P53-transformation-related protein: Kinetics of synthesis and accumulation in sv40-infected primary mouse kidney cell cultures

VIROLOGY

147,275-286

(1985)

P53-Transformation-Related Protein: Kinetics of Synthesis and Accumulation in SVLZO-Infected Primary Mouse Kidney Cell Cultures ARLETTE DUTHU,* JEAN-CLAUDE EHRHART,* SAM BENCHIMOL,? KRISH CHANDRASEKARAN,* AND PIERRE MAY*,l *In&&t de Recherches Scienti&ques SUT le Cancer, 94802 Villejuzf Ced.ex, France, and ~Division of Biological Research, The Ontario Cancer Institute and the Department of Medical Biophysics, University of Toronto, 500 Sherbowne Street, Twonto, Ontario MLX lK9, Canada Received May 31, 1985; accepted August 7, 1985 During abortive infection of Go/Gl-arrested primary baby mouse kidney (BMK) cell cultures with simian virus 40 (SV40), expression of the viral large T antigen is followed by a mitotic host response including the stimulation of host macromolecular synthesis and induction into the cell cycle of Go/Gl-arrested cells. We performed an extensive study of the sequential events taking place after SV40 infection of confluent BMK cell cultures. This study comprised a detailed kinetic analysis of transcription, synthesis, and accumulation of ~53, in conjunction with the time course of large T antigen synthesis and SV40-induced cellular DNA replication. The monoclonal antibodies used for specifically recognizing mouse p53 were PAb 421, PAb 122, PAb 246, PAb 248, and RA3-2C2. Our results consistently show that under our experimental conditions, the stimulation of p53 synthesis and the accumulation of p53 occur well after the onset of T antigen-induced cellular DNA replication. This relatively late activation of p53 expression appears to be controlled at a level other than transcription. In conclusion, we suggest that, at least in certain cases, T antigen’s mitogenic potential is not dependent on its interaction with ~53. 0 1985 Academic

Press. Inc.

organization of the p53 gene in mouse has been investigated in detail. It has been The p53 protein is a transformation-reshown that the mouse genome contains a lated phosphoprotein that is expressed at single functional gene for the p53 protein elevated levels in a variety of transformed (Bienz et ak, 1984; Zakut-Houri et ak, 1983). cell lines of murine, human, or other origin, Recently it was reported that the p53 gene including tumor-derived cell lines (for re- can cooperate with an activated ras gene views, see Crawford, 1984; Klein, 1982). The to transform primary rodent cells (Eliyahu major cellular location of p53 is in the nu- et ah, 1984; Parada et al., 1984) and that it cleus (Gurney et ab, 1980). The presence of is also able to immortalize such cells (Jenp53 is not restricted to transformed or ma- kins et al., 1984a). lignant cells; relatively low levels of p53 In nontransformed mouse cells the p53 have been detected in normal primate and protein has a very short half-life (Oren et murine fibroblasts (Benchimol et al., 1982; ak, 1981) and it might act as a control factor Chen et ab, 1983; Thomas et ak, 1983), and in growth regulation, as suggested by sevin explants of early mouse embryos (Mora eral recent findings. Firstly, when confluent et al, 1980). The full-length cDNA of mouse 3T3 cells arrested in Go/G1 by serum dep53 has been completely analyzed in terms privation are stimulated to resume the cell of its nucleotide sequence (Jenkins et al, cycle by addition of serum, an increase in 1984b; Pennica et al., 1984) and the genomic both the steady-state levels of p53 protein and the synthesis of p53 mRNA is observed ’ Author to whom requests for reprints should be prior to DNA synthesis in late Gl (Reich addressed. and Levine, 1984). In this system, the p53 INTRODUCTION

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0042-6822/85 Copyright All rights

$3.00

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

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protein has a short half-life (about 20 min), and control of its expression appears to take place at the transcriptional level (Reich and Levine, 1984). Second, microinjection of monoclonal antibody against ~53 inhibits the entry of quiescent Swiss 3T3 cells stimulated by serum into S phase (Mercer et aZ., 1982,1984). In SV40-transformed cells, SV40 large T antigen (hereafter referred to as T antigen) is physically complexed with p53 (Harlow et aL, 1981b; Lane and Crawford, 1979; McCormick et aL, 1981; McCormick and Harlow, 1980) and as a consequence the stability and level of the p53 protein are considerably increased (Oren et aL, 1981). In a recent study of p53 synthesis in nondividing and dividing mouse lymphocytes (Milner, 1984), it was reported that in nondividing cells the p53 protein is detected by the monoclonal antibodies PAb 248 and RA3-2C2 but not by the monoclonal antibodies PAb 122 and PAb 421. Conversely, after mitogenic stimulation of the cells by concanavalin A, the ~53 expressed in the dividing cells is recognized by PAb 421 and PAb 122 but not by PAb 248 and RA3-2C2. These results, together with those of Wolf et al. (1985) indicate the existence of distinct immunological types of mouse p53. Time-course studies performed with the established mouse cell line BALB/c 3T3 infected by SV40 have shown that in these cells the rate of synthesis of p53 begins to rise at the same time as T antigen (Carroll et aL, 1980) or slightly later (Linzer et aL, 1979) than that of T antigen. These observations might be relevant to the mechanism by which T antigen acts in these cells. For instance the possibility might be considered that T antigen-p53 complexes could act as intermediate in virus-induced cellular DNA replication (Greenspan and Carroll, 1981; Rigby and Lane, 1983). However, 3T3 cells are fibroblasts that have been immortalized by passage through crisis in culture and they are different in several respects from primary cultured mouse cells (Land et ak, 1983). This led us to study the sequence of events in confluent primary baby mouse kidney (BMK) cell cultures after infection with SV40, reasoning that

ET AL.

these cells most closely resemble normal cells. In addition, confluent BMK cell cultures can be maintained and infected in chemically defined medium without added serum or serum-derived growth factors (Weil, 1978; Weil et ak, 1974). A large body of evidence indicates that during abortive infection of Go/Gl-arrested primary BMK cell cultures with SV40, expression of the viral T antigen is followed by a mitotic host response including the stimulation of host macromolecular synthesis and induction into the cell cycle of Go/Gl-arrested mouse cells (Hiscott and Defendi, 1981; Matter et aL, 1983). During this infection no virus replication is detectable. In this paper we performed an extensive study of the sequential events taking place after infection of contact inhibited BMK cells with SV40. This study comprised a detailed kinetic analysis of transcription, synthesis, and accumulation of ~53, in conjunction with the time course of T antigen synthesis and SV40-induced cellular DNA replication. We show that an activation of ~53 expression occurred in this system only well after the onset of virus-induced cellular DNA replication, suggesting that, at least in certain cases, T antigen’s mitogenic potential of SV40 T antigen is not necessarily dependent on its interaction with ~53. In addition, we report that the activation of ~53 expression is controlled at a level of regulation other than transcription. MATERIALS

AND

METHODS

Cells and Virus Primary BMK cell cultures prepared from lo-day-old Swiss CR-l mice, were grown in large plastic Petri dishes (90 mm diameter) or, where indicated, in 30-mm plastic Petri dishes. They were used for infection 4 days after seeding when they had reached confluence, as described by May et c&L,(1971). The BMK cells were infected with SV40 at a m.0.i. of approximately 15 PFU/cell (wild-type SV40, large-plaque SVLP strain) (Lange et aL, 1981). The beginning of the adsorption period was considered as time zero of the infection.

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SV40 infection of BMK cell cultures per Petri dish, or for 1.5 hr with 150 or 400 caused a slight increase in the amounts of &i of r5S]methionine (NEN) per Petri total DNA and protein in the cultures. De- dish. termination of the DNA and protein levels by the methods of Burton (1956) and Lowry Extraction of Proteins, Immunoprecipitaet aZ., (1951), respectively, showed that tion, and Gel Electrophoresis during the course of SV40 infection, these Proteins were extracted from cells as increases remained below 20% for DNA and 30% for protein, relative to the cor- described by Kress et al. (1979), in the responding values of parallel mock-in- presence of 1 mM each of diisopropyl fluofluofected cultures. This range of variation was rophosphate, phenylmethylsulfonyl not considered to be large enough to affect ride and L-1-tosylamide-2-phenylethyl chloromethyl ketone (Sigma) as prothe interpretation of our experimental tease inhibitors. Immunoprecipitation and data. SDS-polyacrylamide gel electrophoresis were performed as previously described Antisera and Mono&ma1 Antibodies (May et al, 1984) unless specified otherwise. The anti-SV40 tumor serum ((YTu) used Eight percent polyacrylamide gels were was a pool of sera obtained from tumor- used. In some experiments, cell extracts bearing Syrian hamsters which had been were treated in an identical manner except inoculated with SV40-transformed TSV5 that they were first subjected to a clearing clone 2 hamster cells (Kress et al., 1979). precipitation with normal mouse serum, The following hybridoma cell lines were followed by affinity chromatography on used: (i) PAb 414 and PAb 419 (Harlow et protein A-sepharose (Pharmacia). This was used as a aZ., 1981a) both secreting monoclonal an- preimmunoprecipitation tibodies against SV40 T antigen; (ii) PAb preliminary step to reduce nonspecific pre421 (Harlow et ak, 1981a); PAb 122 (Gurney cipitation. In some experiments the imet al., 1980); RA3-2C2 (Coff man and Weiss- mune complexes were purified by precipiman, 1981); PAb 246 and PAb 248 (Yewdell, tation on Formalin-fixed Staphylococcus Gannon, and Lane, in preparation) each aureus (Calbiochem) (Morrison et ak, 1983) secreting monoclonal antibodies against instead of being purified by chromatogramouse ~53. The cell culture media of these phy on staphylococcal protein A-sepharose. The radioactive bands of the SDS-polyhybridomas were used as the corresponding monoclonal antibodies. It should be acrylamide gels were visualized by autonoted that the epitopes recognized by PAb radiography. The gels were exposed to Fuji 246, PAb 248, and PAb 421 are clearly dis- X-ray film at -70” for 1 or 2 days, utilizing a DuPont Cronex hi-plus intensifying tinct (Wade-Evans and Jenkins, 1985). Control sera were either a pool of sera screen. In some [35S]methionine labeling obtained from normal adult Syrian ham- experiments, the bands corresponding to T sters or from normal adult BALB/c mice. antigen and p53 were localized and cut out of the gel. Each excised gel band was completely dissolved in 0.2 ml of Soluene 350 Labeling of Cells (Packard) overnight at 60”, and the [3HJZ’hymidine. The rate of rH]dThd in- amounts of 35Swere then determined by corporation was determined by labeling liquid scintillation counting, using Picocultures in 30-mm plastic Petri dishes for Fluor 15 (Packard) as scintillation solution. 2 hr at the times indicated under Results, as previously described (May et al, 1971). Electrophwetic Protein Blotting (Western [32P]Orthophosphate wr [35S]methionine. Procedure) The cultures were incubated for 1 hr, at 37”, in phosphate or methionine-free DulThe transfer procedure was adapted becco’s medium, and then labeled either for from Towbin et aC (1979), Burnette (1981), 1 hr with 500 &i of 32P04(C.E.A., France) and M. Kress (personal communication).

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Proteins were subjected to electrophoretic transfer from SDS-polyacrylamide gels to a BA83 nitrocellulose sheet (Schleicher & Schtill) in Towbin’s blotting buffer at 6 V/ cm for 16 hr at 4”. The sheet was then soaked for 2 hr at 37” in Tris-saline, pH 7.5, containing 2% bovine serum albumin (BSA, fraction V, Sigma) and 0.1% NP-40 (Nonidet P-40, Shell Chemical Co.). It was further incubated with 2% aTu and 16-20% of PAb 421 culture medium in the same buffer for 20 hr at 4” on a rocking platform. After five washes of 10 min each in the same cold buffer without BSA, the sheet was incubated for 2 hr at 37” in Tris-saline-NP-40, pH 8.2, with 20% fetal calf serum containing about lo5 cpm/ml of 1251Protein A (NEN). The sheet was then rinsed as described above but at pH 8.0. The blot was exposed to Fuji film at -70” for 5 days, utilizing a DuPont Cronex hiplus intensifying screen. Hybridization

ET AL.

for T antigen. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis. The bands of T antigen and ~53 protein were excised and the 35Scontent of each slice was determined by liquid scintillation counting. In parallel cultures, cellular DNA synthesis was studied by following the incorporation of [3H]dThd (Fig. 1). Synthesis of SV40 T antigen, as judged by radioimmunoprecipitation performed with anti-T antigen monoclonal antibody PAb 419, began approximately 6 hr postinfection, then rose strikingly, and reached a maximum at approximately 20 hr p.i. fol-

Probes

A partial cDNA copy of mouse p53 cloned in a plasmid vector PAT 153 was used. This recombinant (designated p53-clone 9) (Benchimol et al, 1984) contains a ~53 cDNA insert of 560 base pairs. It was nicktranslated (sp act lo8 cpm/pg) to be used as a probe. RESULTS

Temporal Relwtionship between T Antigen Synthesis, SV40-Induced Replication of Cellular DNA, Synthesis and Accumulation of p53 following Infection of BMK Cells with SV40 Time-course studies were performed with confluent primary BMK cell cultures. They consisted mainly of epithelioid cells arrested in phase Go. The cultures were infected at 37”, with a multiplicity of infection of 15 PFU/cell24 hr after they had reached confluence (4 days after seeding). The rates of ~53 and T antigen synthesis were determined by labeling the cells with [35S]methionine at the indicated times for 1.5 hr. Soluble cell extracts were prepared and immunoprecipitated using the monoclonal antibody PAb 419 which is specific

0

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FIG. 1. Temporal relationship between synthesis of T antigen, SV40-induced replication of cellular DNA, and appearance of T antigen-associated p53 protein during infection of BMK cells with SV40. Confluent BMK cell cultures (in go-mm-diameter Petri dishes) were infected with SV40 (15 PFU/cell). At the indicated times p.i., the cultures were labeled with 75 &i/ ml of [%l]methionine (2 ml per dish) for 1.5 hr. After being labeled the proteins were extracted from the cells (one dish for each time point) and preimmunoprecipitated with normal mouse serum to lower nonspecific background, and then immunoprecipitated with the monoclonal antibody PAb 419, specific for SV40 T antigen. The immunoprecipitated proteins were heated at 100’ for 5 min, then resolved by 8% SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The gel bands corresponding to T antigen and the p53 protein, respectively, were excised from the gel and the %I contents were determined in a scintillation counter. The amounts of radioactive % recovered from T antigen and p53 protein bands were plotted as a function of time postinfection. In the same diagram, we also plotted as a function of time, the rate of rH]dThd incorporation determined, in a parallel experiment, by labeling cultures (in 30mm-diameter Petri dishes) for 2 hr.

SV40-INDUCED

S PHASE

lowed by a moderate decline (Fig. 1). T antigen-induced cellular DNA replication, measured by [3H]dThd incorporation experiments, began to rise at about the same time or slightly later than T antigen (see May et aL, 1971). We verified that this increase in [3H]dThd incorporation corresponded to the onset of cellular DNA replication, by the same tests as those already used in our previous work (May et ak, 1971). As determined by autoradiography after [3H]dThd labeling, the number of cells synthesizing DNA began to increase above the amount (2-4s) of mock-infected control cultures at about 8-10 hr p.i.; it reached a peak around 18-20 hr p.i. when about 35% of the cells were engaged in DNA synthesis. At all times tested, the rate at which [3H]dThd was incorporated was roughly proportional to the number of cells participating in SV40-induced cellular DNA synthesis. A burst of mitosis was observed around 24-30 hr p.i. (data not shown). The [3H]dThd incorporation reached a first peak at about 18-20 hr, which was followed by a sharp decline and a later more diffuse peak at approximately 44-48 hr p.i. The second peak of [3H]dThd incorporation probably corresponds to a second round of DNA synthesis. Synthesis of the p53 protein coimmunoprecipitated with T antigen began approximately at 18-24 hr p.i. and sharply reached a maximum around 48 hr p.i., followed by a slow decline. Similarly, when the time course of p53 synthesis was studied by immunoprecipitation of cell extracts with anti-p53 monoclonal antibody PAb 421 or PAb 248, followed by SDS-polyacrylamide gel electrophoresis and autoradiography (Fig. 2), synthesis of p53 became detectable at 18 hr p.i. and reached a maximum between 48 and 72 hr p.i. The variation of the times at which the intensity of the p53 band was maximum might be accounted for by the severe loss in synchrony after the first mitogenic host response in SV40-infected BMK cell cultures (May et aL, 1971; Weil, 1978). A faint band of T antigen was detectable between 24 and 72 hr p.i. (visible in original PAb 248 autoradiogram). We interpret this latter observation to mean that newly synthesized ?S-labeled T an-

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tigen is poorly active in binding to the p53 protein (Carroll and Gurney, 1982) and/or that it was diluted with a large amount of cold T antigen. Similar results were obtained when the monoclonal antibodies PAb 246, PAb 122, or RA3-2C2 were used instead of PAb 421 or PAb 248 (data not shown). We also carried out these experiments under essentially the same conditions except that the radioactive labeling of the cells was performed with 32P04instead of [35S]methionine. Either PAb 421 or aTu was used to immunoprecipitate the extracts. The autoradiograms obtained are presented in Fig. 3. As judged by the relative intensity of the bands in the autoradiograms, radiolabeling the cells with 32P04yields similar kinetics of appearance of T antigen and p53 as compared to those obtained using [35S]methionine as isotopic label (Fig. 1 and 2), although the experiment in which the cell extracts were reacted with aTu revealed that this antiserum is poorly reactive with newly synthesized 32P-labeled mouse p53 (Fig. 3). To analyze the steady-state levels of T antigen and of p53 at various times after infection, the proteins were extracted from the cells and immunoprecipitated either by anti-SV40 tumor serum or by monoclonal antibodies specific for T antigen (PAb 414 or PAb 419) or for p53 (PAb 421) and then electrophoresed in SDS-polyacrylamide gels. The fractionated proteins were transferred to nitrocellulose sheets and then identified by immunodetection (Fig. 4). It is worth pointing out that the intensities of the T antigen bands in Fig. 3, panel PAb 421, are a reflection of the phosphorylation rate of the subclass of T antigen that coimmunoprecipitates with ~53, whereas the intensities of T antigen bands in Fig. 4, panel PAb 421, are a reflection of the accumulation of the same subclass of T antigen molecules. Considering that this subclass of T antigen molecules is highly phosphorylated (Fanning et aL, 1981; Greenspan and Carroll, 1981; McCormick and Harlow, 1980), it is not surprising that the incorporation of 32P into coimmunoprecipitable T antigen (Fig. 3, panel PAb 421) might be detected prior to the accu-

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ET AL. Time

Time

-0

12 ---

p.i.,

p.i., hrs.

hrs.

18 -24 --48 -72 --96

-T

1-““--dNPNPNPNPNPNPN

P

NPNPNPNPNPNPNPNP PA b 240

PAb421

FIG. 2. P53 protein synthesis during infection of BMK cells with SV40. Confluent BMK cell cultures (in SO-mm-Petri dishes) were infected with SV40 and labeled with 200 &i/ml [3SS]methionine (2 ml/culture) for 1.5 hr at various times pi. Cell extracts (one dish per time point) were immunoprecipitated with the anti-p53 monoclonal antibody PAb 421 (left panel) or PAb 248 (right panel) as described in the text. In these experiments, the immune complexes were purified by Formalin-fixed StuphyZococcus uureua The immunoprecipitates were resolved in an 8% SDS-polyacrylamide gel and detected by autoradiography. The gel was exposed to Fuji X-ray film for 48 hr at -70’. The bands corresponding to large T antigen and cellular p53 protein are indicated. Lanes N: normal BALB/c mouse serum. Lanes P: PAb 421 or PAb 248 monoclonal antibodies. The PAb 421 and PAb 248 immunoprecipitations correspond to independent experiments.

0

Time p.i., hrs. 6 18 24 48 12 96

o

-

Time p.i., hrs. 6 12 18 24 48 12 96

-II

-P53

- -P53

em OCTU

PAL 421

FIG. 3. Time course at 37” of the appearance of phosphorylated T antigen and ~53. Confluent BMK cell cultures were infected with SV40 (15 PFU/cell). At the times indicated, the cultures were labeled with 250 rCi/ml (2 ml per dish) of 90, for 1 hr. After being labeled, the proteins were extracted from the cells (one dish per time point) and immunoprecipitated with (i) anti-SV40 tumor serum (aTu), or (ii) the anti-p53 monoclonal antibody PAb 421. The immunoprecipitated proteins were heated at 100” for 5 min, then resolved by 8% SDS-polyaerylamide gel electrophoresis and visualized by autoradiography. The gels were exposed to Fuji X-ray film at -70” for 2 days utilizing a Dupont Cronex hi-plus intensifying screen.

SV40-INDUCED

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mulation of this subclass of T antigen, as blot” hybridization (Fig. 5) and Northern revealed by immunoblot (Fig. 4, panel blot analysis (Fig. 6) at different times p.i. Serial dilutions of polyadenylated cytoPAb 421). An important point emerging from the plasmic RNA extracted from mock-inexamination of these time courses is that fected or SV40-infected BMK cell cultures both the stimulation of ~53 synthesis and were spotted on nitrocellulose filter paper the accumulation of p53 took place rela- and hybridized with nick-translated cloned ~53 cDNA. As illustrated in Fig. 5, serial tively late during the abortive infection, occurring well after the onset of T antigen- dilution of each individual BMK mRNA induced cellular DNA replication (Fig. 1). tested produced a progressively decreasing autoradiographic response. Dot blot analysis of mRNA from mock-infected cells and Transcription of the Gene Coding for ~53 in from infected cells harvested at 18 and 28 Mock-Infected and SV&I~ected BMK hr gave a similar response, whereas a relCells atively slight decrease in p53 mRNA level To investigate whether the accumulation was observed at 48 and ‘72hr p.i. These obof ~53, starting about 18-24 hr p.i., was servations allow us to demonstrate unambiguously that there is no virus-induced mediated via a regulation at the transcriptional level, we compared the amount of increase in the levels of ~53. This conclup53 transcripts in SV40-infected BMK cells sion is confirmed by Northern blot hybridand in mock-infected controls both by “dot ization analysis of ~53 mRNA levels in Time pi., hrs. 0 6 16 24 46 72 96

il

-I-

Time

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0 6 16 24 40

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r~-+m

-T

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-P53

-P53 .

OCTU

PAb 414 0

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-P53 -P53

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FIG. 4. Western blot analysis of the time course of T-antigen and ~53 protein levels after SV40 infection of BMK cells. Confluent BMK cell cultures were infected with SV40 (15 PFU/cell). At the times indicated, they were harvested and the cell extracts (two dishes per time point) were immunoprecipitated with (i) antiSV40 tumor serum (aTu), (ii) anti-T antigen monoclonal antibodies PAb 414 or PAb 419, or (iii) anti-p53 monoclonal antibody PAb 421. The immunoprecipitates were heated at 100’ for 5 min, applied to 8% SDS-polyacrylamide gels, and transferred to BA 83 nitrocellulose sheets. The immobilized antigens were visualized by incubation with a mixture of aTu and PAb 421 antibodies and subsequent binding of ‘“I-labeled Stoph$ococncs aureus protein A, followed by autoradiography. Exposure time of this autoradiogram was 5 days.

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ET AL.

RNA

Mock

Poly AB

18h. p.i.

dlka11+

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C

1)@

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d

D

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0.5

p53 cDNA

0.25

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clone

'"

I) 48h.p.i. 25

E

RNA

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FIG. 5. Dot-blot analysis of p53 mRNA in mock-infected and SV40-infected BMK cell cultures. Polyadenylated cytoplasmic RNA from mock-infected or SV40-infected BMK cells was prepared as described previously (Lange et al, 1981), treated with glyoxal at 50” for 1 hr. The RNA was then serially diluted and applied in 4 ~1, in duplicate, to nitrocellulose paper pretreated as described by Thomas (1983). The nitrocellulose dot blots were hybridized with a nick-translated cloned p53 cDNA. An autoradiogram of the blot after hybridization (20 hr) and washing is shown (A: mock-infected cells; B, C, D, E: infected cells; the time p.i. at which the RNA was extracted is indicated on the left). Hybridization and the washing of nitrocellulose membranes were performed as described by Garapin et aL (1978). In parallel, nonpolyadenylated cytoplasmic RNA from infected cells (28 hr pi.) (F) or p53 cDNA (G) were dotted onto the same filter. The quantities of RNA (or DNA) placed on each spot are indicated. Autoradiography was performed by exposing the nitrocellulose sheet to Kodak Royal X-Omat film at -70’ for 3 days with a DuPont Cronex hi-plus intensifying screen.

mock-infected and SV40-infected BMK cells at different times after infection (Fig. 6). The autoradiogram of this Northern blot shows the presence of a single band corresponding to a sedimentation value of approximately 18 S which was the expected value (Reich and Levine, 1984). The intensity of this band did not show noticeable variation at all time points examined. DISCUSSION

SV40 T antigen is a multifunctional protein involved in a large number of the biological effects of this virus (for reviews, see Martin, 1981; Rigby and Lane, 1983; Tooze,

1980; Weil, 1978). In particular, the earliest detectable effects occurring after SV40 infection in both permissive and nonpermissive cells comprise a stimulation of overall cellular RNA and protein synthesis, and the induction of one or several rounds of cellular DNA replication, even in quiescent cells (Hiscott and Defendi, 1981; Matter et CL&1983). Although there is some indirect evidence suggesting that these effects might involve a direct action of T antigen upon cellular DNA sequences, the possibility must also be considered that the interactions responsible for the induction of cellular DNA replication may require cellular proteins (Rigby and Lane, 1983). In

SV40-INDUCED

cSmall -Large

S PHASE

T T

P53--)

II

II,

12345 FIG. 6. Northern blot of p53 mRNA in mock-infected and SV40-infected BMK cell cultures. Polyadenylated cytoplasmic RNA from mock-infected or SV40-infected BMK cells was prepared as previously described (Lange et a& 1981). Poly(A)-containing RNA (5 pg) was dissolved in 3 ~1 of Hz0 plus ‘7 ~1 of sample preparation buffer. The sample preparation buffer was prepared by mixing 10 vol of formamide with 5 X gel buffer. The gel buffer was 20 mM triethanolamine, pH 7.4,2.5 mM EDTA, and 2.2 M formaldehyde. The sample was then incubated at 55” for 15 min. The RNA sample was loaded onto a 0.7% agarose gel and electrophoresed at 3 V/cm for 15 hr. The transfer of RNA from agarose gels to nitrocellulose sheets, the hybridization of nick-translated cloned p53 cDNA (10s cpm/pg) to the RNA that was immobilized on the membrane filters, and the washing of the filters were performed as described by Thomas (1983). Autoradiography was performed by exposing the nitrocellulose sheet to Kodak Royal X-Omat film at -70” for 7 days with a Dupont Cronex hi-plus intensifying screen. The RNA was extracted at the following times postinfection for runs 1-5, respectively: 0,18,28,48, and 72 hr. After removal of the probe from the blot and rehybridization with nick-translated SV40 DNA, it was possible to position the bands of small T- and large T-antigen mRNAs, used as markers (indicated on the right-hand side of the gel). May et al (1980) evaluated the sedimentation values of small T- and large T-antigen mRNA to 21 and 19 S, respectively.

this respect, it has been proposed that the induction of cellular DNA synthesis might be caused by the increase in p53 stability and consequently in ~53 levels, which results from the formation of p53-T antigen complex in SV40-infected cells (Oren et al., 1981). The experiments we present here suggest indications with regard to this latter question. In fact we show that, following SV40

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infection of Go-arrested BMK cells, a wellordered sequence of events occurs which may be represented as (1) T antigen synthesis and onset of the first virus-induced S phase and (2) stimulation of ~53 synthesis and accumulation of ~53. At the time (about 6 hr p.i.) that cellular DNA replication commenced no detectable levels of p53 protein were observed either by radioimmunoprecipitation or by Western blot experiments. The onset of p53 synthesis started later, at approximately 18-24 hr p.i., as shown by radioimmunoprecipitation performed after a relatively short pulse-labeling with [?S]methionine (or with 32P04). The accumulation of p53 started about the same time as judged by Western blot experiments. Comparing our results with the kinetic data reported on SV40-infected BALB/c 3T3 mouse cells (Carroll et al., 1980; Linzer et ah, 1979) (see our Introduction), it is noteworthy that the duration of the period between the first appearance of T antigen and the onset of ~53 expression in SV40infected cultured mouse cells varies widely with the type of mouse cells and/or with the physiological state of the cells. Based on our present results, the SV40induced activation of p53 synthesis in BMK cells appears to occur too late to be involved in the first virus-induced round of cellular DNA replication. It seems unlikely that a very low, undetectable increase of ~53, or the production of a p53 species not reactive with PAb 421, PAb 246, or PAb 248 is responsible for the induction of cellular DNA replication, although these possibilities cannot be completely excluded. We should recall that the latter monoclonal antibodies recognize three distinct antigenic sites on p53 (Wade-Evans and Jenkins, 1985). However, we are not able to assesswhether p53 is involved in the control of the second and subsequent rounds of cellular DNA replication. We interpret our results to mean that T antigen in a form not bound to ~53, is able to trigger at least the first round of cellular DNA replication through a p53-independent pathway in infected BMK cells. This is in agreement with the observation of Mercer et al. (1982), who show that mi-

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croinjection of p53 antibody (ap53) into the nuclei of SV40-infected quiescent Swiss 3T3 mouse cells has no effect on SV40-induced DNA synthesis, while microinjection of ap53 into the nuclei of 3T3 cells at the time of serum stimulation prevents the subsequent entry of cells into the S phase of the cell cycle. The late step of p53 synthesis and accumulation is apparently regulated at levels other than transcription (Figs. 5 and 6). This event could result, at least partially, from the stabilization of p53 through formation of a complex with T antigen. If this is the case, we should consider the possibility that post-translational modifications, occurring preferentially late after infection, might increase the ability of T antigen and/or p53 to associate with each other. In conclusion, comparing (i) the SV40induced mitotic response in BMK cells with (ii) the mitogenic response of serumstarved confluent 3T3 cells to addition of serum (Reich and Levine, 1984), it is apparent that some of the biochemical changes observed are different in the two systems. In SV40-infected BMK cells the activation of p53 expression occurs late after the onset of cellular DNA replication. On the contrary, in quiescent 3T3 cells stimulated to enter the cell cycle by addition of serum, an increase in the synthesis and steady-state levels of p53 protein and p53 mRNA (Reich and Levine, 1984) occurs prior to DNA replication. Therefore, our data suggest that the pathway which links the synthesis of SV40 T antigen to the triggering of (the first round of) cellular DNA replication in infected primary cultures of baby mouse kidney cells is distinct from the sequence of events occurring prior to DNA replication in quiescent cultures of 3T3 cells stimulated to enter the cell cycle by addition of serum. It is possible that the p53-independent mitogenic activity of T antigen may contribute to the properties of certain aminoterminal fragments of SV40 T antigen that are unable to complex p53 but are nevertheless functional in focus-forming assays (Clayton et aZ., 1982; Sompayrac et aL, 1983).

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