Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants on DNA replication in partially lysed cells

Biochimica et Biophysica Acta 826 (1985) 213-223 Elsevier

213

BBA 91525

Stimulatory and inhibitory effects of extracts from mammalian cell-cycle mutants o n D N A r e p l i c a t i o n in p a r t i a l l y l y s e d cells W . B l a n k - L i s s a n d R. S c h i n d l e r * Department of Pathology, University of Bern, Freiburgstrasse 30, 3010 Bern (Switzerland) (Received September 12th, 1985)

Key words: DNA replication: Replication factor: Replication inhibitor: Cell cycle; (Mouse mastocytoma)

Heat-sensitive (arrested at 39.5°C, multiplying at 33°C) and cold-sensitive (arrested at 33°C, multiplying at 39.5°C) cell-cycle mutants of the P-815-X2 murine mastocytoma line were used for the preparation of cell extracts. These were tested for their effects on DNA synthesis in 'gently lysed cells' (obtained by treatment with 0.01% Brij-58) or 'highly lysed cells' (obtained by treatment with 0.1% Brij-58). Gently lysed cells prepared from proliferating P-815-X2 or mutant cells incorporated [3HldTTP efficiently, while highly lysed cells exhibited a low level of [3H]dTYP incorporation which was markedly increased by the addition of extracts from proliferating cells. Extracts prepared from arrested mutant cells, however, were found to inhibit DNA synthesis by gently and highly lysed cells prepared from proliferating cells. After return of arrested mutant cells to the permissive temperature, stimulating activity in cell extracts reappeared at the time of reentry of cells into S phase. Both stimulatory and inhibitory activities were associated with material(s) of molecular weight above 25000, but differed in heat sensitivity, and in sensitivity to immobilized proteinase and ribonuclease. Extracts from arrested cells counteracted the stimulating effects of extracts from proliferating cells with kinetics suggesting competitive interaction between stimulating and inhibitory factors.

Introduction In previous communications [1-4], some characteristics of heat- and cold-sensitive cell-cycle mutants derived from the P-815-X2 murine mastocytoma have been described. When cultured at the respective nonpermissive temperature (39.5°C for heat-sensitive, 33°C for cold-sensitive cells), proliferation of these mutants is reversibly arrested with a cellular DNA content typical of cells in G 1 phase, while at the respective permissive temperature (33°C for heat-sensitive, 39.5°C for cold-sensitive cells), continued exponential cell * To whom correspondence should be addressed. Abbreviations: Mops, 4-morpholinepropanesulfonic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid.

multiplication is observed. The heat-sensitive mutants were found to differ from the cold-sensitive lines in several respects, such as dominant versus recessive expression of the mutant phenotype in heterokaryons obtained by fusion with 'wild-type' cells [1,4] and the capacity to undergo morphological differentiation when brought to the nonpermissive temperature [2]. This indicated that the two types of mutants, when incubated at the respective nonpermissive temperature, enter qualitatively different states of reversible proliferative quiescence. Another difference between these mutants was observed with respect to control of thymidine kinase, which at the nonpermissive temperature decreased to very low levels: after return of arrested heat-sensitive cells to the permissive temperature, thymidine kinase activity increased

0167-4781/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

214

in parallel with reentry of cells into S phase, whereas in cold-sensitive cells, the increase of thymidine kinase levels was delayed by approximately one cell-cycle time, as compared to reentry of cells into S phase [3]. In view of these observations, it appeared of interest to test other biochemical parameters more directly related to D N A replication in proliferating and arrested heat- and cold-sensitive cell-cycle mutants. While changes in D N A polymerase c~, fl and ~, activities have recently been reported [5], the present communication is concerned with the effects of extracts from heat- and cold-sensitive mutant cells on D N A replication ill a subcellular assay system consisting of partially lysed cells prepared by treatment with Brij-58. The characteristics of this assay system, as studied with C H O cells, have been previously described [6]. It was shown that D N A synthesis by 'highly lysed' C H O cells that had been obtained by treatment with 0.1% Brij-58 was only 2 5% of that in 'gently lysed" cells obtained by treatment with 0.01% Brij58, and that the low level of D N A synthesis by highly lysed cells could be increased by a factor of approx. 50 by the addition of C H O cell extract. In the present work, this system was adapted to cells of the P-815-X2 line. It will be shown that extracts from proliferating 'wild-type' and mutant cells, similar to the C H O system, stimulate D N A synthesis of highly lysed cells, whereas extracts prepared from heat- and cold-sensitive mutant cells that have been arrested by incubation at the respective nonpermissive temperature exhibit a pronounced inhibitory activity on D N A replication of gently lysed cells. Materials and Methods

Cell lines and culture techniques The selection of a heat-sensitive and a cold-sensitive cell-cycle variant from a clonal subline (K21) of the P-815-X2 mouse mastocytoma has been previously described [1]. Of the two variant lines thus obtained, clonal sublines were derived by isolation of single cells; in this communication, the effects of cellular extracts prepared from a subclone of the heat-sensitive line (termed 21-Tb), a subclone of the cold-sensitive line (termed 21-Fc), and from the parent clone (K21) or the P-815-X2

line on D N A replication in cells partially lysed by Brij-58 treatment are reported. The cells were grown under continuous agitation in culture medium I [7] supplemented with 10% horse serum. The cultures were diluted every 24 h with fresh medium to obtain 200000 cells/ml. If cell density after 24 h of incubation was less than 267000 cells/ml, a portion of the ceils was centrifuged, resuspended in fresh medium and recombined with the remainder of the culture in order to renew at least 25% of the medium per day. Cell multiplication was determined by measuring cell density with a Coulter counter.

Chemicals Brij-58 and nucleoside triphosphates were purchased from Serva; digitonin, dextran ( M r approx. 110000), E G T A and Mops from Fluka; enzymes immobilized on beaded agarose, i.e. trypsin (from bovine pancreas, 100 units per ml of packed gel), proteinase type VII-A (from Bacillus amyloliquefaciens, 10 units per ml of packed gel), and ribonuclease A (20 units per ml of packed gel), and dithiothreitol, creatine phosphate and creatine phosphokinase from Sigma; trasylol (aprotinin, proteinase inhibitor) from Bayer; Hepes from Calbiochem-Behring; [2-14C]thymidine (58 m C i / m m o l ) , [methyl-3H]thymidine (24 C i / m m o l ) and [methyl-3H]-thymidine 5'-triphosphate (30 C i / m m o l ) from Amersham International.

Preparation of cell extracts The method described by Mackall et al. [8] f~r the release of cytoplasmic proteins from cultured cells was modified as follows. A cultured suspension containing (0.6-2). 10 x cells was centrifuged, and the cells were washed twice with ice-cold phosphate-containing saline (5 mM N a 2 H P O 4, 150 mM NaC1, 2 mM KCI) and resuspended in 10 ml of this solution for determination of cell number. The cells were then recentrifuged and suspended in digitonin solution (17 mM Mops (pH 7.0), 250 mM sucrose, 2.5 mM EDTA, 400 units trasylol/ml and 2 mg digitonin/ml) to obtain a cell density of 1.2.10 ~ cells/ml. After treatment with digitonin solution for 10 rain at room temperature, the suspension was centrifuged (30000 x g for 10 rain at 0°C), and the supernatant was dialyzed for 24 h against several changes of extract buffer (40 mM

215 Hepes (pH 7.8), 80 mM KCI, 4 mM MgCI2, 300 mM sucrose, 2 mM dithiothreitol, 1 mM EGTA, 2 mM ATP, 0.01% NAN3). The resulting extracts were stored in 50-/,1 aliquots at - 7 0 ° C . In the following, cell extract obtained from 107 cells will be designated as 1 relative unit of cell extract.

Filtration through selecti~,e membranes and ammonium sulfate precipitation In one series of experiments, extracts were diluted 7-fold with extract buffer and centrifuged at 0°C in conical Amicon selective membranes CF-25 (exclusion of molecules > 25 kDa; > 95% retention) for 2 h at 80 × g. The filtrates and the retained fractions were stored at - 7 0 ° C . For a m m o n i u m sulfate precipitation, an ammonium sulfate solution saturated at 0°C was added dropwise to ice-cold extracts with continuous stirring, to obtain 60% saturation. Stirring was continued for 45 rain, and samples were centrifuged for 30 min at 32000 × g. The precipitates were redissolved in extract buffer, and the precipitates as well as the supernatants were dialyzed for 24 h against several changes of the extract buffer and stored at - 7 0 ° C . Treatment of cell extracts with immobilized enzymes The gels with immobilized trypsin, bacterial proteinase or ribonuclease A were washed several times with ice-cold water and with extract buffer. For enzymatic digestion, cell extracts were incubated with enzyme gel at 30°C for 2 h with gentle agitation. As controls, gels were incubated in the extract buffer without cell extract. After incubation, the extracts were separated from the enzyme gel by filtration through Whatman No. 1 paper and stored at - 7 0 ° C . Determination of D N A polymerase c~ activity D N A polymerase ~ activity was determined in a reaction mixture containing 2',3'-dideoxythymidine 5'-triphosphate as described [5]. Preparation of partially lysed cells Cultures were prelabeled overnight with [laC]dThd (1 n C i / m l , 58Ci/mol), cooled in ice, collected by centrifugation, washed with saline A containing no phenol red [9], and suspended at 0°C in lysis solution to obtain a cell density of

6 . 1 0 6 cells/ml. The lysis solution contained 40 mM Hepes (pH 7.8), 2 mM dithiothreitol, 2% ( w / v ) dextran, 300 mM sucrose, 1 mM EGTA, 4 mM MgC12, 2 mM ATP, 0.01 or 0.1% ( w / v ) Brij-58 and 80 or 200 mM KCI. Gently lysed cells were obtained by a 10 min treatment at 0°C with lysis solution containing 0.01% Brij-58 and 80 mM KCI, whereas highly lysed cells were obtained by a 10 min treatment with lysis solution containing 0.1% Brij-58 and 200 mM KCI. The suspension of partially lysed cells (up to 3 ml) was layered on top of 6 ml of lysis solution containing no Brij-58, while the concentration of all other components was higher by a factor of 1.1. After centrifugation, the lysed cells were washed with 0.5-1 ml of lysis solution containing 80 mM KC1, but no Brij-58, centrifuged and suspended'in the reaction mixture described below to obtain a density of 4.107 cells/ml.

Determination of [SH]dTTP incorporation into DNA by partially lysed cells The reaction mixture contained, in a total volume of 20/~1, 40 mM Hepes (pH 7.8), 80 mM KCI, 3.3 mM M g C I > 1 mM EGTA, 225 mM sucrose, 1.5 mM dithiothreitol, 0.5% ( w / v ) dextram 1.7 mM ATP, 5 mM creatine phosphate, 0.1 m g / m l creatine phosphokinase, 0.1 mM each of CTP, G T P and UTP, 0.012 mM [3H]dTTP (40 # C i / m l ) , 0.1 mM each of dATP, dCTP, dGTP, 0.005% ( w / v ) N a N 3, and 10/zl of cell extract or of extract buffer. The partially lysed cells were incubated in this reaction mixture for 1 h at 30°C. The reaction was stopped by cooling to 0°C, and 0.5 ml of 0.4 N N a O H containing 200 /xg carrier D N A was added. The acid-insoluble material was precipitated with 1 ml of 1.5 N HCI containing 12% N a 4 P 2 0 7 - 10H20 (PP,), collected on Whatman G F / C filters, washed with 0.1 N HCI containing 0.5% PPi and dried with ethanol and acetone. Radioactivities retained on the filters were determined in a scintillation counter, and 3H/14C ratios were calculated to correct for variations in cell numbers. Confirmation of the semiconservative nature of DNA replication The mode of D N A synthesis by partially lysed cells was analyzed as described by Reinhard et al.

216

[6]. Intact cells were prelabeled for 16 h at 37°C with [14C]dThd ( 0 . 0 1 5 / , C i / m l ) , then for 2 h with B r d U r d in culture medium containing 2- 10 f M BrdUrd, 10 5 M dThd, 10 ~' M F d U r d and 10 4 M dCyd, and used to prepare gently and highly lysed cells. These were incubated for 2 h at 30°C in a reaction mixture containing 6 . 7 . 1 0 ~' M B r d U T P and 3.3 • 10 ~ M [~H]dTTP (50 ffCi/ml). The reaction mixture used for highly lysed cells was further supplemented with cell extract from P-815-X2 cells. The D N A was purified [10] and centrifuged both in neutral or alkaline CsC1 gradients. Fractions were collected from the b o t t o m of the tubes, a n d their r a d i o a c t i v i t i e s were determined.

A utoradiography To label intact cells, cultures were incubated with [3H]dThd and processed as previously described [3]. Partially lysed cells were labeled by incubation for 5 min (highly lysed cells) or 1 h (gently lysed cells) in the reaction mixture containing [~H]dTTP (40 ffCi/ml). Highly lysed cells were incubated for only 5 min because during longer incubation times the nuclei underwent morphological disintegration, precluding a quantitative analysis by autoradiography. The partially lysed cells were then suspended in ice-cold 70 m M KC1 and incubated for 5 min at 0°C, fixed with a mixture of ethanol and acetic acid ( 1 0 : 1 , v/v), centrifuged, transferred onto microscope slides and dried. The preparations were covered with KodakN T B - 2 emulsion, exposed for 10 14 days at 4°C. developed and stained with Giemsa. The percentage of labeled nuclei (labeling index) was determined by scoring at least 500 cells or nuclei per preparation. Results

Characteristics of DNA ,synthesis by gently and highly lysed cells Fig. 1 illustrates the time course of [~H]dTFP incorporation by gently and by highly lysed P-815X2 cells in the presence and absence of extract prepared from P-815-X2 cells. Gently lysed cells exhibited a relatively high capacity for D N A synthesis which was more or less the same in the presence or absence of cell extract. On the other

hand, for highly lysed cells a low level of D N A synthesis was observed which was markedly increased by the addition of extract from proliferating cells. Highly lysed cells thus represent a convenient assay system for stimulating factor(s) present in extracts from proliferating cells. On the other hand, it will be shown below that gently lysed cells may be used to assay for inhibitory factor(s) present in extracts from arrested mutant cells. To show that [~H]dTTP incorporation was attributable to semiconservative D N A replication, partially lysed cells were incubated with B r d U T P and [3H]dTTP, and the D N A was subjected to density gradient centrifugation. The results obtained (not shown) were qualitatively the same as previously reported for partially lysed C H O cells

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Fig. 1. [3H]dTTP incorporation into D N A of gently and highly lysed P-815-X2 cells. The cells were prelabeled with [14C]dThd and partially lysed with Brij-58. Gently lysed (m, @) and highly lysed cells (D, @) were incubated at 30°C with 10 5 M

[~H]dTFP (3.5 Ci/mmol) in the reaction mixture without cell extract (11, D), or with P-815-X2 cell extract at a concentration of 3 relative units/ml (~, ()), and radioactivity in DNA was determined as a function of incubation time. The 3H radioactivity incorporated during incubation of gently lysed cells without cell extract for 60 rain corresponds to 20.7 pmol dTTP/106 gently lysed cells.

217 TABLE I EFFECT OF EXTRACT FROM P R O L I F E R A T I N G CELLS ON DNA SYNTHESIS BY PARTIALLY LYSED CELLS PREPARED FROM CULTURES OF P R O L I F E R A T I N G OR AI~RESTED CELL-CYCLE MUTANTS Cultures of 21-Fc and 21-Tb cells were prelabeled with [~4C]dThd at the permissive temperature (39.5°C and 330( ', respectively), and then arrested by incubation at the nonpermissive temperature (33°C and 39.5°C, respectively) for 4 days. Proliferating cells (i.e. before transfer to the nonpermissive temperature) and arrested cells were subjected to partial lysis, and highly or gently lysed cells were incubated, for 60 min at 30°C with [~H]dTTP in the reaction mixture containing cell extract from proliferating P-815-X2 cells (3 relative u n i t s / m b , or without cell extract. Data are means of results obtained in three independent experiments, with standard errors. Assay system

Partially lysed cells prepared

[ 3H]dTTP incorporation ( p m o l / h per 106 partially lysed cells)

from

without cell extract

with cell extract

Highly lysed

21-Fc (39.5°C) 21-Tb (33°C)

0.68_+0.17 0.89+0.05

5.1 _+2.2 5.4+ 1.6

cells

21_Fc (33oc) 21-Tb (39.5°C)

0.07+0.01 0.04+0.02

0.22_+0.11 0.22_+0.17

Gently lysed

21-Fc (39.5°C) 21 -Tb (33°C)

16.8_+5.2 33.4 _+6.1

cells

21-Fc (33°C) 21-Tb (39.5°C)

0.42_+0.02 0.72_+0.02

18.9_+3.3 25.8 _+6.3 0.38_+0.19 0.58_+0.19

[6] and support the conclusion that [3H]dTTP incorporation by gently or highly lysed P-815-X2 cells reflects semiconservative DNA replication.

Capacity for DNA synthesis of nuclei in partially h,sed cells prepared from proliferating and arrested mutant cells Heat- and cold-sensitive cell-cycle mutants were either maintained at the respective permissive temperature or arrested by incubation for 4 days at the nonpermissive temperature, and used to prepare gently and highly lysed cells. As seen in Table I, partially lysed cells prepared from arrested cellcycle mutants exhibited a comparatively low extent of [3H]dTTP incorporation, whereas partially lysed cells prepared from proliferating mutants

were able to incorporate considerably higher amounts of dTTP. In highly lysed cells prepared from proliferating mutant cells, incorporation was increased by the addition of P-815-X2 cell extract, and some increase was also observed in highly lysed cells prepared from arrested mutant cells. In cultures of arrested 21-Fc or 21-Tb cells, a very low percentage of the cell population was in S phase, as determined by [3H]dThd labeling and autoradiography and summarized in Table I1. Highly lysed cells prepared from proliferating or arrested mutant cells and incubated with [3 H]dTTP exhibited labeling indices (percent labeled nuclei) very similar to those of corresponding intact cells labeled with [3H]dThd, and relative numbers of [~H]d'VFP-labeled highly lysed cells were not increased by the addition of P-815-X2 cell extract. DNA synthesis by highly lysed cells thus was restricted to cells that were already in S phase prior to partial lysis, as previously reported for

TABLE II LACK OF EFFECT OF EXTRACT FROM PROLIFERATING CELLS ON NUMBERS OF D N A - S Y N T H E S I Z I N G NUCLEI IN H I G H L Y LYSFD CELLS PREPARED FROM C U L T U R E S OF P R O L I F E R A T I N G OR A R R E S T E D C E L L - C Y C L E MUTANTS Cultures of 21-Fc and 21-Tb cells were arrested by incubation at the nonpermissive temperature (33°C and 39.5°C, respectively) for 4 days. Proliferating cells (i.e. before transfer to the nonpermissive temperature) and arrested cells were used to prepare highly lysed cells, and these were incubated for 5 min at 30°C with [3H]dTI'P in the reaction mixture containing extract from proliferating P-815-X2 cells (3 relative units/ml), or without cell extract. In addition, aliquots of cultures of proliferating and arrested mutant cells were incubated for 30 min with [~H]dThd. Relative numbers of DNA-synthesizing nuclei in intact and highly lysed cells were determined by autoradiography. Data are means of results obtained in three independent experiments, with standard errors. Highly lysed cells prepared from

21-Fc (39.5°C) 21-Tb (33°C) 21-Fc (33°C) 21-Tb (39.5°C)

Labeling index (%) intact cells

52.6_+1.2 51.1_+0.4 1.5_+0.1 0.8_+0.l

highly lysed cells without cell extract

with ceil extract

50.0+0.5 49.7+0.4

50.4+0.9 51.5+0.4

1.3+0.2 0.6+0.2

1.4+0.2 0.8+0

218

Effects q# extracts from proliferating and arrested mutant cells on DNA ,synthesis by partially Ivsed cells

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sensitive mutant P-815-X2

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Fig. 2. Effects of increasing concentrations of cell extracts from proliferating and arrested cell-cycle mutant and "wild-type' K21 cells on [~H]dTTP incorporation by gently or highly lysed P-gI5-X2 cells. P-815-X2 cells prelabeled with [ 1 4 q d T h d were partially lysed and incubated for 60 min at 30°C with [~H]dTTP in the reaction mixture containing different concentrations of cell extracts. Highly lysed P-815-X2 cells (HLC, open symbols) were used to determine the effects of extracts prepared from proliferating cell-cycle mutants grown at the permissive temperature (©, 21-Fc at 39.5°C: zx, 21-Tb at 33°Cl, whereas gently lysed cells (GLC, closed symbols) were used to test the effects of extracts prepared at 4 days after transfer of 21-Fc (Q), and 21-Tb (*,) cells to the nonpermissive temperature of 33°C and 39.5°C, respectively. Extracts from 'wild-type" K21 cells grown at 39.5°C (D, I), and 33 ° ( O , 0 ) were used in both assay systems. One relative unit of cell extract corresponds to cell extract from 107 cells. The 100% values, i.e. [YH]dTTP incorporation by gently and highly lysed cells in the absence of cell extract, correspond to 31.2 and 2.5 pmol d T T P / 1 0 ~' partially lysed cells, respectively.

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Fig. 3. Stimulation and inhibition of DNA synthesis in partially lysed P-815-X2 cells b~ extracts prepared from cold-sensitive 21-Fc cells at different times after transfer of cultures to the r,,mpermissive temperature of 33°C and back to the permissive temperature of 39.50( '. (_'ell extracts were prepared at 24-h intervals after transfer of a 21-Fc culture from 39.50( ' to 33o( ". After 5 days at 33o(_`, a culture was returned to 39.5°( ', and cell extracts were again prepared at 24-h intervals. Every 24 h, relative numbers of cells in S phase ('I-) were determined by pulse-labeling with [~HldThd and autoradiography. Effects of cell extracts obtained at different incubation times on I)NA synthesis of highly' lysed (el and gently, lysed ( O ) P-815-X2 cells wcrc determined as described for Fig. 2, using an extract concentration of 3 relative units/ml. The controls (100~;~ values) represent [ZHIdTTP incorporation without cell extract. Inhibitory effects (0 100%) are drawn on a larger scale than stimulator,,, effects (above 100,g 1.

219 s e e n in Fig. 2, e x t r a c t s f r o m a r r e s t e d 2 1 - T b a n d

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TIME OF CELL CULTURE, DAYS Fig. 4. Stimulation and inhibition of DNA synthesis in partially lysed P-815-X2 cells by extracts prepared from heal-sensitive 21-Tb cells at different times after transfer of cultures to the nonpermissive temperature of 39.5°C and back to the permissive temperature of 33°C. Cell extracts were prepared at 24-h intervals after transfer of a 2l-Tb culture from 33°C to 39._~°C. After 4 days at 39.5°C, the culture was returned to 33°C. and cell extracts were again prepared at various time intervals. In parallel, relative numbers of cells in S phase (.A-) ',,,'ere determined by pulse-labeling with [3H]dThd and autoradiography. Effects of cell extracts obtained at different incubation times on DNA synthesis of highly lysed (*,) and gently lysed (zx) P-g15-X2 cells were determined as described for Fig. 2, using an extract concentration of 3 relative units/ml. The controls (1009~ values) represent [3H]dTTP incorporation without cell extract. Inhibitory effects (0-100%) are drawn on a larger scale than stimulatory effects (above 100%).

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EXTRACT CONCENTRATION 10g [ RELATIVE UNITS/m[ ] Fig. 5. Counteracting effects of extracts from proliferating and arrested 21-Fc cells on DNA replication in partially lysed cells. P-815-X2 cells prelabeled with [14C]dThd were partially lysed and incubated for 60 min at 30°C with [aH]dTTP in the reaction mixture containing cell extract prepared from proliferating (21-Fc at 39.5°C) and/or from arrested cells (21-Fc at 4 days after transfer to 33°C). Upper panel: highly lysed cells (HLC) were incubated in a reaction mixture containing increasing concentrations of extract prepared from proliferating 21-Fc cells, in the absence (© ©) or in the presence of 0.6 relative units/ml ( O . - - O ) or 1.2 relative units/ml (© -©) of extract from arrested 21-Fc cells. Lower panel: gently lysed cells (GLC) were incubated in a reaction mixture containing increasing concentrations of extract prepared from arrested 21-Fc cells in the absence (e e) or in the presence of 0.6 relative units/ml (e- • .e) or 1.2 relative units/ml ( e I I - - e ) of extract from proliferating 21-Fc cells. The controls (100% values) represent [3H]dTTP incorporation without cell extract.

220

were prepared and tested for their effects on D N A synthesis by gently and highly lysed P-815-X2 cells. In addition, after 4 or 5 days at the respective nonpermissive temperature, cultures were returned to the permissive temperature, and extracts prepared at 24 h intervals were again tested for their effects on gently,' and highly lysed cells. As seen in Figs. 3 and 4, transfer of mutant cells to the nonpermissive temperature resulted m a relatively slow decrease in the capacity of extracts to stimulate DNA synthesis by highly lysed cells, with the concomitant appearance of an inhibitory activity o n gently lysed cells, l r i comparison, cellcycle arrest of mutant cell populations, as determined by autoradiographical determination of relative numbers of cells in S phase, occurred more rapidly. After return of arrested cells to the permissive temperaturc, however, loss of inhibitory activity and reappearance of stimulatory activity in cell extracts from both 21-Tb and 21-Fc cells followed a time course which was similar to that of reentry of cells into S phase.

Antagonistic' effects of extracts from proliferating and arrested mutant cells As illustrated in Fig. 5, the stimulatory effects of an extract from proliferating 21-Fc cells on highly lysed P-815-X2 cells were antagonized by the addition of extract obtained from arrested 21-Fc cells. Similarly, the inhibitory effects of an extract from arrested 21-Fc cells were counteracted by the addition of extract from proliferating 21-Fc cells to the reaction mixture. The nearly parallel shift of the curves obtained by the addition of increasing amounts of extract from arrested or proliferating cells, respectively, suggests that the stimulatory and inhibitory factors in the two types of extract interact in a competitive manner. This assumption is also supported by Lineweaver-Burk plots of the data (not shown), which indicate that at high extract concentrations. stimulating and inhibitory factors interact competitively. Similar results were obtained with extracts from proliferating and arrested 21-Tb cells (data not shown).

TABI.E Ill EFFECTS OF PROTEOLYTIC DIGESTION OF EXTRACTS FROM P R O L I F E R A T I N G A N D ARRESTED MUTANT ('ELLS ON THE CAPACITY TO STIMULATE OR INHIBIT DNA SYNTHESIS IN PARTIALLY LYSED ('ELLS Extracts (6 relative units/ml) prepared from proliferating and arrested 21-Ec and 21-Tb cells as described for Fig, 2. or extract buffer were added to the immobilized trypsin preparation (8.2 units of trypsin per 300 p,I of extract or extract buffer). After incubation at 30°C for 0 and I20 rain, the agarose-bound enzyme was removed by filtration, and the filtrates were tested for their effects on DNA synthesis by gently lysed (GLC) and highly lysed tHLC) P-815-X2 cells. The 100% values represent [3H]dTTP incorporation in the presence of extract buffer that was not exposed to immobilized trypsin. Data are means of results obtained with four separate trypsin digests each, with standard errors. Assay system

('ell extract prepared from

HL("

(}L("

[3 H]dTTP incorporation (% of control) with cell extract (not trypsin-digested)

with cell extract (trypsin-digested for 120 rain)

with extract buffer (trypsin-treated for 120 rain)

21-Fc{3g.5°C) 21-Tb (33°(;)

347+ 7 284_+ l0

33+1 31 + 1

76+5 82 12-_2

21-Fc(3g.5°C) 21-Tb(33°( ` )

88+ 0.4 105+ 2

15+1 18+0.4

72+2

ltI.C

21-Fc(33°C) 21-Tb(30.5°( "}

52+ 3 80+ 2

42+1 21 + 2

81 +6 72+-5

(}L('

21-Fc(33°C) 21-Tb(39.5°( `)

26± 1 31+ 0.4

35+1 21_+1

73-+2 69+3

63+2

221

Physicochemical characterization of stimulatorv and inhibitory activities The stimulatory and inhibitory activities in extracts from proliferating and arrested 21-Tb and 21-Fc cells were subjected to a preliminary characterization. Both activities were retained by Amicon CF-25 membranes, indicating that they were associated with materials of molecular weights above 25000. Furthermore, both activities were precipitable by (NH4)2SO 4 (data not shown). In additional experiments, extracts from proliferating P-815-X2 cells were precipitated with (NH4)2SO 4 at 60% saturation, fractionated by the addition of (NH4)2SO4 solutions of progressively lower concentration, and the fractions obtained were tested for their capacity to stimulate D N A synthesis in highly lysed P-815-X2 cells and for D N A polymerase a activity. The results obtained (not shown) indicate that stimulation of D N A synthesis was not attributable to D N A polymerase a activity. The stimulatory activity from proliferating 21-

TABLE IV EFFECTS OF R I B O N U C L E A S E A N D P R O T E I N A S E DIGESTI ON OF EXTRACT FROM A R R E S T E D 21-Fc CELLS ON I N H I B I T O R Y ACTIVITY FOR DNA SYNTHESIS IN G E N T L Y LYSED CELLS The cell extract was prepared from arrested 21-Fc cells as described for Fig. 2. After adjustment of the pH to 7.4. the extract (6 relative units per ml) and the extract buffer were incubated for 120 min at 30°C with immobilized proteinase (0.24 units per 300 ~1) or ribonuclease (1.5 units per 300 ttl), or for 120 min each with the two enzyme preparations, and the effects of pretreated extract buffers and cell extracts on [3 H]dTTP incorporation by gently lysed P-815-X2 cells were determined. Data are means of results obtained in four independent experiments, with standard errors. Pretreatment of extract buffer or extract with enzymes

[ 3 H]dTTP incorporation (% of control) with extract buffer

with cell extract

No enzyme treatment

100

33 + 5

A. Proteinase digestion Proteinase followed by ribonuclease digestion B. Ribonuclease digestion Ribonuclease followed by proteinase digestion

74 _+3

26 ± 2

6l + 4

45 +_4

70 +_3

48 + 6

66 ± 5

66 ± 5

Tb, 21-Fc and K21 cells was sensitive to heating at both 50 and 70°C. In contrast, the inhibitory activity in extracts from arrested cells was lost after heating at 70°C, but maintained for at least 60 min at 50°C (data not shown). As shown in Table III, treatment of extracts from proliferating mutant cells with immobilized trypsin resulted in loss of stimulatory activity, as tested in highly lysed P-815-X2 cells, with the concomitant appearance of an inhibitory activity which was detectable in both gently and highly lysed cells. On the other hand, the inhibitory activity in extracts from arrested 21-Fc and 21-Tb cells was found to be insensitive to trypsin treatment. Similar to trypsin, immobilized bacterial proteinase had essentially no effect on the inhibitory activity in extracts from arrested mutant cells, and digestion with this enzyme also reduced the stimulating activity in extracts from proliferating cells, although without the appearance of an inhibitory activity (data not shown). While the inhibitory activity in extracts from arrested cells appeared to be insensitive to treatment with these proteinases, it was lost if extracts from arrested 21-Fc cells were subjected to treatment with immobilized ribonuclease, followed by treatment with bacterial proteinase (Table IV). If, however, proteinase was applied prior to ribonuclease treatment, only a partial reduction of inhibitory activity was observed. Discussion

Stimulation of D N A replication in permeabilized cells, isolated nuclei and related subcellular systems by extracts from proliferating cells has been described in several reports (see Refs. 6 and 11). In confirmation of previous studies with C H O cells [6], the results obtained with the murine mastocytoma cells support the conclusion that extracts from proliferating cells stimulated D N A synthesis of highly lysed cells that were already in S phase at the time of partial lysis, whereas nuclei of gently or highly lysed cells that were in other cell-cycle phases or in a state of proliferative quiescence at the time of partial lysis were not induced to initiate D N A synthesis by the addition of extracts from proliferating cells. In agreement with other studies on factors stimulating D N A repli-

222 cation in subcellular systems [11 17], the results of our preliminary characterization of the stimulatory factor(s) from proliferating'wild-type' and mutant mastocytoma cells indicate that this activity resides in protein(s) of a molecular weight above 25 000. In addition, this activity was found to be different from D N A polymerase a. As an unexpected finding, extracts from arrested heat- and cold-sensitive cell-cycle mutant cells not only lacked a stimulatory activity on D N A synthesis in highly lysed cells, but were able to inhibit D N A synthesis in gently lysed cells. This effect was concentration-dependent, and the results of mixing experiments suggest that stimulatory and inhibitory factors interacted in a more or less competitive manner, levels of D N A synthesis observed in partially lysed cells thus depending on relative concentrations of the two types of factors. The method used for preparing the cell extracts by treatment with digitonin has been shown to result in release of cytosolic proteins, but not of lysosomal or mitochondrial enzymes [8], indicating lhat both stimulating and inhibitory activities are located in the cytoplasm. Furthermore, their effects on nuclear D N A synthesis suggest that they have the capacity to enter into nuclei, at least in gently and highly lysed cells. The physicochemical properties of the inhibitory factor(s) in extracts from arrested mutant cells differed from those of stimulating factor(s) in some respects, such as sensitivity to heating at 50°C. In addition, the stimulating, but not the inhibitory activity was sensitive to two proteinases, while the inhibitory activity was abolished by sequential treatment with ribonuclease and a proteinase, suggesting that it n-my represent a ribonucleoprotein. A ribonucleoprotein with a specific function, ribonuclease P, an enzyme involved in maturation of 5'-termini of tRNAs, has been described [18]. Proteins inhibiting D N A polymerase e~ have recently been described as being present in rat liver [19]. After treatment of extracts from proliferating cells with trypsin, an inhibitory effect on D N A synthesis of gently lysed cells similar to that obtained with extracts from arrested cells was observed (Table III). These results are compatible with the assumption that proliferating cells conrain considerably higher levels of stimulatory fac-

tor(s) than arrested cells, whereas the activity of factor(s) inhibiting D N A synthesis may be more or less constant irrespective of the proliferative state of the cells. While the biological function of the inhibitory factor(s) is not known at the present time, a physiological role is suggested by the observation of a competitive interaction with stimulating factors. It may, for instance, be considered that this type of inhibitory activity could be responsible for the relatively slow rate of D N A chain elongation in eukaryotic as compared to prokaryotic systems. After transfer of heat- and cold-sensitive mutant cells to the nonpermissive temperature, the decrease m relative numbers of cells in S phase occurred more rapidly than changes in activities stimulating or inhibiting DNA synthesis (Figs. 3 and 4). On the other hand, after return of arrested cells to the permissive temperature, the increase in stimulatory activity was rather well correlated with the time course of reentry of cells into S phase, suggesting that the stimulatory factor(s) may be indispensable for initiation of S phase. In fact, similar changes with time after changing the incubation temperature were observed for DNA polymerase ~x [5], whereas the increase in thymidine kinase activity was delayed by approximately one cell-cycle time with respect to reentry into S phase after return of arrested cold-sensitive cells to the permissive temperature [3]. It thus appears that stimulatory factor(s) for D N A synthesis, as detected in the highly lysed cell system, and DNA polymerase c~, which represents one of the key enzymes in semiconservative nuclear D N A replication [20], are under coordinate control during transition of cells from proliferative quiescence into S phase, as well as during entry of proliferating cells into proliferative quiescence. Thymidine kinase, however, which as a salvage enzyme is not required for DNA replication, may be under a different type of regulation.

Acknowledgements This work was supported by the Swiss National Science Foundation. The expert technical assistance of Miss B. MiXller is gratefully acknowledged. The authors are indebted to Dr. E. Schneider for carrying out the DNA polymerase ~x assays.

223

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