Proto-oncogene expression in human chondrosarcoma and malignant fibrous histiocytoma


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Proto-oncogene expression in human chondrosarcoma and malignant fibrous histiocytoma
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ix, 182 leaves : ill. ; 29 cm.
Gibson, Jane Carolyn Strandberg, 1962-
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Proto-Oncogenes   ( mesh )
Dermatofibroma   ( mesh )
Chondrosarcoma   ( mesh )
Pathology thesis Ph.D   ( mesh )
Dissertations, Academic -- Pathology -- UF   ( mesh )
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theses   ( marcgt )
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Thesis (Ph. D.)--University of Florida, 1989.
Includes bibliographical references (leaves 160-181).
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Also available online.
Statement of Responsibility:
by Jane Carolyn Strandberg Gibson.
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University of Florida
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Copyright 1989


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.


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


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.




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

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


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



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


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


These data suggest that increased levels of c-myc

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


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.


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


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


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

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


sites may offer clues to regulatory mechanisms involved in

proto-oncogene transcript production.


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



Endothelio- ---> Endothelial
blast Cell

/ 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:

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


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

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


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.


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


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


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


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


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


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


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


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


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


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


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


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


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


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





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



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


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,


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,


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

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



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








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

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


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



Table 1. Continued.

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


(pTK 11)





(H 25-3.8) 6





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


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

(HT .96)

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


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


PMC 41



Sca/Xho I 9


2.0 kb Bam HI
human cDNA

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

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
PO Pl P2


I- I-

o 0
* __ U


------PGEM H 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





- =- I, ~

H25-3.8 P380--A






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

C--FOS 6.4 KB

I '" '* -- --- I




"- I I


- !-

,I I 'IIi


PTK 11 1.25 KB

-I_ I-


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


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



& v 3
se ,

._ __.___ ,vv A 3'





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



PO P1 P2


SCA /)01 I

1 KB

805 BP


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.


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.


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