Biochemica et Biophysica Acta 458 (1976) 109-134 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s B B A 87021
CHROMATIN
STRUCTURE
AND
FUNCTION
IN PROLIFERATING
CELLS*
RENATO BASERGA ° and CLAUDIO NICOLINI b
* Department of Pathology and Fels Research Institute, and a Department of Physiology and Biophysics, Committee of Biophysics and Bioengineering, Temple University Health Sciences Center, Philadelphia, Pa. 19140, (U.S.A.) (Received October 22nd, 1975)
CONTENTS I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
110
II.
Isolated c h r o m a t i n a n d c h r o m a t i n o f living cells
110
III.
IV.
Characteristics o f isolated c h r o m a t i n
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . .
111
A.
T e m p l a t e activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
112
B.
Circular dichroism spectra
113
C.
D y e binding
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
114
D.
Effect o f s h e a r i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
115
. . . . . . . . . . . . . . . . . . . . . . . . .
E.
Linear dichroism
F.
Electron microscopy a n d X - r a y diffraction . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117 117
G.
N e u t r o n diffraction at low angle
H.
Nuclear magnetic resonance
. . . . . . . . . . . . . . . . . . . . . .
117
. . . . . . . . . . . . . . . . . . . . . . . .
118
I.
Viscosity a n d t h e r m a l d e n a t u r a t i o n
. . . . . . . . . . . . . . . . . . . . .
119
J.
Fractionation of chromatin . . . . . . . . . . . . . . . . . . . . . . . . .
119
K.
Reconstitution of chromatin
. . . . . . . . . . . . . . . . . . . . . . . .
122
C h r o m a t i n changes d u r i n g the cell cycle . . . . . . . . . . . . . . . . . . . . .
122
A.
C o n t i n u o u s l y dividing cells
. . . . . . . . . . . . . . . . . . . . . . . .
122
B. C.
Go cells stimulated to proliferate . . . . . . . . . . . . . . . . . . . . . . Effect o f low salt extraction . . . . . . . . . . . . . . . . . . . . . . . .
123 125
D.
Significance o f c h r o m a t i n changes
E.
Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V.
C h r o m a t i n c h a n g e s in t r a n s f o r m e d cells
VI.
Conclusions
References
. . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . .
126 128 128
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
* Dedicated to Professor J a m e s B o n n e t , o n the occasion o f his 65th birthday.
110 I. INTRODUCTION The term "chromatin", as used in this review, refers to the complex of DNA, RNA and proteins that can be isolated from living cells as the diffuse interphase form of the chromosome of eukaryotic cells [1,2]. How this chromatin relates to the chromatin originally described as the basophilic staining material of the nucleus by Flemming [3] in 1882, is a question that is as irrelevant as it is hotly debated. Histologists (and cell biologists with morphological proclivities) consider the use of the term chromatin for the isolated complex as improper and misleading, but the fact is that the material called chromatin by Flemming was (and still is) vague and indefinite. Thus, Bonner has as much right to call chromatin his complex as Flemming had to call chromatin the nuclear substance he described histologically. More important for our discussion is to know whether isolated chromatin reflects in vitro the functional and structural characteristics of living chromatin. Therefore, before discussing the characteristics of chromatin in resting and proliferating cells, we shall spend a few words on the relationship of isolated chromatin to the chromatin of living cells. II. ISOLATED CHROMATIN AND CHROMATIN OF LIVING CELLS The question considered in this section is whether the functional capacity of living chromatin is quantitatively and qualitatively preserved in isolated chromatin. In other words, is the RNA synthesized in vitro by isolated chromatin quantitatively and qualitatively similar to that synthesized by the nucleus of the living cell? Quantitatively, there is a good relationship between the capacity of a cell to synthesize R N A in vivo and chromatin template activity in vitro. Thus, template activity is very low in chromatin isolated from cells such as spermatozoa and chicken erythrocytes that synthesize very little RNA in vivo [4--8], and conversely, is higher in chromatin from cells more actively engaged in transcriptional activity [1,9]. A similar quantitative relationship between in vivo RNA synthesis and chromatin template activity is also observed in proliferating cells, as it will be described later. Furthermore, in resting cells stimulated to proliferate, it is possible to show that the physicochemical changes detectable in isolated chromatin also occur in intact nuclei (see below). The RNA transcripts made by chromatin in vitro, especially when using a bacterial R N A polymerase, are not wholly similar to the transcripts made in vivo. The evidence in this regard is convincing [10-13] and can be summarized in the conclusion offered by Reeder [14] who studied mouse and Xenopus chromatin. " . . . there are sequences transcribed in the living cell that Escherichia coli polymerase can also transcribe ... there are sequences not transcribed in the cell but which E. coli polymerase can transcribe ... A third class of sequences are those not transcribed in vivo and also inaccessible to bacterial polymerase". The use of chromatin, prepared by gentler methods, that can utilize its own DNA-dependent RNA polymerase [15], may in part overcome these objections.
Ill TABLE I CHEMICAL COMPOSITION OF CHROMAT1N (in ug/ug DNA) Tissue of origin Chicken brain Chicken liver Chicken erythrocyte Sea urchin sperm Rat liver Calf thymus HeLa cells Pea growing cotyledon Pig cerebellum Rat kidney Human fibroblasts
Histones
Non-histones
RNA
Reference
0.8 0.80 0.84 1.02 1.00 1.14 1.02 0.76 1.60 0.95 1.00
1.9 1.2 0.36 0.13 0.67 0.33 0.71 0.36 0.50 0.70 0.98
0.17 0.10 c
~D
2~
2~
2~
3~
Fig. 6. Effect of salt extraction on CD spectra of W1-38 chrornatin. The chromatins used in this experiment were chromatin from confluent, resting monolayers (--) and chromatin from ceils 6 h after trypsinization and replating at lower density (- - -). The CD spectra were read directly in 0.25 M NaC1.
1VD. Significance of chromatin changes during the cell cycle A simple explanation for the chromatin changes observed in proliferating cells is that they are due to the amount of R N A in the chromatin preparations. Thus it is well known that R N A synthesis increases in Go cells stimulated to proliferate (see review by Stein and Baserga [182]), the increase being detectable as early as the first 10-15 min after stimulation. In continuously dividing cells, R N A synthesis increases sharply during the S phase [183] when changes in CD spectra and ethidium bromide binding are also most dramatic, and reaches a minimum at mitosis. Again, according to Groner et al. [184], gene activation in eukaryotes may be caused by an unwinding of chromatin DNA, and newly synthesized R N A is hydrogen bonded to the single-stranded DNA. Indeed, Hjelm and Huang [185] believe that the decrease in positive ellipticity caused in chromatin by extraction with low concentrations of salt (0.2-0.35), is largely due to the removal of RNA. Thus, at first blush the R N A explanation seems to be very attractive. On closer examination, however, certain discrepancies become apparent that raise the possibility of other explanations. In the first place, let us take the results of Hjelm and Huang [185], which we accept as absolutely reliable and incontrovertible. These authors have prepared their chromatin by the method of Huang and Huang [131] and with this method they
127
\
_--CI
U 0 ~
-.a
O / I
t 1
~0
6o
~80 nm
3oo
i 13 o12 o,. 0'.6 R (added dye/DNA)
Fig. 7. Effect of 0.25 M NaC1 on CD spectra of protein-free DNA and on its ability to bind ethidium bromide: (a) CD spectra between 250-300 nm of protein-free DNA in 10 mM Tris (--), and in 10 mM Tris plus 0.25 M NaC1 (- - -). (b) Elhidium bromide ellipticity at 308 nm as a function of the ratio added dye/DNA phosphate (abscissa). The ordinate gives the ellipticity at [0] 308 of ethidium bromide, complexed with calf thymus DNA in 10 mM Tris, pH 8 ( ©-- ©), or with calf thymus DNA in 10 mM Tris plus 0.25 M NaC1 ([] - - - 5). Other conditions are given in the text.
report an R N A / D N A mass ratio of 0.03 in chromatin o f chick erythrocytes. Using the more c o m m o n Marushige and Bonner method [186], Seligy and Miyagi [9] found in the same chick erythrocyte chromatin an R N A / D N A mass ratio of 0.003, that is, 1/10 the a m o u n t reported by Hjelm and H u a n g [185]. Most, if not all, the data on chromatin changes during the cell cycle have been obtained with chromatin prepared by the method o f Marushige and Bonner [186] as modified by Paul and Gilmour [17]. While the chromatin prepared by the method of H u a n g and H u a n g [131] may actually be a better chromatin than the one usually studied (see the recent paper by Marzluff and H u a n g [15], this possibility is not relevant to the question o f the role o f R N A in chromatin changes. It should also be noted that the R N A isolated from the salt extract by Hjelm and H u a n g [185] has an unusually high ellipticity, as remarked by the authors themselves. Their two main conclusions, however, are worth reporting, namely: (1) " . . . no influence on the C D of the c h r o m o s o m a l D N A by low salt extractable R N A and/or non-histone proteins is detectable"; and (2) the material in the low salt extract " m a y be closely associated with transcription activity o f the chromatin". The last statement is easily agreed upon, since there are now several observations that low salt extraction o f chromatin or nuclei can modify their template activity (see above). W h a t we consider debatable is the first conclusion, which is the
128 natural corollary of the belief that RNA is responsible for the CD changes caused by low salt extraction. The following counter-arguments can be offered: (1) RNA synthesis remains very high in G2 (183], yet, ellipticity in CD spectra and ethidium bromide binding decrease from S to G2 [143]. (2) Non-histone chromosomal proteins isolated by Bio-Rex chromatography [187] and therefore presumably free of any RNA contamination, can modify the CD spectra of nucleohistone complexes [66]. (3) Nicolini et al. [168] were unable to find any appreciable amount of RNA in the 0.25 M NaC1 extract of WI-38 chromatin prepared by the method of Paul and Gilmour [17]. (4) It is difficult to explain an increase in chromatin template activity purely on the basis of the amount of RNA, unless this RNA has a regulatory function. We would like, in turn, to offer the following conclusions: (a) It is clearly established that extraction of chromatin with low concentrations of salt has remarkable effects on its function and structure; and (b) It is still open to question whether RNA or non-histone proteins, or just something else is responsible for these effects.
1VE. Aging There have been several reports in the literature indicating that normal diploid mamalian cells have a finite life span [188]. This is also true of chick fibroblasts, and for exceptions see review by Pont6n [189]. These studies have shown that human diploid fibroblasts progress through three definite stages. Phase I corresponds to the development of a primary culture; phase II cell populations grow exponentially to form confluent monolayers upon subcultivation, while phase II| cells show evidence of decreased proliferative capacity ultimately leading to complete growth failure upon sub-cultivation. This decreased proliferative capacity of phase Ill populations has been associated with several morphological and biochemical cellular alterations [190-192]. Chromatin derived from confluent phase III cell populations differs in circular dichroism spectra and in ethidium bromide binding capacity from the chromatin of cells capable of undergoing a more marked proliferative response to a stimulus to divide (phase If). These structural alterations of chromatin were associated with differences in 0.25 M NaCI extractable proteins and with differences in population kinetics [193]. These findings are in agreement with the recent report by Ryan and Cristofalo [194] that chromatin template activity is lower in senescent than in young WI-38 fibroblasts. V. CHROMATIN CHANGES IN TRANSFORMED CELLS Table VI summarizes the literature reports on differences observed in chromatin and chromosomal proteins between neoplastic cells and their normal counterparts. Immunological, physicochemical and biochemical differences have been described. Differences extend to whole chromatin and non-histone proteins, but not to histones,
129 TABLE Vl REPORTED DIFFERENCES IN CHROMATIN AND CHROMOSOMAL PROTEINS BETWEEN NORMAL AND TRANSFORMED CELLS Cell types WI-38 and 2RA (SV-40) WI-38 and 2RA Lymphocytes and leukemic cells Rat liver and Novikoff hepatoma Liver and hepatomas Wl-38 and 2RA Brain cells and neuroectodermal tumors Liver and hepatoma Liver and hepatomas WI-38 and 2RA
Differences in chromatin NHCP NHCP NHCP NHCP NHCP NHCP NHCP NHCP NHCP
Reference 195 196 33 197 198 199 200, 201 202 203 204
which seem to be indistinguishable in normal and transformed cells [195]. All reports listed in Table VI, including those from our own laboratory, are open to considerable criticism, not of the data, but of their interpretation. When an animal tumor is compared to its tissue of origin (for instance, hepatoma to liver), the criticism is almost intuitive (see also review by Baserga [36]). The liver has many cell types that have disappeared or are markedly decreased in number in hepatomas, and the differences observed may reflect differences in cell types rather than meaningful differences between normal and neoplastic cells. The use of lymphocytes or cell cultures is an improvement, but not a solution of the problem. The cell lines used (of transformed cells) are so grossly aneuploid that the differences probably reflect changes subsequent to rather than concomitant with transformation. Despite these reservations, these studies do point out some differences in chromatin and non-histone proteins (but not histones) of normal and transformed cells, and suggest that the primary inquiry should be extended to other more rigorous models, wich are presently becoming available. Indeed, preliminary studies have been carried out on a subclone of H6-15 cells, which are temperature-sensitive mutants of SV-40 transformed 3T3 cells. This mutant, isolated by Renger and Basilico [205] expresses the transformed phenotype at low (32 °C) but not at high (39 °C) temperature. Three parameters typical of in vitro transformation are present at the lower temperature, namely, high saturation density, ability to form colonies on monolayers of normal 3T3 cells, and lack of contact-inhibition of D N A synthesis [205]. We have studied the chromatin of this mutant at both temperatures and at different stages of growth and quiescence (lde and Baserga [206]). Circular dichroism and ethidium bromide binding capacity were the two parameters used to characterize chromatin. We found that circular dichroism spectra were strictly dependent on the extent of cell proliferation. During the exponential phase of growth, the CD spectra of chromatin were exactly the same, whether the cells were grown at 32°C (transformed) or at 39 °C (untransformed
130 phenotype) Ethidium bromide binding capacity was also exactly the same, provided the cells were exponentially growing. At confluence, the chromatin from cells at 32 °C (transformed) exhibited a higher maximum positive eltipticity in CD spectra and a higher capacity to bind ethidium bromide than the chromatin of cells at 39 °C (untransformed). However, it could be shown that H6-15 cells at 39 °C are truly resting, while at 32 °C they continue to proliferate although the number of cells per dish remains stationary [205,206]. It therefore seems from these data that the structure and function of chromatin can be readily changed by variations in the rate of cell proliferation, but that the methods used do not allow us to distinguish between cells expressing the transformed phenotype and the same cells expressing the untransformed phenotype. It still remains to be seen whether the data collected in Table VI are valid or whether they simply reflect differences in the extent of cell proliferation. It seems clear, however, that a comparison between normal and tumor tissues can be meaningful only when both normal and neoplastic cells have the same rate of cell proliferation.
V1. CONCLUSIONS The conclusions that we would like to draw from this review are the following: (a) Chromatin structure and function are exceedingly sensitive to changes in the proliferative state of a cell. Differences can be detected between cells in mitosis, G1 and S, and even between Go and G1 cells. (b) These differences are very unlikely to be artifactual, since similar changes can also be demonstrated in intact nuclei. (c) Some of these differences can be abolished by extraction of chromatins with low concentrations of salt. (d) Differences between chromatins of normal and neoplastic cells can also be detected, but they are largely related to differences in the extent of cell proliferation. (e) A number of laboratories have been very busy in trying to elucidate chromatin structure with different technologies. Sometimes a change in a macromolecule caused by a physiological stimulus can tell us as much about its structure as a thousand instruments. The changes occurring in chromatin of proliferating cells could perhaps be profitably used to know more about chromatin structure.
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CHROMATIN
STRUCTURE
AND
FUNCTION
IN PROLIFERATING
CELLS*
RENATO BASERGA ° and CLAUDIO NICOLINI b
* Department of Pathology and Fels Research Institute, and a Department of Physiology and Biophysics, Committee of Biophysics and Bioengineering, Temple University Health Sciences Center, Philadelphia, Pa. 19140, (U.S.A.) (Received October 22nd, 1975)
CONTENTS I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
110
II.
Isolated c h r o m a t i n a n d c h r o m a t i n o f living cells
110
III.
IV.
Characteristics o f isolated c h r o m a t i n
. . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . .
111
A.
T e m p l a t e activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
112
B.
Circular dichroism spectra
113
C.
D y e binding
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
114
D.
Effect o f s h e a r i n g . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
115
. . . . . . . . . . . . . . . . . . . . . . . . .
E.
Linear dichroism
F.
Electron microscopy a n d X - r a y diffraction . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117 117
G.
N e u t r o n diffraction at low angle
H.
Nuclear magnetic resonance
. . . . . . . . . . . . . . . . . . . . . .
117
. . . . . . . . . . . . . . . . . . . . . . . .
118
I.
Viscosity a n d t h e r m a l d e n a t u r a t i o n
. . . . . . . . . . . . . . . . . . . . .
119
J.
Fractionation of chromatin . . . . . . . . . . . . . . . . . . . . . . . . .
119
K.
Reconstitution of chromatin
. . . . . . . . . . . . . . . . . . . . . . . .
122
C h r o m a t i n changes d u r i n g the cell cycle . . . . . . . . . . . . . . . . . . . . .
122
A.
C o n t i n u o u s l y dividing cells
. . . . . . . . . . . . . . . . . . . . . . . .
122
B. C.
Go cells stimulated to proliferate . . . . . . . . . . . . . . . . . . . . . . Effect o f low salt extraction . . . . . . . . . . . . . . . . . . . . . . . .
123 125
D.
Significance o f c h r o m a t i n changes
E.
Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
V.
C h r o m a t i n c h a n g e s in t r a n s f o r m e d cells
VI.
Conclusions
References
. . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . .
126 128 128
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
* Dedicated to Professor J a m e s B o n n e t , o n the occasion o f his 65th birthday.
110 I. INTRODUCTION The term "chromatin", as used in this review, refers to the complex of DNA, RNA and proteins that can be isolated from living cells as the diffuse interphase form of the chromosome of eukaryotic cells [1,2]. How this chromatin relates to the chromatin originally described as the basophilic staining material of the nucleus by Flemming [3] in 1882, is a question that is as irrelevant as it is hotly debated. Histologists (and cell biologists with morphological proclivities) consider the use of the term chromatin for the isolated complex as improper and misleading, but the fact is that the material called chromatin by Flemming was (and still is) vague and indefinite. Thus, Bonner has as much right to call chromatin his complex as Flemming had to call chromatin the nuclear substance he described histologically. More important for our discussion is to know whether isolated chromatin reflects in vitro the functional and structural characteristics of living chromatin. Therefore, before discussing the characteristics of chromatin in resting and proliferating cells, we shall spend a few words on the relationship of isolated chromatin to the chromatin of living cells. II. ISOLATED CHROMATIN AND CHROMATIN OF LIVING CELLS The question considered in this section is whether the functional capacity of living chromatin is quantitatively and qualitatively preserved in isolated chromatin. In other words, is the RNA synthesized in vitro by isolated chromatin quantitatively and qualitatively similar to that synthesized by the nucleus of the living cell? Quantitatively, there is a good relationship between the capacity of a cell to synthesize R N A in vivo and chromatin template activity in vitro. Thus, template activity is very low in chromatin isolated from cells such as spermatozoa and chicken erythrocytes that synthesize very little RNA in vivo [4--8], and conversely, is higher in chromatin from cells more actively engaged in transcriptional activity [1,9]. A similar quantitative relationship between in vivo RNA synthesis and chromatin template activity is also observed in proliferating cells, as it will be described later. Furthermore, in resting cells stimulated to proliferate, it is possible to show that the physicochemical changes detectable in isolated chromatin also occur in intact nuclei (see below). The RNA transcripts made by chromatin in vitro, especially when using a bacterial R N A polymerase, are not wholly similar to the transcripts made in vivo. The evidence in this regard is convincing [10-13] and can be summarized in the conclusion offered by Reeder [14] who studied mouse and Xenopus chromatin. " . . . there are sequences transcribed in the living cell that Escherichia coli polymerase can also transcribe ... there are sequences not transcribed in the cell but which E. coli polymerase can transcribe ... A third class of sequences are those not transcribed in vivo and also inaccessible to bacterial polymerase". The use of chromatin, prepared by gentler methods, that can utilize its own DNA-dependent RNA polymerase [15], may in part overcome these objections.
Ill TABLE I CHEMICAL COMPOSITION OF CHROMAT1N (in ug/ug DNA) Tissue of origin Chicken brain Chicken liver Chicken erythrocyte Sea urchin sperm Rat liver Calf thymus HeLa cells Pea growing cotyledon Pig cerebellum Rat kidney Human fibroblasts
Histones
Non-histones
RNA
Reference
0.8 0.80 0.84 1.02 1.00 1.14 1.02 0.76 1.60 0.95 1.00
1.9 1.2 0.36 0.13 0.67 0.33 0.71 0.36 0.50 0.70 0.98
0.17 0.10 c
~D
2~
2~
2~
3~
Fig. 6. Effect of salt extraction on CD spectra of W1-38 chrornatin. The chromatins used in this experiment were chromatin from confluent, resting monolayers (--) and chromatin from ceils 6 h after trypsinization and replating at lower density (- - -). The CD spectra were read directly in 0.25 M NaC1.
1VD. Significance of chromatin changes during the cell cycle A simple explanation for the chromatin changes observed in proliferating cells is that they are due to the amount of R N A in the chromatin preparations. Thus it is well known that R N A synthesis increases in Go cells stimulated to proliferate (see review by Stein and Baserga [182]), the increase being detectable as early as the first 10-15 min after stimulation. In continuously dividing cells, R N A synthesis increases sharply during the S phase [183] when changes in CD spectra and ethidium bromide binding are also most dramatic, and reaches a minimum at mitosis. Again, according to Groner et al. [184], gene activation in eukaryotes may be caused by an unwinding of chromatin DNA, and newly synthesized R N A is hydrogen bonded to the single-stranded DNA. Indeed, Hjelm and Huang [185] believe that the decrease in positive ellipticity caused in chromatin by extraction with low concentrations of salt (0.2-0.35), is largely due to the removal of RNA. Thus, at first blush the R N A explanation seems to be very attractive. On closer examination, however, certain discrepancies become apparent that raise the possibility of other explanations. In the first place, let us take the results of Hjelm and Huang [185], which we accept as absolutely reliable and incontrovertible. These authors have prepared their chromatin by the method of Huang and Huang [131] and with this method they
127
\
_--CI
U 0 ~
-.a
O / I
t 1
~0
6o
~80 nm
3oo
i 13 o12 o,. 0'.6 R (added dye/DNA)
Fig. 7. Effect of 0.25 M NaC1 on CD spectra of protein-free DNA and on its ability to bind ethidium bromide: (a) CD spectra between 250-300 nm of protein-free DNA in 10 mM Tris (--), and in 10 mM Tris plus 0.25 M NaC1 (- - -). (b) Elhidium bromide ellipticity at 308 nm as a function of the ratio added dye/DNA phosphate (abscissa). The ordinate gives the ellipticity at [0] 308 of ethidium bromide, complexed with calf thymus DNA in 10 mM Tris, pH 8 ( ©-- ©), or with calf thymus DNA in 10 mM Tris plus 0.25 M NaC1 ([] - - - 5). Other conditions are given in the text.
report an R N A / D N A mass ratio of 0.03 in chromatin o f chick erythrocytes. Using the more c o m m o n Marushige and Bonner method [186], Seligy and Miyagi [9] found in the same chick erythrocyte chromatin an R N A / D N A mass ratio of 0.003, that is, 1/10 the a m o u n t reported by Hjelm and H u a n g [185]. Most, if not all, the data on chromatin changes during the cell cycle have been obtained with chromatin prepared by the method o f Marushige and Bonner [186] as modified by Paul and Gilmour [17]. While the chromatin prepared by the method of H u a n g and H u a n g [131] may actually be a better chromatin than the one usually studied (see the recent paper by Marzluff and H u a n g [15], this possibility is not relevant to the question o f the role o f R N A in chromatin changes. It should also be noted that the R N A isolated from the salt extract by Hjelm and H u a n g [185] has an unusually high ellipticity, as remarked by the authors themselves. Their two main conclusions, however, are worth reporting, namely: (1) " . . . no influence on the C D of the c h r o m o s o m a l D N A by low salt extractable R N A and/or non-histone proteins is detectable"; and (2) the material in the low salt extract " m a y be closely associated with transcription activity o f the chromatin". The last statement is easily agreed upon, since there are now several observations that low salt extraction o f chromatin or nuclei can modify their template activity (see above). W h a t we consider debatable is the first conclusion, which is the
128 natural corollary of the belief that RNA is responsible for the CD changes caused by low salt extraction. The following counter-arguments can be offered: (1) RNA synthesis remains very high in G2 (183], yet, ellipticity in CD spectra and ethidium bromide binding decrease from S to G2 [143]. (2) Non-histone chromosomal proteins isolated by Bio-Rex chromatography [187] and therefore presumably free of any RNA contamination, can modify the CD spectra of nucleohistone complexes [66]. (3) Nicolini et al. [168] were unable to find any appreciable amount of RNA in the 0.25 M NaC1 extract of WI-38 chromatin prepared by the method of Paul and Gilmour [17]. (4) It is difficult to explain an increase in chromatin template activity purely on the basis of the amount of RNA, unless this RNA has a regulatory function. We would like, in turn, to offer the following conclusions: (a) It is clearly established that extraction of chromatin with low concentrations of salt has remarkable effects on its function and structure; and (b) It is still open to question whether RNA or non-histone proteins, or just something else is responsible for these effects.
1VE. Aging There have been several reports in the literature indicating that normal diploid mamalian cells have a finite life span [188]. This is also true of chick fibroblasts, and for exceptions see review by Pont6n [189]. These studies have shown that human diploid fibroblasts progress through three definite stages. Phase I corresponds to the development of a primary culture; phase II cell populations grow exponentially to form confluent monolayers upon subcultivation, while phase II| cells show evidence of decreased proliferative capacity ultimately leading to complete growth failure upon sub-cultivation. This decreased proliferative capacity of phase Ill populations has been associated with several morphological and biochemical cellular alterations [190-192]. Chromatin derived from confluent phase III cell populations differs in circular dichroism spectra and in ethidium bromide binding capacity from the chromatin of cells capable of undergoing a more marked proliferative response to a stimulus to divide (phase If). These structural alterations of chromatin were associated with differences in 0.25 M NaCI extractable proteins and with differences in population kinetics [193]. These findings are in agreement with the recent report by Ryan and Cristofalo [194] that chromatin template activity is lower in senescent than in young WI-38 fibroblasts. V. CHROMATIN CHANGES IN TRANSFORMED CELLS Table VI summarizes the literature reports on differences observed in chromatin and chromosomal proteins between neoplastic cells and their normal counterparts. Immunological, physicochemical and biochemical differences have been described. Differences extend to whole chromatin and non-histone proteins, but not to histones,
129 TABLE Vl REPORTED DIFFERENCES IN CHROMATIN AND CHROMOSOMAL PROTEINS BETWEEN NORMAL AND TRANSFORMED CELLS Cell types WI-38 and 2RA (SV-40) WI-38 and 2RA Lymphocytes and leukemic cells Rat liver and Novikoff hepatoma Liver and hepatomas Wl-38 and 2RA Brain cells and neuroectodermal tumors Liver and hepatoma Liver and hepatomas WI-38 and 2RA
Differences in chromatin NHCP NHCP NHCP NHCP NHCP NHCP NHCP NHCP NHCP
Reference 195 196 33 197 198 199 200, 201 202 203 204
which seem to be indistinguishable in normal and transformed cells [195]. All reports listed in Table VI, including those from our own laboratory, are open to considerable criticism, not of the data, but of their interpretation. When an animal tumor is compared to its tissue of origin (for instance, hepatoma to liver), the criticism is almost intuitive (see also review by Baserga [36]). The liver has many cell types that have disappeared or are markedly decreased in number in hepatomas, and the differences observed may reflect differences in cell types rather than meaningful differences between normal and neoplastic cells. The use of lymphocytes or cell cultures is an improvement, but not a solution of the problem. The cell lines used (of transformed cells) are so grossly aneuploid that the differences probably reflect changes subsequent to rather than concomitant with transformation. Despite these reservations, these studies do point out some differences in chromatin and non-histone proteins (but not histones) of normal and transformed cells, and suggest that the primary inquiry should be extended to other more rigorous models, wich are presently becoming available. Indeed, preliminary studies have been carried out on a subclone of H6-15 cells, which are temperature-sensitive mutants of SV-40 transformed 3T3 cells. This mutant, isolated by Renger and Basilico [205] expresses the transformed phenotype at low (32 °C) but not at high (39 °C) temperature. Three parameters typical of in vitro transformation are present at the lower temperature, namely, high saturation density, ability to form colonies on monolayers of normal 3T3 cells, and lack of contact-inhibition of D N A synthesis [205]. We have studied the chromatin of this mutant at both temperatures and at different stages of growth and quiescence (lde and Baserga [206]). Circular dichroism and ethidium bromide binding capacity were the two parameters used to characterize chromatin. We found that circular dichroism spectra were strictly dependent on the extent of cell proliferation. During the exponential phase of growth, the CD spectra of chromatin were exactly the same, whether the cells were grown at 32°C (transformed) or at 39 °C (untransformed
130 phenotype) Ethidium bromide binding capacity was also exactly the same, provided the cells were exponentially growing. At confluence, the chromatin from cells at 32 °C (transformed) exhibited a higher maximum positive eltipticity in CD spectra and a higher capacity to bind ethidium bromide than the chromatin of cells at 39 °C (untransformed). However, it could be shown that H6-15 cells at 39 °C are truly resting, while at 32 °C they continue to proliferate although the number of cells per dish remains stationary [205,206]. It therefore seems from these data that the structure and function of chromatin can be readily changed by variations in the rate of cell proliferation, but that the methods used do not allow us to distinguish between cells expressing the transformed phenotype and the same cells expressing the untransformed phenotype. It still remains to be seen whether the data collected in Table VI are valid or whether they simply reflect differences in the extent of cell proliferation. It seems clear, however, that a comparison between normal and tumor tissues can be meaningful only when both normal and neoplastic cells have the same rate of cell proliferation.
V1. CONCLUSIONS The conclusions that we would like to draw from this review are the following: (a) Chromatin structure and function are exceedingly sensitive to changes in the proliferative state of a cell. Differences can be detected between cells in mitosis, G1 and S, and even between Go and G1 cells. (b) These differences are very unlikely to be artifactual, since similar changes can also be demonstrated in intact nuclei. (c) Some of these differences can be abolished by extraction of chromatins with low concentrations of salt. (d) Differences between chromatins of normal and neoplastic cells can also be detected, but they are largely related to differences in the extent of cell proliferation. (e) A number of laboratories have been very busy in trying to elucidate chromatin structure with different technologies. Sometimes a change in a macromolecule caused by a physiological stimulus can tell us as much about its structure as a thousand instruments. The changes occurring in chromatin of proliferating cells could perhaps be profitably used to know more about chromatin structure.
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