thymidine Kinase Gene: Molecular Cloning and
Nucleotide Sequence of a cDNA Expressible in Mammalian
Cells. Molec. Cell. Biol. 4: 2316.
22. Bravo, R., J. Burckhardt, T.Curran, and R. Muller.
1986. Expression of C-fos in NIH 3T3 Cells is Very
Low But Inducible Throughout the Cell Cycle. 1986.
EMBO J. 5: 695.
23. Brooks, J. 1986. The Significance of Double
Phenotypic Patterns and Markers in Human Sarcomas. A
New Model of Mesenchymal Differentiation. Am.J.
Path. 125: 113.
24. Capon, D.J., P.H. Seeburg, J.P. McGrath, H.S.
Hayflick, V. Edman, A.D. Levinson, and D.V. Goedell.
1983. Activation of Ki-ras 2 Gene in Human Colon and
Lung Carcinomas by Two Different Point Mutations.
1983. Nature 304: 507.
25. Cesarman, E., R. Dalla-Favera, D. Bentley, and M.
Groudine. 1988. Mutations in the First Exon Are
Associated With Altered Transcription of C-myc in
Burkitt Lymphoma. 1988. Science 238: 1272.


99
Table 4. C-myc, c-Ha-ras, and c-fos:actin ratios from slot-
blot analyses of genomic DNA from normal muscle and bone
marrow tissues. Also shown are TK:actin ratios.
SAMPLE
c-mvc c-Ha-ras c-fos TK
actin actin actin actin
MFH patients
MM-1
1.06
1.01
0.964
0.810
MM-2
1.03
1.01
0.898
0.951
MM-3
1.27
1.03
0.876
0.920
MM-4
0.948
0.958
0.979
0.944
MM-5
0.964
0.964
0.918
0.996
CHONDROSARCOMA
patients
MC-1
1.21
1.17
0.952
0.955
MC-2
1.02
1.06
0.842
0.747
MC-3
1.22
1.05
0.868
1.10
MC-4
1.03
1.21
0.952
0.924
Bone marrows
BM-1
0.746
0.918
0.858
0.903
BM-2
1.16
0.990
1.21
1.21
BM-3
0.853
1.08
0.975
1.02
BM-4
0.743
0.792
0.743
0.847
BM-5
1.09
0.903
1.13
1.27
BM-6
0.786
0.731
0.786
0.897


136
8 10
o M W -*
C-MYC
2.4 KB
TK
1.0 KB
ACT1N
2.0 KB
Figure 22. Northern blot analysis of total cellular RNA from
the HFF normal human fibroblast cell line. HFF cells were
made quiescent (GO) in MEM containing 0.1 percent fetal
bovine serum (37 C degrees, 3 days), then released by
addition of MEM supplemented with 10 percent fetal bovine
serum. RNA samples were evaluated for levels of c-myc, TK
and actin transcript at GO, 0.5, 1, 2, and 3 hours after
serum release, and during log phase growth (L) (see materials
and methods).


51
membrane and converts them to lasting responses such as cell
division and possibly memory formation (113).
The c-fos gene was recognized as a cell cycle dependent
gene early after studies with it began. C-fos can be rapidly
activated by the treatment of quiescent cells with PDGF, EGF,
nerve cell growth factor, and serum containing growth factors
(69). This led to speculation that c-fos had something to
do with cellular growth control.
Studies with nerve cells revealed additional information
about the activities of the c-fos gene. It was found that
c-fos expression is controlled by factors which differentiate
and trigger nerve cell activity. In vitro experiments have
indicated that c-fos induction depends on the ability of
neuroactive agents to open calcium channels (119). Calcium
entry is a normal component of neuronal responses to
stimulation. It was found that a dramatic increase in c-fos
gene activity occurs in the brains of mice treated with
metrazole, a drug which causes epilepsy like seizures (119).
The synthesis of c-fos proteins was found to occur primarily
in the nerve tracts stimulated by metrazole. The results
suggested that the c-fos protein mediates the long term
adaptation of nerve cells to metrazole stimulation.


141
and Sea 1/ Bsm I (920 bases) which differ by 50 base pairs
and are 0.5 cm apart on the blot. Lambda DNA digested with
Eco R I/Hind III was run on either side of the gel as size
markers, and to assure the gel ran evenly. Sizes of these
marker bands are also shown in figure 24. Two samples of
DNAse I treated DNAs from P3C cells (0.2 and 0.5 ug/ml, A
and B respectively) were run because both produced optimal
visualization of DNAse I generated bands, as did sample 0.5
ug/ml for HFF cells. Bands corresponding to sites 4 and 6
(figure 25) were observed in both the P3C and HFF cell lines,
and map to distances of 760 and 2025 base pairs 3' of the
Sea I site respectively. Site 5, which is present
exclusively in the P3C cell line, maps to a distance of 960
base pairs downstream of the Sea I site. As a result of
these analyses, site 4 can be placed approximately 45 base
pairs 5' of the Pvu II site in one region known to be a
transcription attenuation site. Site 5 can be placed in a
region 3' of exon 1, approximately 10 base pairs 5' of an Mae
III site. This site is located in a region also known to
contain a transcription elongation block, which extends 15
base pairs 5' and 5 base pairs 3' of the Mae III site. Site
6 was found to be approximately 465 bases 5' of exon 2 in the
same region as an SI nuclease sensitive site described by
Grosso and Pitot (73).


9
have a direct impact on DNA itself. According to Ruddon
(142), this theory depends on three kinds of evidence.
1. Agents which damage DNA are freguently carcinogenic.
It has been shown that chemical carcinogens are usually
activated to generate electrophilic agents. These form
specific reaction products with DNA. In some cases, as with
alkyl 0-6-guanine, the extent of product formation has been
shown to correlate with mutagenicity and carcinogenicity of
the agent (142).
2. Most carcinogenic agents are mutagens. A number of
in vitro test systems using mutational events in
microorganisms have been developed to rapidly screen the
mutagenic potential of various chemical agents. One of the
best known is the Ames test. Ames and his collegues have
shown that about 90 percent of all carcinogens are also
mutagenic (114). Very few noncarcinogens showed significant
mutagenicity in this test system.
3. The incidence of cancer in patients with DNA-repair
deficiencies is increased. In individuals with certain
recessively inherited disorders, the prevalence of cancer is
significantly higher than in the general population (103,
153). The common characteristic shared between these
disorders is the inability to repair some kinds of physical


126
from these cell lines were prepared as previously described,
and analyzed using the pGEM H MYC probe and conditions
established for Southern hybridizations. Gene copy number
was determined by laser densitometry and normalized to muscle
DNA. The P3C cell line was found to contain approximately 10
copies of the c-myc gene, while the UR HCL 1 cell line has a
single copy c-myc gene (figure 17).
Five and 10 ug aliquots of total cellular RNA from the
P3C, UR HCL 1, HFF, and ST 486 cell lines were evaluated for
relative c-myc transcript levels using northern blot
hybridization methods and the p GEM H MYC probe. The blot
was rehybridized with the human beta actin gene probe as a
control for RNA quantitation. P3C cells clearly showed
increased levels of c-myc transcript relative to the other
cell lines (figure 18).
Locations of DNAse I Sites in P3C. UR HCL 1, HFF, and St 486
Cell Lines
Data generated thusfar support the hypothesis that
increases in c-myc transcript are due to gene amplification.
The possibility that other regulatory changes may contribute
to this as well cannot be ruled out. Therefore, potential
differences in chromatin structure were evaluated between


73
Table 1. Continued.
Probe Source*
Name
Use**
Reference
Method of Description
Labelingt
Beta-
actin
1
1
28
N.T.
800 bp Nco 1/
Taq I genomic
fragment from
chicken beta-
actin gene
TK
(pTK 11)
3
1,2
21
N.T/
P.E.
1.25 kb Sma I
Bam H I human
genomic fragment
pGEM-H MYC
4
2,3,4
188
N.T
1020 bp Pst I
human cDNA
fragment cloned
into pGEM 1
p380-8A
5
4
78
N.T.
1.8 kb Sal I/SstI
human genomic
fragment cloned
into puc 19
Carbonic
Anhydrase
(H 25-3.8)
6
4
183
N.T.
3.8 kb Eco R I
human genomic
fragment cloned
into PBR 325
Thyro-
globulin
(HT .96)
6
4
3
N.T.
960 bp Pst I
human cDNA cloned
into puc 8


87
below) were chosen to generate fragments used as internal gel
markers to map DNAse I hypersensitive sites in this region.
First, P3C genomic DNA was restricted to completion with
Sea I (Boerhinger Mannheim). Five ug aliquots of this DNA
were then restricted a second time with either Pvu II (BRL,
Gaithersburg, MD), Bsm I (New England Biolabs), or Mae III
(Boehinger Mannheim).
Bsm I cut a single time within the c-myc gene, therefore
the restriction reaction was allowed to go to completion
overnight at 65 C. Pvu II and Mae III cut at multiple
sites within the c-myc gene. Reactions were therefore
controlled to prevent complete digestion of the DNA.
Partial digestion of DNA with an enzyme which cuts at
multiple sites between a desired site and Sea I will yield
band sizes corresponding to distances between the desired
site and the Sea I site.
Five ug aliquots of Sea I restricted P3C DNA were
restricted with various concentrations of Pvu II (37 C) and
Mae III (55 C) for 30 min reaction times. Concentrations
which yielded optimum visualization of the desired marker
band sizes were used to restrict P3C DNAs for use as markers
in mapping analyses.


61
and purification of the genomic DNA. Its most useful feature
is that it allows mapping of DNAse I sensitive sites in a
single direction (72). Regions of DNAse I sensitivity are
usually the size of a nucleosomal repeat which is
approximately 150-100 base pairs (72) This can make precise
mapping difficult, but resolution can be improved by fine
mapping technigues.
DNAse I hypersensitive sites have been associated with a
wide variety of functions (72). In Saccharomvces cerevisiae.
hypersensitive sites have been seen near centromeres,
silencers, recombination sites, origins of replication,
activation sequences, promoters, and potential sites of
transcription termination (72). Therefore, these sites are
probably associated with cis acting factors (72).
Topoisomerases I and II, RNA polymerase II, and some
transcription factors have been associated with DNAse I
hypersensitive sites (72). The proteins associated with most
sites in genes which have been studied have yet to be
identified.
The mechanisms which are involved in the formation and
maintainance of DNAse I hypersensitive sites are not clear.
Because the functions of these sites are so diverse, several
mechanisms are likely to be involved (72). It is thought


96
dependent gene (75, 134), and was used to normalize
hybridization signal intensities for c-sis, v-erb-B-1, and v-
src, which also are non-cell-cycle-dependent. Hybridization
signal intensities of cell-cycle dependent genes c-myc, c-Ha-
ras, and c-fos were normalized to TK, also a cell-cycle-
dependent gene (93) Ratios of TK/actin were taken as a
molecular measurement of cell division.
The extent of hybridization for slot-blots was estimated
by densitometric scanning of the x-ray film (Bio-rad video
densitometer). Results were reported as the logarithm of the
peak areas for 10, 5, and 2.5 ug for RNA, and 20, 10, and 5
ug for DNA. The mean values for the 3 aliquots of each RNA
and DNA sample were calculated and used to determine a
gene:actin and gene:TK ratio (tables 2-13). All DNA values
are normalized to actin as are RNA values for c-sis, v-erb-
1, and v-src. C-myc, c-Ha-ras, and c-fos RNA levels are
normalized to TK. Significant differences between any two
groups were determined with Students T test.
RNA Slot-Blot Results
Thymidine kinase, c-myc, c-Ha-ras, and c-fos are
undetectable in muscle using this method of detection


71
Preparation of Radiolabeled Probes
Descriptions, sources, and methods of labeling for all
probes used in slot-blot, Southern blot, northern blot, and
chromatin structure analysis are summarized in Table 1.
Figure 5 shows locations of the different c-myc probes as
well as other probes located on chromosome 8 used in mapping
and dilutional analysis of c-myc amplicons in MFHs.
Restriction maps of all other probes in Table 1 are shown in
figure 6.
Nick Translated Probes
Probes were nick translated by adding 250 ng of DNA
to a reaction mixture which contained 80 uCi 32P (dATP), 5.0
ul of 10X (dCTP, dGTP, dTTP), 1.25 ul of lmg/ml bovine serum
albumin (BSA), 5 ul of nick translation buffer (0.5 M
Tris HCL, pH 7.8, 0.1 M 2-mercaptoethanol, and 0.05 M MgC12),
1.5 ul DNAse I/Polymerase I (BRL, Gaithersburg, MD), and
deionized H20 to a final volume of 25 ul. The reaction was
run at 15 C for 45 min. Labeled DNA was separated from
unincorporated nucleotides using a Biogel A-15m (Biorad,
Rockville Center, NY) column.


6
problem with the provirus hypothesis was that there was no
known way for the tumor virus RNA to be converted into DNA
and integrated. Temin and Baltimore independently
demonstrated the presence of a virus coded, RNA directed, DNA
polymerase activity now known as reverse transcriptase (4,
174). As a result of this work, Temin proposed the
"protovirus" theory in which he postulated that genomes of
oncogenic viruses arose during evolution from normal
cellular DNA altered by some exogeneous carcinogen (173).
The normal cellular homologues of viral oncogenes (v-
onc) are known as proto-oncogenes (c-onc). These are thought
to have been evolutionarily conserved in the genomes of most
animal cells over a long period of time. They seem to be
involved in control of cellular growth and proliferation. It
is likely that their activations to oncogenic states occur
from one or more rare events such as translocations,
amplifications, point mutations or other aberrations of key
nucleotide sequences (102). Highly oncogenic viruses
presumably arose from genetic recombination events between
viruses of low oncogenicity and proto-oncogenes. The
combination of these two elements seems to have produced
highly transforming viral genomes. Many of these viruses are
replication defective, and do not form complete viruses


95
z z s z z z
11* 11 11
u A to ^ -* -*>
Figure 11. RNA formaldehyde gel electrophoresis of total
cellular RNA from 6 MFHs. These samples illustrate typical
moderate degredation seen with RNA samples extracted from
tumor and normal tissues.


128
510510510510
<4KB > PP* *
ACTIN
2.1KB
Figure 18. Northern blot analysis of total cellular RNA
from UR HCL 1, P3C, HFF, and ST 486 cell lines. Ten and 5 ug
aliquots of RNA from each cell line were electrophoresed
through 1.2 percent formaldehyde agarose gels, blotted, and
hybridized with the pGEM H MYC and beta actin probes (See
materials and methods). Message sizes were determined by
an RNA ladder.


24
Chondrosarcoma is primarily a tumor of adulthood (39).
The incidence of bone tumors in general is highest
during adolescence with a rate of 3 per 100,000 (61). The
incidence falls to 0.2 per 100,000 at ages 30-35 and rises
slowly thereafter to an incidence rate equal to that of
adolescence (30, 39). Chondrosarcoma is the third most
common type of bone tumor and makes up approximately 13
percent of all malignant bone tumors (85). More than 75
percent of chondrosarcomas occur in the trunk and the upper
ends of the femora and humeri. It is much less common for
these tumors to be located in the distal extremeties such as
the elbows and ankles (39).
Many chondrosarcomas are palpable, but many of those
affecting the trunk or long bones of the extremeties which
have not broken the cortex may cause pain alone to indicate
the presence of the lesion. Roentgenograms provide a very
helpful means for diagnosis. Osseous destruction in the area
of the lesion combined with irregular densities from calci
fication and ossification are commonly observed. Central
chondrosarcomas of long bones commonly produce fusiform
expansion of the shaft associated with thickening of the
cortex (39).


39
located at the inner surface of the plasma membrane and
although the viral proteins are phosphorylated at amino acid
residue 59 which is a threonine, the human p21s do not have a
threonine at 59, nor are they phosphorylated. The ras genes,
which are cell cycle dependent (94), are activated by point
mutations and therefore the modes of action of normal and
transforming p21s are of interest.
Both transforming and normal cellular p21s bind GTP and
GDP egually and have a GTPase activity (116). However, the
transforming version of p21 hydrolyzes GTP about 10 times
more slowly than the normal proteins (77) The normal ras
roteins are thought to interact with a receptor in response
to an external signal, bind GTP and interact with an as yet
unknown molecule to generate a second messenger (77).
Adenylate cyclase is unlikely to be directly involved
because the G proteins associated with it have different
molecular weights from ras p21 (77). Since transforming p21
has reduced GTPase activity, this could result in abnormally
high levels of the second messenger.
Ras encoded proteins are also regulators of inositol
triphosphate. Some of the proteins involved in the inositol
lipid breakdown pathway are GTP binding proteins and it is
possible ras may be one of these. Calcium has long been


140
2027 >
1904 >
1584 >
1375 >
947 >
831 >
564 i*
< 2027
< 1904
< 1584
< 1375
< 947
< 831
< 564
Figure 24. Southern blot of P3C and HFF DNAse I treated DNAs
used in fine mapping of DNAse I hypersensitive sites in the
exon 1/ intron 1 region. Also shown are P3C genomic DNA
marker fragments; Scal/Bsm I, Sea 1/ Mae III, and Scal/Pvu
II. P3C A and P3C B were treated with 0.2 and 0.5 ug/ml
DNAse I respectively (from figure 20). The HFF sample (from
figure 20) was treated with 0.5 ug/ml DNAse I. The blot was
hybridized with the 355 b.p. Sea 1/ Xho I fragment (2.0 X 10
E7 cpm/ml/ 10 E8 cpm/ug) (see materials and methods). Also
shown are lambda DNA markers produced by digestion with Eco R
I and Hind III.


11
Tumor promotion is itself a multistage process
sometimes labeled collectively as "tumor progression". Tumor
promotion is thought to be a stage of cell proliferation and
clonal expansion induced by mitogenic stimuli. The
progression phase is the evolution of genotypically and
phenotypically altered cells resulting from genetic
instability (128). During tumor progression which can take
years in humans, individual tumors develop heterogeneity with
respect to their invasive and metastatic characteristics,
antigenic specificity, state of differentiation, and response
to drugs and hormones (128). It is thought that some major
selection process occurs to favor the growth of one cell
over another, thus a dominant clonal population of cells may
emerge. This may be a result of competition for nutrients,
ability to evade the immune system, and resistance to
chemotherapeutic drugs.
The concepts of initiation and promotion support the
notion that cancer is not a "one-hit" event. Evidence
obtained from studies done with oncogenes and antioncogenes
further supports this concept. Weinberg (102) showed that
when rat endothelial fibroblasts were transfected with the
c-Ha-ras and c-myc oncogenes alone, no transforming effect
was observed. However, when c-myc and c-Ha-ras were


68
a solution containing 4M guanidine isothiocyanate (Ultra-
pure, BRL, Gaithersburg, MD), 25mM sodium citrate, pH 7.0,
and 0.1 M 2-mercaptoethanol. The mixture was then
homogenized using a tissuemizer (Brinkman Instruments), and
0.75 ml of 1 M acetic acid was added. The suspensions were
layered into SW 50 ultra-centrifuge tubes (Beckman
instruments) containing a 1.5 ml pad of 5.7 M cesium
chloride. The samples were centrifuged for 16 hr at 20 C and
35,000 rpm.
Following centrifugation, the pellets were resuspended
in 1.0 ml DEPC treated, sterile H20, and extracted once with
25:25:1 phenol:chloroform: isoamyl alcohol. The RNA was
then precipitated by adding 100 ul of 4 M potassium acetate,
and 2.5 ul ethanol. Recovery of the RNA was accomplished by
centrifugation at 10,000 rpm for 20 min at 4 C. The pellets
were resuspended in 0.2 mM EDTA, visualized on formaldehyde
agarose gels (as described for northern blotting below),
quantitated by absorbance at 280 nm, and stored in aliquots
at -70 C.
Preparation of Genomic DNA


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E7XHF6Y97_0LSTD3 INGEST_TIME 2014-10-07T01:13:16Z PACKAGE AA00025744_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


131
o
o
0>
ug/ml DNAse I
URtiCL 1
o in n
111
o H O o
HFF
o in n
O '-i o o
ST486
Irt CM H
I I I
O O O O
e
!c
<

44 *
Figure 20. Southern blot of P3C, UR HCL 1, HFF, and ST 486
genomic DNA from DNAse I treated nuclei. The concentrations
of DNAse I shown for each cell line were the ones which gave
optimal visualization of bands in the initial analyses. The
genomic DNA from DNAse I treated nuclei was restricted with
Eco R I, electrophoresed through 0.8 percent agarose gels,
blotted, and hybridized with the pmc 41 probe. Controls
shown for each cell line were 0 ug/ml DNAse at 37 C degrees.


156
potential changes in chromatin structure during c-myc
upregulation in normal cells were studied. It has been
shown that c-myc transcript levels peak during the G0/G1
transition when serum starved fibroblasts are released from
their quiescent states (175). Although transcript levels
peaked 1 hr after serum addition in HFF cells as shown by
northern blot analysis, no differences in DNAse I sensitive
sites were observed between fibroblasts in GO, those in log
phase, and after maximal physiologic stimulation (G0/G1). The
DNAse I sites found at each of the time points (GO, 0.5, 1,
2, 3 hours after serum release and during log phase growth)
are identical to those described previously for HFF (sites
1,2,3,4, and 6, figure 27). These data are consistent with
those reported by Blanchard et al. (19) which suggest that
cellular levels of c-myc transcript are primarily regulated
by post-transcriptional mechanisms at the level of message
degredation in normal cells.
C-myc chromatin structure analyses reported here, and
those previously reported for lymphocytes provide important
data as to the nature of regulatory interactions taking place
with amplified c-myc in MFHs. First, chromatin structure
data from quiescent and serum released HFF fibroblasts


7
unless coinfected with a "helper" virus. Recombination
between replication-competent helper viruses and cellular
genes also may have produced highly oncogenic virus strains.
The "oncogene" hypothesis of Huebner and Todaro (88)
postulates that the cells of most or all vertebrates contain
"virogenes". These genes include sequences responsible for
transformation and are transmitted vertically form parent to
offspring. In this hypothesis, the occurrence of cancer may
be determined by the derepression of endogenous viral
oncogenes. Activation of repressed genes could result from
exposure of cells to chemical carcinogens, irradiation,
normal aging, or a combination thereof. This theory provides
an explanation for the known vertical transmission of certain
animal viruses. It also explains the observed necessity of
synergistic interactions between chemical carcinogens and
irradiation for transformation by some oncogenic viruses.
Multistep Carcinogenesis
The idea that development of cancer is a multistage
process arose from early studies of virus induced tumors, and
from discovery of cocarcinogenic effects of croton oil.
Rous discovered that certain virus induced skin papillomas in


124
Table 19. Copy numbers of 5' and 3' regions of the c-myc
gene and flanking regions in mt-8, mt-16, mt-17, mt-18, mt-
20, and mt-22 as determined by Southern blot analyses of
genomic DNA digested with Hind III and hybridized with the c-
myc, p GEM H MYC, p 380-8A, H25-3.8, and HT 0.96 probes. *
Probe
C-myc
pGEM H MYC
p380-8A
H25-3.8
HT 0.96
Size of
hybridized
fragment (kb)
9.6
9.6
6.8
9.4
3.4
Tumor
MT-8
2
2
2
1
1
MT-16
9
9
9
1
1
MT-17
9
8
9
1
1
MT-18
10
10
10
1
1
MT-2 0
10
10
10
1
1
MT-22
10
11
11
1
1
MUSCLE
1
1
1
1
1
* Copy numbers were determined by laser densitometry. Values
were normalized to that of muscle which has a c-myc gene
copy number of 1.


HIND III
142
C-MYC EXON I
EXON 2
\
I 1
1 KB
PROBE
4 5 6
= TRANSCRIPT ELONGATION BLOCK
Figure 25. Locations of c-myc DNAse I hypersensitive sites
4, 5, and 6 for P3C and HFF cell lines as determined from
fine mapping analysis from the 5' direction.


151
tumors with a larger growth fraction would be expected to
have increased c-myc transcript levels regardless of the
factors driving cell division.
Southern blot analyses show that c-myc amplicons in MFHs
(which contain approximately 10 copies) are very large and
contain all three exons. Hybridization with the p380-8A
probe (specific for a region approximately 50 kb upstream
from the c-myc promoter) indicates that the c-myc promoter
region is amplified as well. Use of this probe in similar
analyses by Haluska and Croce (78) has shown this region to
be co-amplified with the c-myc gene in COLO 320 (colon
carcinoma cell line), but not with c-myc in HL-60 cells.
The measurements reported here for c-myc gene
amplification are in accordance with other studies which have
demonstrated c-myc gene amplification of large regions of DNA
in other tumor systems (2). The question of why c-myc
amplification occurs at all has been the focus of much
speculation. In the case of Wilm's tumor for example, high
levels of transcript have been observed from a single
gene (2). If c-myc is a nuclear regulatory protein found in
most normal cells then why would normal growth provide an
ever increasing pressure for selection of cells with elevated
levels of c-myc protein? The type of selective pressures


27
Figure 3.
Histiologic appearance
of MFH.


56
neuroblastomas, a pediatric tumor of embryonal origin that
arises in the peripheral nervous system.
The N-myc and c-myc genes have a very similar overall
structure, exhibit extensive homology in their coding
regions, and encode similar sized nuclear proteins (41, 99).
It has been confirmed that N-myc has transforming activity
equivalent to that of c-myc in the rat embryo fibroblast
assay (150). The N-myc gene has been found to be amplified
in all human neuroblastomas having cytogenetic
characteristics of gene amplification such as homogenously
staining regions or double minutes (147). Patterns of N-myc
amplification in neuroblastomas have been associated with
tumor progression. A greater copy number of the N-myc gene
is associated with a more advanced stage of the tumor.
(152). N-myc activation has thusfar been found to occur only
by amplification and only in a restricted set of tumors.
In addition to neuroblastomas, N-myc amplification has
been observed in a subset of small cell lung carcinomas
(SCLC) and in a few retinoblastomas (98, 106, 124). Like
neuroblastomas, these tumors have neural characteristics.
Considering the oncogenic potentials and similarities of
c-myc and N-myc, the reason for relatively restricted
activation of the N-myc gene as opposed to the c-myc gene is


normal cells. As a result, malignant cells in
culture may grow to a much greater density.
14
c.Transformed cells seem to have much lower require
ments for serum and/or growth factors to survive
in culture than normal cells do.
d. There also seems to be a loss of anchorage
dependence with transformed cells. They may
no longer need to grow attached to solid surfaces,
and can grow in soft agar.
e. It has been observed that when transformed cells in
culture are subjected to biochemical restrictions,
they do not stop growing. An example of this is a
lack of response to serum starvation.
3. In vitro transformed cells may also change their surface
properties. Changes of this nature include; alteration
in structure of surface glycolipids and glycoproteins,
loss of surface fibronectins, increased agglutination
by lectins, changes in surface antigens which may
be tumor specifc and involved in immune responses,
and increases in the degree of amino acid uptake.
4. Cultured malignant cells produce increased levels of the
enzymes involved in DNA synthesis. They also


125
which was specific for the 3' end of the gene. These probes
were specific for overlapping regions, and both were used for
confirmatory purposes.
The p 380-8A probe which is specific for a region
approximately 50 kb upstream from the c-myc promoter
indicates an amplification of approximately 10 fold in mt-16,
mt-17, mt-18, mt-20, and mt-22, while mt-8 has 2 copies of
this region (table 19). Analyses with the thyroglobulin and
carbonic anhydrase probes, show single copy genes (table 19).
These results indicate that the amplfied regions of myc (8q
24) in MFHs are very large, and contain all three exons as
well as regulatory regions. The amplicons do not extend as
far 5' as the carbonic anhydrase gene (8q 22), or as far 3'
as the q terminal region of chromosome 8, where the
thyroglobulin gene is located.
Chromatin Structure Analysis
C-Mvc Gene Copy Number and Transcript Levels in P3C.
UR HCL 1, HFF and ST 486 Cell Lines
C-myc gene copy numbers were determined for the P3C and
UR HCL 1 cell lines. Twenty, 10, 5, and 2 ug of genomic DNA


112
10
2.5
MT-1
MT-2
MT-3
MT4
MT-5
MT
MT7
MT8
MT9
MT10
MT11
MT12
MT13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 13. Slot-blot of total cellular RNA from MFHs
hybridized with the actin probe. Quantities of 10, 5, and
2.5 ug of RNA were slot-blotted onto nitro-plus 2000, and
hybridized with the actin probe (3.0 x 10 E6 cpm/ 10 E8
cpm/ug).


43
The products of five oncogenes; myc, myb, fos, ski (77),
and B-lym (65) are known to be located in the nucleus. The
expression of c-myc, c-fos, and c-myb appears to be dependent
on the proliferative state of the cell (2, 100, 120 175).
Quiescent 3T3 cells for example, have undetectable levels of
c-fos mRNA but within 30 minutes of stimulation by PDGF
(100), the levels are dramatically increased. This is only
transient, and after about 2 hours the high levels disappear
(120). Thus the interaction of PDGF with its receptor not
only facilitates activated intracellular phosphorylation
events and the breakdown of inositol lipids, but also leads
to the generation of a nuclear signal to switch on c-fos
expression. Since phosphorylation of intracellular proteins
occurs within a few minutes of mitogenic stimulation it is
likely that c-fos expression is a direct result of these
events. Like c-fos, the c-myc gene is expressed at very low
levels in quiescent cells, and its transcript levels
increase transiently after stimulation with PDGF, insulin,
and serum (19, 22, 67, 95, 126, 175).
The roles of c-fos and c-myc gene products will be
discussed in more detail below. It will be mentioned for
now that since c-myc and c-fos gene expression follows a
direct relationship to cell cycle, it is generally believed


I certify that I have read this study and that in ray
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Associate Professor of Pathology
and Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Warren E. Ross
Associate Professor of
Pharmacology and Therapeutics
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Hutt-Fletcher
Associate Professor of Pathology
and Laboratory Medicine
Linds e^M.


107
Table 12. C-myc, c-Ha-ras, and c-fos: actin ratios from
slot-blot analyses of genomic DNA from MFHs. Also shown
are TK:actin ratios.
MFH SAMPLE
c-mvc
c-Ha-ras
c-fos
TK
actin
actin
actin
actin
MT-1
0.833
1.26
0.991
0.800
MT-2
1.06
0.638
0.938
0.964
MT-3
1.05
0.956
0.815
1.05
MT-4
1.21
0.897
1.25
1.06
MT-5
1.18
1.31
1.07
0.919
MT-6
1.96
1.24
0.812
0.988
MT-7
1.17
1.06
1.04
0.891
MT-8
1.13
0.952
1.15
0.980
MT-9
1.26
0.916
1.32
1.27
MT-10
1.08
1.39
0.732
1.07
MT-11
1.14
0.834
0.908
1.01
MT-12
1.39
1.27
1.22
1.24
MT-13
1.19
1.15
0.998
1.14
MT-14
1.11
1.09
1.19
1.28
MT-15
1.21
1.27
1.09
1.18
MT-16
1.81
1.21
1.30
1.16
MT-17
2.25
1.20
1.26
1.21
MT-18
2.41
1.03
1.17
1.19
MT-19
1.01
1.11
1.27
1.20
MT-2 0
2.72
1.19
1.02
1.09
MT-21
1.25
1.28
1.20
1.19
MT-2 2
2.69
1.33
1.13
1.14
MT-2 3
1.45
1.31
1.29
0.960


164
35. Courtneidge, S.A., A.D. Levinson, and J.M. Bishop.
1980. The Protein Encoded by the Transforming Gene
of Avian Sarcoma Viurs and a Homologous Protein in
Normal Cells (pp 60 C-src) Are Associated With the
Plasma Membrane. Proc. Natl. Acad. Sci. U.S.A. 77:
3783 .
36. Curran, T., W.P. MacConnell, F. VanStraaten, and I.M.
Verma. 1983. Structure of the FBJ Murine Osteosarcoma
Virus Genome: Molecular Cloning of its Associated
Helper Virus and the Cellular Homolog of the V-fos Gene
From Mouse and Human Cells. Molec. Cell. Biol. 3:
914.
37. Dalla Favara, R., E.P. Gelman, R.C. Gallo, and F.
Wong-Staal. 1981. A Human One Gene Homologous to the
Transforming Gene (V-src) of Simian Sarcoma Virus.
Nature 292: 31.
38. Decker, S. 1981. Phosphorylation of Ribsomal Protein
S6 in Avian Sarcoma Virus-Transformed Chicken Embryo
Fibroblasts Proc. Natl. Acad. Sci., U.S.A. 78:
4112.
39. Dahlin, D.C., and K.K. Unni. 1986. Bone Tumors:
General Aspect- and Data on 8,452 Cases. Fourth
Edition. Springfield, Thomas, U.S.A.
40. DeFeo, D., M.A. Gonda, H.A. Young, E.H. Chang, D.R.
Lowy, E.M. Scolnick, and R.W. Ellis. 1981. Analysis
of Two Divergent Rat Genomic Clones Homologous to the
Transforming Gene of Harvey Murine Sarcoma Viurs.
Proc. Natl. Acad. Sci., U.S.A. 78: 3228.
41. DePinho, R., E Legouy, L. Feldman, N. Kohl, G.
Yancopoulos, and F. Alt. 1986. Structure and
Expression of the Murine N-myc Gene. Proc. Natl.
Acad. Sci. U.S.A. 83: 1827.
42. Der, C.J., T.G. Krontiris, and G.M. Cooper. 1982.
Transforming Genes of Human Bladder and Lung Carcinoma
Cell Lines are Homologous to the Ras Genes of Harvey
and Kirsten Sarcoma Viruses. Proc. Natl. Acad. Sci.
U.S.A. 79: 3637.


41
epidermal growth factor (EGF) receptor gene (46, 82). The
neu (erb-B-2) oncogene, first detected by transfection assays
has homology with erb-B-1 and also encodes a receptor-like
molecule (145). The sis oncogene codes for one subunit of
PDGF (45). Recently, it has been shown that v-fms is derived
from the normal cellular gene encoding the receptor for
colony stimulating factor 1 (CSF-1) (111, 146).
The erb-B-1 oncogene protein is different from the
normal EGF receptor in that the extracellular EGF binding
domain is absent (17). It is possible that this truncated
receptor is in an activated configuration even in the absence
of EGF stimulation. More about the erb-B-1 oncogene product
will be discussed below. It has been predicted that other
known growth factor receptors in addition to those for EGF
and CSF-1 such as those for PDGF could be altered or
inappropriately expressed to yield oncogenic proteins. So
far no spontaneous examples of this have been reported.
Oncogenic changes in a growth factor protein are well
exemplified in the case of the sis oncogene product. The c-
sis protein sequences are homologous to one of the chains of
PDGF, and are normally produced in only a restricted number
of cell types; including bone marrow megakaryocytes (77),
human placental cells, and endothelial cells (8). Receptors


23
Figure 2. Histiologic appearance of chondrosarcoma. This
section was taken from a patient with areas of grade I (less
dense cellularity) and grade III tumor. The appearance of
the grade III area closely resembles that of an MFH (shown
below).


18
of transcript. Examination of gene copy numbers and gene
expression will offer clues to possible mechanisms involved
in activation of proto-oncogenes. The following was a
general basis for this project:
Normal cellular genes, when mutated by several suggested
mechanisms, may contribute to the tumorigenesis and
biologic behavior of chondrosarcoma and malignant
fibrous histiocytoma.
From this, the following hypotheses were derived:
1) Increases in proto-oncogene transcript levels may
be due to gene amplification.
2) There are differences in chromatin structure
between amplified and single copy proto-oncogenes.
To test the first hypothesis, tumor RNA and DNA samples
were evaluated for proto-oncogene transcript levels and gene
copy numbers of c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and
v-src. These genes were studied because of previous
associations with sarcomas in humans and other animals.
It was desirable to study potential regulatory changes
which accompanied proto-oncogene amplification and increased
transcript production. Therefore, the second hypothesis was
tested by studying the locations of DNAse I hypersensitive
sites. These sites represent areas where regulatory inter
actions are thought to occur. Changes in locations of these


100
Table 5. C-sis, v-erb-B-1, and v-src:actin ratios from
slot-blot analyses of genomic DNA from normal muscle and bone
marrow tissues.
SAMPLE
c-sis v-erb-B-1 v-src
actin actin actin
MFH patients
MM-1
0.979
0.907
0.927
MM-2
0.962
1.00
1.04
MM-3
1.01
1.03
1.01
MM-4
0.876
0.856
0.825
MM-5
0.933
0.989
0.893
CHONDROSARCOMA
patients
MC-1
0.856
1.06
0.968
MC-2
0.852
0.974
0.888
MC-3
0.953
1.05
0.108
MC-4
0.925
0.973
0.957
Bone marrows
BM-1
1.06
1.00
0.940
BM-2
1.05
1.14
1.13
BM-3
0.829
0.846
0.919
BM-4
0.861
0.757
0.822
BM-5
1.18
1.19
0.894
BM-6
0.882
1.09
0.844


172
109. Little, J.B. 1977. Radiation Carcinogenesis In Vitro:
Implications For Mechanisms. In Origins of Human
Cancer. H.H. Hiatt, J.D. Watson, and J.A.
Winsten, eds. Cold Spring Harbor Laboratory, Cold
Spring Harbor.
110. Loeb, L.A. 1985. Apurinic Sites As Mutagenic
Intermediates. Cell 40: 483.
111. Manger, R., L. Najita, E.J. Nichols, S. Hakomori, and
L. Rohrschneider 1984. Cell Surface Expression of
the McDonough Strain of Feline Sarcoma Virus Fms Gene
Product (GP 140 fms). Cell 39: 327.
112. Maniatis, T., E.F. Fritsch, and J. Sambrook. 1982.
Molecular Cloning: A Laboratory Manual. Cold Spring
Harbor Laboratory Publishers, Cold Spring Harbor,
New York.
113. Marx, J. 1987. The Fos Gene As a "Master Switch."
Science 237: 854.
114. McCann, J., E. Choe, E. Yamasaki, and B.N. Ames.
1975. Detection of Carcinogens as Mutagens in the
Salmonella Microsome Test: Assay of 300 Chemicals.
Proc. Natl. Acad. Sci. U.S.A. 72: 5135.
115. McCoy, M., J.T. Tode, J.M. Cunningham, E.H. Chang,
D.R. Lowy, and R.A. Weinberg. 1983. Characterization
of a Human Colon/Lung Carcinoma Gene. Nature 302:
79.
116. McGrath, J.P., D.J. Capon, D.V. Goeddel, and A.D.
Levinson. 1984. Comparative Biochemical Properties of
Normal and Activated Human Ras p21 Protein. Nature
310: 644.
117. Miller, E.C. 1978. Some Current Perspectives on
Chemical Carcinogenesis in Humans and Experimental
Animals. Can. Res. 38: 1479.
118. Moelling, K., B. Heimann, P. Beimling, U.R. Rapp, and
T. Sander. 1984. Serine and Threonine Specific
Protein Kinase Activities of Purified Gag-Mil and Gag
-Raf Proteins. Nature 312: 558.


176
150. Schwab, M., H. Varmus, and J. Bishop. 1985. The
Human N-myc Gene Contributes to Tumorigenic Conversion
of Mammalian Cells in Culture. Nature 316: 160.
151. Scott, W.A., and D.J. Wigmore. 1978. Sites in Simian
Virus 40 Chromatin Which Are Preferentially Cleaved by
Endogenous Nucleases. Cell 15: 1511.
152. Seeger, R., G. Brodeur, H. Sather, A. Dalton, S.
Siegel, K. Wong, and O. Hammond. 1985. Association
of Multiple Copies of the N-myc Oncogene With Rapid
Progression of Neuroblastomas. N. Eng. J. Med. 313:
1111.
153. Setlow, R.B. 1978. Repairing Deficient Human
Disorders and Cancer. Nature 271: 713.
154. Shimada, T., K. Inokuchi, and A.W. Nienhuis. 1986.
Chromatin Structure of the Human Dihydrofolate
Reductase Gene Promoter. Multiple Protein Binding
Sites. J. Biol. Chem. 261: 1445.
155. Siebenlist, U., L. Henninghausen, J. Battey, and P.
Leder. 1984. Chromatin Structure and Protein Binding
in the Putative Regulatory Region of the C-myc Gene in
Burkitt Lymphoma. Cell 37: 381.
156. Siebenlist, U., P. Bressler, and K. Kelly. 1988. Two
Distinct Mechanisms of Transcriptional Control Operate
on C-myc During Differentiation of HL-60 Cells. Molec.
Cell. Biol. 8: 867.
157. Slamon, D., T. Boone, R. Seeger, D. Keith, V. Chazin,
H. Lee, and L. Souza. 1986. Identification and
Characterization of the Protein Encoded by the Human N
-myc Oncogene. Science 232: 768.
158. Slamon, D.J., G.M. Clark, S.G. Wong, W.J. Levin, A.
Ulrich, and W.L. Maguire. 1987. Human Breast Cancer:
Correlation of Relapse and Survival With Amplification
of the HER-2/neu Oncogene. Science 235: 177.
159. Slamon, D.J., J.B. DeKernion, I.M. Verma, and M.J.
Cline. 1984. Expression of Cellular Oncogenes in
Human Malignancies. Science: 224: 256.


59
amplification has been found in many tumor types, and cell
lines, including HL-60 (promyelocytic leukemia), and COLO 320
(colon carcinoma) cell lines (2, 32).
The amplified region of the c-myc gene has been closely
studied in HL-60 cells. It has been shown that the amplified
region is very large and contains multiple copies of the
entire c-myc gene. Seguencing data indicates that
amplified c-myc gene units or "amplicons" appear to be
structurally normal (2). High levels of c-myc transcript
have been observed in HL-60 cell lines as well as the COLO
320 line and this has been attributed to gene dosage effects.
As a result of these investigations, it is commonly assumed
that when high levels of c-myc transcript are accompanied by
multiple copies, gene amplification is the cause of increased
expression.
Chromatin Structure Analysis of the C-myc Gene
The chromosomes of eukaryotes replicate, undergo
meiosis and mitosis, recombine, segregate, and are
transcribed. The occurence of these processes is
mediated through the interaction of chromosomal DNA and
proteins (72). In order for these proteins to act, specific


166
52. Enneking, W.F., S.S. Spanier, and M.A. Goodman. 1980
A System for the Surgical Staging of Musculoskeletal
Sarcoma. Clin, and Ortho. Rel. Res. 153: 106.
53. Euphrussi, A., G. Church, S. Tonegawa, and W. Gilbert
1985. B Lineage Specific Interactions of an Ig
Enhancer With Cellular Factors In Vivo. Science
227: 134.
54. Eva, A., K.C. Robbins, P.R. Andersen, A. Srinivasan,
S. Tronick, E. Reddy, P. Elmore, N.W. Galen, A.T.
Lautenberger, J.A. Papas, E.H. Westin, F. Wong-Staal,
R.C. Gallo, and S.A. Aaronson. 1982. Cellular Genes
Analogous to Retroviurs One Genes are Transcribed in
Human Tumor Cells. Nature 295: 116.
55. Eva, A. S.R. Tronick, R.A. Gol, J. H. Pierce, and S.A
Aaronson. 1983. Transforming Genes of Human
Hematopoietic Tumors: Frequent Detection of Ras-
Related Oncogenes Whose Activation Appears to be
Independent of Tumor Phenotype. Proc. Natl. Acad.
Sci. U.S.A. 80: 4926.
56. Eva, Y., H.P. Lee, H. To, J. Shew, R. Bookstein,
P.Scully, and W. Lee. 1988. Inactivation of
Retinoblastoma Susceptibility Gene in Human Breast
Cancers. Science 241: 218.
57. Evans, H.L., A.G. Ayala, and M.M. Romsdahl. 1979.
Prognostic Factors in Chondrosarcoma of Bone. Cancer
40: 818.
58. Evinger-Hodges, M.J., K.A. Dicke, J.V. Gutterman, and
M. Blick. 1987. Proto-Oncogene Expression in Human
Normal Bone Marrow. Leukemia 1: 597.
59. Flockhart, D.A., and J.D. Corbin. 1982. Regulatory
Mechanisms in the Control of Protein Kinases. CRC
Crit. Rev. Biochem. 12: 133.
60. Franza, B.R., F.J. Rauscher, S.F. Josephs, and T.
Curran. 1988. The Fos Complex and Fos-Related
Antigens Recognize Elements That Contain AP-1 Binding
Sites. Science: 239: 1150.


CHAPTER 3
MATERIALS AND METHODS
Slot-Blotting of RNA and DNA
Preparation of Total Cellular RNA
Total cellular RNA was prepared from surgically obtained
tumor, normal muscle, and bone marrow tissue specimens from
patients treated at Shands Hospital, University of Florida.
These specimens included; 20 chondrosarcomas, 23 malignant
fibrous histiocytomas (MFH), 9 normal muscle, and 6 bone
marrow tissue specimens. Approximately 1-3 hr after surgical
removal the tissues were frozen at -70 C until use. Total
cellular RNA was prepared as described by Chirgwin et al.
(26). Before use, all glassware and centrifuge tubes were
rendered nuclease-free with 0.1 percent diethylpyrocarbonate
(DEPC) in deionized water and thouroghly dried. All stock
solutions were freed of RNAse by adding several drops of 0.2
percent DEPC and subseguent autoclaving.
Tumor and normal tissues weighing 1.0-1.2 gr, or
approximately 10 E6 cultured cells were placed into 10 ml of
67


170
89. Hunter, T., and B. Sefton. 1980. Transforming Gene
Product of Rous Sarcoma Virus Phosphorylates Tyrosine.
Proc. Natl. Acad. Sci. U.S.A. 77: 1311.
90. Iwasaki, H., T. Isayama, H.Johzaki, and M. Kikuchi.
1987. Malignant Fibrous Histiocytoma: Evidence of
Perivascular Mesenchymal Cell Origin Immunocyto-
chemical Studies With Monoclonal Anti-MFH Antibodies.
Am. J. Path. 128: 528.
91. Jacobs, C., and H. Rubsamen. 1983. Expression of pp60
C-src Protein Kinase in Adult and Fetal Human Tissue:
High Activities in Some Sarcomas and Mammary
Carcinomas. Can. Res. 43: 1696.
92. Jakobovits, E.B., J.E. Majors, and H.E. Varmus 1984.
Hormonal Regulation of the Rous Sarcoma Virus Src Gene
via a Heterologous Promoter Defines a Threshold Dose
For Cellular Transformation. Cell 38: 757.
93. Johnson, L.F., L. Gollakota, and A. Muench. 1982.
Regulation of Thymidine Kinase Enzyme Level in Serum
-Stimulated Mouse 3T6 Fibroblasts. Exp. Cell Res.
138: 79.
94. Kaczmarek, L. 1986. Proto-Oncogene Expression During
the Cell Cycle. Laboratory Investigation 54: 365.
95. Kelly, K., B.H. Cochran, C.D. Stiles, and P. Leder.
1983. Cell-Specific Regulation of the C-myc Gene by
Lymphocyte Mitogens and Platelet-Derived Growth
Factor. Cell 35: 603.
96. Kelly, K., and U. Siebenlist. 1985. The Role of C-myc
in the Proliferation of Normal and Neoplastic Cells.
Journal of Clinical Immunology 5: 65.
97. Knudson, A.G. 1985. Hereditary Cancer, Oncogenes and
Antioncogenes. Can. Res. 45: 1437.
98. Kohl, N.E., N. Kanda, R. Schreck, G. Bruns, S. Laft,
and F. Gilbert. 1983. Transpostions and Amplificaton
of Oncogene Related Seguence in Human Neuroblastomas.
Cell 35: 359.


CHAPTER 5
DISCUSSION
An amplified c-myc gene and increased levels of c-myc
and c-sis transcript suggest an involvement of these genes
in the pathogenesis and progression of MFHs. No increased
transcript levels or amplified copy numbers of any of the
proto-oncogenes were found in chondrosarcomas. MFHs are more
malignant, have a higher fraction of dividing cells (39), and
are potentially more genetically unstable. It is therefore
not surprising that more proto-oncogene mutations would be
observed in these tumors.
Muscle and bone marrow specimens were compared as
examples of normal non-dividing and dividing mesenchymal
tissues. Results presented here show that transcript levels
of TK, c-myc, c-Ha-ras, and c-fos are undetectable in
skeletal muscle tissues while levels of c-sis, v-erb-B-1, v-
src and actin are present at detectable levels. These
results are expected since normal skeletal muscle is a non
dividing tissue and c-myc, c-Ha-ras, c-fos, and TK are cell
cycle dependent genes, while c-sis, v-erb-B-1, v-src and
146


84
1¡
S/WVY
PMC 41
i
ISOLATE DNA, DIGEST WITH ECO F
X
SOUTHERN TRANSFER, PROBE WITH PMC 41
Figure 7. Illustration of the indirect end labeling
technique. This technique allows 5' or 3' orientation of
the locations of DNAse I hypersensitive sites. It always
yields pieces of DNA which have a restriction site on one
side, therefore allowing analysis of DNA segments in one
direction. Various concentrations of DNAse I are used to
allow partial digestion. If DNAse I cut at sites 1 and 2,
analysis by this method would yield three bands on a Southern
blot. A main band of 9.6 kb which corresponds to the Eco
Rl/Eco R1 fragment would be present along with bands of sizes
corresponding to the lengths of the DNAsel cleavage sites to
the 3' ECO R I site.


50
increase of pp60c-src kinase activity was present in human
skin tumors compared to normal skin (7).
Nuclear Related Proto-oncogenes
Fos. The fos gene was first discovered as the oncogene
of two related murine viruses that cause osteogenic sarcoma.
The name fos refers to its origins in the FBJ and FBR
osteogenic sarcoma viruses. The fos oncogene, like other
oncogenes, causes the transformation of cells and is
derived from a normal cellular gene. The cellular and viral
fos genes have an interesting relationship to each other.
The first 332 amino acids of v-fos and murine c-fos differ in
only five positions but the remaining 49 amino acids are
completely different. The 104 bases at the C-terminus of c-
fos are deleted in v-fos, and although this changes the
reading frame and alters subseguent amino acids, the
mobilities of the proteins are similar (v-fos 55 kd, c-fos,
62 kd) (184) .
The fos gene seems to serve as a kind of master switch
for turning on other various genes in response to a wide
range of stimuli including growth factors. Fos may act as a
sensor which detects incoming signals at the cellular


66
Zajac-Kaye et al. (199) noted similar findings in 5/7
Burkitt lymphoma cell lines. Their data indicated that a 20
base pair region in the first exon (site F, figure 4) was
susceptible to sporadic point mutations. Mutations in this
region abolished binding of a regulatory protein known to
down regulate c-myc transcription.
Relevance to This Project
The c-myc, c-Ha-ras, c-fos, c-sis, v-erb-B-1, and v-
src proto-oncogenes have been studied in other human tumor
systems. The object of this study is to determine whether or
not these genes play a significant role in the biology of
chondrosarcoma and MFH. Studying transcript levels and copy
numbers of these genes will offer clues to possible
involvements in the pathogenesis and progression of these
tumors. Furthermore, chromatin structure analysis will
enhance understanding of mechanisms involved in transcript
regulation.


CHAPTER 1
INTRODUCTION
Mechanisms of Tumor Development
Fundamental requirements for successful prevention and
sometimes treatment of cancer are knowledge and understanding
of its causitive factors. This task is not an easy one by
any means. Agents having abilities to contribute to or
cause cancer are called carcinogens. Studies to determine
the roles these agents play in neoplastic processes have
focused on three general classes of carcinogens: chemical,
physical, and biological.
Carcinogenic chemicals and ionizing radiation are known
to affect DNA at a structural level and to be mutagenic
under certain conditions. Therefore, one of the long
standing theories of carcinogenesis has been that cancer is
caused by genetic mutations.
Evidence that chemicals can induce cancer has been
reported for more than two centuries. The first observations
of chemically induced cancer were made in humans. The first
of these was in 1761 when Hill noticed that nasal cancer was
1


3
are known to react with cellular RNA as in the case of
dimethylnitrosamine (105), but most react with DNA (142).
Reaction of chemical carcinogens with DNA can facilitate the
induction of heritable changes in cells and may lead to
malignant transformation. Thus, it is generally believed
that this is the most likely mechanism for chemical
initiation of carcinogenesis. Representative agents from
virtually all classes of chemical carcinogens have been shown
to affect DNA in some way. The actions of many of these have
been found to result in the formation of base-adducts. The
potential biological conseguences of these are are several:
Base adducts may stabilize intercalation reactions. For
example, if the flat planar rings of a polycyclic hydrocarbon
were stably integrated between the stacked bases of double
helical DNA, the helix would be distorted. This could lead
to a frame-shift mutation which would occur during DNA
replication past the point of intercalation (86).
Many of the base adducts formed by carcinogens involve
modification of N-3 or N-7 positions on purines. This
induces an instability in the glycosidic bond between the
purine base and deoxyribose. The destabilized structure can
then undergo cleavage by DNA glycosylase, resulting in loss
of the base, and creation of an apurinic site in the DNA.


36
membrane, and does not posess receptor activity, it probably
does play some role in the early signalling process (35).
Therefore, the presence of a tyrosine kinase encoded by a
viral oncogene might result in a continuous, deregulated
mitogenic signal for cell division.
Just how the cell responds to these signals is presently
unknown. Many attempts have been made to find the cellular
targets for phosphorylation by pp60src and by growth factor
receptors. One effect of pp60src which is thought to be of
importance is that it leads to increased protein
phosphorylation on serine residues (38). The phosphorylation
of the S6 ribosomal protein on a serine residue is thought to
be a critical event in the mitogenic stimulation of normal
quiescent cells. This may occur via a serine kinase
intermediate which might be activated directly or indirectly
by the pp60src tyrosine kinase (38).
Two different biochemical pathways have been shown to be
important in the mitogenic stimulation of cells and a
possible involvement with both has been shown for pp60src.
Both of these pathways involve the generation of second
messengers. The first involves the generation of cyclic AMP
by membrane bound adenylate cyclase, leading to increased


32
A third mechanism involves enhancer/promoter activity.
Enhancer sequences may increase utilization of
transcriptional promoters to which they become linked. The
affected promoter may be several kilobases away in either 5'
or 3' directions (74). One example of this is the presence
of retrovirus genome fragments downstream from the c-myc gene
in some avian lymphomas (131). Here, the retrovirus elements
appear to act by contributing an enhancer sequence rather
than a promoter. It is entirely possible that point
mutations at key regulatory sites such as promoter regions
rather than coding regions may result in in proto-oncogene
activation. This could facilitate the deregulation of a
proto-oncogene, i.e. one with abnormal transcriptional
control, or one which is inappropriately expressed.
A fourth mechanism involves the c-myc gene in
particular. Work with Burkitt lymphomas has demonstrated the
juxtaposition of the c-myc gene and immunoglobulin genes
following a translocation event. As a result of this
translocation, the c-myc gene loses all or part of its own
regulatory exon and acquires normally unlinked sequences
involved in immunoglobulin production (104). Rearranged c-
myb sequences have been found in certain mouse plasmacytomas


139
1,2,3,4,6) in figure 21. Despite a peak of myc transcript
production 1 hr after serum release, no changes were observed
in the locations of these sites during the transition of HFF
fibroblasts from GO to Gl, or between any time points after
serum release.
Fine Mapping Analysis of DNAse I Hypersensitive Sites in P3C
Cells From a 51 Direction
Fine mapping of P3C c-myc DNAse I hypersensitive sites
(shown in figure 21) in the exon 1/ intron 1 region was done
from the 5' direction to more precisely determine their
locations (particularly site 5) relative to known
transcription elongation block sites. Five ug of P3C and 15
ug of HFF DNA from DNAse I treated nuclei were restricted
with Sea I. These, and P3C marker DNAs restricted with Sea
I/Mae III, Sea I/Bsm I, and Sea I/Pvu II were analyzed by
Southern blot hybridization using the Sea I/Xho I probe (see
materials and methods). The Southern blot from fine mapping
analysis of c-myc DNAse I hypersensitive sites in the 3'
region of exon 1, and in intron 1 is shown in figure 24. The
resolution of bands on this blot was found to be at least 25
base pairs. This is demonstrated by the easy resolution of
bands generated by digestion with Sea I/Mae III (970 bases)


62
that interaction with trans-acting factors may be one
of these mechanisms (72). The base composition of the DNA,
methylation, looping, conformation, and torsional stress may
also have an involvement in this process (72).
Fundamental knowledge of these principles should provide
insight into molecular bases of regulation. Thus, it is a
well accepted fact that DNase I hypersensitive sites
represent regions where potential regulatory interactions are
thought to occur. Specific DNA seguences of this nature have
been shown to be located in promoter regions for such genes
as globin (51), immunoglobulin (53), c-myc (48, 73, 155,
156), heatshock (129, 193, 194), SV 40 early region (47),
and dihydrofolate reductase (154). The remainder of this
review will focus on those involving the c-myc gene.
As previously mentioned, amplification and translocation
are well known and widely studied potential c-myc activation
mechanisms. As a result of translocation to chromosome 14 in
Burkitt lymphoma, c-myc loses all or part of exon 1. This
exon is thought to serve primarily as a regulatory region, as
it is transcribed but not translated (77). Therefore, c-myc
may be deregulated by its loss, and may be influenced by
promoters of other genes proximal to its translocated site.
In the case of HL-60 cells (30-50 copies of the c-myc gene),


105
Table 10. C-myc, c-Ha-ras, and c-fos:TK ratios from slot-
blot analyses of total cellular RNA from MFHs. also shown
are TK:actin ratios which were used as molecular measures
of cell cycle.
MFH SAMPLE
c-myc
c-Ha-ras
c-fos
TK
TK
TK
TK
actin
MT-1
0.455
0.686
0.419
0.501
MT-2
1.24
0.412
1.27
0.560
MT-3
1.14
0.465
2.54
2.03
MT-4
1.00
0.184
1.15
1.01
MT-5
0.870
0.781
0.987
0.636
MT-6
1.31
0.474
0.862
2.19
MT-7
0.690
0.412
0.953
0.651
MT-8
1.68
1.05
0.520
2.35
MT-9
0.935
0.360
0.417
0.602
MT-10
1.00
1.25
0.458
0.393
MT-11
1.09
3.87
0.556
1.31
MT-12
1.21
0.346
0.968
1.29
MT-13
1.26
0.370
0.472
1.46
MT-14
1.04
3.40
0.855
1.10
MT-15
1.15
0.258
0.908
1.51
MT-16
1.92
0.198
0.899
1.63
MT-17
1.97
0.498
0.895
2.13
MT-18
1.96
0.215
0.955
3.69
MT-19
1.05
0.145
1.25
1.30
MT-20
1.93
1.27
1.04
1.33
MT-21
1.14
0.896
1.30
1.06
MT-22
1.96
1.20
1.04
1.71
MT-2 3
1.10
0.987
1.30
1.03


BIOGRAPHICAL SKETCH
Jane Carolyn Strandberg Gibson was born in Madison,
Wisconsin, on September 29, 1962, to James and Kathleen
Strandberg, and is the oldest of their 3 children.
Jane has lived in the Orlando, Florida, area since 1969,
where she graduated from Bishop Moore High School in 1980.
During that same year, she began her college career at the
University of Central Florida in Orlando. In 1982 she moved
to Gainesville and attended the University of Florida were
she received a Bachelor of Science degree in microbiology and
cell science in April,1984.
In the fall of 1984, she began Graduate School as a
student in the Department of Pathology at the University of
Florida. In August 1986, she received her Master of Science
degree in medical sciences-pathology. At that time she
decided to continue her work with proto-oncogene expression
in human sarcomas under the direction of Dr. Byron Croker.
Jane married Ronald Lee Gibson on May 7, 1988. Since that
time she has continued her work toward a Doctor of
Philosophy degree in the Department of Pathology and
Laboratory Medicine, University of Florida College of
Medicine.
182


132
DNA digested with either Hind III (right side of blot), or
both Eco R1 and Hind II together (left side of blot).
Controls at 0 ug/ml DNAse I at 0 and 37 C degrees
demonstrated that DNAse I generated bands are real, and
not due to endogenous nuclease activity. There is a band
seen in the control lane for the P3C cell line. Although
this band may be generated by endogenous nucleases, it ran
differently than the DNAse I band of similar size, and was
not relevant to these analyses.
Locations of DNAse I hypersensitive sites for the c-myc
gene in each of the cell lines studied are shown in figure
21. It was found that five DNAse I hypersensitive sites at
identical locations are present for UR HCL 1 and the normal
human fibroblast line HFF (sites 1,2,3,4,6, figure 21). One
site is located 5' to the first exon and 5' of promoter PO
(site 1). Two sites are located 5' of the first exon and 3'
of the PO promoter region (sites 2,3). Another site is
located in the 3' region of the first exon near a PVU II site
(site 4), and a fifth site was found to be 5' of exon 2 (site
6). The amplified c-myc gene in the P3C cell line also had
four of these sites (1,3,4,6), however a site near the PO
promoter region is not present (site 2), and a new site in
the 5' region of the first intron is seen (site 5). Each of


135
Total cellular RNA from the cells at each of these time
points was evaluated by Northern blot analysis for transcript
levels of c-myc and TK to ascertain that the desired phases
of the cell cycle were represented (figure 22).
Hybridization with the c-myc probe demonstrates a profile of
transcript levels which starts out at a basal level during
GO, peaks 1 hr after serum release, then returns to levels
comparable to those of GO during log phase growth. Levels of
TK at the various time points indicate that transcript levels
are highest during log phase, and lowest during GO.
Hybridization with actin demonstrates consistent actin
transcript levels and RNA quantitation. These results are
expected for transcript levels of these genes during the
cell cycle.
Fifteen ug of genomic DNA isolated from DNAse I treated
nuclei at each time point discussed above were restricted
with ECO R1 and analyzed for Locations of DNAse I
hypersensitive sites as previously described (figure 23).
Mapping of sites was accomplished by band size comparisons to
those generated by digestion of lambda DNA with Eco R 1 and
Hind III. Locations of c-myc DNAse I hypersensitive sites
for HFF at each time point examined were found to be
identical to those previously described for HFF (sites


47
alter the capabilities of its product. Also unclear is
whether or not the homodimer produced from it has abnormal
activity compared to the related but different subunits of
PDGF. Whether the formation of homodimer causes PDGF 2-B to
be processed abnormally, or if the sis oncogene product acts
at an anomalous site inside the cell are questions yet to be
answered. It is possible that cells produce factors which
cooperate with sis in neoplastic transformation (17). All of
these issues only obviate the fact that much more needs to be
done before a full understanding of the sis gene and its
product can be obtained.
The presence of the c-sis gene has been demonstrated in
several tumor types. Eva et al. (54) reported that cell
lines from both human sarcomas and gliomas were analyzed for
the presence of sis message. It was found to be at elevated
levels in 5/6 of sarcoma cell lines and 3/5 of glioma cell
lines studied. Sis message has also been found to be at
elevated levels in the metastases of two stomach carcinomas
(172) .
Protein Kinases


25
Chondrosarcomas usually have a slow clinical evolution.
Metastasis is relatively rare and occurs late. The basic
therapeutic goal is to control the lesion locally and to
prevent local recurrence. Therefore, radical early
surgical treatment is desirable (39). A long followup after
treatment is necessary because recurrence may develop many
years later. The overall survival is approximately 50
percent at 5 years (180).
With respect to prognosis, the correlation between poor
differentiation, rapid growth rate, and metastasis is high.
Clinical study results suggest that a high cure rate is
expected for patients with more differentiated tumors (135).
A grading system exists for chondrosarcoma and is important
in terms of predicting survival and establishing the most
effective treatment protocol (39).
Criteria for grading chondrosarcomas are those of Evans
et al. (57), and include the following: Grade I tumors have
the presence of or domination of cells with small densely
staining nuclei, an inter-cellular background of a chondroid
or myxoid nature, freguent calcification patches, and
multiple nuclei present within a single lacuna. Grade II
tumor characteristics include; areas where a significant
fraction of the nuclei are of a moderate size, a mitotic


114
10 5 2.5
MT-1
MT-2
MT3
MT4
MT5
MT6
MT7
MT8
MT9
MT10
MT11
MT12
MT13
MT14
MT15
MT16
MT17
MT18
MT19
MT20
MT21
MT22
MT23
Figure 14. Slot-blot of total cellular RNA from MFHs
hybridized with the c-sis probe. Quantities of 10, 5, and
2.5 ug of RNA were slot-blotted onto nitro-plus 2000, and
hybridized with the c-sis probe (3.0 x 10 E6 cpm/ 10 E8
cpm/ug).


103
Table 8. C-myc, c-Ha-ras, and c-fos:actin ratios from slot-
blot analyses of genomic DNA from chondrosarcomas. Also shown
are TK:actin ratios.
CHONDROSARCOMA SAMPLE
c-mvc
actin
c-Ha-ras
actin
c-fos
actin
TK
actin
CS-1
0.980
0.825
0.948
0.951
CS-2
0.757
0.933
1.20
1.06
CS-3
0.878
0.929
0707
0.982
CS-4
0.955
0.700
0.642
1.06
CS-5
0.922
1.16
1.14
1.04
CS-6
0.775
0.769
0.656
0.973
CS-7
1.03
0.735
0.816
0.974
CS-8
0.933
0.698
0.872
0.903
CS-9
1.14
0.901
1.18
1.24
CS-10
1.23
1.35
0.956
1.23
CS-11
1.24
0.756
0.783
1.08
CS-12
0.993
0.868
1.03
0.833
CS-13
1.12
1.20
1.33
1.19
CS-14
1.08
0.800
1.24
1.04
CS-15
1.03
0.793
1.25
0.833
CS-16
0.885
1.23
0.799
1.19
CS-17
1.25
1.05
1.32
1.04
CS-18
1.09
1.04
0.774
0.879
CS-19
1.16
0.901
1.31
1.18
CS-2 0
1.05
1.14
1.09
1.17


122
MIO 5 2 1
B
M 10 5 2 1
C
M 10 5 2 1
Figure 16. Southern blot/DNA dilutional analysis of genomic
DNA from MFHs with 2 or greater copies of c-myc. Tumor DNA
samples of 10, 5, 2, and 1 ug (labeled 10, 5, 2 and 1) and
muscle DNA samples of 10 ug (labeled M) were digested with
Hind III, electrophoresed through 0.8 percent agarose gels,
blotted, and hybridized with p GEM H MYC. C-myc gene copy
numbers were calculated to be (A-F) 8.7, 8.4, 9.6, 9.9, 10.6
and 2.2 respectively. Samples A,B,C,D,E, and F correspond to
samples MT-16, MT-17, MT-18, MT-20, MT-22, and MT-8
respectively as shown in figure 15.


144
O-MYC
CONTROL
CONTROL
<0 r*
§
116 KD>
92 KD >
66 KD>
45 KD>
31 KD>
P3C ST 486 UR HCL 1 HFF/GO
HFF/0.5
HFF/1
HFF/2
HFF/L
6 5 2 1
1
2
1
1
Figure 26. Western blot showing relative levels of c-myc protein
in P3C, UR HCL 1, ST 486, and HFF (GO, 0.5, 1.0, 2.0 hr after
serum release, and during log phase growth (L)) cell lines.
Twenty ug of protein were separated by size using PAGE, and
electroblotted onto nitrocellulose. The panels were (L-R)
incubated with the HL-40 (anti c-myc) monoclonal antibody, the K
88.151.G127 (anti met 72) antibody (control for non-specific
binding), and the third panel was stained with 0.1 percent india
ink to control for protein quantitation. The c-myc monoclonal
antibody bound to a 65 kd protein band as determined by molecular
weight markers. Shown below the figure are the relative levels
of c-myc protein as determined by reflectance densitometry.
Values were normalized to myc protein levels in HFF cells at GO
which was given a value of 1.


134
the DNAse I bands for P3C cells were similar in intensity.
This suggests that most if not all of the copies of c-myc
have these changes in chromatin structure. Three DNAse I
hypersensitive sites were observed for the ST 486 cell line.
These were in identical regions as those previously reported
by Siebenlist et al. (156) in Burkitt lymphoma BL 31 cells.
Chromatin Structure of the C-mvc Gene During the G0/G1
Transition in the HFF Normal Human Fibroblast Cell Line
These data show that increases in transcript and
changes in DNAse I hypersensitive sites accompany c-myc gene
amplification in P3C cells. It was of interest to determine
if changes in DNAse I hypersensitive sites are seen in normal
cells during periods when peak levies of c-myc transcript are
produced. Increased levels of c-myc transcript production
have been observed during the G0/G1 transition in quiescent
fibroblasts after serum addition. Quiescent HFF cells
were evaluated at GO 0.5, 1, 2, and 3 hours after serum
release, and during log phase growth. Locations of DNAse I
hypersensitive sites in these cells were mapped from the 3'
direction using Southern hybridization with the pmc 41 probe.


10
or chemical damage to DNA. Such examples include, xeroderma
pigmentosum (deficiency in excision repair), ataxia
telangiectasia (greater sensitivity to X-irradiation, more
prone to leukemia and other cancers), Fanconi1s syndrome
(deficiency in repair of cross-linked bases, repair of X-ray
or UV induced damage), and Bloom's syndrome (increased
propensity to develop cancer, high genetic instability of
chromosomes). The high incidence of cancer in patients with
these diseases constitutes the best available evidence for a
causal relationship between mutagenicity and carcinogenicity
in humans (168, 169, 185).
Tumor initiating agents most likely interact with DNA to
induce mutations, rearrangements or amplifications, producing
a genotypically altered cell. The initiated cell then
undergoes clonal expansion influenced by promoting agents
which act as mitogens for the transformed cell (142). It has
been suggested that promoting activities may be mediated by
cellular membrane events. Direct action of promoters on DNA
has also been proposed (142). As a result, multiple clones
of cells are likely to be initiated by a DNA damaging agent.
Then, through a rare second event, one or a small number of
these clones progresses to malignant cancer.


80
electrophoresed in running buffer which consisted of IX MOPS
and 10 percent formaldehyde (volume/37%) at 120 volts for 3-
3.5 hr.
After electrophoresis, the gels were rinsed several
times in deionized water, then soaked in 10X SSC for 45 min.
Blotting stacks were assembled as for Southern blotting.
Overnight transfers to Zetabind membranes were completed in
2OX SSC. Blots were pre-washed in 0.1 X SSC, 0.5 percent SDS
at 65 C for 1 hr. Pre-hybridization and hybridization
conditions (2.0 X 10 E6 cpm/ml/ 10 E8 cpm/ug), as well as
post-hybridization washes and rehybridization procedures were
identical to those described for Southern analysis.
Chromatin Structure Analysis
Cell Lines Used in Chromatin Structure Analysis
UR-HCL-1. The UR-HCL-1 cell line is a human MFH tumor
cell line obtained from ATCC.
P3C. The P3C cell line is an MFH tumor cell line
obtained from Dr. Byron Croker, Department of Pathology,
University of Florida. The cell line was made by culturing


154
same regions as sites B and C (C for P3C cells) in HL-60 and
Burkitt lymphoma cells. Site 2, which is not seen in P3C
cells, was located in the same region as site B in HL-60
cells. The disappearance of this site has been shown to
accompany decreased c-myc transcript production in
differentiating HL-60 cells post treatment with DMSO. Even
though site 2 and site B map to different sides of the PO
promoter, they can be considered to be located in the same
region because DNAse I hypersensitive sites may include 150-
200 base pairs (about the size of a nucleosomal repeat).
Site 4 in HFF, UR HCL 1, and P3C cells was located in a
region which contains a PVU II site and a c-myc transcript
elongation block in Burkitt lymphoma biopsies and cell
lines (Site E, figure 27) (25). Site 5 was observed
exclusively in P3C cells. This site mapped to a region in
the first intron also known to contain a c-myc transcript
elongation block in Burkitt lymphoma cell lines
(Site F, figure 27) (199). Site 6 in the HFF, UR HCL 1, and
P3C cell lines has not yet been found by DNAse I
hypersensitive site analysis. However, an S-l nuclease
sensitive site in a similar region has been described by
Grosso and Pitot (73).


29
metastasis. Grade is further classified as low and high,
local extent as intracompartmental and extracompartmental,
and the extent of regional or distant metastasis is defined
as either present or absent.
Proto-Oncogenes
During the early 1970s it was discovered that a single
gene carried by a retrovirus could cause cancer in animals.
Soon thereafter, it was thought that the oncogenes acquired
by retroviruses might be derived from normal cellular genes
present in the host. It was later shown by Stehlin et al.
(164) that cDNA specific for the v-src region of Rous
Sarcoma Virus could detect closely related sequences in the
genome of normal chicken cells. This gene, now called c-src
has been found in all other vertibrate species including man
(162) .
Since the discovery of cellular sequences homologous to
v-src, cellular counterparts (c-onc) for the other viral (v-
onc) oncogenes have also been found (16). These cellular
sequences are known as cellular oncogenes or proto-oncognes.
There are now more than forty known proto-oncogenes which are
expressed in most normal mammalian cells (77). The c-ras and


119
The results indicated a positive correlation between c-myc
gene copy number and cell division as represented by TKractin
ratios (p<0.05) (table 18).
The slot-blotting technique used in these studies was
primarily a technique for screening total cellular RNA and
genomic DNA from tumors and normal tissues for proto-oncogene
expression. A restricted range of linearity for
hybridization signal intensities was one technical
limitation of this system, therefore limiting its
reliability as a quantitative tool when signal intensities
exceeded or fell short of the linear range. For example, DNA
copy numbers greater than 2 could not be assessed in 6 MFHs.
Another technical limitation of the slot-blot is that it
cannot be ascertained what size transcript the probe
hybridized to. In these analyses, all probes were confirmed
for correct specificity by restriction digests and Southern
blot analysis prior to use in slot-blot hybridizations.
The beta-actin control was the most validating factor
for the slot-blotting assay used in this study.
Hybridization with this gene is an internal control for both
RNA and DNA slot-blots. As shown in these results, actin
values show little variability between samples on both RNA


To my husband Ron, to my grandparents, to Karen and
Ken, and to Mom and Dad; whose love, support, and belief
me will always be my inspiration.
in


171
99. Kohl, N., E. Legouy, R. DePinho, R. Smith, C.Gee, and
F. Alt. 1986. Human N-myc is Closely Related in
Organization and Nucleotide Sequence to C-myc. Nature
319: 73.
100. Kruijer, W., J.A. Cooper, T. Hunter, and I.M. Verma.
1984. Platelet-Derived Growth Factor Induces Rapid but
Transient Expression of the C-fos Gene and Protein.
1984. Nature 312: 711.
101. Laemmli, Y. 1970. Cleavage of Structural Protein
During the Assembly of the Head in Bacteriophage T4.
Nature 227: 280.
102. Land, H., L.F. Parada, and R.A. Weinberg. 1983.
Cellular Oncogenes and Multistep Carcinogenesis.
Science 222: 771.
103. LeBeau, M.M., and J.D. Rowley. Heritable Fragile Sites
in Cancer. Nature 308: 607.
104. Leder, P., J. Battey, G. Lenoir, C. Moulding, W.Murphy,
H. Potter, T. Stewart, and R. Taub. 1983.
Translocations Among Antibody Genes in Human Cancer.
Science 222: 765.
105. Lee, K.Y., W. Lijinsky, and P.N. Magee. 1964.
Methylation of Ribonucleic Acids of Liver and Other
Organs in Different Species Treated With C14 or H3
Dimethylnitrosamine In Vivo. J. Natl. Cancer Inst.
32: 65.
106. Lee, W., A. Murphree, and W. Benedict. 1984.
Expression and Amplification of the N-myc Gene in
Primary Retinoblastoma. Nature 309: 458.
107. Leibovitch, S.A., M.P. Leibovitch, M. Guiller, J.
Hillion, and J. Harel. 1986. Differentiation of
Proto-Oncogenes Related to Transformation and Cancer
Progression in Rat Myoblasts. Can. Res. 46: 4097.
108. Libermann, T.A., H.R. Nusbaum, N. Razn, R. Kris, I.
Lax, H. Soreq, N. Whittle, M.D. Waterfield, A. Ulrich,
and J. Schlessinger. 1985. Amplification, Enhanced
Expression and Possible Rearrangement of EGF Receptor
Gene in Primary Human Brain Tumors of Glial Origin.
Nature 313: 144.


90
percent BSA, added to one blot, and allowed to incubate at
room temperature for 1 hr with light agitation. As a control
for non-specific binding, an identical blot was incubated
with an anti-met 72 monoclonal antibody (K 88. 151. G 127)
(IGG 1, ascites purified by protein A column), (0.01 mg/ml in
PBST, 2 percent BSA) obtained from Dr. Arthur Kimura. The
blots were then washed 3 times for 5 min each with PBST.
This was followed by incubation with a 1 ug/ml solution of a
horseradish peroxidase conjugated goat-anti-mouse Ig
(Southern Biotechnology Associates) diluted in PBST, 2
percent BSA for 1 hr at room temperature. Blots were then
washed 3 times with PBST as before, and incubated with a 180
ug/ml solution of the substrate, diaminobenzoate (DAB), in
PBST, and 0.01 percent H202 for 2-3 minutes. The reaction
was stopped with excess H20. Quantification of c-myc protein
bands was carried out by reflectance densitometry.
As a control for guantification, a third identical blot
was stained with a 0.1 percent solution of india ink in PBST
for 1 hr at room temperature, then destained with PBST until
the desired resolution was achieved.


5
hormones did not fit with the notion of an infectious origin
of cancer. Early studies attempted to transmit malignant
disease by inoculation of filtered extracts prepared from
diseased tissues. It was later demonstrated by Ellerman and
Bang in 1908 that chicken leukemia could be transmitted by
cell-free filtered extracts (50). They were among the first
to demonstrate the viral etiology of this disease. An example
of a virus thought to cause cancer in humans is seen with
Human T-cell leukemia virus 1 (HTLV-1), a transmissible
virus thought to cause leukemia (133).
Other oncogenic RNA viruses are capable of participating
in transformation. Understanding of the molecular mechanisms
involved in cellular transformation by these viruses is based
on the Nobel prize winning work of Baltimore and Temin (5,
173). In the early 1960s, Temin demonstrated that mutations
in the Rous sarcoma virus (RSV) genome of RSV-infected
chicken cells could be induced at a high rate. It was also
shown that mutation of an RSV gene present in an infected
cell often changed the morphology of the cell, and the virus
genome was stably inherited by progeny cells (173). This led
to the notion that virus genetic information was contained in
a regularly inherited structure of the host cell as a
"provirus" integrated into the host cell's genome. The


89
Twenty ug of protein were combined with an equal volume
of treatment buffer (0.125 M Tris-HCL, pH6.8, 4 percent SDS,
20 percent glycerol, and 10 percent 2-mercaptoethanol),
incubated at 90 C for 1.5 min, ice-quenched, then loaded onto
the gels. Molecular weight markers ranging from 31,000 to
200,000 daltons (Biorad) were loaded as well.
A tank buffer which consisted of 25 mM Tris-HCL, pH 8.3,
0.192 M glycine, and 0.1 percent SDS was used as a running
buffer. Gels were electrophoresed in a Hoefer SE 600
vertical slab unit at 30 ma/1.5 mm gel thickness.
Western Blotting and Immunoperoxidase Assay
Western blotting of proteins was carried out at 0.6 amps
for 45 min at 4 C. Proteins were blotted onto 0.2 um pore
size nitrocellulose (Schleicher & Schuell, Keene, NH) using
methods described by Towbin (176). Transfer was carried out
using a Hoeffer TE 52 Transphor unit. Following transfer,
the blots were air-dried, then incubated for 3 hr in PBST (IX
phosphate buffered saline (PBS), 0.05 percent Tween 20) and 2
percent BSA. An anti- human c-myc monoclonal antibody (HL-
40) (IGG 1, ascites purified by protein A column) obtained
from Dr. Henry Neiman was then diluted (0.1 mg/ml) in PBST, 2


93
A
B
C
D
E
F
Densitometer readings for slot-blot of alkaline
degraded total cellular RNA from HL-60 cells
as shown above.
A 8.71 E5
E 8.64 E5
C 9.03 E5
D 8.41 E5
E 8.66 E5
F 1.09 E5
Figure 10. Slot-blot of alkaline degraded HL-60 total
cellular RNA hybridized with the c-myc probe (3.0 X 10 E6
cpm / 1 X 10 E 8 cpm/ug). Densitometric values (peak areas
sq um/ug) correspond to hybridization signals for 2.5 ug of
RNA neutralized after (a) 0 min (b) 0.5 min (c) 1 min (d) 2
min (e) 5 min and (f) 30 min. Densitometric values (shown
below blot) for lanes A-E indicated that moderately degraded
RNA was as reliably quantitated by slot-blotting as totally
intact RNA. Extensively degraded RNA (lane F) was not, and
no samples showing this level of degredation were used in
these analyses.


4
This open space can then be filled by any base, resulting in
a base transition (purine-pyrimidine base change) (110).
Interaction with some carcinogens has been shown to
favor a conformational transition of DNA from its usual
double-helical B form to a Z DNA form (125). This could
alter the ability of certain genes to be transcribed, since
B-Z conformational transitions are thought to be involved in
regulating chromatin structure (142).
Both X-rays and ultraviolet radiation also produce
damage to DNA. As with chemical carcinogens, this damage
induces DNA repair processes, some of which are error prone
and lead to mutations. Studies have shown that the
development of malignant transformations in cultured cells
after irradiation requires fixation of the initial damamge
into a heritable change. This is experimentally accomplished
by allowing clonal proliferation and expression of the
transformed phenotypes (109).
In addition to chemical and physical carcinogens,
biological carcinogens exist as well. It was long suspected
that various forms of cancer, particularly certain lymphomas
and leukemias, were caused or at least cocaused by
transmissable viruses. The known carcinogenic effects of
certain chemicals, irradiation, chronic irritation and


75
33:3
23.1
22
12
11.2
12
13
21.3
22.1
22.2
22.3
23
24.1
24.2
24.3
CA-2
<
o
Imyc
Itg
H25-3.8
ir
P0P1 P2
SCA l/XHO I
P380-8A
8
h h
(0 w
a a
PGEMH MYC
HT.96
CMYC
Figure 5. Locations of the different c-myc probes as
described in table 1. Also shown are other probes located on
chromosome 8 which were used in mapping of c-myc amplicons in
MFHs.


97
Table 2. C-myc, c-Ha-ras, and c-fos:TK ratios from slot-
blot analyses of total cellular RNA from normal muscle and
bone marrow tissues. Also shown are TK:actin ratios which
were used as molecular measures of cell cycle.
SAMPLE
c-mvc
TK
c-Ha-ras
TK
c-fos
TK
TK
actin
MUSCLE
MFH patients
MM-1
*
*
*
*
MM-2
*
*
*
k
MM-3
*
*
*
*
MM-4
*
*
:k
*
MM-5
*
*
*
*
CHONDROSARCOMA
patients
MC-1
*
*
*
*
MC-2
*
*
*
*
MC-3
*
k
*
*
MC-4

*
k
*
BONE MARROWS
BM-1
1.18
1.28
1.05
0.909
BM-2
0.945
1.15
0.935
1.01
BM-3
0.848
0.831
0.834
1.02
BM-4
0.804
0.972
0.935
1.13
BM-5
0.551
1.05
1.08
1.28
BM-6
0.957
0.813
0.968
1.15
* C-myc, c-Ha-ras, c-fos, and TK were undetectable in normal
muscle tissues by these analyses.


173
119.Morgan, J.I., D.R. Cohen, J.L. Hempstead, and T.
Curran. 1986. Mapping Patterns of C-fos Expression
in the Central Nervous System After Seizure. Science
237: 192.
120. Muller, R., R. Bravo, J. Burckhardt, and T. Curran.
1984. Induction of C-fos Gene and Protein by Growth
Factors Preceedes Activation of C-myc. Nature 312:
716.
121. Murphy, W., H. Potter, T. Steward, and R. Taub. 1983.
Translocations Among Antibody Genes in Human Cancer.
Science 222: 765
122. Mushinski, J.F., M. Potter, S.R. Bauer, and E.P.
Reddy. 1983. DNA Rearrangement and Altered RNA
Expression of the C-myb Oncogene in Mouse Plasmacytoid
Lymphosarcomas. Science 220: 795.
123. Nau, M., B.Brooks, J. Batley, E. Sausville, A. Gazdar,
I. Kirsch, 0. McBride, V. Bertness, G. Hollis, and J.
Minna. 1985. L-myc, A New Myc Related Gene Amplified
and Expressed in Human Small Cell Lung Cancer. Nature
318: 69.
124. Nau, M., S. Brooks, D. Carney, A. Gazdar, J. Battey,
E. Sausville, and J. Minna. 1986. Human Small Cell
Lung Cancers Show Amplification and Expression of the
N-Myc Gene. Proc. Natl. Acad. Sci. U.S.A. 83: 1092.
125. Needle, S. 1981. New Twists To DNA and DNA-
Carcinogen Interactions. Nature 292: 292.
126. Nishikura, K., and J.M. Murray. 1987. Antisense RNA
of Proto-Oncogene C-fos Blocks Renewed Growth of
Quiescent 3T3 Cells. Molec. and Cell. Biol. 7: 639.
127. Nishizuka, Y. 1984. Turnover of Inositol Phospholipids
and Signal Transduction. Science 225:
128. Nowell, P.C. 1976. The Clonal Evolution of Tumor Cell
Populations. Science 194: 23.
129. Parker, C.S. and T. Topol. 1984. Drosophila RNA
Polymerase II Transcription Factor Binds to the
Regulatory Site of an HSP 70 Gene. Cell 37: 273.


31
those afforded by native promoters of the normal proto
oncogenes. In vivo, the c-myc and c-erb-B-1 proto-oncogenes
present in several avian hematopoietic neoplasias have become
activated after adjacent integration of an avian leukosis
proviral DNA segment. This viral segment provides a strong
transcriptional promoter which replaces indigenous promoters
of these genes (83, 131).
A second mechanism of activation involves overexpression
due to amplification of the proto-oncogene (gene dosage
effects). The c-myc proto-oncogene is amplified 30-50 times
in HL-60 promyelocytic leukemia cells (32), and in a
neuroendocrinal tumor the the colon (1). A c-Ki-ras gene is
amplified 3-5 times in a human colon carcinoma cell line
(115), and 60 fold in an adrenocortical tumor of mice
(149). Human neuroblastomas were found to contain 30-100
copies of the N-myc gene (150). This was later confirmed,
and shown to be associated with patient survival (152). A
human chronic myelogenous leukemia cell line was discovered
to have multiple copies of the c-abl gene (33). In each of
these cases, gene dosage effects are thought to be
responsible for increases in transcript levels and gene
product.


21
Primitive
Uncommited
Mesenchymal
Cell
->
Endothelio-
blast
yofibroblast
Chondro-
Osteoblast
-> Endothelial
Cell
Pericyte
Smooth Muscle
>Chondrocyte
'Fibroblast
Lipoblast
Schwannoblast
Fibrocyte
> Lipocyte
Schwann Cell
Rhabdomyoblast^Rhabdomyocyte
Figure 1. Hypothetical model of mesenchymal differentiation as
proposed by Brooks. *
* Taken from Brooks, J.J. 1986. The Significance of Double
Phenotypic Patterns and Markers in Human Sarcomas. A New
Model of Mesenchymal Differentiation. Am. J. Pathol. 125:
113-123.


165
43. Derynck, R., A.B. Roberts, M.E. Winkler, E.Y. Chen,
and D.V. Goeddel. 1984. Human Transforming Growth
Factor Alpha: Precursor, Structure, and Expression
in E.Coli. Cell 38: 287.
44. Distel, R.J., R. Hyo-Sung, B. Rosen, D.L. Groves, and
B.M. Spiegelman. 1987. Nucleoprotein Complexes That
Regulate Gene Expression in Adipocyte Differentiation
Direct Participation of C-fos. Cell: 49: 835.
45. Doolittle, R.F., M.W. Hunkapiller, L.E. Hood, S.G.
DeVare, K.C. Robbins, S.A. Aaronson, and H.N.
Antoniades. 1983. Simian Sarcoma Virus One Gene
V-sis is Derived From the Gene Encoding a Platelet-
Derived Growth Factor. Science 221: 275.
46. Downward, J., Y. Yarden, E. Mayes, G.Scrace, N. Totty
P. Stockwell, A. Ullrich, J. Schlessinger, and M.D.
Waterfield. 1984. Close Similarity of Epidermal
Growth Factor Receptor and V-erb-B Oncogene Protein
Seguences. Nature 307: 521.
47. Dynan, W.S., and Tjian, R. 1983. The Promoter
Specific Transcription Factor SP 1 Binds to Upstream
Seguences in the SV 40 Early Promoter. Cell 35: 79
48. Dyson, P.J., T.D. Littlewood, A. Forster, and T.H.
Rabbits. 1985. Chromatin Structure of
Transcriptionally Active and Inactive Human C-myc
Alleles. EMBO J 4: 2885.
49. Eick, D., and G.W. Bornkamm 1986. Transcriptional
Arrest Within the First Exon is a Fast Control
Mechanism in C-myc Gene Expression. Nucleic
Acids Res. 14: 8331.
50. Ellerman, V., and O. Bang. 1908. Experimentelle
Leukamie Bei Huhnern. Centrallbl f. Bacteriol. 46:
595.
Emerson, B.M., and G. Felsenfeld. 1984. Specific
Factor Conferring Nuclease Hypersensitivity at the 5
End of the Chicken Adult Beta-Globin Gene. Proc.
Natl. Acad. Sci. U.S.A. 81: 95.
51.


138
2 HR
3 3 3-
3 HR
UG/ML DNASE I
3 3 3 m g
LOG PHASE
3 3 3 N §
M * ft*
*ft
Figure 23. contd.


Table 1. Continued.
Probe
Name
Source*
Use**
Reference
Method of
Labeling+
Description
Beta-
actin
7
2
75
P.E.
2.0 kb Bam HI
human cDNA
fragment
PMC 41
8
5
73
N.T.
1.6 kb Cla 1/
Eco RI human
genomic fragment
cloned into PBR
322
C-myc
Sca/Xho
I 9
6
1
P.E.
355 bp Sea 1/
Xho I human
genomic fragment
* Sources: 1)
Oncor,
Inc, Gaitersburg, MD,
2) Oncogene
Sciences, Minela, NY, 3) Dr. Harvey Bradshaw, 4)
Dr. Ken Soprano, 5) Dr. Carlo Croce, 6) American Type
Culture Collection, Rockville, MD, 7) Dr. Larry Kedes,
8) Dr. Robert Gallo, and 9) Made from c-myc plasmid
obtained from Oncor, Inc. (above)
** Uses: 1) Slot-blot hybridization, 2) northern blot
hybridization, 3) titration of c-myc gene copy number
in MFHs, 4) Mapping of c-myc amplicons in MFHs,
5) mapping DNAse I hypersensitive sites in cell lines
from the 3' direction, and 6) mapping DNAse I
hypersensitive sites in cell lines from the 5'
direction (fine mapping analysis).
+ Method of labeling: N.T. = nick translation, P.E. =
random primer extension.


147
actin are non-cell-cycle dependent. It has been reported
that actin transcript levels vary during stages of the cell
cycle (69). However, later studies with guiescent
fibrob"asts have shown that beta actin message levels were
consistent during all phases of the cell cycle after serum
release (175). These studies suggest that levels of actin
transcript production during phases of the cell cycle may
depend on cell type and culture conditions. Data reported
here indicates that actin levels are consistent during the
cell cycle in normal HFF fibroblasts. Therefore, actin was
used in normalization of results for slot-blot and northern
blot analyses.
These results for the v-erb-B-1 and v-src genes are
somewhat in agreement with those reported by Claycomb and
Lanson (27) and Leibovitch et al. (107) which show that c-
myc, c-Ha-ras, c-sis, c-src, and v-erb-B-1 transcripts are
present in skeletal muscle cells in culture while those of TK
and c-fos are not.
Transcript levels of cell cycle dependent proto
oncogenes and TK as well as non-cell-cycle dependent proto
oncogenes and actin were found to be detectable in all bone
marrow samples. It would be reasonable to suspect that all
of these proto-oncogenes including cell-cycle dependent genes


42
for PDGF have been found mainly on mesenchymal and glial
cells (165). In the case of virally transformed fibroblasts,
the v-sis sequences are fused to the env sequences of the
virus and this allows export of the abnormal PDGF-like
molecule to the membrane.
Abnormal expression of any mitogenic factor such as sis
may make it a possible candidate for a role in oncogenesis,
providing the cells which produce it have the appropriate
receptors. It is thought that high levels of sis expression
cause transformation, presumably by autocrine stimulation via
the PDGF receptor. The sis oncogene protein will be
discussed in further detail below.
Many tumor cells release transforming growth factors
(TGF). One class of these, TGF alpha is closely related in
sequence to EGF and interacts with the EGF receptor (43).
Other evidence suggests that TGF molecules function normally
as necessary mitogens for embryonic development (163, 179).
Inappropriate expression in adult cells could be a step in
transformation.
Nuclear Proteins


13
cancer in vivo. These types of studies have greatly
increased understanding of cancer cell biochemistry.
Unfortunately, many biochemical characteristics of cultured
cells are dissociable from their abilities to produce tumors
in animals (142). Furthermore, individual cells of
malignant tumors from animals and humans exhibit extensive
biochemical differences. These differences are reflected in
cell surface composition, enzyme levels, immunogenicity,
and response to cancer drugs.
Some general characteristics of transformed malignant
cells growing in culture include the following (142):
1. Histiologic characteristics of malignant cells in
vivo. The nuclei are increased in both size and number,
and there is a great deal of variation in the sizes and
and shapes of the cells. Also, there are increased
nuclear:cytoplasmic ratios, and the formation of
clusters of cells may be observed.
2. Differences in growth characteristics are common:
a. Transformed cells in culture are immortalized.
Malignant transformed cells can be passaged in
culture for an indefinite period of time,
b. Transformed cells tend to pile up in culture and are
not subject to contact inhibition seen with


108
Table 13. C-sis, v-erb-B-1, and v-src: actin ratios from
slot-blot analyses of genomic DNA from MFHs.
MFH SAMPLE
c-sis
v-erb-B-1
v-src
actin
actin
actin
MT-1
1.15
1.37
0.946
MT-2
1.22
0.851
0.784
MT-3
0.759
1.13
0.947
MT-4
1.27
0.943
0.860
MT-5
1.17
1.01
1.02
MT-6
1.40
0.946
1.07
MT-7
0.868
0.978
1.08
MT-8
1.02
0.987
0.674
MT-9
1.35
0.976
1.03
MT-10
0.830
1.41
1.36
MT-11
1.34
0.891
0.777
MT-12
0.789
1.29
1.28
MT-13
1.17
1.28
1.14
MT-14
1.30
1.23
1.24
MT-15
1.13
1.36
1.17
MT-16
1.20
0.881
1.03
MT-17
1.22
1.24
1.05
MT-18
1.10
1.11
1.08
MT-19
1.30
1.24
1.35
MT-2 0
1.25
1.16
1.27
MT-21
1.33
1.22
1.27
MT-2 2
1.28
1.28
1.24
MT-2 3
1.32
1.15
1.26


145
release, and during log phase growth had the same amounts of
c-myc protein as in GO. Protein levels in UR HCL 1 cells and
fibroblasts 1 hr after serum release (presumably G0/G1
transition) were twice this level (2), while ST 486 and P3C
cells had 5 and 6 times as much protein respectively.


12
transfected together, multiple foci of transformed cells were
obtained. These cells had the capabilities to grow very
rapidly in culture and seed tumors in nude mice. Acting
together, c-myc and c-Ha-ras could do what neither gene could
do on its own.
Additional evidence for multistep carcinogenesis is seen
with the retinoblastoma (rb) gene. Retinoblastoma is a
childhood ocular tumor which requires both alleles of the rb
gene to be mutated in order for the disease to occur.
Knudson (97) has proposed that the rb gene behaves as an
"anti-oncogene", in that one normal allele is sufficient to
protect against the disease. His "two hit" model for this
disease suggests that two mutagenic events occur at 13ql4
of chromosome 13. These two events can be in the form of 2
germline events, 1 germline and 1 somatic, or two somatic.
The rb gene is now thought to have an involvement in other
human malignancies including osteosarcoma (79) and mammary
carcinoma (56,80), and its activation provides an example of
multistep cancer in humans.
The Neoplastic Phenotype and Steps of Tumor Progression
Much effort has gone into comparing phenotypic
characteristics of in vitro transformed cells with those of