Wild-type p53 is not a negative regulator of simian virus 40 DNA replication in infected monkey cells.

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Wild-Type p53

Is

Not a

Negative Regulator

of Simian

Virus

40 DNA

Replication

in Infected

Monkey

Cells

ANDREASVON DERWETHAND WOLFGANG DEPPERT*

Heinrich-Pette-Institut fair Experimentelle VirologieundImmunologie,

Martinistrasse52, D-2000 Hamburg 20, Germany Received25June1992/Accepted 10November 1992

Toanalyze theproposed growth-inhibitoryfunction ofwild-typep53,wecomparedsimianvirus 40(SV40)

DNA replication in primary rhesus monkey kidney (PRK) cells, which express wild-type p53, and in the established rhesus monkey kidneycell lineLLC-MK2,which expressesa mutatedp53 thatdoesnotcomplex with large T antigen. SV40 DNA replication proceeded

identically

in both cell types during the course of infection.

Endogenously

expressed

wild-type

p53thus does not

negatively

modulateSV40DNAreplicationin vivo. We suggestthat inhibition ofSV40DNAreplication by wild-type p53in invitroreplicationassaysisdue togrosslyelevated ratios ofp53tolargeTantigen,thusdepletingthereplication-competentfreelargeTantigen inthe assay mixturesbycomplex formation.Incontrast,the ratio ofp53tolargeTantigenin in vivoreplication islow, leaving the

majority

oflargeTantigenin afree, replication-competent state.

Mutations in thetumorsuppressor genep53constitute the most common genetic alterations known in human cancer (18, 21, 29). Recent evidence suggests that p53 acts as a tumor suppressor and that mutations in p53 abolish this repressor activity (27, 30). Very little is understood about

p53 functions atthe molecular level. p53 is involved in cell cycleregulation (38),but seemingly contradictoryevidence has beenprovided forboth apositive anda negative regu-latory role forp53 inthese processes (7).Whereas there is compelling evidence for the need for p53 expression for

progression of normal and even some tumor cells through

thecell cycle (8, 33, 34, 45, 56), it has been unequivocally

demonstrated thatexpressionoftransfectedwild-typep53in tumorandtransformed cells leadstocessationof cellgrowth (36). Thisgrowth-inhibitory function ofwild-type p53 now

commonly is seen as the basis of its tumor suppressor activity (20,30). Althoughthis isanattractivehypothesis, it has to be reconciled with the set of complementary data

describing agrowth-promotingfunctionforwild-typep53in normal cells. Therefore, it seems absolutely necessary to understand both the growth-promoting and

growth-inhibi-tory activities ofwild-typep53 at themolecular level. Inthisregard, analysis of cellular transformation and lytic

replicationofavariety ofDNAtumorviruses might be very helpful, since major regulatory proteins of these viruses interact withwild-type p53 both during lytic infection and

during cellular transformation (5, 29, 42, 49). One of the

best-analyzed viral systems is simian virus 40 (SV40), in which expression of the large T antigen (large T) leads to bothcomplex formation(17, 28) and metabolic stabilization of p53 (9). In vivo as well as in vitro studies suggested a regulatory function forp53in SV40 DNA replication. It has beenshown that addition of purified wild-typep53to invitro DNA replication assay mixtures inhibits SV40 DNA repli-cation (2, 13) and that transient expression of transfected wild-type p53 in SV40-transformed monkey (Cos) cells in-hibits in vivo replication of plasmids containing the SV40

origin ofreplication (ORI) (2). Furthermore, the inhibitory effect oflargeT-p53 complexes isolated from infected

mon-*Correspondingauthor.

key cells has beendirectly demonstratedin invitro

replica-tion assays(39).Thisinhibitory functionof

p53

isdueatleast in partto aninhibition of the DNA helicaseactivityoflarge

T (25, 58, 64). On the basis of these data, it has been postulated that wild-type p53playsanegativeregulatoryrole in SV40 DNAreplicationinvivo,possibly by

down-regulat-ing SV40 DNAreplicationduringlatetimes of thelytic cycle (15,25, 58,64). Thispostulated negative regulatoryeffect of

p53inSV40 DNAreplicationoftenisconsideredamodel for theproposedantiproliferativeeffect ofwild-typep53,asit is consistent with the finding that mutant p53 has lost its inhibitory effect on SV40 DNA replication inboth in vitro andin vivo assays(13, 39),inanalogytothepostulatedloss of a growth-inhibitory function of mutant p53 in cellular

proliferation (35, 37). On the other hand, the postulated

inhibitory role ofendogenously expressedwild-typep53has notyet beendemonstrated directly, i.e.,inlyticallyinfected cells, because of the lack ofa suitable cellulartestsystem. Suchasystemcould beprovided bymonkeycellsexpressing

amutant

p53,

asSV40 DNAreplicationinsuch cells should be devoid of any negative regulation byp53. That is, such cells shouldproduceenhanced levels ofSV40 DNA,asSV40 DNA replication should be released from the proposed

down-regulation byendogenouslyexpressed wild-typep53.

SV40replication usuallyis studied withkidneycells of the African green monkey (Cercopithecus aethiops), although its natural hostis the rhesus monkey(Macacamulatta) (61). The major reason for using such cells (e.g., TC7 or CV1

cells)is thatSV40 in rhesus monkey cells reportedly estab-lishes a persistent infection (41), whereas infections of African green monkey cells are lytic. Analyzing this phe-nomenon, we recently found that primary rhesus kidney (PRK) cells derived from a SV40-free monkey colony could belyticallyinfectedwith SV40 (63). As reported previously, cells of the established rhesus monkey kidney cell line LLC-MK2,on the other hand, were persistently infected by SV40 (41, 63). We found that a major cellular difference between PRK and LLC-MK2 is that LLC-MK2 cells

ex-pressed a mutant p53, whereas PRK cells, by definition, expressed a wild-type

p53.

This system of homologous monkey cellsdiffering in the phenotypes of theirp53proteins wastherefore usedtodirectly compare the effects of endog-886

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enously expressed wild-type and mutant p53 proteins on SV40 DNA replication in vivo.

MATERIALSANDMETHODS

Cells and virus. TC7cells (46), LLC-MK2 cells (22), and PRKcells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). Rhesus monkey kidneys for primary cultures were kindly provided by the Deutsches Primatenzentrum, Gottingen, Germany. Primarycultureswere preparedwith 0.2% Wor-thington collagenase type I (Seromed) in DMEM supple-mented with 20% FCS. Infection was carried out by inocu-lation of monolayer cultures of TC7, LLC-MK2, and PRK cells withSV40strain776 at aninput multiplicityofinfection (MOI)of 1.

Quantitative evaluation of rates of viral DNA synthesis. At various times postinfection, cells were labeled with

[methyl-3H]thymidine(Amersham) (30

puCi/ml/106

cells) for 1 h. Viral DNA wasthen extracted by the method of Hirt (19). DNA in thesupernatant of Hirt extracts was precipitated with etha-nol and suspended in TE buffer (10 mM Tris hydrochloride, 1 mM EDTA [pH 7.4]). [3H]thymidine incorporation into

newly synthesized viral DNA was determined by liquid scintillation counting of aliquots of DNA samples after

trichloroacetic acidprecipitation. Ratesof viral DNA repli-cationwereexpressed ascounts perminute per microgram of viral DNA per hour oflabeling.

Quantitative evaluation of amounts of total viral DNA. Amounts of total viral DNA were quantitated by dot blot

hybridization.Viral DNA fromnonlabeled infected cells was extracted by the method of Hirt (19) at various times postinfection (p.i.). Aliquots of DNA samples and defined amountsof SV40 DNAas areference were adjusted to 0.25 NNaOH(10minat2°C)and then diluted withSSC buffer to a final concentration of 0.125 N NaOH and 0.125x SSC

(0.125x SSC is18 mM NaClplus1.8 mMsodium citrate[pH 7.2]).DNAs weretransferredto aGeneScreen Plus Nylon66 membrane (New England Nuclear)with a DotBlot I Mini-fold apparatus (Schleicher & Schuell) and then hybridized

with random-primed labeled SV40 DNA.Labelingwas car-ried outby the method ofFeinbergand Vogelstein (12)as describedbyManiatisetal. (31).Toevaluate theamountof viral DNA, blots were excised and

32p

counts per minute were determined byliquidscintillation analysis.

Characterization of the replicative forms of SV40 DNA.

Cellswere

pulse-labeled

at36h

p.i.

with

[methyl-3H]thymi-dine(Amersham)(200 ,uCi/ml/10 cells)for 15 min, and viral DNAwasextractedbythemethod of Hirt(19). Aliquotsof SV40 DNAwerefractionatedon0.8% neutralagarose

gels

in

running buffer composed of 5 mM sodium acetate, 1 mM

EDTA, 0.03% sodium dodecyl sulfate

(SDS),

and 80 mM

Tris-hydrochloride (pH

7.5)

as

previously

described

(59).

Thegelswere

electrophoresed

for 48 hat a constant

voltage

of 1 V/cm. To detect steady-state levels of viral

DNA,

we stainedgelswithethidiumbromide. For

fluorography

anal-ysis, gels

were

dehydrated

with acetic acid-methanol

(3:1)

for4 to5h,incubated with2.5%2,5-diphenyloxazole

(PPO)

in aceticacid for12h,and soaked inwaterfor 3to5h. The

gelswerethendried underavacuumat50°Cand

exposed

on X-rayfilmsat -70°C.

Determination ofvirus yields. Virus

yields

were

quanti-tatedbytitration of tissue culturesupernatantsfrom infected cells harvested at various times

p.i.

for infectious units

(MOI). MOIson TC7cellswere determined

by

immunoflu-orescenceanalysisfor

expression

ofSV40

large

T.TC7cells

grown on coverslips were infected with serial dilutions of virus-containing supernatants and prepared for immunoflu-orescence 24 h p.i. as described previously (43, 45). The percentageof large T-positive cells was calculated for each dilution step. The dilution step leading to 90 to 100% large T-positive cells was defined as having an MOI of 1.

Labeling of proteins. Cells were labeled as described previously (55) before extraction with [35S]methionine (Am-ersham), with 50 ,uCi per plate per ml for pulse-labeling and 100

p,Ci

per plate per ml for pulse-chase-labeling experi-ments.

Extraction of proteins.Cells were washed three times with phosphate-buffered saline (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4 [pH 7.4]) and then extracted for 30 min at 2°C with 1 ml of lysis buffer [120 mM NaCl, 5 mM dithiothreitol, 1 mM ethylene

glycol-bis(P-aminoethyl

ether)-N,N,N',N'-tetraacetic

acid (EGTA), 1% aprotinin,

0.5%Nonidet P-40,10%glycerol, 50 mM Tris-hydrochloride

(pH 8.0)]. Extracts were cleared by centrifugation(10 min, 20,000 x g, 2°C), and proteins were immunoprecipitated from thesupernatants.

Immunoprecipitation, SDS-polyacrylamide gel electro-phoresis, and Western immunoblotting.

Immunoprecipita-tions of p53 andlarge T in 1-ml cellular extract samples were

performed for 4 h at 2°C with 100 ,ul of settled protein A-Sepharose (Pharmacia) and 500 ,ul of cell culture superna-tant of the monoclonal anti-p53 antibody-producing hybri-doma cell line PAb122 (16), PAb421 (66), or PAb240 (14). Large T wasimmunoprecipitatedwith 500

pl

of cellculture

supernatant of the anti-large T antibody-producing hybri-domacell linePAb419(52).

Immunocomplexeswerewashedextensively,andproteins

wereeluted from protein A-Sepharoseand thensubjected to

SDS-polyacrylamide gelelectrophoresis asdescribed previ-ously (9). Proteins were analyzed on 10% polyacrylamide

slabgelsby the systemof Laemmli(26).Westernblotting(3)

wasperformed with 4 ml of cell culture supernatant

contain-inganti-p53 monoclonal antibodyPAb421(66)oranti-largeT monoclonal antibody PAb1O8 (16) and [3H]protein A as describedpreviously (50).

RESULTS

Down-regulation of SV40 DNA replicationin

lytically

SV40-infected monkey cells during the courseof infection. Down-regulation of SV40 DNA replication in lytically infected

monkeycells could beanactiveprocessofviralreplication.

Alternatively, it might simply be the result of cytopathic

viral effectsonthe hostreplication machinery.To discrimi-nate between these alternatives, we analyzed the rates of SV40 DNA replication during the course of infection

([3H]thymidine incorporated per microgram of SV40 DNA perhour oflabeling) inlyticallyinfected monkeyTC7cells andrelated thisinformationtothe releaseofinfectious virus from thesecells andtothe appearance ofcytopathiceffects

(see Materials and Methods for

details). Figure

1 demon-strates that the maximal rate of SV40 DNA

replication

occurred around 36 hp.i. and that theratedeclined

sharply

from thenon.Virus release started around 36 h

p.i.

in these cells and reached its maximum about 72 h

p.i.

Cytopathic

effectswere notdetectable before 60 h

p.i.

This

analysis

thus

stronglysuggests that

down-regulation

ofSV40DNA

repli-cation isnotdueto

cytopathic

effects but reflectsanactive processduringviral

replication.

Comparisonof

p53

phenotypesin PRK and

LLC-MK2

cells.

Analysis of the

postulated

role of

wild-type p53

in

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IRATEOF VIRALDNA VIRUSRELEASE"* SYNTHESIS*

o-e __s_.

* 105cpm/pgviralDNA/1hlabeling **MOI

oI,

12 24 36 48 60 72

hp.i. FIG. 1. Course ofSV40 infection in lytically infected monkey cells. Rates of viral DNAsynthesis and virus releasewere deter-minedatthe timesindicatedasdescribed in Materials and Methods.

CPE, cytopathiceffect.

regulationofSV40DNAreplicationand the release from this down-regulation bymutant p53 invivo requires a

homolo-gouscellular system expressing authenticwild-type or

mu-tantp53 protein,respectively. Suchasystemisprovided by

PRK cells and the established rhesus monkey kidney cell lineLLC-MK2. PRKcells, by definition, expresswild-type p53,whereasp53 expressedinLLC-MK2cells ismutant,as wasdemonstrated by the following experiments.

Western blot analysis of steady-state levels of p53 in LLC-MK2cellsand ofp53in PRK cells revealed thatp53in LLC-MK2 cells accumulated to relatively high levels (Fig. 2A,lane a),whereasp53 in PRKcellswasundetectable by

thisanalysis (Fig. 2A,laneb).The enhanced level ofp53in LLC-MK2cells is duetotheenhanced metabolicstabilityof this protein compared with that of p53 in PRK cells, as

demonstrated by pulse-chase analyses of [35S]methionine-labeledp53 from both cell lines.p53 inLLC-MK2 cellswas

foundtobemetabolicallystable (Fig. 2B,panel a),whereas

A

B

a b a b

P 15 30 60 90 P 15 30 6090

FIG. 2. Steady-statelevels (A) and metabolicstability(B) of p53 inLLC-MK2 and PRK cells. (A)p53 from LLC-MK2 (lane a) and PRK (lane b) cells was immunoprecipitated with PAb122 and analyzed by polyacrylamidegelelectrophoresisand then by

West-ernblotting with PAb421and [3H]proteinAfor quantitative

evalu-ationoftheprotein. (B) The metabolicstability of p53wasanalyzed

bypulse-chase labeling. LLC-MK2 (panel a)and PRK(panel b) cells

werepulse-labeled with [35S]methionine(100,uCiperplateperml)

for30 min (lanes P) and then chased for 15 to 90 min. p53 was

immunoprecipitated with monoclonal antibody PAb122.

[image:3.612.63.300.72.264.2]

a

b

c

d

FIG. 3. Reactivity of p53 in PRK and LLC-MK2 cells with

monoclonal antibodies PAb240 and PAb421. Cells were pulse-labeled with[35S]methionine (50 ,uCi perplateperml)for 1 h and then immunoprecipitated with either the p53-specific antibody PAb421(lanea,LLC-MK2;lanec,PRK)orPAb240,which

recog-nizes only p53 inamutantconformation(lane b, LLC-MK2;laned, PRK).

p53in PRK cells(Fig. 2B,panel b)showed thefast turnover characteristic ofwild-type p53 (27, 29).Theenhanced level and metabolic stability of p53 in LLC-MK2 cells already

were indicative ofa mutant p53. The mutantphenotype of p53inLLC-MK2 cellswasfurthercorroborated by demon-strating that this p53 reacted with monoclonal antibody PAb240 (Fig. 3,laneb),which isspecificforp53inamutant conformation(14),whereasp53from PRK cells didnot react withPAb240 (Fig. 3, laned).

Formationofcomplexesofp53andlargeT isanimportant functional property ofwild-type p53 which is supposed to play an important role in regulating large T and/or p53 activities during lytic replication, especially in the down-regulationofSV40DNAreplication (2, 13, 25, 44, 58, 64).To analyze the ability of p53 proteins expressed in PRK and LLC-MK2 cells to form a complex with large T, infected cells were labeled with [35S]methionine for 1 h at 24 h p.i. andextracted,andlargeT-p53 complexeswere immunopre-cipitated with the large T-specific monoclonal antibody PAb419 (52). Figure 4A, lane b, demonstrates that p53 in infected PRK cellswas readilydetected in acomplexwith

large T, whereas p53 from LLC-MK2 cells was not in a

complexwithlargeT(Fig. 4A, lane a). Analysisof cellular extractsafterpulse-chase-labelingofinfectedcells with the p53-specific monoclonal antibody PAb122 (16) showed the characteristic time courseofentry oflarge T intocomplex with p53in PRKcells (Fig. 4B, panel b) and demonstrated that p53 in PRK cells became metabolically stabilized, as

reported for p53 in African green monkey kidney cells

infected withSV40 (11, 32).Incontrast,large T in LLC-MK2 cells didnot enteracomplex with thep53 in these cells,even

afterprolongedchaseperiods (Fig. 4B, panel a). Since p53 in these cellswas alsometabolically stable inuninfected cells

(Fig. 2B, panel a), a further increase inmetabolic stability uponSV40 infectionwasnottobe expected. Itis important

to notethatmetabolicstabilization of p53 inPRKcells after

SV40 infectionresulted in similar steady-statelevels of p53

at about48hp.i., asin LLC-MK2 cells (see Fig. 7B, panel

1).Theproperties of the p53 proteins expressed inPRKand in LLC-MK2cells aresummarized in Table 1.

Comparison of SV40 DNA replication in PRK and in LLC-MK2cells. Theexpression ofabona fide wild-typep53 in PRKcells andamutantp53 that doesnotform complexes with large T in LLC-MK2 cells, as described above, ren-12

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A

B

a

b

a

b

T-Ag 4p_

p53

T-Ag - - - o

p53 _,in14W_W_,_ 0

P

15 30 60 90

P

15 30 60 90

FIG. 4. Complexformationbyp53andlarge T (T-Ag) afterSV40 infection of PRK and LLC-MK2 cells. (A) Infected LLC-MK2 (lane a) and PRK(lane b)cells werepulse-labeled (asdescribedin the legend to Fig. 3) 24 h p.i., and large T wasimmunoprecipitated with the large T-specific monoclonal antibodyPAb419. (B) Pulse-chase-labeling (as described in the legend to Fig. 2) 24 h p.i., followed by immunopre-cipitation with the p53-specific monoclonal antibody PAb122from cellular extracts of LLC-MK2 (panel a) and PRK (panel b) cells, was performed toanalyzethe time course of entry of large T into complexes with p53 as well as the metabolic stabilization of p53.

dered these cells anideal system for the analysis ofpossible effects of endogenously expressed wild-type or mutant p53 on SV40 DNAreplication. In preceding analyses, we had established thatlarge Texpression and itsinteractions with cellular targets, especially with structural systems of the nucleus, were identical in these two types of cells (63a), thus excluding any obvious differences in viral replication.

ToanalyzeSV40 DNAreplicationin PRKand LLC-MK2

cells,thesecellswereinfected withSV40at anMOI of 1 and

labeledwith [3H]thymidine (30 ,uCi/ml) for 1 hat the times indicated in Fig. 5. SV40 DNA thenwas extracted by the method of Hirt (19) and purified. Rates of viral DNA

synthesis (counts per minute per microgram of DNA per hour oflabeling) during infection werethendetermined. In

addition,SV40 DNAwasharvested fromparallel,unlabeled culturestodetermine totalamountsof viralDNAbydot blot analysis. A graphical evaluation of these data is shown in

Fig.5. Figure5Ademonstrates identicalratesofviral DNA

synthesis in both types of cells during the course of infec-tion. As in lytically infected African green monkey cells

(compare Fig. 1and5A), DNAsynthesisstarted about 12 h

p.i.andpeakedabout 36 hp.i.inboth types of cells. Therate of DNA synthesis thendropped, regardless of whether the cells expressedawild-type (PRK)or a mutant

(LLC-MK2)

p53. It is importantto statethat the cells at36 hp.i.didnot show any sign of

cytopathic

effects, indicating that the reduction in the rate of SV40 DNA synthesis, as in TC7

cells, was not simply due to cell damage induced byviral infection but reflected a cellularly or virally controlled reaction. Analysis of the total amounts of SV40 DNA

synthesized duringthecourseof infection in these cells

(Fig.

[image:4.612.145.478.72.243.2]

SB) showed a similar increase in the accumulation of viral

TABLE 1. Propertiesofp53inPRK andLLC-MK2cells

Reactivity of Formation of

Cell line p53half-life p53with: _withcomplexes_large_T PAb421 PAb240 byp53

PRK 30 min + - +

LLC-MK2 4h + +

-DNA in these cells until about 48 h p.i. Amounts of viral DNAin PRK cells stayed constant from then on, whereas a further increase was observed in LLC-MK2 cells. This apparentincrease in theyield of SV40 DNA, however, is due tothe fact thatmostof thenewly replicated SV40 remained in LLC-MK2 cells at the times at which it was analyzed, whereas PRK cells constantlyreleased infectious virus (Fig.

5C). Takingthis intoconsideration,the total amounts of viral DNA synthesized in the two types of cells were largely

similar.

SV40 DNAreplicationstartson superhelical form I DNA molecules within the ORI. Bidirectional replication then generates intermediate Cairns structures (ICS). Elongation ofnascentDNA on ICSproceeds until replication is about

90%complete.At thispoint, replication forks pause,

gener-ating the latest Cairns structures (LCS). The remaining parental DNA strands in the LCS arecompletely unwound, andtwocircular SV40monomers(SV40 form II* DNA) are

separated; each contains a short gap in the termination region of the nascent DNA strand (51; for a review, see reference 6). To characterize the replicative forms of SV40 DNAsynthesizedin PRK andLLC-MK2cells, infected cells werelabeled for 15 min with

[3H]thymidine

at 36 hp.i. and extracted by the method of Hirt (19). Viral DNA was fractionated by neutral agarose gel electrophoresis (60)and visualizedbyethidiumbromidestainingandfluorographyas described in Materials and Methods. By

applying

these

conditions,themobilities of the various forms of SV40DNA in lyticallyinfected TC7 cells have been previously charac-terized in detail(51).The resultsareshown in Fig. 6. Inthe ethidium bromide-stainedgelsofSV40DNAfromboth cells

(lanes A), representing steady-state levels of SV40 DNA

intermediates,

the most

prominent

bandwas form I DNA

(superhelical). In addition, minor bands of form II + II* DNA (relaxed circles), and catenated dimers were visible.

Fluorographic analysis of

replicating

forms of SV40 DNA

(Fig. 6B) demonstrates that

pulse-labeling

of viral DNA resulted inradiolabelingof formIDNA,form II +II*

DNA,

and theLCS,whileaconsiderableamountof theradiolabel was present in the ICS. ICS migrate as a smear between form I and the LCS (59, 60,

65),

representing

a random

population of nascent SV40 DNA at various stages of

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A ?3

-G

CZ

z 0

B

co

8

0

12

-e-- ^

10

24 4X 72 h

FIG. 5. Comparison ofSV40DNA replication in PRK and

LLC-MK2 cells. (A) Rates of viral DNAsynthesis per microgram of viral

DNA wereanalyzed by measurement of 3H incorporation into viral

DNAafterpulse-labeling with 30,uCiof [3H]thymidine for 1 h at the

indicated time points (see Materials and Methods). (B) Total

amounts of viral DNA were evaluated by dot blot hybridization with

32P-labeled SV4o DNA atvarioustimes afterinfection(seeMaterials

andMethods). (C) Virus release was measured by quantitation of

theamountsofinfectious virusreleasedincellculturesupernatants

harvestedfrominfected cells at thetimepointsindicated. TheMOI

ofthe supernatants was determined as descrinbedinMaterials and

Methods.

continued replication. No difference between replicative intermediates ofSV40 DNA from LLC-MK2 cells (lane a) andthose fromPRKcells(lane b) could be detected, further supporting our conclusion that SV40 DNA replication in these two types ofcells did not show any qualitative and

quantitativedifferencesandproceeded via thesame

replica-tiveintermediates.

Large T/p53 ratios in SV40-infected cells. In SV40 DNA replication, the large T intrinsic DNA-dependent helicase activity is required forboth initiation and elongation func-tions (54). This activity is inhibited by p53 complexing to

largeT (13, 25, 58, 64).

p53,

inaddition, competes for the

A

B

,-,

JA-.a.

I.;

- LCS

-CD

-11 + 11* ICS

a b a b

FIG. 6. Comparison of replicative forms of SV40 DNA repli-catedin LLC-MK2 and PRKcells. LLC-MK2(lanes a)and PRK (lanesb) cellswerepulse labeled 36hp.i.with

[3H]thymidine

(200

p.Ci/106

cells) for 15 min, and viralDNAwasextracted.Aliquotsof viralDNA werefractionatedby neutralagarosegelelectrophoresis asdescribedinMaterialsand Methods. (A)Thegelswerestained with ethidiumbromide for detection ofsteady-state levels of viral DNA. (B) Fluorographic analysis revealed the different forms of pulse-labeled newly replicated SV40DNA.Replicativeforms(ICS, LCS, catenated dimers

[CD],

and II + II*) and terminatedform I SV40DNAareindicated (seetext).

association ofDNApolymerasewithlargeT(15). Thus,it is evident thatlargeT-dependent SV40 DNAreplication

pre-dominantly requires the activity oflarge T not

complexed

withp53 (freelargeT).Ininvivo SV40 DNAreplication,the presenceofsufficientamountsoffreelargeTis ensuredby

twoindependentmechanisms.Firstly,theamountoflargeT present in infected cells at all times of infection greatly exceedstheamountofp53

(e.g.,

Fig. 4A,lane

b);

secondly, preferentiallyold andnotnewly synthesized largeT associ-ateswithp53(62) (Fig. 4B, panel b). SV40-infectedcells thus containalargeexcessof freelargeToverlarge Tcomplexed

withp53 even at48 h p.i., i.e., at atime when SV40 DNA

replicationisalready down-regulated.This isdocumentedin

Fig. 7.

Large

T-p53

complexesfrominfectedPRKandLLC-MK2 cells at 48 h p.i. were separated from free large T by

sequential immunoprecipitation,

firstwith

p53-specific

mon-oclonalantibodyPAb421(Fig.7, panels1)and then withthe large T-specific monoclonal antibody PAb419 (panels 2). Aftera2-hpulse-label of infected cells, immunoprecipitation forp53demonstrated that aconsiderable amount of large T wascoprecipitatedwith

p53

from PRK cells (Fig. 7A, panel 1, lane b), whereas no large T in complex with p53 was

immunoprecipitatedfromLLC-MK2cells (Fig. 7A, panel 1, lanea). The amountsof p53 appeared to be similar in both cell typesatthat timep.i.Immunoprecipitation of free large T from clearedextracts (Fig. 7A, panel2), however, dem-onstrated thatmuch largeramountsof freelargeTthan large Tin complex with

p53

were found in PRK cells (Fig. 7A, panel 1, lane b).

Western blot analysis ofsteady-state levels of p53,

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2

A

-0m

T

p53

a b a b

B

T

p53

[image:6.612.119.248.77.247.2]

a b a b

FIG. 7. Comparison of large T/p53ratios in infected LLC-MK2 (lanes a) and PRK (lanes b)cells. Forty-eight hours p.i., large T-p53 complexeswereseparated from freelarge T by sequential immuno-precipitation, first with anti-p53monoclonal antibody PAb421

(pan-els 1) and then with anti-large T monoclonal antibody PAb419 (panels 2). Proteins were sequentially immunoprecipitated from extracts of either 2-h pulse-labeled ([35S]methionine, 50 pCi per

plateperml) cells (A)orunlabeled cells (B).Theproteins in panelB weresubjectedtoWestern blot analysis.

tained by sequential immunoprecipitations of infected unla-beledPRKandLLC-MK2 cells (Fig. 7B, panel1), revealed thatas aresult ofmetabolic stabilizationof p53 in PRK cells,

equal levels of p53werepresentinLLC-MK2 (Fig. 7B, panel 1,lanea) and PRK(Fig.7B,panel 1, lane b) cells. However,

inbothcelltypes,thelevelsof free large T (Fig. 7, panels 2)

exceededby far the levels of total p53 (Fig. 7, panels 1)or

thelevel ofcomplexed large Tin PRK cells (Fig. 7A, panel 1,laneb). Freelarge T in LLC-MK2 cells(Fig. 7B, panel 2, lane a) reflects the total amount of large T in these cells, whereas freelargeT from PRK cells(Fig. 7B, panel 2, lane b)reflectstotal large T minus largeTcoprecipitatedwith p53 inthe first step of the sequential immunoprecipitation. The fact thatapproximately equal levels of free largeT(Fig. 7B, panel 2) were present in both cell types clearly documents that the proportion of the large T molecules blocked for replication functions by complexformationwith p53 makes

uponlyasmallfractionof the large Tmolecules availablein

infectedcells invivo. Infact,the amountof free large T in infectedPRKcells(Fig. 7, panels 2, lanes b)wasverysimilar

totheamountoffree(total) largeTinLLC-MK2 cells (Fig. 7,panels 2, lanesa).

DISCUSSION

Inthisstudy, weasked whether viral DNAreplication in

SV40-infected monkey cells would be released from the postulated negative regulatoryeffect of wild-typep53incells endogenously expressing mutated p53, i.e., whether such cells would overreplicate SV40DNA. Suchoverreplication ought to be expected if down-regulation of SV40 DNA replication in the late phase of viral infection indeed is mediatedby wild-type p53.Thisquestion isimportant inso-far as p53-mediated inhibition of SV40 DNA replication

constitutes theonlymodelsystem foranegative regulatory

role of p53 in a proliferation-associated function (DNA

replication)inwhichit ispossibletodelineate theunderlying

molecular events. Analysis of this question required a ho-mologous system of cells expressing either endogenous wild-type or mutant p53. Such a system was provided by PRK and LLC-MK2 cells. Whereas p53 in PRK cells by definition is wild type, our analysis of p53 in LLC-MK2 cells, a permanent cell line established from PRK cells (22), clearly showed that this p53 was mutant; the most prominent feature of p53 in this cell line, as far as this study was concerned,

was that thisp53failed to complex with large T in vivo. On the basis of the available data, one can assume that this mutant p53 is unable to inhibit SV40 DNA replication in vitro. Although only a limited number ofp53 mutants have been tested for their inhibitory activity on SV40 DNA replication in vitro (13, 25, 57, 58, 64), all but one of them were

inhibitory.

The only exception described so far is an oligomerization-defective mutant p53 recently constructed by Sturzbecher et al. (57). Such a mutant p53 has not yet been found among any p53 mutants arising in vivo. Given this caveat, comparison of SV40 DNA replication in LLC-MK2 cells with that in wild-type p53-expressing PRK cells should have allowed us to detect differences due to the different p53 phenotypes. However, no such differences could be detected. In particular, the comparison of the rates ofviral DNA synthesis during the course of infection dem-onstrated that these rates were similar in both types of cells throughout infection, i.e., that down-regulation of SV40 DNA replication occurred in both types of cells. As a consequence, similar steady-state levels of SV40 DNA were produced in both types of cells during late times of infection. As in lytically infected TC7 cells, a decrease in the rate of

SV40 DNAsynthesis during later times p.i. (36 h p.i.) was observed in both types of cells; therefore, this decrease was independent of whether the cells expressed a mutant or wild-typep53.Importantly, this reduction in the rate of viral DNA synthesis occurred at times after infection when no signs of cell damage were visible. Therefore, this reduction indeed reflected a cellularly or virally controlled process. However, this control apparently was not exerted by thep53

proteins expressed in the infected cells or was not directly dependent on the p53 phenotype. We thus conclude that endogenously expressed wild-type p53, although it accumu-lates to relatively high concentrations in infected cells be-cause of its metabolic stabilization after infection (Fig. 7), has no direct inhibitory effect on SV40 DNA replication in vivo. The roles ofp53and of largeT-p53 complexes inSV40 DNA replication thus remain elusive.

Animportant clue forunderstanding the apparent discrep-ancy between the analysis of the role of wild-type p53 in SV40 DNA replication described here and previous analy-ses, both in vitro and in vivo (2, 13, 25, 39, 58, 64), might be provided by considering the quantitative aspects of inhibi-tion by wild-typep53 in the latter assays. Significant inhibi-tion of in vitro SV40 DNA replication by wild-type p53

required at least equimolar concentrations ofp53 and large T, withcomplete inhibition ofSV40 DNA replication being observed only when p53was added in at least equal ratios (39, 64) or in excess over large T (2, 13, 58). Infected cells, in contrast, contain an excess of free large T even at times after infection when SV40 DNA replication was already down-regulated (Fig. 7). Free large T performs important functions in SV40 DNA replication which cannot be per-formed by large T complexed to p53 (25, 58). Therefore, a likely explanation for the inhibitory effect of wild-type p53 onSV40 DNA replication in in vitro assays might be that the added excess p53reduced the level of free large T below a threshold necessary for sustaining SV40 DNA replication.

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This explanation may also be relevant for resolving the apparentparadox thatwild-typep53 in normal cellsseems to be absolutely required for cell cycle progression (8, 33, 34, 45, 56) but reconstitution of wild-type p53 expression in tumor cells lacking p53 expression or expressing mutated p53 (35-37), on the other hand, leads to growth arrest. In fact, one has to be aware that the proposed antiproliferative effect of wild-type p53 in normal cells has not yet been demonstrated. Therefore, it seems possible that

endoge-nouslyexpressed wild-type p53 (andeven some mutantp53 proteins

[8,

56]) indeed exerts a positive regulatory effecton cellularproliferation and that reconstitution ofwild-typep53

expression in tumor cellsrepresents ahighly artificialsystem for analyzing the effects of endogenous wild-type p53 on cellular DNA synthesis. Therefore, the experiments

de-scribed in this study, by analogy, may suggest that the antiproliferative effects of wild-type p53 transfected into tumorcellsdo not reflect a regulatory function of p53atthe level of cellular DNA synthesis under physiological growth conditions. Considering the strict regulation of p53 levels in

normalcells, both at the level of mRNA formation andatthe level of p53 protein synthesis and stability (8, 10, 42, 47,56), it is to be expected that reconstitution of wild-type p53 expression in tumor cells may lead very easily to

overex-pression of wild-type p53 and that growth inhibition by wild-typep53 might depend on the level of its expression (4). Since the contact sites of p53 with large T (24, 53) represent mutational hot spots on p53 (1, 23, 30, 40, 48), it is tempting to speculate that the association of p53 with large T mimics an interaction of p53 with a so far unknown cellular target. We therefore want to suggest that, in analogy to the func-tional elimination of free large T by excess p53 in in vitro SV40 DNA replication assays, which leads to cessation of

SV40 DNA replication, overexpression of reconstituted wild-type p53 might functionally eliminate an important target for p53 in cell cycle control and thereby lead to a

nonphysiological growth arrest. Although this model is

purely speculative at the moment, the system of a homolo-gous pair of cell types described in this study, differing in that one expresses wild-type p53 and the other expresses mutantp53, might be helpful in defining functions for wild-type p53 in SV40 replication. Definition of such function shouldgreatly enhance our understanding of the role(s) of

p53inproliferation of normal cells and tumor cells. ACKNOWLEDGMENTS

We thank the Deutsches Primatenzentrum for providing rhesus monkey kidneysfor preparations of primary cultures.

This study was supported by grants DFG De 212/8-1 from the DeutscheForschungsgemeinschaft and W29/90/De 1 from the Deut-sche Krebshilfe (Dr. Mildred Scheel Stiftung furKrebsforschung) and by the Fonds der Chemischen Industrie. The Heinrich-Pette-Institut is supported by the Freie und Hansestadt Hamburg and by theBundesministerium furGesundheit.

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