Eur. J. Biochem. 77, 545-553 (1977)
Metabolic Behaviour of Nonhistone Chromosomal Proteins in Proliferating and in Resting Fibroblasts Lalju Petrov DJONDJUROV, Emilia Christova IVANOVA, and Roumen Georgiev TSANEV Institute of Biochemistry, Bulgarian Academy of Sciences, Sofia (Received April 12, 1977)
The metabolism of nonhistone chromosomal proteins was studied in two lines of cells showing a different degree of contact inhibition : human diploid fibroblasts, which are easily contactinhibited, and Chinese hamster fibroblasts, which had been made to stop proliferating by fasting. By following the 3H/14C ratio of [3H]tryptophan-labelled nonhistone chromosomal proteins and [14C]thymidine-labelledDNA in chase experiments three main groups of these proteins could be detected with respect to their metabolic behaviour: (a) a metabolically stable group which is acid-insoluble and represents the bulk of nonhistone chromosomal proteins in proliferating cells ; this group is conserved when the cells enter a resting phase; (b) a metabolically labile group which is acid-soluble and is observed as a minor fraction in proliferating cells; (c) a metabolically labile group which is acid-insoluble and accumulates in resting cells; this fraction is much larger in contact-inhibited cells. Stimulation of cell proliferation by trypsinization decreases the amount of nonhistone chromosomal proteins in resting cells to the basic level observed in proliferating cells. Until recently nonhistone chromosomal proteins were believed to represent a rapidly metabolized group [l]. However, in a previous study we have found that fibroblasts cultured in vitro contain a fraction of metabolically stable nonhistone chromosomal proteins completely preserved in resting cells and partly eliminated when the cells are stimulated to proliferate by trypsinization [2]. Metabolically stable nonhistone chromosomal proteins were found also in rat liver where a fraction of these proteins labelled after partial hepatectomy was preserved after a long chase [3]. The existence of metabolically stable nonhistone chromosomal proteins was also proved in the case of continuously proliferating HeLa cells [4]. Unlike the results obtained with fibroblasts and liver cells, in HeLa cells all these proteins were reported to be completely preserved. The question arose whether this difference reflected a difference between growthregulated and non-regulated cells or a difference between continuously proliferating cells and resting cells stimulated to proliferate. In the present paper we have studied the metabolic stability of nonhistone chromosomal proteins in two lines of fibroblasts exhibiting a different degree of contact inhibition. For this purpose the conservation of [3H]tryptophan in these proteins was followed in resting and in proliferating cells and during the transition between these two states. It was found that both metabolically stable and metabolically
labile nonhistone chromosomal proteins existed, but in proliferating cells the bulk of these proteins were metabolically stable. MATERIALS AND METHODS Cells Two cellular lines were used: Chinese hamster fibroblasts (BII dii, FAF-28, see [ 5 ] ) and diploid human lung fibroblasts (of passages 18 to 24). Both cellular lines were grown as monolayer cultures. The cells were cultivated in 250-ml flasks with 30 ml of minimum essential medium (Eurobio, France) in the case of Chinese hamster fibroblasts (plated at a density of 9.104 cells/cm2) or of basal medium (Eurobio, France) in the case of human fibroblasts (plated at a density of 6.104 cells/cm2). In both cases the medium was supplemented with 20 % calf serum. Under these conditions if the medium was not changed the human diploid fibroblasts entered a stationary phase after 4 days of proliferation (early stationary phase). A late stationary phase was reached if the medium was not changed for another period of 4 days. The resting cells were stimulated to proliferate either by trypsinization (0.25 % Difco trypsin free of Mg2+and Ca2+)or by their mechanical detachment and replating in a fresh medium. The stimulated cells started DNA synthesis 10- 12 h after replating.
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Metabolic Behaviour of Nonhistone Chromosomal Proteins in Fibroblasts
When synchronized cultures were used Chinese hamster fibroblasts were grown in 1000-ml flasks in medium 199 containing 10 % calf serum. Synchronization was achieved by detachment of metaphase cells arrested by colcemid [6] as follows: the cells were treated with colcemid (0.05 pg/ml) for 4 h, then trypsinized for 45 s with 0.1 % trypsin at 4 "C and detached by vigorous shaking in chilled medium 199 with serum. This resulted in a suspension of 94 - 98 % metaphase cells which were collected by centrifugation at 600 x g and plated in 100-ml flasks at a density of 5 x lo6 cells per flask. In all experiments the completion of mitosis and its duration in colcemid-blocked cells were controlled by phase-contrast microscopy. Labelling and Chase Short-Term Experiments with Proliferating Cells. Chinese hamster fibroblasts were pulse-labelled for 30 s with [3H]tryptophan, 10 pCi/ml (Amersham, 6.4 Ci/mmol). The incorporation of the labelled compound was stopped by adding 10 times the volume of a chilled medium rich in unlabelled tryptophan (200 mg/l). After washing the cells with the same medium they were grown in it for different periods up to 30 min. After the chase they were frozen in liquid nitrogen. Long-Term Experiments with Proliferating Cells. One day after plating the cells of both cell lines were labelled for 22-24 h with [14C]thymidine, 0.05 pCi/ ml (Amersham, 57 mCi/mmol). To obtain a better leveling of the labelled DNA the cells from all flasks were collected by trypsinization, pooled and replated at the same initial density. One day later the medium was replaced by a tryptophan-free medium and the cells were labelled for 5 h (Chinese hamster fibroblasts) or for 7 h (human diploid fibroblasts) with [3H]tryptophan, 2 pCi/ml. After labelling the cells were washed twice with a medium rich in unlabelled tryptophan (200 mg/l) and allowed to grow in the same tryptophan-rich medium. During the chase period the human fibroblasts entered a stationary phase after 4 days if the medium was not changed. Experiments with Synchronized Cells. Chinese hamster fibroblasts were labelled with [3H]thymidine (0.1 pCi/ml) during the period of active proliferation. After synchronization (see above) they were pulselabelled for 30 min with [14C]tryptophan (0.2 pCi/ml) at different intervals after plating : immediately and 30 min after plating (metaphase and telophase cells respectively), at the 3rd hour (GI cells), at the 8th hour (S cells) and at the 15th hour (Gz cells). At each interval part of the cells were collected immediately after labelling to determine the initial level of 14C incorporation. The remaining cells were washed with a medium containing an excess of unlabelled tryptophan (200 mg/l) and allowed to grow further in the
same medium. During this chase period the cells were maintained in a state of active proliferation by daily replating. Samples containing about 2 x lo7 cells were withdrawn 6, 12, 24, 48 and 72 h after the beginning of the chase. All cells were collected by centrifugation at 600 x g , washed with the same medium without calf serum and used for isolation of chromatin. Resting Cells. Human fibroblasts with prelabelled [14C]DNA obtained as described above (for longterm experiments) were grown for 8 days without changing the medium (late stationary phase) and were then labelled for 7 h with [3H]tryptophan (5 pCi/ml) in the same medium. The chase was performed also in a conditioned medium containing 200 mg/l unlabelled tryptophan. For electrophoretic fractionation of nonhistone chromosomal proteins the cells were labelled with [3H]tryptophan only (10 pCi/ml) during the same period and in the same way as their corresponding double-labelled counterparts. Isolation of Chromatin and Nonhistone Chromosomal Proteins The cells were washed with a fresh culture medium and suspended for 5min in a hypotonic medium (10 mM Tris . HCl, 10 mM KCl, 1.6 mM MgClZ, pH 8.1). Nonidet P-40 was then added in a final concentration of 1 %, the cell suspension was left in the cold for 5 min and centrifuged. After two such treatments pure nuclei were obtained without detectable cytoplasmic contaminations as checked by phasecontrast microscopy. The nuclear pellet was extracted twice with 0.075 M NaCl, 0.025 M EDTA, twice with 0.037 M NaCl, 0.012 M EDTA and dialysed against deionized water. In some experiments 1 mM sodium bisulfite was present in all solutions. Histones together with a small fraction of acidsoluble nonhistone chromosomal proteins were extracted with 125 mM HzS04 and the acid-insoluble nonhistone chromosomal proteins were dissolved in 0.1 M NaOH. Analytical Procedures DNA was determined by the method of Burton [7] and proteins by the procedure of Lowry [8]. For radioactivity counting the sample was dissolved in 10 vol. of a toluene/Triton X-100 scintillation mixture [9]. Double-labelled (3H and 14C) material was differentially counted in a Packard Tricarb 3200 liquid scintillation spectrometer. Gel Electrophoresis [3H]Tryptophan-labelled nonhistone chromosomal proteins were fractionated in polyacrylamide gel
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L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev
according to Laemmli [lo]. The gels were stained with Coomassie brilliant blue and scanned at 520nm. They were then sliced at 1-mm intervals, the slices were treated with Protosol and their radioactivity measured.
experiments. It demonstrates that some labelled proteins continue to enter the nucleus from the cytoplasm during the chase period. The same regularity in the metabolism of nonhistone chromosomal proteins is found with diploid human fibroblasts (Fig. 1B). The difference between the two cellular lines is that in the human fibroblasts only 30% of the label was lost with a half-life of 12 h as compared to 45 % in the Chinese hamster fibroblasts with a half-life of 9 h. The different metabolic behaviour of the acidsoluble and the acid-insoluble fractions in proliferating cells can be demonstrated also in short pulse experiments. As seen in Fig.2, the minor acid-soluble frac-
RESULTS AND DISCUSSION Proliferating Cells
When Chinese hamster fibroblasts were labelled during the phase of exponential growth two distinct fractions of nonhistone chromosomal proteins could be distinguished with respect to their metabolic stability. As seen in Fig. 1A the 3H/14C ratio of the total nonhistone chromosomal proteins decreases by 45 % during the first 24 h of the chase and then remains constant or increases slightly. During the whole chase period the cells continue to proliferate, although with a slightly diminishing rate as shown by the slope of the curve of the specific radioactivity of DNA. These data show the synthesis in proliferating cells of two classes of nonhistone chromosomal proteins which associate with the chromatin: one which is metabolized during the first 24 h and a second which is metabolically stable. When these proteins are divided into two groups, acid-soluble (extracted with the histones) and acid-insoluble, it is seen that the metabolically labile proteins go into the acidsoluble group which loses 70% of its label, while the acid-insoluble proteins are metabolically stable (Fig. 1A), The slight increase in the 3H/'4C ratio of the metabolically stable group was reproduced in all
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Fig. 2. Accumulation and turnover of the acid-soluble ( @ - - O ) and of the acid-insoluble fractions ( b e ) of nonhistone chromosomal proteins in short pulse experiments. The logarithms o f the specific radioactivity, A,, were measured. Proliferating Chinese hamster fibroblasts were pulse-labelled for 30 s with [3H]tryptophan, washed with a medium containing unlabelled tryptophan and allowed to grow in the same medium
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Fig.1. Turnover of nonhistone chromosomal proteins in proliferating fibroblasts. (A) Chinese hamster fibroblasts; (B) human diploid fibroblasts. The relative changes of the 3H/'4C ratio in the total chromatin (A-A), in the acid-insoluble (M and ) in the acid-soluble fractions (G---o) and the logarithm of the specific radioactivity of DNA ( A , ) were measured. Proliferating cells with prelabelled [I4C]DNA were labelled for 5 h (A) or for 7 h (B) with [3H]tryptophan and then allowed to grow in the presence of unlabelled tryptophan without further changes of medium
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Metabolic Behaviour of Nonhistone Chromosomal Proteins in Fibroblasts
Histones
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Fig. 3. Electrophoretic pattern of the acid-soluble chromosomal proteins of proliferating Chinese hamster fibroblasts. Staining intensity, As20 (-) and labelling before (o----o) and after a 48-h chase (04). The cells were labelled for 5 h with [3H]tryptophan and chased for 48 h in a medium rich in unlabelled tryptophan. Chromatin was isolated at zero time and after a 48-h chase. The acid-soluble fraction of the chromatin was precipitated with 3 vol. of ethanol and fractionated according to Laemmli [9]
tion synthesized during a 30-s pulse with [3H]tryptophan shows a rapid decrease in its specific activity during a 30-min chase, while the labelling of the major fraction of acid-insoluble proteins continues to increase. Under conditions of cell proliferation in both cellular lines the metabolically stable proteins represent the major fraction of nonhistone chromosomal proteins, since the acid-soluble group extracted together with the histones makes no more than 10% of the total of these proteins. As shown in Fig.3, the acid-soluble proteins represent a highly heterogeneous group with a high specific radioactivity after a 5-h pulse with [3H]tryptophan. The presence of acid-soluble nonhistone proteins in the chromatin of proliferating cells is in agreement with the finding that this protein fraction strongly increases in metaphase chromosomes [ll]. In our experiments the amount of acid-soluble nonhistone chromosomal proteins in resting fibroblasts was very small. The question might be asked whether these proteins are not a cytoplasmic contamination due to the intimate contact of the chromosomes with the cytoplasm in dividing cells. The latter possibility is suggested by experimental evidence that acid-soluble cytoplasmic proteins are adsorbed onto metaphase chromosomes during the isolation procedure [12]. However, our experiments with synchronized cells are not in agreement with this possibility. As seen in Fig. 4, the amount of 14C-labelled nonhistone chromosomal proteins turned over during the chase
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Fig.4. Relative changes of the I4Cl3H ratio of' chromatin during the chase period of synchronized Chinese hamster fibroblasts prelabelled with [3H]thymidine and pulse-labelled for 30 min with ('4C]tryptophan in metaphase (A-A), telophase (M), GI ( a d ) ,S (-1 and G2 ( M i .During the chase period the cells were maintained in active proliferation by daily replating
period is minimal when labelling was at metaphase : 15 % as compared with 50 % loss of the label incorporated in telephase, GI, S and Gz cells. These data show that metabolically labile nonhistone proteins are synthesized during the whole cell cycle and they are present in the chromatin at periods when the chromosomes are not in a direct contact with the cytoplasm. Thus, under our conditions of chromatin isolation it seems very likely that the acid-soluble
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Fig. 5 . Electrophoretic pattern of the acid-insoluble nonhistone chromosomal proteins of proliferating Chinese hamster ,fibroblasts. Staining intensity, A520(-) and labelling before (---a) and after a 48-h chase ( 0 0 ) .The cells were labelled and chased as in Fig. 3. The acidsoluble proteins were extracted and the remaining material was fractionated as in Fig. 3
metabolically labile fraction of nonhistone proteins is not a cytoplasmic contaminant, but represent a real constituent of the chromatin of proliferating cells. Thus, if we eliminate the acid-soluble fraction all remaining nonhistone chromosomal proteins of proliferating cells are metabolically stable. This explains the results of Seale [4] obtained on continuously proliferating HeLa cells after the elimination of all acid-soluble proteins. The acid-insoluble metabolically stable nonhistone chromosomal proteins of proliferating cells are also a heterogeneous group of proteins not only with respect to their molecular weights but also concerning their labelling pattern after a 5-h pulse with [3H]tryptophan. As seen in Fig.5, this group can be separated by dodecylsulfate gel electrophoresis into about 20 bands each of which has a different specific radioactivity. This may reflect both differences in tryptophan content and different migration rates into the chromatin of the newly synthesized proteins. A 48-h chase leads to a more than 4-fold decrease in the incorporated radioactivity due to a dilution corresponding to more than two cell cycles which are expected to take place during 48 h. Resting Cells
The nonhistone chromosomal proteins of resting cells exhibit a different metabolic behaviour. When human diploid fibroblasts with prelabelled [I4C]DNA enter a late stationary phase their nonhistone proteins
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Fig. 6 . Accumulation and turnover o j nonhistone chromosomal proteins in resting human diploidfibroblasts. (A) The relative changes of the 3H/'4C ratio in the total chromatin (0-0) and of the acid-insoluble fraction ( G - - - O ) ; (B) logarithms of the specific radioactivity of DNA. Human diploid fibroblasts with prelabelled [14C]DNAwere labelled for 7 h with [3H]tryptophan during the late stationary phase and chased in a medium rich in unlabelled tryptophan
incorporate [3H]tryptophan at a significant rate. Considering the almost complete arrest of DNA synthesis (Fig. 6 B) and the constant nonhistone protein/DNA ratio, this demonstrates the presence of metabolically labile proteins. As seen in Fig.6A the
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Fig. 7. Electrophoretic pattern of the acid-insoluble proteins of' resting human diploid,fibrohlasts. Staining intensity, A520 (-) and labelling and after a 48-h chase (00). The cells were labelled for 7 h with [3H]tryptophan and chased in a medium rich in before (&---O) unlabelled tryptophan. Chromatin was isolated at zero time and after a 48-h chase. After the extraction of the acid-soluble proteins the remaining material was fractionated as in Fig. 3
label continues to enter the chromatin during the first two days of the chase showing the low migration rate of some proteins into the nucleus. Then the 3H/'4C ratio begins to decrease, as would be expected for proteins which are continuously turned over. The same figure shows that the behaviour of the acidinsoluble fraction is identical to that of the total nonhistone chromosomal proteins. In fact the [3H]tryptophan radioactivity of the acid-soluble fraction of resting cells is negligibly small as compared to that of the acid-insoluble fraction. The electrophoretic pattern of nonhistone chromosomal proteins of resting human diploid fibroblasts is shown in Fig.7. It is seen that the labelling is mainly due to several fractions and that one highly labelled fraction only is responsible for the continuously increasing labelling during the first two days of the chase. The presence of electrophoretic fractions which remain practically unlabelled is consistent with the existence in resting cells of metabolically stable proteins which have been synthesized during the period of proliferation. Such proteins are located mainly in the high-molecular-weight region.
Transition Periods The transition of the human diploid fibroblasts from a proliferative into a resting phase is accompanied by a significant accumulation of nonhistone chromosomal proteins in the chromatin. Fig.8 shows that the ratio of total nonhistone proteins to DNA increases from about 2 in proliferating cells to about 5 in cells which have reached a late stationary phase. After trypsinization this ratio falls to the initial level
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Fig. 8. Changes of' the nonhistone chromosomal proteinslDNA ratio in human diploid fibroblasts entering a stationary phase. The cells were plated at zero time and allowed to grow without changing the medium. At the time indicated by the arrow the cells were trypsinized, replated and allowed to grow under the same conditions
and then again increases in the stationary phase. The continuous accumulation of nonhistone proteins in the chromatin during several days when the cells stop proliferating is responsible for the slightly increasing 3H-labelled nonhistone protein~/['~C]DNA ratio during the chase period (Fig.1). This again shows the slow but continuous migration into the chromatin of proteins which have been synthesized several days before. The strong decrease in the nonhistone proteins/ DNA ratio caused by trypsinization affects also the group of labelled nonhistone chromosomal proteins which have been synthesized during the proliferative period. This is clearly seen from the fall in the 3H/14C ratio shown in Fig. 10B. The percentage loss of
L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev
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Fig. 9. Correlation between the trypsin-induced loss of total nonhistone chromosomal proteins and the loss of these proteins synthesized during the proliferative period. The percentage loss of total acidinsoluble nonhistone chromosomal protein was plotted against the percentage loss of labelled acid-insoluble nonhistone chromosomal proteins, measured as the loss of ['Hltryptophan. Human diploid fibroblasts with prelabelled [14C]DNA were labelled with ['Hltryptophan during the proliferative phase as in Fig. 1 . They were then grown without changing the medium for different periods of time to obtain cells with different nonhistone proteins/DNA ratio, as shown in Fig. 8. At the corresponding times the cells were collected and divided into two groups. In the first group the nonhistone proteins/DNA and the 3H/14Cratios were determined. The second group was stimulated to proliferate by trypsinization. The same ratios were determined 48 h after replating and the corresponding loss of label and of nonhistone chromosomal proteins was calculated
labelled proteins is proportional to the percentage loss of total nonhistone chromosomal proteins which, on the other hand, increases with the increasing nonhistone proteins/DNA ratio proportionally with the time the cells have spent in a state of quiescence. Thus, the loss of labelled proteins varies from zero in untreated continuously proliferating cells to about 75% in resting cells stimulated to proliferate by trypsinization in a late stationary phase (Fig. 9). These experiments show that nonhistone chromosomal proteins which have been synthesized during the period of proliferation and were conserved in quiescent cells, are lost when cells are stimulated to proliferate by trypsinization. Thus, it is evident that after trypsinization these proteins are eliminated both from the fraction present in proliferating cells and from that accumulating during the resting period. Although the percentage loss of the first fraction is the greater the longer the cells have spent in a resting stage, the nonhistone proteins/DNA ratio always decreases to the same basic level of 2 characteristic of proliferating cells. These data suggest that the metabolically stable nonhistone proteins which are synthesized and combine with chromatin during the proliferative period
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may be slowly exchanged with proteins synthesized during the period of quiescence in such a way that they still remain associated with the chromatin but can be eliminated by trypsinization. The data in Fig.8 and 9 show that in about 5 days 75% of the metabolically stable nonhistone chromosomal proteins of proliferating cells are converted into a 'trypsin-sensitive' group. The possibility that one and the same protein may be in a different state in the chromatin is also indicated by the similarity in the electrophoretic pattern of tightly bound and loosely bound nonhistone chromosomal proteins [13]. The slow conversion of the metabolically stable nonhistone chromosomal proteins into proteins that are eliminated in trypsin-stimulated cells favours the idea of a dynamic equilibrium between identical protein molecules existing in a different state within the chromatin. The question arises whether these proteins are eliminated only when cells are stimulated to proliferate by trypsinization or whether they will be normally exchanged and lost during a time of quiescence much longer than that studied in our experiments. Unlike human diploid fibroblasts, Chinese hamster fibroblasts do not accumulate nonhistone chromosomal proteins to such a large extent. Their nonhistone proteins/DNA ratio increases from 1.6 in proliferating cells to 2.1 only when the cells are prevented from proliferation by fasting. Thus, the two lines of fibroblasts differed in their capacity to accumulate nonhistone chromosomal proteins. It would be interesting to correlate this difference with their different capacity of contact inhibition. The human diploid cells were easily contact-inhibited while the hamster cells were able to form multilayer cultures. It is tempting to assume that some nonhistone proteins accumulating in the chromatin are responsible for the inhibited capacity to proliferate in the late stationary phase. The difference between the two lines would thus reside in the decreased capacity of the Chinese hamster fibroblasts to synthesize this proliferation-inhibiting fraction. Such a suggestion is supported by the fact that, in order to induce proliferation in late stationary fibroblasts, it is necessary to eliminate these proteins by trypsin treatment. This is evident from experiments where we have compared the effect of trypsin with the effect of a mechanical detachment and replating of resting cells in an early stationary phase (nonhistone proteins/DNA about 3 ) and in a late stationary phase (nonhistone proteins/DNA about 5). As seen in Fig. 10A, no nonhistone chromosomal proteins are eliminated from cells in a late stationary phase after a mechanical replating, in contrast to trypsinization which reduces their content to the basic level characteristic of proliferating cells. At the same time DNA synthesis is only slightly increased in the first case,
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Metabolic Behaviour of Nonhistone Chromosomal Proteins in Fibroblasts
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trypsin treatment of resting cells in a late stationary phase eliminates about 80 % of the labelled nonhistone chromosomal proteins while mechanically replated cells do not lose the label. These experiments show that stimulation of cell proliferation affects some nonhistone chromosomal proteins which accumulate in the late stationary phase and this occurs as a consequence of the trypsin treatment. In the absence of these proteins (early stationary phase) cell proliferation can be stimulated without a detectable decrease in the nonhistone proteins/DNA ratio. Thus, diploid fibroblasts in an early and in a late stationary phase are in a different physiological state, probably determined by some nonhistone chromosomal proteins. This is consistent with other data showing the decreased proliferation capacity of W138 fibroblasts after a prolonged quiescence [14,151. Our data show that, with respect to their metabolic behaviour, there are three main fractions of nonhistone chromosomal proteins in the fibroblasts, as follows. (a) A fraction which makes the basic level of nonhistone chromosomal proteins and which is conserved in proliferating cells. This metabolically stable fraction is acid-insoluble and represents the bulk of these proteins in proliferating cells. In resting cells these proteins are conserved but become sensitive to trypsin stimulation. (b) A minor fraction of metabolically labile rapidly labelled nonhistone chromosomal proteins (no more than 10% of the total) which is extracted with acids together with the histones and is detectable in proliferating cells. (c) A metabolically labile fraction which accumulates during the resting period of contact-inhibited cells and shows a low turnover rate. All our data lead to the conclusion that the metabolic behaviour of nonhistone chromosomal proteins depends on the physiological state of the cells while the contact inhibition may depend on the capacity of the cells to synthesize some special nonhistone chromosomal proteins.
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Fig. 10. Behaviour of nonhistone chromosomalproteins upon stimulation of cell proliferation in a stationary culture of human diploid ,fibroblasts. Cells in early stationary phase (A-A) and cells mechanically replated; cells in in late stationary phase (0-0) late stationary phase stimulated by trypsinization (*---@). (A) Changes in the nonhistone chromosomal protein/DNA ratio ; (B) changes in the 3H/'4C ratio; (C) relative changes in the specific radioactivity of DNA. Cells with prelabelled [14C]DNAand [3H]tryptophan-labelled nonhistone chromosomal proteins as in Fig. 1 were grown without changing the medium for 4 days (early stationary phase) or for 8 days (late stationary phase) and then stimulated to proliferate by a mechanical replating or by trypsinization
while in trypsin-treated cells the normal stimulatory effect is observed (Fig. 1OC). On the other hand, mechanical replating of cells in an early stationary phase is quite successful as seen in Fig. 1OC. In this case, however, no decrease in the protein content of chromatin occurs (Fig. 10A) showing that a nonhistone proteins/DNA ratio of 3 is still compatible with cell proliferation. Here again it can be seen that the transition of the proliferating cells into a stationary phase is accompanied with a strong accumulation of nonhistone proteins in the chromatin (Fig. 10A). The difference between trypsintreated and mechanically replated cells is demonstrated also in Fig.10B showing the 3H-labelled nonhistone pr~teins/['~C]DNA ratio. It is seen that
REFERENCES 1. McClure, M. E. & Hnilica, L. S. (1972) Subcell. Biochem. I , 311 -332. 2. Tsanev, R., Djondjurov, L. & Ivanova, E. (1974) Exp. Cell Res. 84,137- 142. 3. Russev, G., Anachkova, B. & Tsanev, R. (1975) Eur. J . Biochem. 58,253 -257. 4. Seale, R. L. (1975) Biochem. Biophys. Res. Commun. 63, 140148. 5. Djondjurov, L., Markov, G. & Tsanev, R. (1972) Exp. Cell Res. 75, 442-448. 6. Stubblefield, E., Klevecz, R. & Deaven, L. (1967) J . Cell. Physiol. 69, 345- 354. I. Burton, K. (1956) Biochem. J . 62, 315-323. 8. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J . Biol. Chem. 193, 265-275.
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L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev 9. Anderson, L. A. & McClure, W. 0. (1973) Anal. Biochem. 53, 173- 179. 10. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685. 11. Sadgopal, A. & Bonner, J. (1970) Biochim. Biophys. Acfu, 207,227 - 239. 12. Comings, D. E. & Tack, L. 0. (1973) Exp. Cell. Res. 82, 175-191.
13. Fujitani, H. & Holoubek, V. (1973) Biochem. Biophys. Res. Commun. 54,1300- 1305. 14. Augenlicht, L. H. & Baserga, R. (1974) Exp. Cell Res. 89, 255 - 262. 15. Rossini, M., J.-Ch. Lin & Baserga, R. (1976) J. Cell. Physiol. 88, 1 - 12.
L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev*, Institut PO Biokhimiya, Bulgarska Akademiya na Naukite, 1113 Sofiya, Bulgaria
* To whom correspondence should be addressed.
Metabolic Behaviour of Nonhistone Chromosomal Proteins in Proliferating and in Resting Fibroblasts Lalju Petrov DJONDJUROV, Emilia Christova IVANOVA, and Roumen Georgiev TSANEV Institute of Biochemistry, Bulgarian Academy of Sciences, Sofia (Received April 12, 1977)
The metabolism of nonhistone chromosomal proteins was studied in two lines of cells showing a different degree of contact inhibition : human diploid fibroblasts, which are easily contactinhibited, and Chinese hamster fibroblasts, which had been made to stop proliferating by fasting. By following the 3H/14C ratio of [3H]tryptophan-labelled nonhistone chromosomal proteins and [14C]thymidine-labelledDNA in chase experiments three main groups of these proteins could be detected with respect to their metabolic behaviour: (a) a metabolically stable group which is acid-insoluble and represents the bulk of nonhistone chromosomal proteins in proliferating cells ; this group is conserved when the cells enter a resting phase; (b) a metabolically labile group which is acid-soluble and is observed as a minor fraction in proliferating cells; (c) a metabolically labile group which is acid-insoluble and accumulates in resting cells; this fraction is much larger in contact-inhibited cells. Stimulation of cell proliferation by trypsinization decreases the amount of nonhistone chromosomal proteins in resting cells to the basic level observed in proliferating cells. Until recently nonhistone chromosomal proteins were believed to represent a rapidly metabolized group [l]. However, in a previous study we have found that fibroblasts cultured in vitro contain a fraction of metabolically stable nonhistone chromosomal proteins completely preserved in resting cells and partly eliminated when the cells are stimulated to proliferate by trypsinization [2]. Metabolically stable nonhistone chromosomal proteins were found also in rat liver where a fraction of these proteins labelled after partial hepatectomy was preserved after a long chase [3]. The existence of metabolically stable nonhistone chromosomal proteins was also proved in the case of continuously proliferating HeLa cells [4]. Unlike the results obtained with fibroblasts and liver cells, in HeLa cells all these proteins were reported to be completely preserved. The question arose whether this difference reflected a difference between growthregulated and non-regulated cells or a difference between continuously proliferating cells and resting cells stimulated to proliferate. In the present paper we have studied the metabolic stability of nonhistone chromosomal proteins in two lines of fibroblasts exhibiting a different degree of contact inhibition. For this purpose the conservation of [3H]tryptophan in these proteins was followed in resting and in proliferating cells and during the transition between these two states. It was found that both metabolically stable and metabolically
labile nonhistone chromosomal proteins existed, but in proliferating cells the bulk of these proteins were metabolically stable. MATERIALS AND METHODS Cells Two cellular lines were used: Chinese hamster fibroblasts (BII dii, FAF-28, see [ 5 ] ) and diploid human lung fibroblasts (of passages 18 to 24). Both cellular lines were grown as monolayer cultures. The cells were cultivated in 250-ml flasks with 30 ml of minimum essential medium (Eurobio, France) in the case of Chinese hamster fibroblasts (plated at a density of 9.104 cells/cm2) or of basal medium (Eurobio, France) in the case of human fibroblasts (plated at a density of 6.104 cells/cm2). In both cases the medium was supplemented with 20 % calf serum. Under these conditions if the medium was not changed the human diploid fibroblasts entered a stationary phase after 4 days of proliferation (early stationary phase). A late stationary phase was reached if the medium was not changed for another period of 4 days. The resting cells were stimulated to proliferate either by trypsinization (0.25 % Difco trypsin free of Mg2+and Ca2+)or by their mechanical detachment and replating in a fresh medium. The stimulated cells started DNA synthesis 10- 12 h after replating.
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Metabolic Behaviour of Nonhistone Chromosomal Proteins in Fibroblasts
When synchronized cultures were used Chinese hamster fibroblasts were grown in 1000-ml flasks in medium 199 containing 10 % calf serum. Synchronization was achieved by detachment of metaphase cells arrested by colcemid [6] as follows: the cells were treated with colcemid (0.05 pg/ml) for 4 h, then trypsinized for 45 s with 0.1 % trypsin at 4 "C and detached by vigorous shaking in chilled medium 199 with serum. This resulted in a suspension of 94 - 98 % metaphase cells which were collected by centrifugation at 600 x g and plated in 100-ml flasks at a density of 5 x lo6 cells per flask. In all experiments the completion of mitosis and its duration in colcemid-blocked cells were controlled by phase-contrast microscopy. Labelling and Chase Short-Term Experiments with Proliferating Cells. Chinese hamster fibroblasts were pulse-labelled for 30 s with [3H]tryptophan, 10 pCi/ml (Amersham, 6.4 Ci/mmol). The incorporation of the labelled compound was stopped by adding 10 times the volume of a chilled medium rich in unlabelled tryptophan (200 mg/l). After washing the cells with the same medium they were grown in it for different periods up to 30 min. After the chase they were frozen in liquid nitrogen. Long-Term Experiments with Proliferating Cells. One day after plating the cells of both cell lines were labelled for 22-24 h with [14C]thymidine, 0.05 pCi/ ml (Amersham, 57 mCi/mmol). To obtain a better leveling of the labelled DNA the cells from all flasks were collected by trypsinization, pooled and replated at the same initial density. One day later the medium was replaced by a tryptophan-free medium and the cells were labelled for 5 h (Chinese hamster fibroblasts) or for 7 h (human diploid fibroblasts) with [3H]tryptophan, 2 pCi/ml. After labelling the cells were washed twice with a medium rich in unlabelled tryptophan (200 mg/l) and allowed to grow in the same tryptophan-rich medium. During the chase period the human fibroblasts entered a stationary phase after 4 days if the medium was not changed. Experiments with Synchronized Cells. Chinese hamster fibroblasts were labelled with [3H]thymidine (0.1 pCi/ml) during the period of active proliferation. After synchronization (see above) they were pulselabelled for 30 min with [14C]tryptophan (0.2 pCi/ml) at different intervals after plating : immediately and 30 min after plating (metaphase and telophase cells respectively), at the 3rd hour (GI cells), at the 8th hour (S cells) and at the 15th hour (Gz cells). At each interval part of the cells were collected immediately after labelling to determine the initial level of 14C incorporation. The remaining cells were washed with a medium containing an excess of unlabelled tryptophan (200 mg/l) and allowed to grow further in the
same medium. During this chase period the cells were maintained in a state of active proliferation by daily replating. Samples containing about 2 x lo7 cells were withdrawn 6, 12, 24, 48 and 72 h after the beginning of the chase. All cells were collected by centrifugation at 600 x g , washed with the same medium without calf serum and used for isolation of chromatin. Resting Cells. Human fibroblasts with prelabelled [14C]DNA obtained as described above (for longterm experiments) were grown for 8 days without changing the medium (late stationary phase) and were then labelled for 7 h with [3H]tryptophan (5 pCi/ml) in the same medium. The chase was performed also in a conditioned medium containing 200 mg/l unlabelled tryptophan. For electrophoretic fractionation of nonhistone chromosomal proteins the cells were labelled with [3H]tryptophan only (10 pCi/ml) during the same period and in the same way as their corresponding double-labelled counterparts. Isolation of Chromatin and Nonhistone Chromosomal Proteins The cells were washed with a fresh culture medium and suspended for 5min in a hypotonic medium (10 mM Tris . HCl, 10 mM KCl, 1.6 mM MgClZ, pH 8.1). Nonidet P-40 was then added in a final concentration of 1 %, the cell suspension was left in the cold for 5 min and centrifuged. After two such treatments pure nuclei were obtained without detectable cytoplasmic contaminations as checked by phasecontrast microscopy. The nuclear pellet was extracted twice with 0.075 M NaCl, 0.025 M EDTA, twice with 0.037 M NaCl, 0.012 M EDTA and dialysed against deionized water. In some experiments 1 mM sodium bisulfite was present in all solutions. Histones together with a small fraction of acidsoluble nonhistone chromosomal proteins were extracted with 125 mM HzS04 and the acid-insoluble nonhistone chromosomal proteins were dissolved in 0.1 M NaOH. Analytical Procedures DNA was determined by the method of Burton [7] and proteins by the procedure of Lowry [8]. For radioactivity counting the sample was dissolved in 10 vol. of a toluene/Triton X-100 scintillation mixture [9]. Double-labelled (3H and 14C) material was differentially counted in a Packard Tricarb 3200 liquid scintillation spectrometer. Gel Electrophoresis [3H]Tryptophan-labelled nonhistone chromosomal proteins were fractionated in polyacrylamide gel
547
L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev
according to Laemmli [lo]. The gels were stained with Coomassie brilliant blue and scanned at 520nm. They were then sliced at 1-mm intervals, the slices were treated with Protosol and their radioactivity measured.
experiments. It demonstrates that some labelled proteins continue to enter the nucleus from the cytoplasm during the chase period. The same regularity in the metabolism of nonhistone chromosomal proteins is found with diploid human fibroblasts (Fig. 1B). The difference between the two cellular lines is that in the human fibroblasts only 30% of the label was lost with a half-life of 12 h as compared to 45 % in the Chinese hamster fibroblasts with a half-life of 9 h. The different metabolic behaviour of the acidsoluble and the acid-insoluble fractions in proliferating cells can be demonstrated also in short pulse experiments. As seen in Fig.2, the minor acid-soluble frac-
RESULTS AND DISCUSSION Proliferating Cells
When Chinese hamster fibroblasts were labelled during the phase of exponential growth two distinct fractions of nonhistone chromosomal proteins could be distinguished with respect to their metabolic stability. As seen in Fig. 1A the 3H/14C ratio of the total nonhistone chromosomal proteins decreases by 45 % during the first 24 h of the chase and then remains constant or increases slightly. During the whole chase period the cells continue to proliferate, although with a slightly diminishing rate as shown by the slope of the curve of the specific radioactivity of DNA. These data show the synthesis in proliferating cells of two classes of nonhistone chromosomal proteins which associate with the chromatin: one which is metabolized during the first 24 h and a second which is metabolically stable. When these proteins are divided into two groups, acid-soluble (extracted with the histones) and acid-insoluble, it is seen that the metabolically labile proteins go into the acidsoluble group which loses 70% of its label, while the acid-insoluble proteins are metabolically stable (Fig. 1A), The slight increase in the 3H/'4C ratio of the metabolically stable group was reproduced in all
o.2
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Fig. 2. Accumulation and turnover of the acid-soluble ( @ - - O ) and of the acid-insoluble fractions ( b e ) of nonhistone chromosomal proteins in short pulse experiments. The logarithms o f the specific radioactivity, A,, were measured. Proliferating Chinese hamster fibroblasts were pulse-labelled for 30 s with [3H]tryptophan, washed with a medium containing unlabelled tryptophan and allowed to grow in the same medium
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Fig.1. Turnover of nonhistone chromosomal proteins in proliferating fibroblasts. (A) Chinese hamster fibroblasts; (B) human diploid fibroblasts. The relative changes of the 3H/'4C ratio in the total chromatin (A-A), in the acid-insoluble (M and ) in the acid-soluble fractions (G---o) and the logarithm of the specific radioactivity of DNA ( A , ) were measured. Proliferating cells with prelabelled [I4C]DNA were labelled for 5 h (A) or for 7 h (B) with [3H]tryptophan and then allowed to grow in the presence of unlabelled tryptophan without further changes of medium
548
Metabolic Behaviour of Nonhistone Chromosomal Proteins in Fibroblasts
Histones
Slice number
Fig. 3. Electrophoretic pattern of the acid-soluble chromosomal proteins of proliferating Chinese hamster fibroblasts. Staining intensity, As20 (-) and labelling before (o----o) and after a 48-h chase (04). The cells were labelled for 5 h with [3H]tryptophan and chased for 48 h in a medium rich in unlabelled tryptophan. Chromatin was isolated at zero time and after a 48-h chase. The acid-soluble fraction of the chromatin was precipitated with 3 vol. of ethanol and fractionated according to Laemmli [9]
tion synthesized during a 30-s pulse with [3H]tryptophan shows a rapid decrease in its specific activity during a 30-min chase, while the labelling of the major fraction of acid-insoluble proteins continues to increase. Under conditions of cell proliferation in both cellular lines the metabolically stable proteins represent the major fraction of nonhistone chromosomal proteins, since the acid-soluble group extracted together with the histones makes no more than 10% of the total of these proteins. As shown in Fig.3, the acid-soluble proteins represent a highly heterogeneous group with a high specific radioactivity after a 5-h pulse with [3H]tryptophan. The presence of acid-soluble nonhistone proteins in the chromatin of proliferating cells is in agreement with the finding that this protein fraction strongly increases in metaphase chromosomes [ll]. In our experiments the amount of acid-soluble nonhistone chromosomal proteins in resting fibroblasts was very small. The question might be asked whether these proteins are not a cytoplasmic contamination due to the intimate contact of the chromosomes with the cytoplasm in dividing cells. The latter possibility is suggested by experimental evidence that acid-soluble cytoplasmic proteins are adsorbed onto metaphase chromosomes during the isolation procedure [12]. However, our experiments with synchronized cells are not in agreement with this possibility. As seen in Fig. 4, the amount of 14C-labelled nonhistone chromosomal proteins turned over during the chase
0
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Time of chase (h)
Fig.4. Relative changes of the I4Cl3H ratio of' chromatin during the chase period of synchronized Chinese hamster fibroblasts prelabelled with [3H]thymidine and pulse-labelled for 30 min with ('4C]tryptophan in metaphase (A-A), telophase (M), GI ( a d ) ,S (-1 and G2 ( M i .During the chase period the cells were maintained in active proliferation by daily replating
period is minimal when labelling was at metaphase : 15 % as compared with 50 % loss of the label incorporated in telephase, GI, S and Gz cells. These data show that metabolically labile nonhistone proteins are synthesized during the whole cell cycle and they are present in the chromatin at periods when the chromosomes are not in a direct contact with the cytoplasm. Thus, under our conditions of chromatin isolation it seems very likely that the acid-soluble
549
L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev
S l i c e number
Fig. 5 . Electrophoretic pattern of the acid-insoluble nonhistone chromosomal proteins of proliferating Chinese hamster ,fibroblasts. Staining intensity, A520(-) and labelling before (---a) and after a 48-h chase ( 0 0 ) .The cells were labelled and chased as in Fig. 3. The acidsoluble proteins were extracted and the remaining material was fractionated as in Fig. 3
metabolically labile fraction of nonhistone proteins is not a cytoplasmic contaminant, but represent a real constituent of the chromatin of proliferating cells. Thus, if we eliminate the acid-soluble fraction all remaining nonhistone chromosomal proteins of proliferating cells are metabolically stable. This explains the results of Seale [4] obtained on continuously proliferating HeLa cells after the elimination of all acid-soluble proteins. The acid-insoluble metabolically stable nonhistone chromosomal proteins of proliferating cells are also a heterogeneous group of proteins not only with respect to their molecular weights but also concerning their labelling pattern after a 5-h pulse with [3H]tryptophan. As seen in Fig.5, this group can be separated by dodecylsulfate gel electrophoresis into about 20 bands each of which has a different specific radioactivity. This may reflect both differences in tryptophan content and different migration rates into the chromatin of the newly synthesized proteins. A 48-h chase leads to a more than 4-fold decrease in the incorporated radioactivity due to a dilution corresponding to more than two cell cycles which are expected to take place during 48 h. Resting Cells
The nonhistone chromosomal proteins of resting cells exhibit a different metabolic behaviour. When human diploid fibroblasts with prelabelled [I4C]DNA enter a late stationary phase their nonhistone proteins
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Fig. 6 . Accumulation and turnover o j nonhistone chromosomal proteins in resting human diploidfibroblasts. (A) The relative changes of the 3H/'4C ratio in the total chromatin (0-0) and of the acid-insoluble fraction ( G - - - O ) ; (B) logarithms of the specific radioactivity of DNA. Human diploid fibroblasts with prelabelled [14C]DNAwere labelled for 7 h with [3H]tryptophan during the late stationary phase and chased in a medium rich in unlabelled tryptophan
incorporate [3H]tryptophan at a significant rate. Considering the almost complete arrest of DNA synthesis (Fig. 6 B) and the constant nonhistone protein/DNA ratio, this demonstrates the presence of metabolically labile proteins. As seen in Fig.6A the
550
Metabolic Behaviour of Nonhistone Chromosomal Proteins in Fibroblasts
Slice number
Fig. 7. Electrophoretic pattern of the acid-insoluble proteins of' resting human diploid,fibrohlasts. Staining intensity, A520 (-) and labelling and after a 48-h chase (00). The cells were labelled for 7 h with [3H]tryptophan and chased in a medium rich in before (&---O) unlabelled tryptophan. Chromatin was isolated at zero time and after a 48-h chase. After the extraction of the acid-soluble proteins the remaining material was fractionated as in Fig. 3
label continues to enter the chromatin during the first two days of the chase showing the low migration rate of some proteins into the nucleus. Then the 3H/'4C ratio begins to decrease, as would be expected for proteins which are continuously turned over. The same figure shows that the behaviour of the acidinsoluble fraction is identical to that of the total nonhistone chromosomal proteins. In fact the [3H]tryptophan radioactivity of the acid-soluble fraction of resting cells is negligibly small as compared to that of the acid-insoluble fraction. The electrophoretic pattern of nonhistone chromosomal proteins of resting human diploid fibroblasts is shown in Fig.7. It is seen that the labelling is mainly due to several fractions and that one highly labelled fraction only is responsible for the continuously increasing labelling during the first two days of the chase. The presence of electrophoretic fractions which remain practically unlabelled is consistent with the existence in resting cells of metabolically stable proteins which have been synthesized during the period of proliferation. Such proteins are located mainly in the high-molecular-weight region.
Transition Periods The transition of the human diploid fibroblasts from a proliferative into a resting phase is accompanied by a significant accumulation of nonhistone chromosomal proteins in the chromatin. Fig.8 shows that the ratio of total nonhistone proteins to DNA increases from about 2 in proliferating cells to about 5 in cells which have reached a late stationary phase. After trypsinization this ratio falls to the initial level
0
2
4 6 8 Time after plating (days)
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Fig. 8. Changes of' the nonhistone chromosomal proteinslDNA ratio in human diploid fibroblasts entering a stationary phase. The cells were plated at zero time and allowed to grow without changing the medium. At the time indicated by the arrow the cells were trypsinized, replated and allowed to grow under the same conditions
and then again increases in the stationary phase. The continuous accumulation of nonhistone proteins in the chromatin during several days when the cells stop proliferating is responsible for the slightly increasing 3H-labelled nonhistone protein~/['~C]DNA ratio during the chase period (Fig.1). This again shows the slow but continuous migration into the chromatin of proteins which have been synthesized several days before. The strong decrease in the nonhistone proteins/ DNA ratio caused by trypsinization affects also the group of labelled nonhistone chromosomal proteins which have been synthesized during the proliferative period. This is clearly seen from the fall in the 3H/14C ratio shown in Fig. 10B. The percentage loss of
L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev
80
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Loss of nonhistone chromosomal proteins (%)
Fig. 9. Correlation between the trypsin-induced loss of total nonhistone chromosomal proteins and the loss of these proteins synthesized during the proliferative period. The percentage loss of total acidinsoluble nonhistone chromosomal protein was plotted against the percentage loss of labelled acid-insoluble nonhistone chromosomal proteins, measured as the loss of ['Hltryptophan. Human diploid fibroblasts with prelabelled [14C]DNA were labelled with ['Hltryptophan during the proliferative phase as in Fig. 1 . They were then grown without changing the medium for different periods of time to obtain cells with different nonhistone proteins/DNA ratio, as shown in Fig. 8. At the corresponding times the cells were collected and divided into two groups. In the first group the nonhistone proteins/DNA and the 3H/14Cratios were determined. The second group was stimulated to proliferate by trypsinization. The same ratios were determined 48 h after replating and the corresponding loss of label and of nonhistone chromosomal proteins was calculated
labelled proteins is proportional to the percentage loss of total nonhistone chromosomal proteins which, on the other hand, increases with the increasing nonhistone proteins/DNA ratio proportionally with the time the cells have spent in a state of quiescence. Thus, the loss of labelled proteins varies from zero in untreated continuously proliferating cells to about 75% in resting cells stimulated to proliferate by trypsinization in a late stationary phase (Fig. 9). These experiments show that nonhistone chromosomal proteins which have been synthesized during the period of proliferation and were conserved in quiescent cells, are lost when cells are stimulated to proliferate by trypsinization. Thus, it is evident that after trypsinization these proteins are eliminated both from the fraction present in proliferating cells and from that accumulating during the resting period. Although the percentage loss of the first fraction is the greater the longer the cells have spent in a resting stage, the nonhistone proteins/DNA ratio always decreases to the same basic level of 2 characteristic of proliferating cells. These data suggest that the metabolically stable nonhistone proteins which are synthesized and combine with chromatin during the proliferative period
551
may be slowly exchanged with proteins synthesized during the period of quiescence in such a way that they still remain associated with the chromatin but can be eliminated by trypsinization. The data in Fig.8 and 9 show that in about 5 days 75% of the metabolically stable nonhistone chromosomal proteins of proliferating cells are converted into a 'trypsin-sensitive' group. The possibility that one and the same protein may be in a different state in the chromatin is also indicated by the similarity in the electrophoretic pattern of tightly bound and loosely bound nonhistone chromosomal proteins [13]. The slow conversion of the metabolically stable nonhistone chromosomal proteins into proteins that are eliminated in trypsin-stimulated cells favours the idea of a dynamic equilibrium between identical protein molecules existing in a different state within the chromatin. The question arises whether these proteins are eliminated only when cells are stimulated to proliferate by trypsinization or whether they will be normally exchanged and lost during a time of quiescence much longer than that studied in our experiments. Unlike human diploid fibroblasts, Chinese hamster fibroblasts do not accumulate nonhistone chromosomal proteins to such a large extent. Their nonhistone proteins/DNA ratio increases from 1.6 in proliferating cells to 2.1 only when the cells are prevented from proliferation by fasting. Thus, the two lines of fibroblasts differed in their capacity to accumulate nonhistone chromosomal proteins. It would be interesting to correlate this difference with their different capacity of contact inhibition. The human diploid cells were easily contact-inhibited while the hamster cells were able to form multilayer cultures. It is tempting to assume that some nonhistone proteins accumulating in the chromatin are responsible for the inhibited capacity to proliferate in the late stationary phase. The difference between the two lines would thus reside in the decreased capacity of the Chinese hamster fibroblasts to synthesize this proliferation-inhibiting fraction. Such a suggestion is supported by the fact that, in order to induce proliferation in late stationary fibroblasts, it is necessary to eliminate these proteins by trypsin treatment. This is evident from experiments where we have compared the effect of trypsin with the effect of a mechanical detachment and replating of resting cells in an early stationary phase (nonhistone proteins/DNA about 3 ) and in a late stationary phase (nonhistone proteins/DNA about 5). As seen in Fig. 10A, no nonhistone chromosomal proteins are eliminated from cells in a late stationary phase after a mechanical replating, in contrast to trypsinization which reduces their content to the basic level characteristic of proliferating cells. At the same time DNA synthesis is only slightly increased in the first case,
5 52
2
Metabolic Behaviour of Nonhistone Chromosomal Proteins in Fibroblasts
7.0
. 0
=
trypsin treatment of resting cells in a late stationary phase eliminates about 80 % of the labelled nonhistone chromosomal proteins while mechanically replated cells do not lose the label. These experiments show that stimulation of cell proliferation affects some nonhistone chromosomal proteins which accumulate in the late stationary phase and this occurs as a consequence of the trypsin treatment. In the absence of these proteins (early stationary phase) cell proliferation can be stimulated without a detectable decrease in the nonhistone proteins/DNA ratio. Thus, diploid fibroblasts in an early and in a late stationary phase are in a different physiological state, probably determined by some nonhistone chromosomal proteins. This is consistent with other data showing the decreased proliferation capacity of W138 fibroblasts after a prolonged quiescence [14,151. Our data show that, with respect to their metabolic behaviour, there are three main fractions of nonhistone chromosomal proteins in the fibroblasts, as follows. (a) A fraction which makes the basic level of nonhistone chromosomal proteins and which is conserved in proliferating cells. This metabolically stable fraction is acid-insoluble and represents the bulk of these proteins in proliferating cells. In resting cells these proteins are conserved but become sensitive to trypsin stimulation. (b) A minor fraction of metabolically labile rapidly labelled nonhistone chromosomal proteins (no more than 10% of the total) which is extracted with acids together with the histones and is detectable in proliferating cells. (c) A metabolically labile fraction which accumulates during the resting period of contact-inhibited cells and shows a low turnover rate. All our data lead to the conclusion that the metabolic behaviour of nonhistone chromosomal proteins depends on the physiological state of the cells while the contact inhibition may depend on the capacity of the cells to synthesize some special nonhistone chromosomal proteins.
2.0
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, .
-
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48 72 Time after plating (h)
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Fig. 10. Behaviour of nonhistone chromosomalproteins upon stimulation of cell proliferation in a stationary culture of human diploid ,fibroblasts. Cells in early stationary phase (A-A) and cells mechanically replated; cells in in late stationary phase (0-0) late stationary phase stimulated by trypsinization (*---@). (A) Changes in the nonhistone chromosomal protein/DNA ratio ; (B) changes in the 3H/'4C ratio; (C) relative changes in the specific radioactivity of DNA. Cells with prelabelled [14C]DNAand [3H]tryptophan-labelled nonhistone chromosomal proteins as in Fig. 1 were grown without changing the medium for 4 days (early stationary phase) or for 8 days (late stationary phase) and then stimulated to proliferate by a mechanical replating or by trypsinization
while in trypsin-treated cells the normal stimulatory effect is observed (Fig. 1OC). On the other hand, mechanical replating of cells in an early stationary phase is quite successful as seen in Fig. 1OC. In this case, however, no decrease in the protein content of chromatin occurs (Fig. 10A) showing that a nonhistone proteins/DNA ratio of 3 is still compatible with cell proliferation. Here again it can be seen that the transition of the proliferating cells into a stationary phase is accompanied with a strong accumulation of nonhistone proteins in the chromatin (Fig. 10A). The difference between trypsintreated and mechanically replated cells is demonstrated also in Fig.10B showing the 3H-labelled nonhistone pr~teins/['~C]DNA ratio. It is seen that
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L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev 9. Anderson, L. A. & McClure, W. 0. (1973) Anal. Biochem. 53, 173- 179. 10. Laemmli, U. K. (1970) Nature (Lond.) 227,680-685. 11. Sadgopal, A. & Bonner, J. (1970) Biochim. Biophys. Acfu, 207,227 - 239. 12. Comings, D. E. & Tack, L. 0. (1973) Exp. Cell. Res. 82, 175-191.
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L. P. Djondjurov, E. C. Ivanova, and R. G. Tsanev*, Institut PO Biokhimiya, Bulgarska Akademiya na Naukite, 1113 Sofiya, Bulgaria
* To whom correspondence should be addressed.
