Nuclear proteins during growth and differentiation of mouse neuroblastoma cells

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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
        Page iv
    Table of Contents
        Page v
        Page vi
    List of Tables
        Page vii
    List of Figures
        Page viii
        Page ix
    Abstract
        Page x
        Page xi
    Chapter 1. Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
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    Chapter 2. Methods
        Page 33
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    Chapter 3. Results
        Page 45
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        Page 48
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        Page 50
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    Chapter 4. Discussion
        Page 106
        Page 107
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        Page 118
        Page 119
    References
        Page 120
        Page 121
        Page 122
        Page 123
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        Page 138
    Biographical sketch
        Page 139
        Page 140
        Page 141
        Page 142
        Page 143
Full Text















NUCLEAR PROTEINS DURING GROWTH AND DIFFERENTIATION OF
MOUSE NEUROBLASTOMA CELLS












By

MICHELLE LOUISE SIMON


A DISSERTATION PRESENTED TO TH1E GRADUATE COUNCIL
OF THE IUIVETSITY 0, FLORIDA
IN PARTIAL FULFILLIE" F TOE IREQUIRZxENTS FOR TYE
DEGREE Of DOCTOR OF PHILOSOPHY








UNIVEPSITY OF FLORIDA



















This dissertation, and the work it represents, is dedicated

lovingly to my parents, Maxine and Julius Simon, who have guided and

supported me with wisdom, love and patience.















ACKNOWLEDGMENTS

The author wishes to express her appreciation to Dr. Owen Rennert

for all his patient support, guidance, and wise counsel she has had

during her academic career at the University of Florida. The opportunity

to be accepted as a member of the GEM Division has been an exciting and

stimulating experience. She is grateful to all the students, staff and

faculty of the GEM Division for their interest and friendship.

She wishes to express sincere gratitude and appreciation to Drs.

Gary and Janet Stein for the opportunity to carry out the studies reported

here and for their guidance and help. She is very appreciative of all

the members of the laboratory, especially Dr. Judy Thompson and Ms. Jeudi

Davis, with whom many hours were spent working on scientific problems.

The author wishes to express her appreciation to the faculty and

staff of the Department of Neuroscience and especially to Dr. Frederick

King.

--- The author gratefully acknowledges the contributions of many faculty

and friends at the University of Florida, especially Dr. Carl Feldherr

and Dr. Kelly Selman for their interest and helpful discussions about

scientific philosophy and experimental design, and for their very real

aid with phase and electron microscopy;

--- Dr. Marieta Heaton for the chance to share an interest in, and

excitement about, developmental neurobiology;

--- Dr. William Luttge for his guidance and advice as Graduate Coordinator

and as a member of her academic advisement committee;











Dr. Robert King for his friendship and invaluable editorial assistance;

Ms. Oonagh Kater for her tireless hours of editing and typing, and for

her support through many last minute dashes to meet deadlines;

her husband, Dr. Steven Zornetzer, for his patience and support

through so much of this work;

and the Graduate Council of the University of Florida, the National

Institute of Mental Health, the Center for Neurobiological Sciences

of the University of Florida, and to the American Cancer Society,

for financial support.










TABLE OF CONTENTS

ACKNONLEDG4MENTS .................................................. iii

LIST OF TAZLES .................................................... vii

LIST OF F IC =' S ................................................... viii

ABSTRACT ............................................................ x

CHA"TER T INTRODUCTION ............................................. 1

Regulation of Genetic Tnformation ................................ 2
Regulation of Gene Expression in Eukaryotic Cells................ 3
Histones ......................................................... 4
Nonhistone Proteins .............................................. 5
Early Neuronal Maturation ........................................ 6
Gene Regulation in Nervous Tissue ................................ 10
Nuclear Protein Synthesis ........................................ 12
Brain Nuclei and Brain Chromatin ................................. 15
Neuroblastoma C1300 Cell Lines ................................... 22

CHAPTER TI YEefIDS .................................................. 33

Cell Culture ...................................................... 33
Cell Growth and Differentiation .................................. 33
Preparation of Nuclei and Chromatin .............................. 36
In Vitro Transcription of Chromatin .............................. 37
Extraction of Histones ........................................... 39
Metabolism of Nuclear Proteins ................................... 39
Polyacrylamide Gel Electrophoretic Fractionation of Chromatin
Proteins ...................................................... 40
DNA Synthesis in Mouse Neuroblastoma Cells ....................... 43

IA"TE iii PStLTS ................................................. 45

Morphology ....................................................... 45
Cell Nuber and Percent "Differentiation......................... 51
lodel of Crowth .................................................. 57
Tsolation of Nuclei and Chlroratin ................................ 60
Transcriptional Activity of Chromatin ............................65
Electroohoretic Fractionation of Nuclear and Chromatin Proteins..74
letabollsm of Tocal Chromatin Polypeptides ....................... 85
"fetabolism of Histones ........................................... 97
DNA Synthesis .................................................... 101

CNA"TER IV DISCUSSION ............................................... 106

Morphology and Growth ............................................ 106
Cell Nuver and Uifferentiation .................................. 108
Isolation of Nuclei and Chromatin ................................ 109
Transcriptional Activity of U- 'romatin ............................ il
Chromatin Proteins ............................................... 113


v











Metabolism of Chromatin Proteins ................................. 114
Post-translational Modifications of the Nonhistone Polypeptides..116
Post-translational Modifications of the Histone Polypeptides ..... 117
DNA Synthesis and Histone Synthesis .............................. 117
Summary .......................................................... 118

REFERENCES ........................................................... 120

BIOGRAPHICAL SKETCH ................................................... 139




















LIST OF TABLES


TABLE PAGE
No. No.

1 Nuclei from brain: mass ratios of protein and
RNA to DNA ................................................. 16

2 Brain chromatin: mass ratios of protein and
RNA to DNA ................................................. 17

3 Thermal stability of brain chromatin ....................... 18

4 Modification of Bodian silver protargol staining
technique .................................................. 35

5 Relative amounts of protein from intact nuclei ............. 64

6 Relative amounts of protein from chromatin ................. 66

7 Transcription assay ........................................ 77

8 Transcription assay ........................................ 78

9 Relative amounts of histone fractions in nuclei ............ 88

10 Relative amounts of protein and incorporation of
3H-tryptophan into nonhistones ............................. 92

11 Histone synthesis .......................................... 100

12 Acetylation of histones .................................... 103

13 Phosphorylation of histones ................................ 105









LIST OF FIGURES

Figure 1. Neuroblastoma cells in MEM with 10% FCS ................. 46

Figure 2. Neuroblastoma cells in MEM without FCS .................. 46

Figure 3. Neuroblastoma cells in MEM plus 0.5 m db cAM .......... 47

Figure 4. Neuroblastoma cells in MEM plus 1.0 mM db cAMP .......... 47

Figure 5. Silver stain of neuroblastoma cells in MEM plus 10% FCS..48

Figure 6. Silver stain of neuroblastoma cells in MEM without FCS...48

Figure 7. Silver stain of neuroblasstoa cells in MEM with 1.0 rmM
db cA T .................................................. 49

Figure 8. Silver stain of human diploid fibroblast ................. 49

Figure 9. SilVer stain of rat brain section ........................ 50

Figure 10. Clone ,2(f1) neuroblastoma cells .......................... 52

Figure 11. Clone 2(D1) neuroblastoma cells .......................... 52

Figure 12. Differentiation of neuroblastoma cells .................. 54

Figure 13. Differentiation of neuroblastoma cells .................. 55

Figure 14. Cell number over tire ................................... 56

Figure 15. Reversibility of "differentiation" ....................... 58

Fiue 16. Electron micrograph of material pelleted after cell lysis 61

Fgre 17. Higher power EM view of pelleted material ............... 62

Figure 18. Comparison of pattern of proteins from intact nuclei .... 69

Figure 19. Comparison of the pattern of proteins from chromatin .... 71

Figure 20. Comparison of the pattern of proteins prepared with and
without TPCK ............................................ 73

Figure 21. Ability of chronatin to support 14C-AP incorporation into
acid-insoluble material ................................. 76

igre 22. Migration of proteins on Bhorjee and Pederson gels ...... 77

Figure 23. Migration of proteins on Laemmli gels ................... 80

viii










Figure 24. Total nuclear proteins on Bhorjee and Pederson gels ...... 82

Figure 25. Chromatin proteins on Bhorjee and Pederson gels .......... 84

Figure 26. Fractionation of histones on Panyim and Chalkley gels .... 87

Figure 27. Incorporation of tryptophan into total chromatin proteins9l

Figure 28. Incorporation of tryptophan into total chromatin proteins94

Figure 29. Incorporation of acetate into chromatin proteins ......... 96

Figure 30. Incorporation of phosphate into chromatin proteins ....... 98

Figure 31. Incorporation of leucine into histones ................... 99

figure 32. Incorporation of acetate into histones .................. 102

Fi 33. Incorporation of phosphate into histones ................ 104
















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



NUCLEAR PROTEINS DURING GROWTH AND DIFFERENTATION
OF MOUSE NEUROBLASTOMA CELLS

by

Michelle Louise Simon

June, 1976

Chairman: Owen M. Rennert, M.D.
Major Department: Neuroscience

Mouse neuroblastoma cells in culture can be induced by several

methods to "differentiate" into cells which exhibit morphological, bio-

chesiical and electrophysiologic properties of neurons. In an attempt to

develop a model for the understanding of the regulation of gene expression

which must occur during the process of "differentiation,' the composition

and transcriptional capacity of chromatin and the metabolism of nuclear

histones and nonhistones were compared in nuclei isolated from mouse

neuroblastoma 1300 cells grown under different conditions. Serum with-

drawal or addition of 1 mM dibutyryl cAMP resulted in reversible morpho-

logic "differentiation" in 70 to 90% of the cells, although cell number

continued to increase in cultures exposed to dibutyryl cAMP. Chromatin

isolated from purified nuclei was transcribed in a cell-free RNA synthe-

sizing system using E. coll RNA polymerase. Chromatin from cells grown

in serum exhibited a significant increase in template activity over that

observed in chromatin from "differentiated" cells. The synthesis,










acetylation, and phosphorylation of histones and nonhistones were ex-

amined by pulse-labelling cells for 60 minutes with appropriate pre-

cursors. Chromosomal proteins were analyzed on SDS- and acetic acid-

urea polyacrylamide gels. Differences in the synthesis of chromosomal

proteins (histones and nonhistones) from cells grown under different

conditions were observed. Significant differences were not detectable

in acetylation or phosphorylation of the chromosomal polypeptides. A

functional role for the observed variations in chromosomal proteins

which accompany mouse neuroblastoma cell "differentiation" is discussed.



































Chairman















CHAPTER I

INTRODUCTION

The numerous types of cells which exist in the mature multicellular

organism are thought to result from the differential expression of

information contained in the genetic material of the cells. Each cell

in the organism contains at least one complete set of genes, yet the

alterations in the morphological and functional properties of the cells

are the ultimate product of the expression of only a portion of the

genome. The progressive alterations in the biochemical makeup of cells

during their development are known collectively as cellular differentia-

tion and are the result of complex interactions of the nucleus, the

cytoplasm, and the environment of the individual cell. Mouse neuroblastoma

cells in in vitro culture show alterations in morphological and bio-

chemical characteristics which are similar to those described for de-

veloping neurons. These cells serve as a useful model to investigate

the nuclear events involved in the differential expression of genetic

information.

The establishment of the differentiated state is often accompanied

by a decreased capacity for cellular proliferation. This relationship,

however, varies with respect to its application to different tissues and

cell types. In some tissues only limited groups of "stem" cells can

divide and replace or add to the number of cells of the tissue. The

stem cells themselves are differentiated in the sense that they are

specialized to divide and produce a limited number of types of progeny.











The central nervous system of vertebrates, in fact, shows the most

complex degree of specialization of cell morphology and function of all

eukaryote tissues. The cellular components are all derivatives of the

neuroectoderm and its subsequent subdivisions, the neural crest and the

neural tube. Yet, each neuron is essentially unique in its position in

space and its relationship to other cells, both by direct contact with

cells in its immediate environment and via neurohormones with cells in

distant regions of the nervous system. Correlated with such special-

ization is an extreme limitation in the ability of the neuron to recover

from injury or to proliferate. How does such specialization of morphology

and function result from the developmental processes involved in the

maturation of the nervous system? How do cells use the information in

their environment and in their genetic material to develop a defined

cell shape, specific contacts, and specific products, such as neuro-

transmitters and neurohormones? Such activities reflect the highly

organized utilization of the information contained in the genome of the

cell via mechanisms common to all cell types which result in the ultimate

synthesis of specific enzym-Is and structural proteins.
Regulation of Genetic Information

The regulation of the expression of genetic information in eukaryotic

cells may occur at several stages in the progression of the transcription

of the genes encoded in DNA into RNA, the processing and transport of

IA from the nucleus into the cytoplasm, and the translation of the mRNA

into specific proteins in the cytoplasm. Although control may be exerted

in mammalian cells at all levels, we will focus on a discussion of re-

gulation of gene expression at the transcriptional level and review










studies on nervous tissue which describe early neuronal differentiation

and biochemical studies of nuclear proteins. An in vitro system which

has provided much information about cellular and biochemical processes

occurring in neuron-like cells will be described and the results of

studies of the nuclear proteins of these neuroblastoma cells will be

presented.

Regulation of Gene Expression in Eukaryotic Cells

The genetic material in eukaryotic cells, coded in DNA, exists in

the interphase nucleus, as seen with the light and electron microscopes,

as dispersed chromosomal material--a heterogeneous collection of fibrils

and dense material. The more dense regions have been called heterochro-

matin and are thought to represent tightly coiled, inactive regions of

the genome. The less dense regions, known as euchroatin, are thought

to represent regions of the DNA that are actively being transcribed.

These same terms, heterochromatin and euchromatin, have been applied to

various fractions of material composed of nucleic acids and protein

which have been extracted by several biochemical methods from cell

nuclei. The wide usage of the term chromatinn" to describe such frac-

tions implies that such material, in fact, represents the chromosomes.

Actually, it is an operational definition, referring to whatever material

has been extracted from the cells. Although the material may reflect

the composition of genetic material in vivo, caution should be exercised

in interpreting data from studies in which different methods of isolation

of chomatin are employed.

Information from numerous studies on biochemically defined chromatin

suggests that the proteins associated with the DNA are involved intimately

in both the maintenance and establishment of the structure of the genetic











material and in regulating the "readout" or transcription of specific

genes. The nuclear proteins fall into two major categories: histones

and nonhistones, or acidic proteins. The composition, distribution and

suggested role of both classes of proteins have been extensively reviewed

recently and only a brief summary will be presented (Stellwagen and

Cole, 1969; DeLange and Smith, 1971; Phillips, 1971; Spelsberg et al.,

1972b; Elgin and Weintraub, 1975; Stein et al., 1974, Stein et al., 1976

(submitted for publication); Cameron and Jeter, 1974; and MacGillivray

and Rickwood, 1974).

Histones

The histones are a defined group of acid-extractable proteins of

relatively small molecular weight (about 11,000 to 22,000 for monomeric

forns) which are present in the somatic cell nuclei of all multicellular

organisms. In general they contain large amounts of basic amino acids

(arginine and lysine) and lack tryptophan, and cysteine (found only in

H3 histone). They are extremely stable and carry a net positive charge

at pH 8. The amino acid sequence and structure of the histone subclasses

are amazingly well conserved throughout the plant and animal kingdoms.

Each of the five subclasses of histones shows a skewed arrangement of

amino acid residues such that the basic amino acids are found in clusters.

This asymmetry is apparently involved in the mode of interaction of the

specific histones with one another and with other components of the

genome, including the DNA and nonhistone proteins.

In addition to slight differences in amino acid sequence, histcnes

are among the most highly modified proteins. Post-translational modifi-

cations involve the covalent addition or removal of acetyl, phosphate or

methyl groups or the modification of the sulfur group in cysteine.








These post-synthetic modifications of the histone polypeptides have been

implicated in structural and functional alterations in the genome. Such

modifications would lead to alterations in histone-DNA, histone-histone,

and histone-nonhistone interactions.

Involvement of histones in the regulation of gene expression has

been suggested since the 1940's. However, the marked conservation of

histone structure throughout tissues and species, the lack of tissue

specificity with respect to the distribution of histones, and their

limited heterogeneity suggest that histones by themselves do not regulate

defined gene loci. The major type of evidence linking histones to gene

regulation involves their role in the structural organization of the

genome and their ability to inhibit DNA-dependent RNA synthesis--i.e.,

to function as structural and repressor macromolecules. Early studies

showed that increasing amounts of histones complexed with DNA resulted

in a progressive decrease in DNA-dependent RNA synthesis. In fact,

transcription is completely inhibited when the template consists of DNA

and histone in a 1:1 ratio (Huang and Bonner, 1962; Allfray et al.,

1963). Consistent with this, histone synthesis in proliferating systems

has been found to be tightly coupled to DNA synthesis and occurs only

during a restricted period of the cell cycle both in cells in vivo and

in vitro.

Nonhistone Proteins

In contrast, the nonhistone proteins represent a far more hetero-

geneous class of proteins which are synthesized throughout the cell

cycle. They do exhibit species and tissue specificity and differ in

their distribution and metabolism in the same cell type throughout the

cell cycle and functional state of their tissue of origin. These proteins










as a group contain relatively large quantities of acidic amino acids. A

few are extractable in dilute mineral acid along with the histones. The

class of nonhistone proteins includes polypeptides which function as

structural, regulatory and enzymatic macromolecules. The evidence for

nonhistone proteins playing a specific role in the regulation of gene

activity includes their ability to confer specificity on the trans-

criptional capacities (quantitative and qualitative) of isolated DNA

plus histone material ("reconstituted chromatin"). When chromatin is

reconstituted by mixing DNA plus histone plus nonhistone protein fractions,

the specificity of the RNA which is recovered from the assay system is

dependent on the source of the nonhistone protein fraction. For example,

when chromatin is reconstituted from tissue A DNA and histone and tissue

B nonhistone fractions, the RNA is similar to RNA transcribed from

tissue B chromatin (Gilmour and Paul, 1970; Spelsberg and Hnilica, 1970;

Stein and Farber, 1972).

Post-translational modifications of nonhistone proteins have also

been implicated in the selective binding of nonhistone chromosomal

proteins to homologous DNA.

Several systems are being intensively studied in order to elucidate

the role of the chromosomal proteins in the regulation of the readout of

specific gene sequences, which include erythropoietic systems and the

transcriptional regulation of the globin gene (Paul et al., 1973), HeLa

cells and W138 fibroblasts and the regulation of the histone genes

(Stein et al., 1975), and chick oviduct cells and the regulation of the

ovalbumiu gene (0'Malley and Schrader, 1976).

Early Neuronal Maturation

Most of the large neurons of the central nervous system arise from








the outer region of the wall of the neural tube known as the primary

germinal centers (Phelps and Pfeiffer, 1975). The neural tube itself

originates as an invagination of the neural ectoderm or neural plate

very early in development (Karfunkel, 1974; Burnside and Jacobson, 1968;

Schroeder, 1970). Cell shape changes, alterations in cellular adhe-

sivity, and regional proliferation have been described as participating

in neurulation and neuronal differentiation. The underlying molecular

events in such developmental processes involve recognition of changes in

the external environment of the cells by the cell membrane and its

constituents and the translocation of signals to the nucleus to alter or

initiate the production of mRNAs which code for components which can in

turn result in changes in cell shape and so on. In order to study such

events a relatively homogeneous population of cells is required. One of

the greatest barriers to such research in nervous tissue in vivo is

the ability to obtain sufficient material to analyze.

We will describe the major early changes in morphology of neuro-

blasts as they leave the mitotic population of the neural tube and then

review briefly what is known about biochemical differentiation. Cowdry

(1914) reported on an intensive, light-microscopic study, using several

histologic stains, of developing components of the neural tube of the

chick embryo. He reported that the first observable alteration in the

cytoplasmic constituents of neural tube cells involved the appearance of

neurofibrils (which show up after silver impregnation of tissue) in the

cytoplasm of cells at the outer margin of the neural tube. These neuro-

fibrils formed first in a definite restricted zone of the cytoplasm on

the side of the nucleus away from the lumen of the neural tube. Nissl

substance, intensely basophilic material arranged in clumps, appeared










much later. Mitochondria were numerous in the peripheral regions of the

cells.

More recent studies utilizing both light and electron microscopy

(Bellairs, 1959; Fujita and Fujita, 1963; Eschner and Glees, 1963;

Lyser, 1964; Meller et al., 1966; and Decker, 1974) confirm the late

appearance of Nissl substance and indicate that it is the light micros-

copic counterpart of the rough endoplasmic reticulum which becomes

organized into arrays of ribosome-studdedemembranes as differentiation

progresses. Early changes in the maturing neuroblasts include the

decrease in yolk droplets present in the cytoplasm, increased numbers of

mitochondria (especially in elongating regions of the cells), and appear-

ance of feltworks of filaments (60-130 A in diameter). Correlations

made between light microscopic, silver-impregnated material and electron

microscopic studies, suggest that filaments may correspond to silver-

staining material. In fact, silver granulations in the cytoplasm on the

side of the nucleus away from the neural tube lumen are the earliest

changes which have been reported. Neurofibrils appear later.

Cells develop a long external process whose distalmost end appears

identical to growth cones described in slightly older material and in

tissue culture studies. As differentiation progresses, the cell becomes

monopolar as its internal process is withdrawn. The nucleus becomes

more uneven in texture and granular endoplasmic reticulum becomes more

extensive. The Golgi material seems to become aggregated and organized

around the nucleus. Centrioles may or may not be present. Secondarily,

the cells become bipolar as a dendrite-like process which is morpho-

logically indistinguishable from the initial axon-like process develops.

Nissl substance appears simultaneously with the development of bi- and










multipolarity. No endings are yet recognizable on either cell bodies or

developing processes.

As mentioned earlier, subsequent development varies from region to

region. Forest (1969) and Peusner (1974) have described the development

of neurons or "post-mitotic neuroblasts" in the brainstem, Hinds (1968a,

b) in the olfactory bulb, Rakic (1973) in the cerebellum, and Berry and

Rogers (1965), and Butler and Caley (1972) in the cerebral cortex.

Jacobson (1974), in an interesting essay, has suggested that all

neurons may be considered to belong to one of two major subpopulations.

One of these, usually composed of larger neuron types, seems to exhibit

invariant morphology and connectivity, and the other seems to be more

flexible in its relationships. He suggests that this dichotomy reflects

basic differences in the regulation of genetic information to produce

constant phenotypes in the larger neurons and more variable ones in the

smaller neurons as a result of the latter cell population's ability to

respond more readily to alterations in the cellular environment. This

is probably a gross oversimplification of regulatory mechanisms, but

perhaps sets up a framework within which to compare regulatory events in

neurons.

Few studies utilizing direct biochemical assays have been reported

for the early nervous system. Cytospectrometric work has indicated that

quantities of RNA increase steadily with development for the first five

days in the cells of the neural tube and then fall at different rates in

dorsal (sensory) and ventral (motor) regions (Hughes, 1955). Cells

taken from chick embryos at 7 and 13 days have been studied for changes

in energy metabolism as reflected by oxygen uptake, lactic acid pro-

duction and content of ATP (Dittmann et al., 1973). The rate of oxygen












uptake per cell doubled over the time studied and lactic acid production

of the cell decreased. ATP per cell remained unchanged, as assayed by

the luciferase method. The bulk of other studies on the developing

nervous system which attempt to identify specific changes in cell

proteins--enzymic and structural--utilize histochemical and immunologic

techniques and are usually carried out on much older tissue. For example,

acetylcholine esterase (AChE) and choline acetyltransferase (CAT) ac-

tivities increase in parallel with morphologic differentiation of neuronal

processes (Kim et al., 1974). Histochemical appearance of the enzymes

was evident slightly later than the biochemical enzyme assays indicated.

AChE appeared earlier than CAT, whose appearance corresponded to the

development of synaptic components as determined with the electron

microscope.

Little information is available in the literature which identifies

the biochemical substrate of silver-staining (Sechrist, 1969) which has

been suggested to represent the earliest evidence of neuronal differ-

entiation, rather than the increase in endoplastic reticulum suggested

by Fujita and Fujita (1963), or the accumulation of neurofibrillary

material (lyser, 1964) as the axonal process begins to develop. Sechrist,

in fact, claims that such evidence of neuron-specific differentiation is

present in prenitotic cells in the neural tube.

Gene Regulation in Nervous Tissue

We have suggested above that nervous tissue offers a unique oppor-

tunity to study the regulation and diversity of developmental processes

within a single tissue with limited cellular proliferation and numerous

specific gene products. The major types of studies which have been re-

ported to date involve the differences in various regions of the brain










of the neurotransmitter enzyme systems with age or with experience or

hormonal treatment. No direct investigations of the regulation of gene

expression have been conducted, although studies have been made of the

qualitative and quantitative composition of cytoplasmic proteins and of

brain nuclei and brain chromatin. Transcriptional activity of brain

chromatin has also been investigated. However, the heterogeneity of the

material used in such studies prevents an unambiguous interpretation of

the data.

Studies of brain chromatin rely heavily on methods of isolation of

cell nuclei. Most published studies have started with homogenates of

whole brain or of relatively large regions of brain. The complex struc-

ture of nervous tissue which consists of interdigitating cells of several

size classes and degrees of branching, of cells joined by several types

of specialized junctions, and satellite-type cells which do possess the

capacity for movement and proliferation in addition to the more stable

neuronal populations, introduces further complications and difficulties

in both the design of experiments and the interpretation of data. In

addition, attempts have been made to fractionate cell types or nuclear

types prior to further characterization. A major drawback in all such

studies is the lack of a reliable means of accurately identifying the

composition of each fraction.

Studies of nervous tissue compare the attributes of various cell

types, based on morphologic criteria such as large nuclei ("neuronal")

and small nuclei ("glial"). Cursory inspection of histological prepar-

ations of nervous tissue reveals the vast heterogeneity of cell (and

nuclear) sizes. Hand dissection of neurons (Hyden and McEwen, 1966) has

limited usefulness because of technical complexity, limited amounts









of material and sampling problems (only the largest neurons can be

obtained). In non-mammalian vertebrates, there are populations of

extremely large neurons which can be studied reliably in this fashion

(Edstrom et al., 1969; Edstrom & Sjostrand, 1969).

Several bulk separation techniques have been applied to nervous

tissue in attempts to separate cell types and isolated nuclear popu-

lations (Freysz et al., 1968; Satake et al., 1968; Rose, 1968; Rose and

Sinha, 1969; Johnson and Sellinger, 1971; Sellinger et al., 1971;

McEwen et al., 1972; McEwen and Zigmond, 1972; and Hadjiolov et al.,

1965). However, Dounce's observation that "no single method at present

is available for the isolation of all nuclei ..., in spite of implications

by some investigators to the contrary" (1963, p. 127), is certainly

still true and definitely applicable to nervous tissue.

Nuclear Protein Synthesis

Although protein synthesis is thought to occur exclusively in the

cytoplasm, nuclear protein synthesis has been reported for several cell

types, including neurons. Allfrey et al. (1964) reported that nuclei of

thymocytes are capable of synthesizing protein by ribosome-dependent

mechanisms which require amino acid activating enzymes, endogenous ATP,

and soluble and stable RNA. This system was reported to differ from

cytoplasmic protein synthesis in that it was resistant to RNase and

required sodium, rather than potassium.

Burdman and Journey (1969) identified a similar system operating in

nuclei isolated from rat brain. Incorporation of leucine was linear for

60 minutes, after which time the preparations showed "loss of stability."

Both "neuronal" and "glial" fractions, separated on sucrose density

gradients (L~vtrup-Rein and McEwen, 1966), incorporated leucine to dif-

ferent extents. In these nuclei, incorporation of amino acid decreased










with increased sodium and was inhibited by both puromycin and cyclohex-

imide.

Two further studies of this phenomenon (Burdman et al., 1970;

Burdman, 1972b) surveyed the incorporation of amino acid in vivo and in

vitro into various fractions of proteins of the nuclei. These protein

fractions were extracted with various concentrations of NaCI from intact

nuclei and included "saline soluble," "residual acidic," "chromatin

acidic," and histone fractions. The histones were found to be almost

inert with respect to in vitro incorporation of leucine. "Residual

proteins" were the most rapidly labelled, and "chromatin acidic proteins"

had the highest acitivity. The ability of nuclei to incorporate such

radioactivity decreased with age. Nuclei from adult animals had only

10% of the activity of nuclei from three-day-old rats. In contrast,

when labelled leucine was administered to intact animals and brains were

removed at various times up to four hours later, the time course of

incorporation was similar in all age groups. Rates of incorporation

could nut be compared across ages since the amounts of label which were

administered varied. This system was inhibited by hypertonic sucrose

and required sodium only for some amino acids (not for valine, leucine,

alanine or threonine). Actinomycin D had only a slight inhibitory

effect and chloramphenicol had none.

Information from the age studies suggests that the purity of the

nuclear fractions may have been different. Three-day-old rats have very

little myelin, yet adult animals have large amounts, and this alone

might be expected to alter the extraction efficiency. No mention is

ever made of this problem in development studies.

Goldstein (1970) reviewed the evidence for nuclear protein synthesis










for many cell types. The major problems involved in most of the studies,

protein migration from cytoplasm to nucleus and problems in nuclear

isolation techniques, render conclusions about the possibility of nuclear

protein synthesis impossible. Only two cell types in addition to neurons

yield plausible data: isolated nuclei of thymocytes and pigeon erythro-

cytes. Both of these cell types have been considered atypical. The

thymocyte has an unusually large nucleus with a small volume of cytoplasm

and can produce ATP from glycolysis, the TCA cycle, and oxidative phos-

phorylation in the nucleus. It is involved in the immunologic recognition

and defense system of the body and possesses a remarkable ability for

specific differentiation. Perhaps the neuron, too, represents an "atypical"

cell.

However, a study by Dravid and Wong (1972) may offer an explanation

of neuronal "nuclear" protein synthesis. These authors noted that when

sterile techniques were used, the amount of incorporation of amino acid

decreased significantly. The incorporation measured in previous studies,

therefore, was probably due to bacterial contamination. Caution must

therefore be exerted in interpreting results of in vitro incubation

procedures which do not use aseptic techniques.

In spite of this study emphasizing the importance of sterile tech-

niques, no reference is made to this work in the later studies of

Burdman and coworkers (Burdman, 1972 a, b; Burdman et al., 1973; Szijan

and Burdman, 1973 a, b; 1974). A recent study of nuclear protein syn-

thesis following in vitro incorporation of isolated nuclei from brain

(Fleischer-Lambropoulos and Reinsch, 1975) made no mention of the use of

sterile techniques or of possible contamination of nuclear fractions

either with bacteria or with cytoplasmic components.










In summary, no definitive evidence yet exists which suggests that

neurons differ from other cell types in their dependence on the cytoplasm

as the site of protein synthesis, including proteins which may be in-

volved in the regulation of nuclear events.

Brain Nuclei and Brain Chromatin

Studies which were directly designed to study characteristics of

the genome and its regulation in brain tissue can be grouped into several

categories: 1) composition and properties of isolated chromatin and

nuclei from brain (see Tables 1-3); 2) differences in the spectrum of

nuclear proteins in brain versus other tissues; 3) age-related changes

in brain nuclear proteins; 4) region-related differences in nuclear

proteins of brain; 5) experience- and sex-related differences in such

proteins; 6) alterations in RNA synthesis (template activity or hybrid-

ization properties) with age or regional differences; and 7) hormonally

induced events in the nucleus.

Interpretation of the results of almost all the studies is limited

by problems resulting from the use of whole brain homogenates (or homo-

genates of large regions of brain) as a source of nuclei or chromatin.

Numerous cell types contribute to such fractions. Reflections of such

heterogeneity can be seen in the range of values which have been reported

for various measurements made on "clean" nuclear preparations (Table 1)

or on chromatinn" (Table 2). These problems are further compounded by

the variations in methodology used to prepare such fractions and to

separate out the various groups of proteins. No well-designed studies

have yet systematically compared the major methods in use to determine

ways to compare such results. Because of these problems, it is most

useful to summarize such studies of brain nuclear proteins in a very






TABLE I

Nuclei from brain: mass ratios of protein and RNA to DNA

Source Protein/DNA Histone/DNA Nonhistone/DNA RNA/DNA Reference

Adult cat cortex 0.26-0.37 0.50-0.52 Hadjiolov et al., 1965

Rat cerebellum 2.2 0.70 1.63, 0.23 0.011 McEwen et al., 1972
other 4.8
hippocampus 0.86 2.54, 1.56 0.18
med.+ post. ctx. 1.05 2.64, 1.99 0.19
amygdala + ctx. 0.99 2.49, 2.29 0.20
Rat brain Austoker et al., 1972
*Fraction I 5.20 0.60
II 5.14 0.66
I1 4.32 0.49
IV 2.47 0.36
V 2.40 0.32

Rat brain 2.2-7.5 0.08-0.45 McEwen and Zigmond, 1972

Rat brain 2.5-3.5 Dravid and Wong, 1972

Rat brain 3.24 Duerre and Gaitonde, 1971

Rat brain 5.10 Piha and Jokela, 1972

Rat cortex 3.12 Fujitani and Holoubek, 1974
cerebellum 2.20
rest 2.73

Rat hippocampus 5.38 0.292 Fleischer-Lambropoulos and
cortex 5.01 0.263 Reinsch, 1975
medulla 4.21 0.204
cerebellum 2.97 0.105
rest 4.47 0.249

*These fractions have different compositions with respect to size groupings of nuclei.








TABLE

Brain chromatin" mass ratios of protein and RNA

Source Protein/DNA Histone/DNA

Chicken embryo-4 day 2.8-3.2 0.90-0.91
8 day 2.9-3.0 0.88-0.99
1.6-2.1 0.78-0.93
Adult hen 2.7-3.1 0.76-0.80
1.8 0.72


2

to DNA
Nonhistone/DNA


NNAi/DNA

0.28-0.30
0.19-0.24
0.08-0.12
0.17-0.19
0.08


Reference

Dingman and Sporn, 1964


Mouse

Chicken embryo-li day

Pig cerebellum
pituitary

Rat large nuclei
small


1.8-4.5

3.45


(1.04-1.48)

0.50
0.45


0.13
0.108


Kurtz and Sinex, 1967

Graziano amd Huang, 1971

Shaw and Huang, 1970


0lpe et al., 1972


Rat

Newborn rat


1.66-2.54


1.42 0.84

0.85-1.14 0.81-1.46
2.31


Rat cortex
cerebellum
rest

Ox cortex

Rat

Rat

Female rat-neuronal
glial


0.06-0.10

0.07-0.08


Duerre and Gaitonde, 1971

Bondy and Roberts, 1969


Fujitani and Holoubek, 1974



0.09 Singh and Sung, 1972

Piha and Jokela, 1972

Duerre and Lee, 1974

Fleischer-Lambropoulos et
al., 1974






























TABLE 3

Thernal stability of brain chromatic

Source Tm Tm Standard DNA Reference


Chicken

Rat cerebellum
pituitary


76-79

76.5
77.1

77


41-42

43


Dingman and Sporn, 1964

Shaw and Huang, 1970


Bondy and Roberts, 1969











general fashion. Studies involving interactions of hormones with nervous

tissue most nearly approach the ideal situation.

In general, histones of brain are identical to those of various

other tissues. All the histone classes are present and vary very little

across different regions of the brain or with age (Dingman and Sporn,

1964; Bekhor et al., 1964; and Duerre and Gaitonde, 1971). They appear

to be the most stable components of brain nuclei--and, in fact, are

almost inert with respect to new synthesis or turnover in both short and

long term studies (Piha et al., 1966; Burdman et al., 1970, 1973;

Bondy, 1971; Burdman, 1972). This is consistent with the tight coupling

of histone synthesis with DNA synthesis which is thought to occur in all

tissues of multicellular organisms. Relatively little DNA synthesis

and/or cell division occurs in mature nervous tissue. Enzymes which

modify histones, such as acetylases (Caspary and Sewell, 1968; Bondy et

al., 1970) and methylases (Miyake and Kakimoto, 1973; Duerre and Lee,

1974), have been described for brain.

The nonhistone proteins of brain, in contrast, appear to differ in

their distribution on polyacrylamide gels from those of various other

tissues of the body (Dingman and Sporn, 1964; Bekhor et al., 1964;

MacGillivray et al., 1972; Uyemura, 1974; and Elgin, 1972) to varying

degrees depending on the methods used to isolate and fractionate them.

Although Burdman and coworkers claim that these proteins, as well as the

histones, can be synthesized in isolated brain cell nuclei, it is more

likely that they are synthesized in the cytoplasm and then migrate into

the nucleus (see the previous section on nuclear protein synthesis).

The nonhistone proteins as a group appear to turn over much more rapidly

than do the histones (Burdman et al., 1973) and are very heterogenous










(Graziano and luang, 1971; Piha and Jokela, 1972; Jokela and Piha, 1972;

Davis et al., 1972; Olpe etil., 1972, 1973; Kohl et al., 1973). When

comparisons are made between nonhistone proteins from young animals

(usually within a few days of birth as the earliest age examined) and

from adults, some differences are seen both in the spectrum of proteins

present and in their rates of labelling following in vivo injections of

isotopically labelled amino acids (Kurtz and Sinex, 1967; Dravid and

Burdman, 1968; Shaw and Huang, 1970; Szijan and Burdman, 1973b; and

Biessmann and Rajewsky, 1975). These differences have been related to

the various degrees of development or activity of the brain regions

involved; however such results should be looked at much more closely.

Many components of nervous tissue, especially non-neuronal ones gliaa,

blood vessels, choroid, and meninges), vary with age and may affect

isolation and fractionation procedures.

It has also been suggested that alterations in the composition of

nonhistone proteins (or in post-translational modifications of them) may

reflect differences in neurons versus glial cells (Fleischer-Lambropoulos

et al., 1974) in amount or type of experience (Glassman et al., 1972) or

in sex of the animals (Yu, 1975). Although Yu found no differences in

several regions of brain in the histones or nonhistone proteins among

male, female or neonatally androgenized female rats, it was suggested

that such differences do, in fact, exist but the capacity of the gel

system or of the extraction procedure to detect such differeneces was

insufficient.

Nonhiztone nuclear proteins of brain include various types of

enzymes, such as RNA and DNA polymerases (Singh and Sung, 1972; Szijan

and Burdman, 1974) as in other tissues (Elgin at al, 1971).










DNA-dependent RNA synthesis in brain, using either endogenous polymerase

or E. coli polymerase, has been reported to be both higher and lower

than in other tissues (Dingman and Sporn, 1964; Bondy and Roberts, 1969;

Smith et al., 1969; Austoker etal., 1972; Singh and Sung, 1972; Burdman

et al., 1973). These values, if lower than other tissues, are inter-

preted to reflect the non-proliferating nature of nervous tissue; and,

if higher than other tissue, to reflect the greater number of genes or

more active genome being expressed in nervous tissue. The differences

are interpreted therefore to reflect functional differences in the

chromatin.

Most preparations of chromatin from cerebellum seem to differ

significantly in content and transcriptional properties from other

regions of the brain (see Tables land 2). This has been interpreted to

reflect the less active gene expression in this region of the brain,

perhaps because the preparations are more homogeneous. Isolated nuclear

fractions from cerebellum seem to be made up mostly of small-sized

nuclei, perhaps from granule cells which are numerous and densely packed

in this region of brain.

Hormones are thought to act very specifically in certain areas of

the brain to influence developmental and metabolic events which are

reflected in behavioral and sexual differences in animals. Several

laboratories are investigating the steroid binding properties of the

hippocampus and hypothalamus (McEwen et al., 1972; Chytil and Toft,

1972; and Grossner et al., 1973). It is thought that such hormones are

taken up specifically by certain cells, bound by cytoplasmic receptor

molecule(s), and transferred to the nucles where they interact with

DNA-associated proteins (nonhistones) to selectively alter gene expression.










Alterations in RNA and protein synthesis have also been reported following

hormome administration (Garfield and Moscona, 1973) to chick neural

retina. Studies in non-neural tissues include those of Mauer and Chalkley,

1967; O'Malley et al., 1968; Cox and Carey, 1971; and Comstock et al.,

1972). Other studies in non-neural tissue, such as the chick oviduct,

suggest that the hormones interact selectively with nonhistones to

initiate changes (Tucker et al., 1971; Spelsberg et al., 1972a; Stein et

al., 1974; and O'Malley and Means, 1974).

Studies of pituitary cells in culture (Watanabe et al., 1973 a, b)

have demonstrated similar effects of steroids on the ACTH-secreting

capacity of these cells. Specific hormone receptor molecules are being

sought. These studies, using a cultured tumor cell line, are particu-

larly interesting since they circumvent many of the problems we have

mentioned which occur in the use of intact brain tissue.

Neuroblastoma C1300 Cell Lines

In addition to the neural retina and pituitary cells in vitro

mentioned in the previous section, several continuous lines of nervous

system-like cells have been developed, mainly from mouse tumors. These

cell lines are designated glioma or glioblastoma and neuroblastoma to

indicate their similarity to and probable origin from glial and neuronal

cells, respectively. These cell lines have been extensively studied and

compared to normal nervous tissue (see McMorris et al., 1973; Sato,

1973; and McMorris and Ruddle, 1974), and have "helped bring the tech-

nology of molecular and cellular biology to the neurosciences" (Haffke

and Seeds, 1975, p. 1655).

The origin of the original mouse tumcr from which the cultured

neuroblastoma cell lines have been derived, and with which we will be











concerned, is somewhat obscure. The earliest reference to the C1300

mouse tumor in the literature appears to be that of Corer (1947) who

used the tumor in a study of the antibody response in mice to inoculated

tumor cells. The C1300 tumor was described as a "round cell tumor,

possibly a neuroblastoma." Additional early references to the C1300

tumor are useful to review since they describe the growth character-

istics of the tumor in vivo and the apparent lack of knowledge of the

origin of the tumor--in spite of which it has been readily accepted as a

neuronal model system and often reported to be of neuronal origin by

recent investigators.

Snell et al., (1948) described the C1300 as a "neurogenic sarcoma,

A strain origin." Eichwald and coworkers (Eichwald et al., 1950; Eichwald

and Chang, 1951) transplanted the tumor into the anterior chamber of the

eye of C57Brown and ABC mice and observed different degrees of metastases

in the different host strains. Moore (1951) reported that the C1300

tumor arose in the abdominal cavity of an A strain mouse and when trans-

ferred into homologous strains "took" 100% of the time. Tumors became

palpable in the new host animals in seven days and grew until the host

died at four to five weeks. The tumor contained closely packed round

cells. This tumor was also noted to be the most susceptible of the five

tumor types tested to the Russian Far East encephalitic virus.

Klein (1951) reported on an extensive study of mouse tumors and

stated that tumor C1300 was found by Dr. Clondman at the Jackson Labora-

tory in 1940 as "a huge irregular red and white tumor filling the ab-

dominal cavity of an A strain mouse." In these studies the tumor was

transplanted subcutaneously and appeared as a diffusely growing anaplastic

tumor with mainly polyhedral and round cells. After interperitoneal










inoculations, huge solid tumors developed and the hosts survived for a

median time of twenty days. The tumor was unique among those tested in

that it followed two clinical courses: either to produce a solid tumor

or bloody exudate.

Dunham amd Stewart (1953) in an extensive summary of transplantable

and transmissible animal tumors listed the C1300 as a tumor of nervous

tissue, with the comment, "Classification in doubt: 'possibly a neuro-

blastoma.'" Hauschka and coworkers (1956) reported that the modal

chromosome number, based on unpublished data of Dr. A. Levan, was 66 to

70 (the diploid value for the mouse is 40).

From this short review it is apparent that the C1300 tumor appeared

spontaneously in the mouse A strain. Schubert and coworkers (1973) dis-

cuss possible alternatives which would account for the heterogeneity

which is observed both in the tumor behavior and in studies, to be

discussed below, of cloned cell lines in vitro. The most simple explana-

tion thet they feel is consistent with observations on other neoplasms

and cell lines is that the tumor was originally derived from a single

neoplastic event and subsequently underwent alterations in the genetic

material during the thousands of cell generations in vivo and in vitro.

The variations in phenotype seen in the various clones from the tumor

probably all represent activities present in the original neoplastic

cell which have been selectively lost in some derivative lines.

Three labs simultaneously reported the in vitro culture of the

C1300 tumor and proposed its usefulness as a model of neuronal cells, in

much the same way as Murray and Stout (1947) had two decades before for

human neuroblastoma (Augusti-Tocco and Sato, 1969; Klebe and Ruddle, 1969;

and Schubert et al., 1969). Mouse tumor cells in suspension cultures










were described as round cells (like the in vivo tumor), 40 P in diameter

with one to three nucleoli, diffuse endoplastic reticulum, and associated

virus particles. The chromosome complement was described as being

approximately tetraploid. In contrast, cells cultured in conditions

which allowed attachment to a substrate developed into monolayers with

large (40 to 140 p diameter cell bodies) flattened cells with one to

eight nucleoli and processes extending out from the cell body for two to

three millimeters. In addition, cells in the monolayer cultures stained

with Bodian silver techniques and seemed to have fewer virus particles.

Clones derived from the original tumor cells in culture were reported to

have measurable activities of tyrosine hydroxylase (TyH), choline

acetyltransferase (CAT), acetylcholine esterase (AChE) and microtubular

protein.

An amazing number of studies has been reported dealing with the

morphological characteristics (Augusti-Tocco et al., 1973; Schubert et

al., 1969; DeLellis et al., 1970; Seeds et al., 1970; Nelson et al.,

1971 a, b; Prasad and Hsie, 1971; Prasad, 1971 a, b, 1972 d; Prasad and

Sheppard, 1972; Prasad et al., Byfield and Karlsson, 1973; Chang and

Goldman, 1973; Ross et al., 1973; Daniels and Hembrecht, 1973, 1974;

Hinckley and Telser, 1974; Breakefield et al., 1975; Ross et al.,

1975), electrophysiological properties (Harris and Dennis, 1970; Nelson

et al., 1969, 1971 a, b; Peacock et al., 1972; Nelson, 1973; Schubert et

a.1., 1973; Spector et al., 1973), surface components (Brown, 1972;

Augusti-Tocco et al., 1973; Schachner, 1973; Truding and Morell, 1973;

Kimelberg, 1974; Stefanovic ettal., 1974; Akeson and Herschman, 1974 a,

b; Matthews et al., 1976), nucleic acid m tabolism (Schubert and Jacob,

1970; Seeds et al., 1970; Rosenberg et al., 1971; Prasad et al.,











1972, 1973; Rosenberg, 1973; Augusti-Tocco et al., 1973; Bondy etal.,

1974), and general protein synthesis (Schubert and Jacob, 1970; Schubert

et al., 1973) of various clonal lines of the C1300 tumor under several

conditons of culture (see Schrier et al. 1974, for list of clones and

their properties).

Early in the tissue culture work, as mentioned above, it was noted

that the morphology of the cells, as well as biochemical characteristics,

shifted toward a more neuron-like spectrum when the cells were shifted

from suspension to monolayer culture (Schubert et al., 1969; Olmsted and

Rosenbaum, 1969; Olmsted etal., 1970; Schachner, 1973; Augusti-Tocco et

al., 1973; Casola et al., 1974) or were treated with various agents,

including exposure to X-ray (Prasad 1971 a, b, 1972 b, d; Prasad and

Mandal, 1972, 1973; Prasad et al., 1972), bromdeoxyuridine (Schubert and

Jacob, 1970; Land and Goldstein, 1971; Brown, 1972), prostaglandin El

(Gilman and Nirenberg, 1971; Prasad, 1972 a, b, d, e; Prasad and Handal,

1972; Sheppard and Prasad, 1973; Sahu and Prasad, 1975), dibutyryl

cyclic AMP (Furmanski et al., 1971; Prasad, 1972 b, c, d; Prasad et al.,

1972 b, 1973; Waymire et al., 1972; Prasad and Gilmer, 1973; Prasad and

Mandal, 1972; Sheppard, 1972; Truding and Morell, 1973; Chalazonitis and

Greene, 1974; Yavin et al., 1975), phosphodiesterase inhibitors (Gilman

and Nirenberg, 1971; Prasad, 1972 b; Sheppard and Prasad, 1973; Bondy et

al., 1974), removal of serum from the medium (Seeds et al., 1970; Kates

et al., 1971; Ciesielski-Treska et al., 1972; Furmanski and Lubin, 1972;

Hermetet et al., 1972 a, b; Nissen et al., 1972, 1973; Sheppard and

Prasad, 1973; Stefanovic et al., 1974; Akeson and Herschman, 1974 a, b;

Yavin et al., 1975), or exposure to hypertonic media (Ross et al.,

1973; Rosenbaum, 1973).










Numerous other compounds have also been used (Kates et al., 1971;

Miller and Levine, 1972; Peacock et al., 1972; Prasad, 1972 d; Furmanski

and Lubin, 1972; Prasad and Sheppard, 1972; Byfield and Karlsson, 1973;

Schneider, 1974; Hinckley and Telser, 1974; Kimhi et al., 1976). Such

alterations in the biochemical and morphological characteristics of the

neuroblastoma cells have been likened to neuronal differentiation;

however, Schubert and coworkers (1973) discuss the problem of describing

the changes in the neuroblastoma cells, involving neurite extension, as

differentiation. The alterations are often reversible, and there are

great difficulties in deciding whether the concept of "cellular differen-

tiation" is appropriate. As discussed by Weiss (1939) and Grobstein

(1959), "differentiation" refers to an irreversible, terminal process.

Because of the semantic and conceptual problems with the use of the word

"differentiation" and comparison of the phenomenon in vivo and in vitro,

we will use "differentiation" to refer to the alterations in neuro-

blastoma cells under different culture conditons and define it as Schubert

et al. (1973) do: "a process directed toward a given end but not

necessarily reaching that final stage of ultimate functional complexity

and specialization," which is perhaps closer to the concept of modulation

discussed by Weiss (1939) to indicate properties which appear in response

to an external alteration in the cellular environment and which may be

reversible when that alteration is reversed. Most of the studies dis-

cussed here use the extension of one or more processes at least as long

as the diameter of the cell body as a criterion of "differentiation."

Many of the studies mentioned above, and others, have investigated












the activity of enzymes which are involved with neurotransmitter synthesis

or degradation in normal nervous tissue. As a general finding, levels

of activity of these enzymes increased when the cells were grown under

conditions which induced morphological "differentiation." Clones with

various combinations of catecholaminergic (TyH: Augusti-Tocco and Sato,

1969; Amano et al., 1972; Schubert et al., 1969; Waymire et al., 1972;

Breakefield et al., 1975; aromatic L-amino acid decarboxylase: Schubert

et al., 1969; monoamine oxidase and catechol-0-methyl transferase:

Blume et al., 1970; Prasad and Mandal, 1972) and cholinergic (AChE:

Blume et al., 1970; Kates et al., 1971; Hermetet et al., 1972 b, c;

Rosenberg, 1973; Schubert et al., 1973; Lanks et al., 1974; CAT:

Rosenberg et al., 1971; Prasad and Mandal, 1973; Steinbach et al.,

1974) enzymes were found, as well as either enzyme group alone. Seroto-

nergic systems seem to be less active in these cells, but are present

(Knapp and Mandell, 1974).

In addition, neurotransmitter-sensitive adenylcyclase systems are

also present in various clones (Prasad and Gilmer, 1973; Sahu and Prasad,

1975) similar to the ones which have been described in normal nervous

tissue (Von Hungen and Roberts, 1974).

Although the "differentiated" neuroblastoma cells have been reported

to have electrophysiological properties similar to those of normal

neurons (see references above), including tetrodotoxin-sensitive action

potential generating systems, only one laboratory has reported ultra-

structural evidence of synapses between adjacent cells (Nelson et al.,

1971 a, b). In fact, in more recent work (Ross et al., 1973, Rosenbaum,









1973) it has been pointed out that only when the cells are grown in

hypertonic media are junctions between cells and vesicles indistinguish-

able from synaptic vesicles visible. Breakefield and coworkers (1975)

have recently investigated an "adrenergic clone" (with TyH and dopamine

B-hydroxylase) which appears to have catecholamine storage granules

identical to those of normal neurons.

Several studies of glucose metabolism and oxygen uptake have been

reported which suggest that, like maturing neurons, "differentiating"

neuroblastoma cell cultures take up oxygen and rely more on aerobic than

anaerobic metabolic pathways than do the "non-differentiating" cultures

(Nissen et al., 1972, 1973; Ciesielski-Treska et al., 1972; Tholey et

al., 1974; Rosenberg, 1973; Sakamoto, 1971; Sakamoto and Prasad, 1972).

Neuroblastoma cells are able to synthesize 14-3-2 protein (Herschman

et al., 1973) which is thought to be a neuron-specific protein (Moore,

1973). The synthesis of phospholipids (Eichberg e t al., 1975), fatty

acids (Yavin et al., 1975), glycospingolipids and glycosaminoglycans

(Stoolmiller et al., 1973) and phosphoproteins (Casola et al., 1974)

have also been studied in neuroblastoma clones.

The characteristics of heterokaryons (cells with a nucleus resulting

from nuclear fusion, see Harris, 1970, and Ephrussi, 1972) from fusion

of neuroblastoma cells with mouse L cells (fibroblast type) or glioma

cell lines have been studied by several laboratories (Nelson, 1973;

Minna et al., 1971; Minna, 1973; Daniels and Hambrecht, 1973, 1974;

McMorris and Ruddle, 1974; Amano et al., 1974; Sharma et al., 1975) or

between mouse neuroblastoma cells and sympathetic ganglion cells (Chalazo-

nitis et al, 1975; Green et al., 1975). In general, characteristics

(enzyme content, electrophysiological properties) have been demonstrated










in the heretokaryons which are absent in the non-neuroblastoma parent

cell line. Attempts have been made to interpret such events in terms of

the regulation of expression of the genes of the two parent cell lines

or to correlate certain characteristics with retention of specific

chromosomes (chromosomes of the slower-dividing parent cell line tend to

be lost with successive heterokaryon generations). However, the karyotypes

of the parent lines themselves are extremely variable, especially after

many generations in culture (Ciesielski-Treska et al., 1975). Shannon

and Macy (1972) characterize the chromosome number of the Neuro 2a clone

(available from the American Type Culture Collection) as 59 to 193 based

on an analysis of 53 cells in one culture (A/J mice normal karyotype =

40).

Another series of studies deals with the interaction of neuroblastoma

cells with other tissue culture cell lines. For example, Schubert and

coworkers (1973) describe studies in which neuroblastoma cells are grown

with myoblasts and Monard and coworkers (1973 a, b) report differences

in the ability of other cells to induce "differentiation" of the neuro-

blastoma cells either when grown with them or when neuroblastoma cells

are exposed to conditioned media in which the other cell types have been

grown. Great caution should be exercised in interpreting the results of

these studies. Methodological problems, such as depletion of factors in

the conditioned media which would then act in a manner similar to serum-

free media to induce "differentiation," are difficult to avoid. In

addition, the other cell lines used are usually ones which, if not

originally neoplastic themselves, have been in culture for many gener-

ations and may be altered from their in vivo tissue of origin.

The neoplastic nature of model systems, including the neuroblastoma










one, cannot be overlooked. The ability of these cell lines to proliferate

indefinitely, their neoplastic origin, and their aneuploidy argue for

the abnormality of regulatory processes basic to the survival of these

cells both in vivo and in vitro.

To describe all the combinations of enzyme activities, suscepti-

bility of neurite extension and/or increases in enzyme activity to

various inhibitors, the spectrum of generation times, or the time course

of neurite development which have been reported in the literature

requires more space than is available here. All of these characteristics

vary to some extent from clone to clone. Perhaps two characteristics of

the cells would be useful to be used as early neuron-specific markers.

These are (1) cell surface properties, expecially nervous-tissue specific

antigens (Akeson and Herschman, 1974 a, b) and (2) microtubule protein

which is thought to be involved in the extension and maintenance of the

processes (Olmsted and Rosenbaum, 1969; Olmsted et al., 1970). Process

formation, which is the usual criterion used to identify "differentiated"

cells, seems to he correlated consistently with the surface antigens to

a much greater degree than with any other characteristic assayed.

Therefore, it seems reasonable to use the development of the surface

antigen or of microtubule protein as an indication of specific gene

expression. A major problem, however, involves the difficulty in

obtaining sufficient material to analyze the synthesis of a specific

protein and the potential instability of the cell clones when cultured

through the number of generations necessary to obtain sufficient quan-

tities of material to carry out such an analysis. We will probably have

to be content with less complete studies until our methods are refined

sufficiently to allow subcellular fractionation on a microscale.












Although the neuroblastoma cell lines have been compared to normal

adult brain tissue (Amano et al., 1972) and to fractions of brain

tissue thought to be enriched for neuronal-type cells (Kimelberg, 1974),

no effort has been made to compare them with cells from the early stages

of nervous system development. The studies of early neural tube in vivo

and in vitro provide an ideal "system to determine the degree of sim-

ilarity between the tumor-derived cell lines and normal brain tissue

(Kim and Wenger, 1972 a, b; Kim et al, 1974).

To date no systematic study of the regulation of the nuclear pro-

cesses involved in the induction and maintenance of "differentiation" in

the mouse neuroblastoma cells has been conducted. We have therefore

undertaken an investigation and comparison of nuclei isolated from

neuroblastoma cells grown under conditions in which the majority of

cells are either round and dividing or neuron-like with limited division

and branching processes.
















CHAPTER II

METHODS

Cell Culture

Neuro 2a clone of mouse neuroblastoma C1300 cells were obtained

from the American Type Tissue Collection and were maintained in mono-

layer culture in minimal essential medium (Eagle) with Earle's salts and

non-essential amino acids (MEM), 50 U/ml pennicillin-G (potassium salt),

10 pg/ml streptomycin sulfate and 10% fetal calf serum (FCS) (vol/vol).

Cells were grown in plastic tissue culture flasks or 150 mm Petri dishes

(Falcon Plastics) or in one-liter glass Blake flasks (Bellco Glass).

Medium was changed every three to four days and cells were subcultured

with 0.05% trypsin (wt/vol) in calcium- and magnesium-free Earle's

balanced salt solution. Tissue culture medium and serum were purchased

from Grand Island Biological Company.

Stocks of cells were frozen in liquid nitrogen for storage in MEM

supplemented with 20% FCS (vol/vol) and 5% glycerol (vol/vol).

Cell Growth and Differentiation

To determine cell growth characteristics, 25 cm2 plastic flasks

were inoculated with approximately 105 cells in HEM with 10% FCS. After

an adequate number of cells had attached, usually within two hours, five

ml of new medium containing 0, 10, or 20% FCS or 10% FCS with 0.5 or 1.0

mM N6, 02'-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (db

cAMP; grade II, sodium salt, Sigma Chemicals) were added. Cells were

counted every two to six hours in the same three fields in each flask.

33










Fields were identified by means of a black plexiglass template which

fitted over the flask. Cell number and the percentage of cells with at

least one process as long as the cell diameter ("differentiated" cells)

were determined. Counting of cells was carried out in a 37' C warm room

to minimize the effect of temperature fluctuation on cell growth.

After 24 hours, medium in flasks from each growth condition was

replaced with fresh MEM plus 10% or 20% FCS and incubated for an ad-

ditional 24-hour period.

The viability of attached cells and cells in suspension was as-

sessed by the trypan blue dye exclusion method (Paul, 1970). Cells were

incubated for five minutes in 1% trypan blue (wt/vol) in balanced salt

solution and the percentage of cells excluding the dye was determined by

standard hemacytometer counting methods.

For morphological studies, cells were fixed in situ in the plastic

flasks or were grown on glass slides in Petri dishes. Cultures were

washed free of medium with balanced salt solution and were then fixed

with one of several fixatives: acetic acid-ethanol (1:3), 2% glutaralde-

hyde (vol/vol) in 0.1 M sodium phosphate buffer (pH 7.6), 10% phosphate-

buffered formalin (vol/vol), formalin-acetic acid-ethanol (5:5:72), or

2% ammonium bromide (wt/vol) -6% formalin (vol/vol). Cells were then

stained with one of several cell stains: hematoxylin, toluidine blue,

cresyl violet or Ciemsa (Armed Forces Institute of Pathology Manual).

In addition, three modifications of the Bodian silver protargol method

specific for neurons was used (Kim, 1971; J.J. Bernstein, Department of

Neuroscience, University of Florida, personal cominunication--see Table

4; Sevier and Munger, 1965).

Human diploid fibroblasts (ATCC 1121, a gift of Dr. Joyce Remsen,




















TABLE 4

Modification of Bodian silver protargol staining technique (J.J.
Bernstein, Department of Neuroscience, University of Florida) for
tissue and cells on slides.

1. Hydrate slides and wash twice in distilled water.
2. Place in 1% protargol solution for 24 hours at 37' C.
3. Place in reducing solutionbc with agitation for 1 minute.
4. Rinse in running distilled water for several minutes.
5. Place in 1% gold chloride (wt/vol) for 2 minutes.
6. Wash in distilled water for I minute.
7. Place in 2% oxalic acid (wt/vol) for 5 minutes.
S. Wash in two changes of distilled water for 1 minute each.
9. Place in 5% sodium thiosulfate (wt/vol) for 4 minutes.
10. Rinse in running distilled water for at least 5 minutes.
11. Counterstain with cresyl violet or directly dehydrate
through 100% ethanol (vol/vol).
12. Place in xylene:ethanol (equal volumes) for 1 minute.
13. Place in two changes of zylene for I minute each.
14. Mount coverslip.

aPlace slides in staining dish in distilled water. Sprinkle
protargol on surface of the water (1 g/l00 ml). Do not mix.
Protect from light.

bReducer is made fresh for each staining dish. Combine solutions
A, B, and C immediately before use. Solution A: 4.8 g gelatin in
160 ml Pearson 0'Neil buffered water; solution B: 0.08 g silver
nitrate in 40 ml distilled water; solution C: 0.16 g Iydroquinone
in 16 ml Pearson 0'Neil buffered water.

cPearsen O'Neil buffered water (pH 4.1): 14 ml of 0.01% acetic
acid (vol/vol)--0.003% sodium acetate (wt/vol) solution plus
distilled water to make one liter.










Department of Biochemistry, University of Florida) and frozen sections

of rat brain were used for comparison with the neuroblastoma cells.

To compare the Neuro 2a clone with another cloned line of mouse

neuroblastoma cells, clone 2 (DI) was obtained from Dr. Jack Waymire

(Department of Psychobiology, University of California, Irvine). These

cells were grown on slides in Ham's F12 medium with 10% FCS.

Preparation of Nuclei and Chromatin

Nuclei and chromatin were prepared at 40 C following mechanical

harvesting of cells with a rubber policeman from Blake flasks or Petri

dishes according to procedures described by Stein and Borun (1972) and

Stein and Thrall (1973). Cells were washed three times with Earle's

balanced salt solution and lysed with 80 0m1 NaCl, 20 mM EDTA, 1% Triton

X-100 (vol/vol) at pH 7.2. Nuclei were pelleted by centrifugation at

1000 x g for five minutes and washed three times with the lysing medium.

Nuclei were then washed twice with 0.15 M NaCI in 0.01 N Tris at pH 8.0.

Isolated nuclear preparations were examined with phase contrast micros-

copy.

Cell lysis and washing of nuclei were carried out both in the pres-

ence and absence of L-t-tosylamide-2-phenyl-ethyl-chloromethyl-ketone

(TPCK, Sigma Chemicals), 50 pg/ml, to inhibit proteolytic degradation

(Taber et al., 1973). Comparisons were made of the molecular weight

profiles of nuclear and chromatin proteins (as described below) prepared

with and without the inhibitor.

Nuclei were prepared for electron microscopic examination following

centrifugation at 1000 x g by fixation in 2% osmium tetroxide (vol/vol)

in 0.1 M sodium cacodylate, pH 7.4 for 30 minutes, washed, and rapidly

dehydrated in ethanol. Nuclei were then infiltrated for several hours









and embedded in Spurr's low viscosity medium (Spurr, 1969). One micron

sections were stained with 0.1% toluidine blue (wt/vol) containing 1%

sodium borate (wt/vol). Thin sections were stained with 7.5% uranyl

acetate (wt/vol) in 50% ethanol (vol/vol) (Watson, 1958) and Sato's lead

stain (Sato, 1968) and examined with a Siemens IA electron microscope.

Tissue blocks were sectioned and examined by Dr. Kelly Selman (Division

of Anatomy, Department of Pathology, University of Florida).

Chromatin was prepared from isolated nuclei by lysis in distilled

water with gentle homogenization with a Dounce glass homogenizer and "A"

pestle. The nuclear material was allowed to swell in an ice bath for 30

minutes and was then pelleted by centrifugation at 20,000 x g for 15

minutes.

Possible contamination of chromatin by cytoplasmic proteins during

the harvesting and isolation procedures was examined by mixing the cyto-

plasmic fraction prepared from cells labelled for 30 minutes with L-

leucine-[4,5-3H] (5 pCi/ml final concentration, New England Nuclear)

prior to harvesting with nuclei isolated from unlabelled cells. Chro-

matin was then prepared as described above. The amount of tritium in

the final chromatin preparation was compared to that added to the un-

labelled nuclei.

In vitro Transcription of Chromatin

The ability of chromatin from neuroblastoma cells grown in medium

containing 0 or 10% FCS or 10% FCS plus 1.0 mh db cAMP to support RNA

synthesis in vitro was determined using E. coli RNA polymerase prepared

according to the procedure of Burgess (1969). Chromatin was resuspended

in 0.01 M Tris (pH 8.0) by several strokes with a Teflon pestle in a

glass homogenizer and dialyzed against 1000 volumes of 0.01 M Tris (pH

8.0) for 12 hours at 40 C. Assay of RNA synthesis was carried out by









the method of Bonner et al., (1968) or by a modification of the method

of Murphy et al. (1973).

Using the method of Bonner et al., the reaction mixture, in a final

volume of 250 4i, contained 20 U1 of E. coli RNA polymerase, 0.02 mole

of sodium phoshate (pH 7.0), 1 VMole MgCl2, 0.25 pMole MnC12, 3 VMoles

2-mercaptoethanol, 0.1 pMole each of guanosine 5'-triphosphate (GTP),

cytidine 5'-triphosphate (CTP), and uridine 5'-triphosphate (UTP). 0 to

180 V1 of chromatic (or equivalent amounts of calf thymus DNA), and 0.1

VCi of adenosine 5'-triphosphate-tetrasodium-[8-14C] (14C-ATP, 44 mCi/mM,

Schwarz Mann). After incubation at 370 C for ten minutes, the reaction

was stopped with the addition of four to five ml of cold 10% trichloro-

acetic acid (wt/vol, TCA). The precipitates were collected after five

minutes on presoaked Millipore filters (HA 0.45 p) and washed three

times with cold 10% TCA. The filters were air dried and dissolved in 1

ml of Cellusolve (ethylene glycol monoethyl ether) and counted in 15 ml

of scintillation cocktail containing Cellusolve, toluene and Liquifluor

(New England Nuclear) in a ratio of 1:3:0.16.

In a later experiment, the substrate cocktail was modified to omit

the phosphate and contained 4 mMoles MgCl2, 1.0 mMoles MnCl2, 0.02

moles EDTA (disodium salt), 0.009% 2-mercaptoethanol (vol/vol), 0.4

mMoles each of GTP, UTP, and CTP and 0.1 pCi 14C-ATP.

Using the modified method of Murphy et al. the reaction mixture, in

a final volume of 750 p1, contained 10 pl oE E. coli RNA polymerase, 50

pMoles of Tris (pH 7.9), 10 mMoles MgCl2, 0.5 mMole EDTA, 50 mMoles KCI,

0.04 eMole each of UTP, CTP, and GTP, 0.1 pCi 14C-ATP, and chromatin

containing three to five pg of DNA. After incubation at 37' C for 20

minutes, the reaction mixture was rapidly chilled to 40 C and 0.1 ml of









bovine serum albumin (BSA, 1 mg/ml) was added. The reaction was stopped

by addition of an equal volume of cold 10% TCA. After 30 minutes the

precipitates were collected on Millipore filters and processed as de-

scribed above.

DNA was determined either by the diphenyla-ine reaction of Burton

(1956) or by the indole reaction described by Ceriotti (1952, 1955),

using calf thymus DNA or salmon sperm DNA as a standard. Protein was

determined by the method of Lowry et R1. (1951), using bovine serum

albumin or calf thymus histone as a standard.

Extraction of Histones

Nuclei were extracted with 0.25 N HCI for 20 hours at 4' C and

centrifuged at 8000 x g for 30 minutes. The nuclear pellets were re-

extracted twice for 30 minutes and the supernatants pooled. Nine

volumes of acetone were added to the combined HUd extracts and histones

were precipitated at 4' C for 16 hours. The histones were then col-

lected by centrifugation at 10,000 x g for 30 minutes, washed with 30 ml

of ethyl ether, followed by centrifugation at 10,000 x g for 30 minutes

and evaporated to dryness in a vacuum desicator.

Metabolism of Nuclear Proteins

The synthesis of chromatic proteins was studied in neuroblastoma

cells grown in medium with and without FCS and with FCS plus db cAMP.

Growth medium was removed from the monolayers and replaced with leucine-

or tryptophan-free 14EM containing 5 pCi/ml of L-leucine-[4,5-3H] (46

Ci/mM, New England Nuclear) or L-typtophan-3H (2.7 Ci/mM, New England

Nuclear), respectively. The cells were incubated in the presence of the

labelled am;no acid for 60 minutes prior to washing and harvesting.

To study acetylation of chromatic proteins, growth medium was










removed and replaced with MEM containing 50 to 100 pCi/ml of sodium

acetate-3ki (0.74 Ci/mM, New England Nuclear) for 60 minutes prior to

harvesting.

To study phosphorylation of chromatin proteins, cells were incubated

in phosphate-free MEM containing 60 to 100 pCi/ml of 32P as H332P04 for

60 minutes.

Histone synthesis and phosphorylation were examined in cells which

were incubated in leucine- and phosphate-free MEM containing 5 uCi/ml

leucine-3H and 60 to 100 pCi/ml of 32p for 60 minutes.

Chromatin was prepared and histones extracted as described above

and the proteins fractionated on polyacrylamide gels which were processed

as described in the following section.

Polyacrylamide Gel Electrophoretic Fractionation of Chromatin Proteins.

Total chromatin proteins. Two SDS-polyacrylamide gel systems were

used to anaylze the molecular weight profile of total chromatin proteins:

that of Bhorjee and Pederson (1972) using phosphate buffer and that of

Laemli (1970) using Tris-glycine buffer. Chromatin was prepared from

cells from three Petri dishes or Blake flasks and was dissociated in 1.5

ml of the appropriate sample buffer (see below) in a Dounce homogenizer

fitt-d with a Type "A" glass pestle. The sample was dialyzed overnight

against 1000 volumes of the buffer at 220 C and sucrose was added to a

final concentration of 15% (wt/vol). Samples were heated in a boiling

water bath for two minutes before beiag applied to polyacrylamide gels

prepared in glass tubes which had been treated with 1% Photoflo (vol/vol,

Kodak).

Gels were prepared using acrylamide, N,N'-methylene-bisacrylamide

(bis), and N,N,N',N'-tetramethylethylene-diamine (TEMED) from Eastman










Kodak Company. Ammonium persulfate was obtained from Aldrich Chemicals

and 2-mercaptoethanol from Matheson, Coleman and Bell.

Gels prepared according to the method of Bhorjee and Pederson were

7.5 cm x 0.6 cm and contained 7.5% acrylamide (wt/vol), 0.25% his (wt/vol),

0.1% SDS (wt/vol), 0.1 M phosphate (pH 7.0), 0.5 M urea, 0.005 M EDTA,

0.05% TEMED (vol/vol), and 0.1% ammonium persulfate (wt/vol). The

separating gel was covered with 0.1% SDS (wt/vol), 0.005% EDTA (wt/vol),

and 0.1% ammonium persulfate (wt/vol) and allowed to polymerize at room

temperature for 45 minutes. A one cm stacking gel was prepared with

final concentrations of 2.5% acrylamide (wt/vol), 0.09% his (wt/vol),

0.1% SDS (wt/vol), 0.01 M sodium phosphate (pH 6.0), 0.5 M urea, 0.005 M

EDTA, 0.07% TEMED (vol/vol), and 0.08% ammonium persulfate (wt/vol).

Samples were suspended in 0.1% SDS (wt/vol), 0.1% 2-mercaptoethanol

(vol/vol), 0.01 M sodium phosphate (pH 7.0), and 15% sucrose (wt/vol).

Bromophenol blue in the sample buffer was used as a dye marker. Samples

were electrophoresed for six to seven hours with 8 mA/gel in a running

buffer of 0.1% SDS (wt/vol), 0.1 M sodium phosphate (pH 7.0), and 0.005

11 EDTA at room temperature.

Laemli gels were 10 cm x 0.6 cm and contained 8% acrylamide (wt/vol),

0.2% bis (wt/vol), 0.1% SDS (wt/vol), 0.375 M1 Tris MCI (pH 8.8), 0.025%

TEMED (vol/vol), and 0.025% ammonium persulfate (wt/vol). The stacking

gel consisted of 0.25 ml of 3% acrylamide (wt/vol), 0.08% his (wt/vol),

0.1% SDS (wt/vol), 0.125 M Tris HC (pH 8.8), 0.025% TEMED (vol/vol) and

0.025% amonium persulfate (wt/vol). Samples were suspended in 0.0625 M

Tris HC1 (pH 6.8), 2% SDS (wt/vol), 5% 2-mercaptoethanol (vol/vol), and

15% sucrose (wt/vol). Samples were electrophoresed for five hours at 2

m/gel in a running buffer of 0.025 M Tris HCl (pH 8.3), 0.192 M glycine

and 0.1% SDS (wt/vol).










Following completion of electrophoresis, gels were immediately

removed from glass tubes and fixed in 12% TCA (wt/vol) in 40% ethanol

(vol/vol) and 7% acetic acid (vol/vol) at room temperature for 12 hours.

Cells were then washed with 40% ethanol--7% acetic acid and stained for

five hours at 37' C with 0.25% Coomassie brilliant blue R (wt/vol, Sigma

Chemicals) in 40% ethanol--7% acetic acid. Gels were destained electro-

phoretically in a Canalco Quick Gel Destainer or diffusion destained

with 10% ethanol--7% acetic acid. Gels were stored in 7% acetic acid.

The linear migration of proteins in proportion to the log molecular

weight in both types of gels was verified using solutions of proteins of

known molecular weight obtained from Sigma Chemicals and Pharmacia.

Histones. Histone polypeptides were fractionated electrophoretically

according to charge and molecular weight on polyacrylamide gels prepared

with acetic acid and urea according to the method of Panyim and Chalkley

(1969). Gels contained 15% acrylamide (wt/vol), 0.5% bis (wt/vol), 2.5

M urea, 0.5% TEMED (vol/vol), 0.125% anonium persulfate (wt/vol) and

5.4% acetic acid (vol/vol). Gels were 9.0 cm x 0.6 cm and were pre-

electrophoresed for four to five hours at 2 mA/gel. Samples, in 0.9 M

acetic acid with 15% sucrose (wt/vol), were run for four hours at 2

mA/gel. Gels were fixed and stained simultaneously in 0.1% Amido black

(wt/vol) in 20% ethanol (vol/vol)--7% acetic acid (vol/vol) for 12 hours

and destained electrophoretically for 15 minutes.

Gels were scanned in a Beckman Acta II spectrophotometer at 590 nm

for the SDS-polyacrylamide gels and at 620 run for the acetic acid-urea

polyacrylamide gels. The areas under the optical density profiles were

integrated with a compensating polar planimeter (Keuffel and Esser









Company) to determine the amounts of protein in discrete molecular

weight regions of the gels. Comparisons of the density of the Coomassie

blue staining pattern were made on gels containing different amounts of

protein to verify that the relationship between the protein concen-

tration and the amount of dye binding was linear in the ranges of protein

concentration commonly used.

For analysis of radioactive isotope incorporation into the various

polypeptide bands, gels were frozen on dry ice and sliced into 1 amm

fractions with a Hoeffler gel slicer. Slices were solubilized in 200 V1

of 35% hydrogen peroxide overnight at 370 C and then counted in a Triton

X-100--toluene--Liquifluor cocktail (1:2:0.13) in a Beckman liquid

scintillation counter.

Gels were photographed using a strong yellow VII filter (Kodak G15)

and Kodak Ektapan film.

DNA Synthesis in Mouse Neuroblastoma Cells.

To study DNA synthesis, cells were inoculated into 150 mm plastic

Petri dishes and the incorporation of thymidine-methyl-3H into TCA-

insoluble material was determined. After 24 hours in HEM with 10% FCS,

medium was changed to HEM plus fresh 10% FCS, MEM without FCS, or to HEM

plus 10% FCS and 1.0 mM db cAMP. Cells were incubated for 24 hours and

then incubated for 60 minutes with 3H-thymidine (final concentration 1

uCi/ml; specific activity 55 ci/mMol, Nuclear Dynamics). Cells were

then mechanically harvested in cold balanced salt solution. Aliquots

were taken to determine cell number, incorporation of 3H-thymidine into

TCA-soluble and -insoluble components of whole cells and of isolated

nuclei, and amount of DNA in the various fractions. DNA was determined

by the diphenyamine reaction of Burton (1956) using paraldehyde in the






44




diphenylamine reagent (Richards, 1974) and calf thymus DNA as a

standard.

















CHAPTER III

RESULTS

Cell Growth and Differentiation

Morphology

The morphology of living and fixed cultures of mouse neuroblastoma

cells was investigated with the aid of phase contrast and bright field

microscopy. Figures 1 through 7 represent cells grown under the various

culture conditions. Cresyl violet is commonly used as a general cell

and nuclear stain for nervous tissue. The Bodian silver protargol

method is specific for neurons. A great deal of morphological diversity

is seen in any one culture flask. In general, three populations of

cells are present: small relatively regular round cells, flattened

larger cells with processes and very large flattened cells with numerous

vacuoles. Cells which had been maintained for 24 hours or longer without

serum or in the presence of db cAMP tended to be larger than cells grown

in serum-containing medium with numerous long branching processes. All

cultures of neuroblastoma cells stained with silver stains and contained

large amounts of argyrophillic material. Comparison of silver-stained

neuroblastoma cells with human fibroblasts (Figure 8) revealed that the

fibroblasts took up very little silver and only into the nuclei, while

the nuclei of neuroblastoma cells stained intensely and the cytoplasm

contained uniformly dispersed argyrophillic material. No evidence of

fibrillar material in the cell bodies or processes of the neuroblastoma

cells was visible or even resembled the fibers visible with the same

-stain in rat brain (Figure 9).
















ylA V,
N.


-.-
~ ,

N)'


I V
9
* 9
4 .t




-9' .
U


'k


4-1


*A


I


40


Figures 1 and 2. Mouse neuroblastoma cells from clone Neuro 2a fixed
and stained with cresyl violet. Cells were grown in 25 cm2 plastic
flasks in MEM with 10% FCS for 24 hours. Medium was then changed to
fresh MFM with 10% FCS (Figure 1) or to MEM without FCS (Figure 2) and
cells were incubated for 24 hours. The bar represents 100 v (magnifi-
cation X 160).













Ah! ~~


o 0


A. -~.



a.
4~, f1.


4

Figures 3 and 4. Mouse neuroblai
plus 0.5 MIn db cUMP (Figure 3) o
were stained with cresyl violet.
ication X 160).


4,,t
%W















stoma cells clone Neuro 2a grown in MEM
r 1.0 mM db cAMP for 24 hours. Cells
The bar represents 100 p (magnifi-


All
Ve
















AI


'A


Figures 5 and 6. Neuroblastoma cells grown on glass microscope slides
and stained with Bodian silver protargol method (see Table 1). Follow-
ing incubation for 24 hours in MEM with 10% FCS, cells were incubated
for 24 hours in fresh t0I4 with 10% FCS (Figure 5) or MEM without serum
(Figure 6). The bar represents 100 pJ (magnification X 850).

































Figure 7. As in Figures 5 and 6 but cells incubated in NM plus 1.0 mM
db cAMP.


Figure 8. Human diploid fibroblast ATCC 1121 grown for 48 hours in MEM
with 10% FCS and stained with Bodian silver protargol method (magni-
fication X 850).















































Figure 9. Rat brain section embedded in parafilm and stained and photo-
graphed as Figures 5 through 7. Note the fine neuronal processes and
large neuronal cell bodies.









A comparison of the C1300 Neuro 2a neuroblastoma cells with a clone

obtained from another laboratory was made in order to determine whether

more recently cloned lines exhibit less morphological heterogeneity.

Observations of hematoxylin and Giemsa-stained material (Figures 10-11),

indicated that clone 2(D,) consists of populations of cells similar to

those seen in C1300 cultures; however, the large vacuolated cells are

present in fewer numbers than in the Neuro 2a cell line.

Cell Number and Percent "Differentiation"

When mouse neuroblastoma C1300 cells were grown in medium containing

10% or 20% FCS, the majority of the cells remained round and relatively

regular and cultures rapidly approached confluency. With either removal

of serum or addition of db cAMP to the medium, cells became flattened,

enlarged and developed one or more branching processes.

Figures 12 and 13 show the increase in what we have called "dif-

ferentiation"--the presence of at least one process as long as the

diameter of the cell body--over time following replacement of growth

medium (MEM with either 10% or 20% FCS) with medium lacking serum or

containing db cAMP. The shapes of the curves for "differentiation" in

..10% and 20% FCS are almost identical for the first 24 hours. Both serum

withdrawal and addition of db cAMP result in similar percentages of

differentiated cells by 24 hours.

In Figure 14 the number of cells counted in each flask over time is

indicated. Since the same fields were counted in each flask, these data

reflect increases in cell number and density with time. Although the

percent of cells with processes increases with time in the cultures

grown in the presence of db cANP, the cell number also continues to

increase at the same rate as in the control cultures.





















































11

Figures 10 and 11. Mouse neuroblastoma clone 2(DI) grown in Ham's F12
medium and stained with Giemsa method. Magnification X 100 (Figure 10)
and X 300 (Figure 11).


-jib





































Figure 12. Differentiation of neuroblastoma cells. Small plastic
tissue culture flasks were inoculated with cells in MEM plus 10% FCS at
time 0. At 2 hours (arrow) medium was changed. Labelled points at 54
hours represent percent differentiation in flasks whose medium was
replaced at 26 hours as follows: (A) MEM with no serum replaced with
MEM plus 10% FCS; (B) IEM plus 10% FCS replaced with fresh MEM plus 10%
FCS; (C) MEM plus 20% FCS replaced with fresh MEM plus 20% FCS; and (D)
MEM with no serum replaced with MEM plus 20% FCS. Vertical bars re-
present standard error of the mean for four flasks at each point out to
26 hours.































p


Time(hourS)































U
20


L 40-


0


Serum, 10%
--No u .rum
....db cAMP i.j ryl,,!
- --d b Av., I .,.


0 48 12 1602 A2-3640444

Time (hours)





Figure 13. Differentiation of neuroblastoma cells. Cells were grown in
MEM plus 10% FCS for four hours and then medium was changed (arrow).
Vertical bars represent the SEM of counts from four flasks of cells.







---Serum 10%
No serum
.... db CAMP, 1,0mM

- db cAMP, 0.5mM


600-



5OOj


4 12 20 2 36 42
rime (hours)
Figure 14. Cell number over time. The actual number of cells in each flask in the experiment
summarized in Figure 13 is indicated. Points represent the average number of cells in three
fields counted in four flasks.


Or


-a 400-




= 300-
U
L

E



4
0


(o









When cultures grown in the absence of serum or in the presence of

db cAMP for 24 hours were fed fresh medium containing serum (Figure 15),

the level of "differentiation" decreased to control levels. In ad-

dition, when cultures which had been maintained in medium containing

serum were re-fed, the level of differentiation decreased. Cultures

exposed to medium without serum had many fewer cells and were visibly

less dense than cultures grown with serum with or without db cAMP. No

increase in the number of cells recoverable from the overlying medium

was detected in cultures without serum.

Under all conditions, greater than 95% of the cells, both attached

to the surface of the flask and recoverable from the overlying medium,

were judged viable by dye exclusion.

Model of Growth

Using data consisting of cell counts for four populations of cells

(10% FCS, no serum, 1.0 mM db cAMP, and 0.5 nM db cAMP) the growth of

each if the four cell populations was modeled by a stationary birth and

death process in collaboration with Rose Ray, Biostatistics Unit, Depart-

ment of Statistics, University of Florida. Each population type is re-

plicated four times; for each of the 16 flasks of cells, the cells in

three fields were counted at 4, 8, 12, 16, 20, 24, 32 and 48 hours (48

hours is missing for the 0.5 mM db cAMP condition). At each time the

number of non-differentiated (X) and the number of differentiated (Y)

cells were counted.

The growth of each of the four cell types was modeled by the fol-

lowing birth and death process:

In any small amount of time dt the probability that a
non-differentiated cell divides is Xdt, the probability that
a non-differentiated cell differentiates is Vdt. The param-
eter A is the birth rate, the parameter V is the differen
tiation rate. These parameters are considered to be constant
















I2
E

10 0 1 E IL
L L
3 L: v
L
0 j 0 M 0) 0





c 60
L


40




20-




24 hours 4 hours






Figure 15. Reversibility of "differentiation." Cells were inoculated
into 25 cm2 flasks in MEM with 10% FCS. Medium was changed after 24
hours and the percent "differentiated" cells in each condition was
determined. The medium in flasks represented by the solid bars was
renewed and medium in flasks represented by striped bars was replaced
with medium containing 10% FCS. At the end of 24 hours, percent "dif-
ferentiation" was determined. Vertical lines represent the SEM of
counts from four flasks.









throughout the 48 hours of the experiment. In addition, each
cell is considered to behave independently of the others.
The probability that more than one event (i.e., cell differen-
tiation or cell division) occurs in a small amount of time dt
is essentially zero. The differentiated cells do not divide
and do not revert to the non-differentiated state.

This model contains only two unknown parameters, X as the birth

rate and p the differentiation rate. These parameters were estimated by

the method of least squares for each of the four populations; that is,

for each of the four populations the sums

E [Xi-X(,P)]2


Z [Yi- (X,P) ] 2



were minimized. Here Xi(A,p) is the expected number of non-differen-

tiated cells for this model if X,p are the parameters; similarly, i(,)

is the expected number of differentiated cells if A,p are the parameters.

These estimators are unbiased and normally distributed for large samples.

It was found that more accurate comparisons of the four populations

could be made with a slight reparameterization of the model. The actual

parameters estimated were b= X-p and p. The parameter b can be described

as the growth rate for the non-differentiated population of cells, i.e.,

the X population is increasing at rate X and decreasing at rate p; b=

A-p is the net growth rate.

The estimates for the four populations are as follows:

Population b (A-p) Variance p Variance

1 10% FCS .029 3.68 x 10-6 .007 2.07 x 10-5

2 No serum -.007 6.58 x 10-6 .021 1.05 x 10-4

3 1.0 m db cAMP .009 2.32 x 10-6 .028 6.04 x 10-5

4 0.5 m1 db cAMP .012 3.61 x 10-6 .025 6.36 x 10-5










Students t-test was used to compare the four populations.

A total of 12 comparisons were made. There is strong evidence that

populations 1, 2 and 3 have different growth parameters, b= A-P. Popu-

lations 3 and 4 appear to have the same growth rate. There is no strong

evidence for any population differences with respect to the differ-

entiation rate p. Population I appears to have a lower differentiation

rate than the other three populations. The comparisons are as follows:

Comparisons for b= X-p (growth rate)

t P
pop. 1 vs pop. 2 11".45 .00005
pop. 1 vs pop. 3 8.49 .00005
pop. 1 vs pop. 4 6.33 .00005
pop. 2 vs pop. 3 -5.33 .00005
pop. 2 vs pop. 4 -6.14 .00005
pop. 3 vs pop. 4 -i.52 .2000

Comparisons for i (differentiation rate)

t p
pop. 1 vs pop. 2 -1.24 NS
pop. 1 vs pop. 3 -2.32 .05
pop. 1 vs pop. 4 -1.92 .20
pop. 2 vs pop. 3 -.54 NS
pop. 2 vs pop. 4 -.90 NS
pop. 3 vs pop. 4 .29 NS

The time invariant birth and death model was found to give only a

mediocre fit to the data. There appears to be some change in the growth

rate at approximately 20 hours.

Isolation of Nuclei and Chromatin

Examination of the pelleted material obtained after lysis of cells

in NaCi-EDTA-Triton X-100 was carried out with both phase contrast and

electron microscopy. Typical regions of the pellets are shown in Figures

16 and 17. Electron microscopic examination indicates that the pellet

is composed primarily of intact nuclei without nuclear membrane components.

The nuclear material, the classic chromatin of the microscopists, is


































7



~4~4i ;<~~


~' ~&~pJC ~

~
~j4


Figure 16. Electron micrograph of material pelleted after lysis of
cells as described in the text. Note the prominent nucleoli and lack of
nuclear membrane components. Magnification approximately X 15,000.































































Fig ure 17. Higher power view of pelleted material shown in Figure 16.
Note the lack of nuclear membrane material. Magnification approximately
x 45,000.










uniformly dispersed and nuclei contain one or more prominent nucleoli.

Non-nuclear material is present to some degree; however, no well-defined

cellular organelles other than nuclei are identifiable.

Following the mixing of 3H-leucine-containing cytoplasmic fractions

with unlabelled nuclear fractions, less than 1% of the tritium was

recovered in the final chromatin preparation.

A B
3H-cpm cytoplasmic 3H-cpm final B/A
fraction chromatin

From cells
grown with 104,276 617 0.006
10% FCS

From cells
grown with 72,407 233 0.003
no FCS

Several attempts were made to characterize the protein and DNA

content of the chromatin prepared from cells grown with and without 10%

FCS. DNA and protein determinations were carried out directly on the

chromatin pellet following lysis of nuclei in water and on fractions of

the pellet following extraction of DNA and histones. Values of the DNA

to protein ratio ranged from 1:0.25 to 1:2.66 for chromatin from cells

grown in serum and from 1:2.17 to 1:6.96 for chromatin from cells grown

without serum. Since very small volumes of material were available for

each determination, the wide variation in results may result from diffi-

culties in accurately pipetting very small volumes of extremely viscous

material.

When histone to non-histone chromosomal protein ratios in chromatic

from cells grown in different media were estimated from the areas under

the optical density profiles of SDS-polyacrylamide gels (as described

below), values ranged from 1.12 to 1.56 (see Table 5). Each set of gels






TABLE 5

Relative amounts of protein from intact nuclei of neuroblastoma cells electrophoretically fractionated on Bhorjee
and Pederson SDS-polyacrylamide gels as described in the text.


Fraction and MW X 10-3

A-B C-E F-J K L-0 P-R Histone/
Nonhistone
Sample 98.2-145.0 64.2-98.2 38.4-64.2 31.5-38.4 17.9-31.5 10.0-17.9

1. Nuclei--with serum 32.9 31.6 25.1 (32.8) 10.4 (67.2) .94
with TPCK

2. (re-scan) 33.7 32.0 25.6 (32.6) 8.6 (67.5) .98

3. 32.8 30.2 24.4 30.5 12.4 69.5 .79

4. with serum
without TCPK 33.9 29.4 24.8 (27.2) 11.9 (72.8) 1.04

5. 31.8 28.9 25.8 (27.5) 13.3 (72.4) .94

6. without serum 27.5 30.5 26.5 (29.5) 15.4 (70.5) 1.04
with TPCK

7. without serum
without TPCK 30.5 29.5 26.3 26.8 13.7 73.2 1.09

8. 34.2 31.5 25.5 (30.9) 8.8 (69.1) .98


Relative amounts of protein were determined by integrating the areas under the regions of the optical density
profiles of the gels, scanned at 590 nm. Values for regions A-J and L-0 are expressed as percent total non-
histone protein and for K and P-R as percent total histone. Values in 0's are uncertain since the optical
density of the histone bands exceeded the upper detection limits for the spectrophotometer. Each line of data
represents one gel.










yielded slightly different values; however the histone to non-histone

ratio was always slightly greater for material from "differentiated"

cultures. This variation reflects differences in background staining of

the gels and limitations of defining limits of each band of polypeptides.

Comparison of gel profiles of polypeptides from intact nuclei and

from chromatin does not reveal any clear differences in polypeptide band

patterns as seen in the scans represented in Figures 18 and 19. When

DNA determinations were made on the various supernatant and precipitate

fractions obtained during the preparation of chromatin, DNA was only

found in the isolated nuclear pellets and in the chromatin. The final

supernatant following lysis of nuclei in distilled water did not contain

any DNA.

When comparisons were made between nuclear and chromatin material

prepared in the presence or absence of TPCK, a serine protease inhibitor,

no differences were seen (Figure 20). Visual inspection of the gels

revealed an apparent quantitative difference in one band; however an-

alysis of the optical density scans and comparisons of the areas under

various peaks did not reveal any consistent differences (Figure 20 and

Tables 5 and 6). No difference in the recovery of histones prepared in

the presence or absence of TPCK was noted.

Transcriptional Activity of Chromatin

The transcriptional activity of the material prepared from cells

grown with or without serum or with serum and db cAMP was determined by

assaying the ability of isolated chromatin to serve as a template for

DNA-dependent RNA synthesis in a cell-free system. The incorporation of

14C-ATP by E. coli RNA polymerase into acid-insoluble material was

measured under three assay conditions in which the amount of DNA template











TABLE 6

Relative amounts of protein from chromatin of neuroblastoma cells electrophoretically fractionated on Bhorjee
and Pederson SDS-polyacrylamide gels as described in the text.


Fraction and MW X 10-3

A-B C-E F-J K L-0 P-R Histone/
Nonhistone
Sample 98.2-145.0 64.2-98.2 38.4-64.2 31.5-38.4 17.9-31.5 10.0-17.9

A
1. Chromatin--with serum 37.5 27.8 18.9 15.9 (1.39)
with TPCK

2. 36.4 26.5 21.2 15.8

3. with serum
without TPCK 33.4 27.9 23.4 15.2

4. 34.4 25.4 22.3 17.9

5. without serum
with TPCK 31.7 24.4 22.4 21.5 (1.56)

6. without serum
without TPCK 36.3 27.8 22.2 13.8


To be continued/









Table 6 (Continued)




Fraction and MW X 10-3

A-B C-E F-J K L-0 P-R Histone/
Nonhistone
Sample 98.2-145.0 64.2-98.2 38.4-64.2 31.5-38.4 17.9-31.5 10.0-17.9

B
1. Chromntin--with serum
with TPCK 36.8 26.0 27.0 (22.9) 10.3 (77.1) 1.12

2. with serum 43.5 25.0 21.0 (14.4) 10.6 (85.6) 1.39
without TPCK

3. without serum
with TPCK 35.6 26.9 28.1 (18.8) 9.5 (81.2) 1.31

4. without serum
without TPCK 36.1 29.1 25.8 (20.6) 9.0 (79.4) 1.56


Relative amounts of protein were determined by integrating the areas under the regions of the optical density
profiles of the gels, scanned at 590 nm. Values for regions A-J and L-0 are expressed as percent total non-
histone proteins and for K and P-R as percent total histone. Values in O's are uncertain since the optical
density of the histone bands exceeded the upper detection limits for the spectrophotometer. Each line of data
represents one gel.

Data sets A and B represent two different experiments and sets of gels.


























Figure 18. Comparison of pattern of Coomassie blue staining of proteins
fractionated electrophoretically on Bhorjee and Pederson gels from
intact nuclei of mouse neuroblastoma cells grown in MEM with 10% FCS
(Serum) or without FCS (No serum). Nuclei were isolated from cells in
the presence of TPCK.


















TPCK
--Serumn
Noserum










II I


0tI




























Figure 19. Comparison of the patterns of staining of proteins frac-
tionated electrophoretically on Bhorjee and Pederson gels from chromatin
prepared from isolated nuclei of neuroblastoma cells grown with (Serum)
and without (No serum) FCS. Chromatin was isolated without the use of
TPCK.


















I I Chromatn
I, II, II
I I No TPCK
I' I
I, I ---Serum
I I Noserum

I'
I III
I I
I I
I I I II

I III
I Ii
I I g II
I I


I I ~I
I I,
I II

II
I II
I V
I I
II
o r
I I
III I~ I I I
D Ii rI I
III II. I
III III
I I I~
11.1 I ~ll I

As It
II II;, I
~II. ~ II
I I
I .
,




ToD





























Figure 20. Comparison of the staining pattern of proteins fractionated
electrophoretically on Bhorjee and Pederson gels from samples of chromatin
prepared with and without TPCK from neuroblastoma cells grown in MEM
with 10% FCS.













Chromatin
Serum

--- No TPCK
TPCK









ni





Top
0 I










Top










was rate limiting. The results of these assays are shown in Figure 21

and Tables 7 and 8. Under all conditions, template activity for chromatin

from cells grown in the presence of serum was several-fold higher than

that of chromatin from cells exposed to no serum conditons or to db

cA4P.

Electrophoretic Fractionation of Nuclear and Chromatin Proteins

Total proteins from isolated nuclei and from lysed nuclei (chromatin)

were investigated in two different SDS-polyacrylamide gel systems. The

migration of proteins of known molecular weight in a linear fashion pro-

portional to the log molecular weight is shown in Figures 22 and 23 for

each type of gel. Histone HI migrates in an anomalous fashion in the

Bhorjee and Pederson gels (Hayashi et al., 1974) and appears in the

region denoted by K in Figure 27. Figures 24 and 25 show the Bhorjee

and Pederson gel patterns of material solubilized from isolated nuclei

and from chromatin prepared in the presence of and in the absence of

TPCK.

When different amounts of protein were applied to a series of gels,

the areas under the optical density profiles corresponded linearly to

the protein concentration applied within the range of 5 to 30 pg of

protein as determined by the method of Lowry et al. (1951).

Tables 5 and 6 indicate the distribution of protein, expressed as

percent of the total, in different regions of the gels. No consistent

differences appear between intact nuclei and chromatin, or chromatin

prepared with and without TPCK. Variability from one set of gels to

another (Table 5 and Table 6, A and B each represent a different set of

gels), as well as from one scan to another of the same gel (Table 5, 1

and 2) is apparent. Although this method of analysis has a limited

degree of resolution, we chose to apply it to an investigation of the

































Figure 21. The ability of chromatin from isolated nuclei of mouse
neuroblastoma cells to support the incorporation of 14C-ApfP into acid-
insoluble material by E. coli RNA polymerase was determined as described
in the text. The actual data are presented in Table 7. Chromatin was
prepared from cells which had been grown in MEM with 10% FCS (Serum),
MEM without FCS (No serum), and in MEM with 10% FCS plus 1.0 mM dbcAMP
(db cAIMP). Incorporation of ATP using naked DNA as a template was
considered to represent 100% template capacity. Preparations of chro-
matin and assay mixture minus RNA polymerase (No enzyme) revealed little
endogenous activity.





76











-12-





Fa10-


-A A DNA
c l Serum
No serum
0 0 db cAMP
C6-
L No enzyme
U
.c

n4_

u


ug DNA or Chromatin DNA










TABLE 7

Transcription assay using Burgess fraction IV E. coli RNA polymerase.
Incorporation of 14C-ATP into acid-insoluble material with either DNA
or chromatin as a template.

Template material 14C-ATP cpm

With enzyme

I Murphy et al. assay conditions
Calf thymus DNA
0 jg 127
5.5 1554
11 1698
22 1651
55 1605
77 1790
100 1854

Chromatin from cells with serum
1.0 pg DNA 671
2.0 1179

Chromatin from cells without serum
0.6 pg DNA 245
1.2 294

II Repeat with new batch enzyme
Chromatin from cells with serum
2.0 4g DNA 1537
4.0 3612

Chromatin from cells without serum
3.0 pg DNA 440
12.0 409

III Bonner et al. assay conditions
Chromatin from cells with serum
0 pg DNA 1601
6.0 4376
12.0 6768
30.0 10867
60.0 14899
108.0 14335

Chromatin from cells without serum
2.4 pg DNA 3608
4.8 3122
12.0 4140
24.0 6721
43.2 11393



















TABLE 8

Transcription assay using Burgess fraction IV E. coli RNA polymerase.
Incorporation of 14C-ATP into acid-insoluble material with either DNA
or chromatin as a template and a modification of Bonner et al. assay
conditions.

14C-ATP % Template
Template material Without enzyme With enzyme Transcribed

Salmon sperm DNA
0 pg 228
1.28 6627
2.56 11072
5.12 12697
10.24 24197

2.56 261 28.7(2)a,b 8732 + 757.4(2) 100.0

Chromatin from cells
with serum
2.34 pg DNA 60 0.2(2) 2311 t 30.1(4) 26.5

Chromatin from cell
without serum
2.43 pg DNA 178 9.3(2) 232 1.4(4) 2.7

Chromatin from cells
with serum plus db
cAt4P
2.38 pg DNA 110 6.5(2) 222 19.0(4) 2.5


a % Template transcribed calculated using cpm incorporated with
naked DNA as 100%.
b Numbers in parentheses represent numbers of samples. Data represent
mea SE.






79









20 A



B



10



1.5


L
U

X eD

0

E


.2 4 .6 18 10
Relative Moility








Figure 22. Migration of proteins of known molecular weight in 7.5%
polyacrylamide gels prepared according to the method of Bhorjee and
Pederson as described in the text. Ten micrograms of protein were
applied to the gels in the sample buffer with a final concentration
of 1 pg/ml: (A) phosphorylase A, 94,000; (B) bovine serum albumin,
68,000; (C) ovalbumin, 43,000; (D) horse myoglobin, 17,200; (E)
cytochrome C, 12,380.



















20










uB
TOc




oC
X eD





o .
L oF




00





.2 A .6 .8 W

Relative Mobility






Figure 23. Migration of proteins of known molecular weight in 8%
polyacrylamide gel prepared according to the method of Laemmli as
described in the text. Approximately 10 pg of protein were applied to
each gel. Gels containing mixtures of the proteins were also run:
(A) phosphorylase A, 94,000; (B) bovine serum albumin, 68,000; (C)
ovalbumin, 43,000; (D) aldolase, 40,000; (E) chymotrypsinogen, 25,700;
(F) trypsin, 23,300; (G) RNase, 13,700.







































Figure 24. Total nuclear proteins on SDS-acrylamide gels prepared
according to the method of Bhorjee and Pederson. Gels were stained with
Cooiiassie brilliant blue. From left to right, material prepared from
total nuclei of cells grown in the presence of 10% FCS isolated with (a)
and without TPCK (b), and material prepared from total nuclei of cells
grown in serum-free MEM prepared with (c) and without TPCK (d).






















































a b


c d


Itt













I-b




































Figure 25. Bhorjee and Pederson gel patterns of chromatin prepared from
nuclei isolated from cells grown in the presence of serum and isolated
with TPCK (a) and without TPCK (b) and chromatin prepared from nuclei
isolated from cells grown in serum-free MEM and prepared in the presence
(c) and absence of TPCK (d).














































a b


c d










composition and metabolism of the chromatin proteins from neuroblastoma

cells grown with and without serum and with serum plus db cAMP.

Laemeli gels were run of chromatin samples in an attempt to separate

the high molecular weight bands to a greater degree than was possible

with the Bhorjee and Pederson gels. This gel system separates high

molecular weight polypeptides slightly better than the Bhorjee and

Pederson gels; however, proteins of less than 25,000 molecular weight

migrate with the buffer front (see Figure 23). No clearly definable

differences were detectable among the chromatin samples from cells grown

in serum, no serum, or serum and db cAMP.

All five major classes of histone fractions are clearly visible on

acetic acid-urea polyacrylamide gels prepared according to the method of

Panyim and Chalkley (Figure 26). The electrophoretic mobilities of the

histone fractions do not differ significantly as a function of the

medium in which the cells were grown. The relative amounts of histone

protein present in each of the five histone fractions is also similar

(Table 9). No attempt was made to compare the acid-extractable non-

histone polypeptides which migrate more slowly in the acetic-acid urea

gels than do the histones.

Metabolism of Tocal Chromatin Polypeptides

To study the composition and synthesis of the non-histone chromatin

proteins from neuroblastoma caIls grown with and without serum and with

db cANT, cells were labelled with either 3H1-leucine or 3H-tryptophan and

the proteins from isolated chromatin were fractionated electrophoretically

on Bhorjee and Pederson gells. Since histones do not contain tryptophan

residues, incorporation of radioactivity is thought to reflect non-

histone chromosomal protein synthesis. Figure 27 illustrates the electro-

phoretic profiles of total chromosomal polypeptides and the incorporation





































Figure 26. Electrophoretic fractionation of histones from nuclei of
cells grown in the presence of 10% FCS (a), in serum-free medium (b),
and in medium with serum and 1.0 mM db cAMP (c) on acetic acid-urea-
polyacrylamide gels prepared according to the method of Panyim and
Chalkley as described in the text.































-Hl

~ H3


H2b
H2


H4


























TABLE 9

Relative amounts of histone fractions in nuclei from neuroblastoma cells
grown with and without serum and with serum plus db cAMP.


Histone Fractions


Nuclei from cells
grown with serum

Nuclei from cells
grown without serum

Nuclei from cells
grown with 1.0 mM
db cAMP


Hi H3 + H2B + H2A H4

.18, .16, .19 .65, .67, .68 .18, .18, .15


.17, .17, .19 .63, .66, .68 .21, .18, .15


.21, .17, .18 .60, .66, .69 .20, .18, .13


Gels were stained with Amido Black, destained electrophoretically and
scanned at 620 nm. Relative amounts of protein in each histone fraction
were determined by integrating areas under each peak of the gel scan and
are expressed as a fraction of the total protein. Data represent values
from three gels, one each from three different experiments.










of radioactivity into various molecular weight fractions of nonhistone

chromosomal proteins. Peaks P, Q and R represent histone fractions H2B

+ H3, H2A, and H4 (for discussion of histone nomenclature see Bradbury,

1975). As mentioned previously, histone Hi migrates anomalously in the

37,000 molecular weight region of the gels present in region K (Hayashi

et al., 1974). The relative amounts of protein in each region of the

gels are similar for chromatin prepared from cells grown with and without

serum (Table 10). An analysis of Figure 27, A and B, indicates variations

occur in specific fractions. Peak A, which consists of nonhistone

chromosomal polypeptides that migrate in in the 120,000 to 130,000

molecular weight region of these gels, is more pronounced in chromatin

from cells grown without serum or with db cAMP. Variations may also be

evident in the E complex, which contains non-histone chromosomal poly-

peptides of molecular weight 64,000 to 78,000.

In the 120,000 to 145,000 molecular weight region of the gel, a

two- to three-fold increase in the incorporation of 3H-tryptophan is

evident in the nonhistone polypeptides which are synthesized and assoc-

iated with the chromatin of cells grown in the presence of serum as

compared to similar material from cells grown without serum. A corres-

ponding increase in the specific activity of these polypeptides is

indicated in Table 10. There is an apparent increase in the specific

activity of peak K in chromatin from cells grown in the presence of

serum; however, the presence of Hl histone in this region makes it

impossible to assign a specific activity to the nonhistone chromosomal

protein component.

In another experiment, the incorporation of 31H-tryptophan into

chromatin components of cells grown with 1.0 mM db cAMP and 10% FCS was