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Biochimica et Biophysica Acta, 3 9 5 ( 1 9 7 5 ) 3 6 1 - - 3 7 2 @ Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 9 8 3 2 1

NON-HISTONE CHROMOSOMAL PROTEINS FROM VIRUS-TRANSFORMED AND UNTRANSFORMED 3T3 MOUSE FIBROBLASTS

C.A. G O N Z A L E Z * a n d K.R. R E E S

Department of Biochemical Pathology, University College Hospital Medical School, London WCIE 6JJ (U.K.) (Received February 10th, 1975)

Summary A peak in the non-histone chromosomal protein polyacrylamide gel electrophoresis profiles has been detected which is higher in log phase 3T3 and 3T3/SV40 cells than in density-inhibited 3T3 cells. Radioactive incorporation is substantially higher into this peak in log phase 3T3 than in 3T3/SV40 and density-inhibited 3T3 cells. Reversion of 3T3/SV40 cells with dibutyryl cyclic AMP and theophylline produces increased radioactive incorporation into the peak. Electrophoresis of non-histone chromosomal proteins extracted at different stages of the cell cycle in density inhibited 3T3 cells following serum stimulation shows a cyclic variation in the a m o u n t of this peak with maximum accumulation in late G1. In contrast the height of an equivalent peak in synchronously growing 3T3/SV40 cells remains constant throughout the cell cycle. It is postulated that the protein(s) of this peak may have a regulatory role in cell growth. Introduction The non-histone chromosomal proteins have been implicated in chromatin metabolism [1--4], gene activation [5] and in determining tissue specificity [6,7]. Following serum stimulation of density-inhibitedconfluent monolayers, there is an increase in chromatin template activity and an increase in the synthesis of nuclear acidic proteins [S]. Similar increases in synthesis occur in lymphocytes treated with phytohaemagglutinin [9], in the oestrogen-stimulated rat uterus [10] and in isoproterenol-stimulated mouse salivary glands

* Present address: Catedra de B i o q u i m i e a , I n s t i t u t o de Medicina Experimental, U.C.V. Apartado 50587, Sabana Grande, Caracas, Venezuela.

362 [11]. These observations have led to the suggestion that the nuclear acidic proteins may control gene activation that eventually leads to DNA synthesis and cell division [12]. In the present investigation, the possible role of the non-histone chromosomal proteins in growth control has been further explored by comparing their electrophoretic patterns and radioactive incorporation in 3T3 mouse fibroblasts with those in 3T3 cells transformed by SV40 under various growth conditions. Materials and Methods

Origin and production of cells 3T3 cells were an established line of Swiss mouse embryo cells obtained from the American Type collection. 3T3/SV40 cells: a line of SV40 transformed Swiss mouse embryo cells obtained by Todaro and Green [13] which were purchased from Meloy Laboratories Inc., Springfield, Va., U.S.A. An antibiotic mixture was added to all media to contain penicillin 50 units, streptomycin 50 pg, neomycin 25 #g and bacitracin 1.25 units/ml. The cells were grown in monolayer cultures in 10 ounce roller flasks. 3T3/SV40 cells were seeded at 5" 106 per bottle in Dulbecco's modification [14] of Eagle's minimum essential medium [15] supplemented with 10% calf serum. 3--4 days later 40 • 106 --50 • 106 cells were harvested. For subculture they were dispersed with 10 ml of 0.02% w/v EDTA in phosphate-buffered saline (Oxoid) per bottle, sedimented by centrifugation at 200 × g for 5 min, resuspended in 10 ml fresh Dulbecco's modification of Eagle's minimum essential medium containing 10% calf serum and counted in a haemocytometer. 3T3 cells were seeded at 10 s per bottle in Eagle's minimum essential medium containing 10% calf serum and 10% tryptose phosphate broth. A medium change every 2 days provided stimulation for optimum growth. They were subcultured just before confluence, as described above, unless density inhibited cells were required. In this case cultures were used 3--4 days after they had become confluent. Chemicals A 3 H-labelled amino acid mixture with radioactive concentration 1 Ci/l, U.~ 4 C-labelled protein hydrolysate (57 Ci/A carbon) and [2 -3 H] thymidine (50 Ci/mole) were purchased from the Radiochemical Centre, Amersham. Acrylamide and N'N'-methylene bisacrylamide were purchased from B.D.H. and recrystaUised as described by Loening [16]. Scintillation fluid consisted of 8% (w/v) naphthalene (Fisons) and 0.4% (w/v) 2,5-bis-(5'-tert-butyl benzoxazolyl2')-thiophene (Ciba) dissolved in a 6.5 : 3.5 (v/v) mixture of toluene and 2 methoxyethanol (Fisons). Bovine pancreatic ribonuclease, deoxyribonuclease (DN-EP), calf thymus DNA, yeast RNA, bovine serum albumin, dibutyryl cyclic AMP and theophylline were purchased from Sigma. Urea, Aristargrade was obtained from B.D.H. Determination of protein, DNA and R N A Protein was determined by the method of Lowry et al. [17], with bovine serum albumin as standard, DNA by the method of Burton [18] with calf

363 t h y m u s DNA as standard and RNA by the orcinol method [19] with yeast R N A as standard.

Radioactive labelling of cultures Protein. In the 24 h radioactive labelling experiments, the 3T3 cells in the logarithmic phase o f growth and 3T3/SV40 cells were labelled with either the 14 C-labelled protein hydrolysate or the 3 H-labelled amino acid mixture at a concentration of 1 mCi/1. Density inhibited 3T3 cells were radioactively labelled in fresh Eagle's minimal essential medium supplemented with 1% calf serum and with a one-tenth amino acid concentration. DNA. 0.2 mCi/l of [3 H] thymidine was added to the cell cultures, 30 min prior to harvesting. A 0.5 ml aliquot was precipitated with 20 vol. of 5% w/v trichloroacetic acid and the precipitate was treated as described by Froelich and Rachmeler [ 2 0 ] .

Isolation of nuclei Nuclei were isolated b y a procedure described by Penman [21]. The nuclei were virtually free o f cytoplasmic contamination as judged b y phase-contrast microscopy. They had an R N A / D N A ratio of 0.30 and a protein/DNA ratio of 3.34.

Preparation of chromatin The nuclei were resuspended in 25 vol. of 0.15 M NaC1, 0.01 M Tris buffer pH 7.4, 0.005 M EDTA, frozen in --15°C ethylene glycol bath, thawed and shaken vigorously in a Vortex mixer. The suspension was centrifuged for 10 min. at 1250 × g and the above procedure repeated twice on the pellet. The final pellet which was designated chromatin was extracted three times with 0.2 M H2 SO4 b y resuspending, homogenising by hand in a glass homogeniser and centrifuging at 1250 × g for 5 min. 50% w/v trichloroacetic acid was added to the combined H2 SO4 extract to give a final concentration of 20% trichloroacetic acid to precipitate the histones which were sedimented by centrifugation at 10 000 × g for 10 min. Electrophoresis of this fraction showed five main histone peaks and a slight contamination with proteins of higher molecular weight, which have been previously described [22]. The residue remaining after H~ SO4 extraction was homogenised in 0.3--0.6 ml 8 M urea containing 0.1% dodecylsulphate and 0.01 M sodium phosphate buffer (pH 7.0) and the suspension was centrifuged at 1250 × g for 10 min. The clear supernatant constituted the non-histone chromosomal proteins. In the chromatin of the 3T3/SV40 cells the total protein/DNA ratio was 2.92, in 3T3 cells in log phase 3.10 and in the density-inhibited cells 2.03. The histone/non-histone chromosomal protein ratio was 1.09 in 3T3/SV40 cells, 1.19 in 3T3 cells in the logarithmic phase of growth and 1.93 in non-dividing cells.

Electrophoresis of non-histone chromosomal proteins No further treatment o f the chromosomal protein extract was required for satisfactory gel analysis. An aliquot of the extract containing 10 #g of protein (50--200 ttl) was applied to the gel, after the addition of 5 pl of 0.2% (w/v)

364

bromphenol blue. 7.5% polyacrylamide gels, 10 cm in length, were prepared as described by Maizel [23] with the modification that a 1 cm spacer gel, 3.75% polyacrylamide was layered on top. Electrophoresis was carried out for 16 h at 40 V (constant voltage) by which time the bromphenol blue had reached the bottom of the gel. The buffer compartments both contained 0.1 M sodium phosphate buffer (pH 7.0) containing 0.1% (w/v) dodecylsulphate and 10% (v/v) glycerol and would accomodate 8 gels (Quickfit Instrumentation, England). Following electrophoresis the gels were immersed for 30 min in 12% (w/v) trichloroacetic acid containing 10% (v/v) glycerol, the gels were then transferred to a solution of 0.25% (w/v) Coomassie blue in 12% (v/v) acetic acid, 12% (v/v) methanol and 10% {v/v) glycerol for 15 h. The gels were then destained by successive washes in a solution containing 15% (v/v) methanol, 10% (v/v) acetic acid and 10% (v/v) glycerol until they were transparent at the tip. The gels were scanned at 260 nm in a Joyce-Loebl U.V. Scanner. The gels were then frozen in aluminium foil troughs, sliced (1 mm) on a Mickle Laboratory Engineering Co. gel slicer and the slices dissolved by the procedure of Goodman and Metzura [24] in plastic counting vials. 15 ml of scintillation fluid (Bray and Gordon [25] and Wolfe [26] ) were added to the vial and the radioactivity determined in an Intertechnique ABAC, SL40 scintillation spectrometer. Recovery of radioactivity layered in the gel was never less than 95%. The counting efficiency was approximately 50% for ~4C and 18--20% for 3 H under conditions of dual isotope counting. Results

The absorbance profiles of the non-histone chromosomal proteins from 3T3 cells, both density inhibited and in the logarithmic phase of growth, as well as 3T3/SV40 cells are shown in Fig. 1. It is possible to recognise 22 peaks in all three profiles and they have been numbered in a sequence as previously described [27,28]. Good reproducibility was found in a number of subsequent experiments. Qualitatively these profiles are indistinguishable but quantitative differences between them may be recognised. Band 11, with a molecular weight of approximately 70 000, is the major peak in the dividing cells (log phase 3T3 and 3T3/SV40 cells) and contrasts with the profile from density-inhibited 3T3 cells, where band 11 has little absorbance representation. The non-histone chromosomal proteins were extracted from cells which had been radioactively labelled for 24 h and various mixtures were co~electrophoresed. The results are shown in Fig. 2 and it may be seen that density inhibited 3T3 cells give a radioactive profile which corresponds very closely to that of 3T3/SV40 cells {Fig. 2A). Interestingly, peak 11 is a major peak in the 3T3 cells in log phase but not in 3T3/SV40 cells (Fig. 2B). Since this band is an important component of the absorbance profile in both lines of cells when dividing, this result suggests that the protein{s) of peak 11 are turning over more slowly in the transformed cells. Sheppard [29] has shown that transformed cells growing in dibutyryl cyclic AMP and theophylline reverted to the growth characteristics of untransformed cells. 3T3/SV40 cells were sub-cultured into eight roller flasks at a

365

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Fig. I. A b s o r b a n c e profiles o f n o n - h i s t o n e c h r o m o s o m a l p r o t e i n s f r o m d e n s i t y - i n h i b i t e d ( t o p ) , log phase 3 T 3 cells (centre) a n d 3 T 3 / S V 4 0 ceils ( b o t t o m ) . 1 0 0 /~g samples p r e p a r e d as d e s c r i b e d in the M e t h o d s s e c t i o n were e l e e t r o p h o r e s e d in 7.5% p o l y a c r y l a m i d e gels a t 4 0 V f o r 16 h a n d stained w i t h C o o m a s s i e blue. A r r o w (a) i n d i c a t e s m i g r a t i o n o f d e o x y r i b o n u c l e a s e a n d (b) r i b o n u c l e a s e .

density of 2 .106 cells per flask and 10 -4 M dibutyryl cyclic AMP and 10 -3 M theophylline were added to four of them. After 48 h the medium was changed in cell flasks to a growth medium containing 1 mCi/1 of 3 H-labelled mixed amino acids, and dibutyryl cyclic AMP and theophylline were again added to

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the previously treated flasks. 24 h later the cells were harvested. During this 72 h period the treated cells had divided four times. The non-histone chromosomal proteins were extracted and electrophoresed. The profile in Fig. 3 shows

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Fig. 4. T h e e f f e c t o f s e r u m s t i m u l a t i o n o f d e n s i t y - i n h i b i t e d 3 T 3 cells. A b s o r b a n c e p r o f i l e s o f n o n - h i s t o n e c h r o m o s o m a l p r o t e i n s , a n a l y s e d b y p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s a t v a r i o u s t i m e s a f t e r s t i m u l a t i o n . T h e n u m b e r s o n t h e r i g h t i n d i c a t e h o u r s a f t e r s e r u m s t i m u l a t i o n . (a) M i g r a t i o n o f d e o x y r i b o n u c l c a s e a n d (b) r i b o n u c l e a s e .

that the revertant cells have a protein profile which resembles that of log phase 3T3 cells (Fig. 2B). Density-inhibited 3T3 cells were stimulated to divide b y the addition of fresh growth medium. The rate o f D N A synthesis and cell numbers were determined and the results were identical to those previously described [20,30].

368 Therefore, the period 0--15 h has been designated G1, 15--25 h S and 25--35 h G2 and M. The non-histone chromosomal proteins were extracted and electrophoresed and the absorbance profiles obtained throughout the time course of the experiment are given in Fig. 4. It may be seen that although the profiles vary throughout the cycle, all fractions to a greater or lesser extent are continually present. Peak 11 showed a large increase with maximum absorbance representation in late G1 (10 h). Thereafter, the absorbance decreased with a minimum at 30 h and reappears as the cells cease to divide and are again density inhibited (40 h). It was of interest to determine whether peak 11 in 3T3/SV40 cells underwent similar cyclic changes. Growth of Chinese hamster ovary cells have been synchronised by treatment with dibutyryl cyclic AMP [31]. Therefore, in order to synchronise the growth of the 3T3/SV40 cells, they were grown to confluence (approx. 8 • 106 cells) and then for 48 h in the presence of 10 -4 M dibutyryl cyclic AMP and 10 .3 M theophylline. The medium was then changed to a growth medium and the ceils allowed to grow for a further 30 h. The effect of dibutyryl cyclic AMP and theophylline on the growth and subsequent behaviour of the culture following the medium change was monitored by measuring the incorporation of [3 H ] t h y m i d i n e into DNA and by cell counting. The results of these measurements are shown in Fig. 5, and it may be seen that in the presence of dibutyryl cyclic AMP and theophylline there is some incor-

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Fig, 5. The effect o f 10 -4 M d i b u t y r y l cyclic AMP and 10 -3 M t h e o p h y l l i n e o n c o n f l u e n t 3 T 3 1 S V 4 0 cells. ( A ) Cell n u m b e r s . • •, untreated; o o , treated with d i b u t y r y l cyclic AMP and theophylline; • o, t r e a t e d cells f o l l o w i n g release f r o m c y c l i c A M P i n h i b i t i o n . (B) [ 3 H ] t h y m i d i n e incorporation into D N A . • •, untreated; o o, t r e a t e d ; • ~, t r e a t e d cells f o l l o w i n g release f r o m cyclic A M P inhibition.

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21 24 Fig. 6. Non-histone c h r o m o s o m a l proteins of 3T3/SV40 cells at different stages of the cell cycle. The numbers on the right indicate hours after m e d i u m change (after release from cyclic AMP inhibition). (a) The migratio n of d e o x y r i b o n u c l e a s e ; (b) ribonuclease.

potation of [3 H] thymidine b u t that this is smaller than the incorporation into the untreated cells. This is followed b y a period in which no cell division occurs in the treated cells but cell numbers have doubled in the control group. Removal of dibutyryl cyclic AMP and theophylline is followed b y incorporation of [3 H ] t h y m i d i n e into DNA which reaches a maximum b y 9--15 h and the cell numbers double by 24 h. Essentially similar results were obtained in t w o sub-

370

sequent experiments. These results were considered to demonstrate that a reasonable degree of synchrony had been achieved in the culture. The proteins were extracted at various times after the medium change and analysed by polyacrylamide gel electrophoresis. The absorbance profiles are shown in Fig. 6 and in contrast to those of 3T3 cells stimulated to divide (Fig. 4), peak 11 is evident throughout the cell cycle. This may be clearly seen by comparing the height of peak 11 with those of peaks 7 and 8 in Figs 4 and 6. Other peak changes may also be observed in Fig. 6 notably peaks 12 and 13 increase during early S phase and subsequently decrease. Discussion Our results indicate that for 3T3 cells there is no major qualitative alteration in the electrophoretic profile of non-histone chromosomal proteins associated with the growth rate of cells, demonstrable by the methods employed in this study. There are, however, marked quantitative differences, and in particular, peak 11 was very pronounced in proliferating cells. In untransformed cells, e.g. Syrian hamster cells, these proteins are synthesised approximately equally in all phases of the cell cycle [32]. This contrasts with the differential rates of synthesis which occur in these proteins when density inhibited cells are stimulated to proliferate by fresh serum [12]. In the present study quantitative differences in the absorbance of the proteins were observed during the cycle of growth following serum stimulation of densityinhibited 3T3 cells. In particular there was an accumulation of peak 11 towards the end of G1. Comparison of the radioactive profiles of proliferating and density-inhibited 3T3 cells showed a high radioactive incorporation in peak 11 of the dividing cells. These profiles were similar to those reported for stationary and exponentially growing Syrian hamster cells [12,32]. To investigate the nature of the relationship between the synthesis of peak 11 and the growth status of cells, the absorbance and radioactive profiles of the non-histone chromosomal proteins of 3T3/SV40 and 3T3 cells were examined. No qualitative differences could be detected between the absorbance profiles of the proliferating 3T3 and 3T3/SV40 cells. Immunological techniques however have distinguished between the non-histone chromosomal proteins in fibroblasts and their viral transformed counterparts [33]. On the other hand, the radioactive profiles of the 3T3/SV40 proteins were similar to the profiles of the resting untransformed cells. This contrasts with the quantitative difference in radioactive profiles observed between WI38 diploid human lung fibroblasts and their SV40 transformed counterparts WI38-VA13 [34]. In view of the pronounced absorbance representations of peak 11 in the 3T3/SV40 cells, it was surprising that the radioactive profiles showed little incorporation into this peak. Physiological reversion of the 3T3/SV40 cells by combined treatment with cyclic AMP and theophylline increased the radioactive incorporation into peak 11. This result suggests that the rate of synthesis of peak 11 is related to the growth characteristics of the cell. The low rate of radioactive incorporation into peak 11 in the 3T3/SV40 cells as compared with those of proliferating 3T3 cells indicates that in transformed cells the rate of synthesis of the proteins of this peak is reduced. This is

371

supported by the finding that in the synchronously growing 3T3/SV40 cells the absorbance of peak 11 remained unchanged throughout the cell cycle. If it is proposed that the proteins in peak 11 play a role in events leading to cell division, their continued presence in 3T3/SV40 cells may be due to a reduced rate of degradation which might be a consequence o f viral transformation. In HeLa cells [28] and Chinese hamster ovary cells [27], neither o f which show density-dependent growth control, a peak which is present throughout the cell cycle has been found in a position equivalent to peak 11, but which is continuously synthesised. This difference may be related to the fact that these two cells lines are not virus-transformed. Possibly the continued presence of proteins o f peak 11 may be associated with a lack o f density-dependent growth inhibition and a reduced or absent G1 phase as has been reported for BHK [35] and other transformed cell lines [36,37]. It is widely agreed that factors controlling cell proliferation are expressed at the cell surface and in this context it is of interest that surface protein alterations [38] are parallel to the non-histone chromosomal protein changes which have been described in thispresent investigation.

Acknowledgements We should like to thank the Cancer Research Campaign for a grant which permitted this investigation to be carried out, Miss Christine Johnson for excellent technical assistance, and Drs. P. Riley, J. White and Linda Garvican for helpful discussions.

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