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Absence of p53 in a mouse mammary tumor model promotes tumor cell proliferation without affecting apoptosis

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Loss or mutation of p53 may have multiple biological and genetic effects that result in accelerated tumor progression. Loss of p53 in some tumors has been correlated with a marked decrease in tumor cell apoptosis. p53 loss may also accelerate tumor
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  Vol.8,829-838, August1997 CellGrowth&Differentiation Absenceofp53inaMouseMammaryTumorModelPromotesTumorCellProliferationwithoutAffectingApoptosis1 JeffreyM.Jones,LauraAttardi, LucyA.Godley,RodolfoLaucirica,DanielMedina,TylerJacks,HaroldE.Varmus,andLawrenceA.Donehower PrograminCellandMolecularBiology[J.M.J.J,DivisionofMolecular Virology [J.M.J.,LA.D.],DepartmentofPathologyandTheMethodistHospital[R.U,DepartmentofCellBiology[D.MI,BaylorCollegeof Medicine, Houston,Texas77030; Center forCancerResearch, MassachusettsInstitute forTechnologyandHowardHughesMedical Institute,Cambridge,Massachusetts02139[L.A.,T.J.];and NationalCancerInstitute,NIH,Bethesda,Maryland20892[LA.G.,H.E.V.] Abstract Lossormutationofp53mayhavemultiplebiological and genetic effects thatresultinacceleratedtumorprogression.Lossofp53insome tumors has beencorrelatedwitha markeddecrease intumorcellapoptosis.p53lossmayalsoacceleratetumorgrowth through anincrease incellproliferationrates.Toexaminetheeffectsofp53lossontumorprogressioninacontrolledexperimentalcontext,wepreviously crossedp53-deficient micetomammarytumor- susceptible Wnt-1 transgenic  TG) mice. Theresultingfemale Wnt-1TG offspring ofthiscrossalldeveloped mammarytumors,regardless ofp53status (p53+1+, p53+/ or p53-/-). However,female p53-/- Wnt-1TG micedevelopedtumorsmuchsoonerthantheir p53+/+ counterparts. Inthisreport,wedemonstratethattheaveragegrowthratesoftumorsmissing (p53-/-) orlosingp53 (p53+/ with lossof heterozygosity) areacceleratedcomparedtotumors with bothwild-type p53 alleles (p53+/+). This acceleratedgrowthrateappearstobedue primarily to increasesinrates oftumorcellproliferation.Tumorcellapoptoticlevelsweremodestandwerenot measurablydifferentinthepresenceorabsenceof wild-typep53.Theseresultsdiffersubstantially from othermouse tumormodelsinwhichp53losswas closelycorrelated with acceleratedgrowthrates throughattenuatedapoptosis.Thus,themechanismsbywhichp53lossinfluencestumorprogressionmay differ,depending onthetissuetypeand/ortheoncogenic pathwaysinvolved. Received 4/30/97;revised5/30/97;accepted5/30/97.Thecostsofpublicationofthisarticleweredefrayedin part bythe paymentof pagecharges.Thisarticlemustthereforebeherebymarked advertisement inaccordancewith18U.S.C.Section1734solelytomdi-catethisfact. 1 ThisworkwasfundedbygrantsfromtheUnitedStatesArmyBreast Cancer ProgramandNational CancerInstitute (toLA.D.). 2 Towhomrequestsforreprintsshouldbeaddressed,atDivisionofMolecularVirology,BaylorCollegeofMedicine,OneBaylorPlaza, Houston,TX77030.E-mail:larryd@bcm.tmc.edu. IntroductionTheimportanceofp53asatumorsuppressorgeneismdi-catedbyits beinglostormutatedinalmostone-halfofall humancancers(1).Humanbreastcancershavebeenfound tohavemutatedp53allelesin28%ofallcases(2).With respecttothe roleofp53dysfunctioninbreasttumors,this numbermaybeanunderestimate,becauseitdoesnotin-dudetumorsfoundtocontainp53proteinrenderednon-functionalthroughcytoplasmicsequestration(3,4)orbybindingtotheoverexpressedoncogenicproteinMdm2(5-7).p53isacellcyclecheckpointproteinthatregulatespro-gressionthroughG1(8,9)andG2-M(10-12)phasesofthe cellcycle.p53levelsaregreatlyincreasedincellsfollowingtreatmentbyavarietyofDNA-damagingagents,particularly thosethatintroduceDNAstrandbreaks(8,9,13).Up-regu-latedp53caninduceeithercellcyclearrestinlateG1(8,9,13,14)orapoptosis(1 5-i7). ThedecisiontoenterapoptosisratherthanarrestinG1maybeinfluencedbyanumberofendogenousandexogenousfactorsincludingcelltype(18),p53levels(19),andenvironmentalgrowthfactorconcentra-tions(14,20).Wild-typep53isapotentinducerofapoptosis intumorcellsfollowingtreatmentwithionizingradiationoranticanceragents(21).Moreover,wild-typep53mayinhibit tumorgrowthandprogressionbyinducingapoptosiswhencellsarerapidlydividingfollowingalteredexpressionofanoncogeneortumorsuppressorgene(22-25).Multiplegroupshaveshowninmousetumormodelsthatlossofwild-typep53convertsatissueorslow-growingtumorwithhighlevelsofapoptoticcellstoarapidlygrowing tumorwithminimalnumbersofcellsundergoingapoptosis (26-28).Forexample,Symonds eta . (26)havedemon-stratedthattransgenicmicethatexpresshighlevelsofa truncated largeTantigen(whichinactivatesRbproteinbutnotp53)inthechoroidplexusdevelopslowlygrowingcho-roidplexustumorswithhighlevelsofapoptosis.Whenthesemicearecrossedtop53-deficientmice,theprogenywiththeTantigentransgene, but missingp53 (p53-I-), develop rapidlygrowingchoroidplexustumorswithalowapoptoticindex.Thisindicatesthatp53-inducedapoptosismaybetheprimarymechanismgoverningtheslowgrowthrateofthese tumors. Loss ormutationofp53inhumantumorshas been cor-relatedwithincreasedaggressivenessand poor prognosis(2,29,30).Althoughthisincreasedaggressivenessmaybeduein part toattenuatedapoptosis,lossofp53mayaffecttumorprogressionbyadditionalmechanisms.Onepossibleeffectofp53lossisanincreaseingenomicinstability.Cell culturestudieshavedocumentedthatlossofp53isclosely associatedwithchromosomalinstability(31-34).Iflossofp53inatumorcellconfersgenomicinstability,thentherateofappearanceoffurtheroncogeniclesionsislikelytobeincreased.Someoftheseoncogeniclesionscouldresultina  E E D+i+ ; III +1- LOH   Fig. 1. Growthratesof mammary tumorsarising in Wnt-1TG p53- deficient femalemice.Tumorsize was measuredonceperweek,andthetumorvolume wasdetermined asdescribedbyMiller eta . (48).Timezero Isthefirst dateatwhichthetumor wasobservedand measured.Fourp53 genotypesare represented: p53+1+,n = 17; p53+ -, n = 13; p53+/- LOH, n = 10; p53-I-,n = 10. Ba,s, SE foreach genotype. TimeinWeeks BY) Absence ofp53inaMouse Mammary TumorModel significantgrowthadvantageandacceleratetumorprogres-sion.Thereappearstobeahighdegreeofcorrelationbe- tween p53mutationandincreasedchromosomalinstability inavarietyofhumantumor types (35-38).Anotherresultofp53lossinatumormightbeanincreaseintherateofcellproliferationduetoahigherpercentageoftumorcellsenteringandprogressingthroughthecellcycle.Wild-type p53isknowntoactivateexpressionofthe p2l Fl cyclin-dependentkinaseinhibitor(39),aprotein importantinmediatingG1 arrest(40).Lossof p53 maypre- ventactivationofp21andothercellcycleinhibitorsand allowahigherfractionofcellstotraverseG1andenterS phase.Insupportofthis,wehaveshownthatearlypassagemousefibroblastsfromp53-/-embryosproliferatemorerapidlyand haveahigherpercentageofcellsinSphase and mitosisthanp53+1+fibroblasts culturedunderidenticalconditions(34).Humantumorswithoverexpressedmutantp53 alsotendtoexpressmarkersthatsuggestahigherrate ofproliferationthantumorswithintactp53(41-44).Toexaminetheeffectsofp53 presenceorabsenceon tumorprogressioninacontrolledexperimentalbreastcancermodel,wehaveusedtheoffspringofmammarytumor-sus-ceptible Wnt-1TG3 micecrossedtop53-deficientmice.The Wnt-1TG micecontainmultiplecopiesofthe Wnt-1 onco-geneexpressedectopicallyathighlevelsinthemammarygland (45).Thep53-deficientmice,generatedbygenetar- getinginembryonicstemcells,containone(p53+/-)or two(p53-/-)disruptedgerm-line p53 alleles andaresuscepti-bletoawidearray oftumortypes(46).Bycrossingthep53-deficientmicetothe Wnt-1TG mice,wewereableto obtainatumormodelwithdistinctadvantages:(a) Wnt-1TG femalesalldevelopthesametypeofmammarytumor(type Badenocarcinoma),regardless ofp53status(p53+1+,p53+/-, orp53-/-);  b) themice all developtumorswithinarelativelyshortamountoftime (2-9 monthsafterbirth);(c) mammarytumongenesiscanbe monitoredbothinthepres-enceandabsenceof p53 (the p53+/+ Wnt-1TG females serve ascontrolstomonitorp53-independentmechanismsoftumorigenesis);and  d) themammarytumors are s.c.and therefore,canbedetectedearlyandmeasuredeasilyfor ratesofgrowth (47). Inanearlierstudyof Wnt-1TG p53-deficientmice(47), we showedthatintheabsenceofp53, Wnt-1 females alldevel-oped mammary tumorsby 15weeksofage,whereasp53+/+ andp53+/- Wnt-1 females hadlongersurvival ratesanddidnotalldeveloptumorsuntil42weeksofage. Wealsofoundthatmammarytumorsfrom p53-/- miceand fromp53+/-micethatlosttheirremainingwild-typeallele (LOH)hadsignificantlyhigherratesofchromosomalinsta- bilitythanp53+/+tumors.Intheearlierstudy,wefocusedontumorinitiation  i.e., whenthetumors first appeared).Inthisstudy,wehaveattemptedtoaddresstheissueoftumor growth afterinitialformation.Doesthepresenceorabsenceofp53influencetumorgrowthrates,andifso,whatarethe 3 The abbreviations usedare:TG,transgenic;LOH,lossofheterozygoslty; TUNELterminaldeoxynucleotidyltransferase-mediatednickendlabel-ing;MMIV,mousemammary tumorvirus. biologicalmechanisms forthedifferentialgrowth?Here,weprovideevidencethatthelossofp53does acceleratetumorgrowth ratesdunngmammarytumongenesis.However,the increasedgrowthrate appearstobeduelargelytoincreased cellproliferationratherthantoreducedratesoftumorcellapoptosis.Thisresultcontrastswithsomeoftheother mousetumormodels(26-28)andindicatesthat p53loss maypromotetumorprogressionbymultiplebiologicalmechanisms. Results Loss ofp53PromotesAcceleratedMammaryTumor Growth Rates. Todirectlyassess tumorgrowthrate,wemonitored Wnt-1TG femalesofallthreep53genotypesfortheappearanceofmammarytumorsbypalpationandvisual examination.Whenasmalltumor was noted,itsdiameter wasmeasuredin two dimensionswithcalipersandthenmeasuredweeklyforanother3weeks.Tumorvolumes were thencalculatedfromthesemeasurementsashasbeen de- scnbedbyMilleret a . (48).Thetumor-bearinganimalswere thensacrificed,andpartofthetumorwasanalyzedfor histopathology.HighmolecularweightDNAwasprepared fromp53+/-tumors,andthestatusoftheremainingwild-type p53 allelewasassessedbySouthernblothybridizationasdescribedpreviously(46).Roughlyone-halfofthe Wnt-1TG p53+/-tumorsdeletetheremainingwild-type p53 allele,andtherestretainit.Wehaveshownpreviouslythatinthosetumorsthatretainthewild-typeallele,the p53 gene remains wildtypeinsequence (47).A compilationofthetumorgrowthdataforeachofthep53genotypesisshownin Fig.1 . Notethatafter 4weeksofgrowth,thep53-/-tumorsdisplayedsignificantly highertumorvolumesthanp53+/+ tumors  P = 0.04 asmeasuredby t test).p53+/-tumorswithLOHalsohadsignificantlyhighertumorvolumesat4 weeks thanp53+/+tumors  P = 0.02). p53+/- tumors  CellGrowth&Differentiation 831 withoutLOHshowedonaveragemarginallyincreased growthratecomparedtop53+/+tumors,butthisdifferencewasnotsignificantby t test. Apoptosis Levels Are LowandIndependentofp53 Status.Themeasurementsofthemammarytumorsre-vealedthatthelossorabsenceofp53significantlyenhancedtumorgrowthrates.Onepotentialmechanismforthisen-hancedtumorgrowthcouldbearesultofareductioninapoptoticcellfractionasseeninothermousetumormodelsmissingp53(26,27).Anetgrowthadvantagewouldresultiffewertumorcellsdieinthep53-deficienttumorscomparedtothetumorswithwild-typep53.Toaddressthispossibility, weexaminedformalin-fixedmammarytumorsectionsbythe TUNELapoptosisassay.Fig.2, A and B, showsexamplesofthisstaininginap53+/+tumor(Fig.2A)andap53-/-tumor(Fig. 2B). TheresultsoftheTUNELassaysarerepre-sentedbythegraphinFig.2C.Theredoesnotappeartobeanycorrelation between thep53statusofatumorandthedegreeofapoptosisobserved.Apoptoticcellsappearedto be randomlydistributedthroughoutthetumorsections.Wealso performed theTUNELassayonhyperplasticmammaryglandtissuefromthep53-/-,p53+ -,andp53+/+ Wnt-1 females.Normalmammaryglandswerenot available,becausethe Wnt-1 transgeneinducesaveryearly hyperpiasiathroughouteachofthemammaryglands(45).It ispossiblethatinhyperplastictissue,wild-typep53mightdelaytumorformationthroughincreasedapoptosisofab-normallyproliferatingmammaryglandcells.Thiscouldex-plainthedelayinonsetofmammarytumorsinp53+/+andp53+1- Wnt-1 micecomparedtop53-/- Wnt-1 mice. However,resultsoftheTUNELassaysrevealedthatapop-tosisin Wnt-1TG femalemammaryglandswasverylow(onaverage,below0.5%oftotalcells)andnotappreciablydif-ferentamongp53-I-,p53+/-,andp53+/+hyperplastictissues(Fig.3). These resultssuggestthatdifferencesinapoptoticratesinthepresenceandabsenceofp53arenotamajorfactoreitherintheinitiationorprogressionofthemammarytumors.ToconfirmtheresultsoftheTUNELassay,wealsoexam-medthetotaltumorDNAforapoptoticDNAfractionsusingaDNAIaddenngassay.DNAfromcellsundergoingapoptosisformsaladderofbandsthatareroughmultiplesof170bp(49).Usingthisassay,wewereagainabletoshowthattheapoptoticDNAfractionin Wnt-1 mammarytumorsofallthreep53genotypeswasrelativelylowanddidnotdifferappreciablyaccordingtop53status(Fig.4).TheapoptoticDNAfractionrangedfrom2-7%,ascalculatedfollowingSouthernblothybridization(see“MaterialsandMethods”).TransferofhighmolecularweightDNAtotheblotswasinefficientincomparisontolowmolecularweightapoptoticDNA;thus,calculationsofapoptoticfractionsarelikelytobeoverestimates.Thecriticalpointisthattherewerenosignif- icantdifferencesintheapoptoticDNAfractions between p53+/+andp53-/-tumors(asmeasuredbyttest). TheAbsenceofp53PromotesCellProliferation. Ifap- optosisisnotplayingamajorroleintheincreasedgrowthratesofthep53-deficient Wnt-1TO tumors,theobviousalternativeisthattumorcellsaredividingmorerapidlyintheabsenceofp53.Totestthispossibility,wefirstexaminedthe numberofmitoticcellsinthemammarytumorsasasimple markerfortherelativenumberofcellsundergoingdivision.Thiswas performed bycountingmitoticfiguresinH&E- stainedtumorcross-sections.Thetotalnumberofmitotic figureswasdeterminedin10highpowermicroscopicfields (x400)foreachtumor. Thefieldswererandomlyselectedneartheedgeoftheexpandingtumorbecausethisareausuallyhasthehighestgrowthrates.Thetotalnumberof mitoticfiguresper10fieldswasalsoconvertedtopercent- agesofmitoticfigurespertotalnumberofcellsinthe10fieldstocontrolforanypossiblesizedifferences between tumorcellsofdifferentp53genotypes.However,withbothmeasurements,therelativemitoticindexwassignificantly higherinthep53-/-andp53+/- LOHtumorsthaninthe p53+/+tumorsasmeasuredbyttest (P = 0.001and0.006,respectively;Fig.5).Thep53+/-tumorcellshadonaverage highermitoticindexesthanp53+/+tumorcells,althoughthedifferenceherewasnotstatisticallysignificant.Thesecondassay used toassesscellproliferationinthetumorsampleswasflowcytometricanalysisforDNAcon-tent.Thisassayprovidesanapproximationofthepercentageofthetumorcellsineachphaseofthecellcycle.Thisassaywas performed ontumorcellnucleiobtainedbycutting50-smsectionsfromparaffin-embeddedsamplesasde- scribed previouslybyDemirel eta . (50).ThreerepresentativeflowcytometryprofilesforthetumorsareshowninFig.6, A-C. NotethehigherpercentagesofcellsinSphaseand G2-Mphaseinthep53-/-samplewhencomparedtothe p53+/+tumorcells.Notealsothatthep53+/-LOHtumor hassubstantialaneuploidyamongitstumorcellpopulation.Thisaneuploidprofilewasconsistentlyobservedforallfivep53+/-LOHtumorsexamined.Onlyoneofsevenp53-I-tumorsexhibitedananeuploidprofile(datanotshown).Thisresultwasconsistentwithchromosomalinstabilitystudies performed onthistumormodelpreviously(47).Theresultsforallfourp53genotypesarecompiledinFig.60.Notethatthep53+/-LOHtumorshaveonaveragethehighestlevelsofcellsinSphase.Althoughthisestimateforthep53+1-LOHSphasefractionmaybeslightlydistortedduetoane-uploidy(Fig. 6c), wehaveattemptedtobeconservativeintheinterpretationsoftheprofiles.p53+/-LOHtumorcellsshowedsignificantlyhighernumbersofcellsinSphaseon average(17.3%)thandidp53+/+cells(6.9%)byttest (P = 0.001).Thep53-/-tumorcellsalsoexhibitedsignificantlyhigherfractionsofS-phasecells(11.7%)thanp53+/+tumor cells (P = 0.03byttest).Theflowcytometryresultsshowthatabsenceorlossofp53resultsinahigherpercentageoftumorcellsproceedingthroughSphaseandG2-M. Discussion Inanearlierstudyusingthe Wnt-1TG p53-deficientmodel, weshowedthattheabsenceofp53acceleratestumongen- esis(47).One-halfofp53-/- Wnt-1TG femalesdevelopedmammarytumorsby11.5weeksofage,whereasone-halfofp53+/+andp53+/- Wnt-1 femalesdevelopedmammary tumorsby23weeksofage (47). Inthisstudy,weshowthatnotonlydoestheabsenceofp53promoteearlytumorfor- mation,butonceformed,tumorslackingp53growsignifi-cantlyfasteronaveragethantumorsintheirp53+/+coun-  B. S- ‘.5’ ., . * .9.. S *I 3 ‘S., C. .1 +1+  s / noLOH +/. LOB -/- 832 Absenceofp53inaMouseMammaryTumorModel A. 4 - * I   -. * ;,   #{149};-‘- ‘--.- .   -  h:  #{149} “.,, ... -.-.. --  wf .- #{149} i .. .k l:: -, .-; r #{149} -  .., ‘ ,-‘ ‘ -‘   ,- -. ..,- -. - b,,;-   .S #{149} #{149} , . S ...   S.$5  S   ft r , , . 55,   ,  &: #{149}:   - .S- ;: Fig.2. TUNELassayonmammarytumorsinthepresenceandabsenceofp53.ThetwopicturesareexamplesoftheTUNELassayperformedonmammarytumorsam-pies. A,p53+1+ tumoratx400withbrown-stainedapoptoticcells. B, p53-/-tumoratx400withbrown-stainedapoptoticcells. C, estimatesofaveragelevelsofapoptosisinmammarytumorsinthepresenceandabsenceofp53,showingtheresultsoftheTUNELassaywhenperformedonindividualmammarytumors.Each column representsaseparatetumor.Theapproximatelevelofapoptosisacrosseachtumorisindicatedbythenumberofpluses. +, veryfewap-optoticcellsperhighpowerfield(0-1); + +, 2-10apoptoticcellsinafield; +++, 10-20apoptoticcellsineachfield. Columns showingintermediatevaluesbetween +- and ++ indicatetumorsdisplayinganover-alllevelofTUNELstainingcorrespondingto + tumorsbutexhibitingoccasionalpatcheswithhigherlevelsofstaining.  A. B.  - . 0 ‘9’,.’ -   A, ) t. c’--   :;   4. v-: ‘-   ‘ 5’ 5. _,,, \ 1’   ---.- -   I I A C. S,  : ‘  *   0.75#{149}0.5- 0.25- +1++1--I- 4,, c cJ 0 0 0.0 0 4,, CellGrowth&Differentiation 833 Fig.3. Apoptosisin Wnt-1 hyperplasticmammaryglandsinthepresenceandabsenceofp53asmeasuredbytheTUNELassay. A, hyperplasticmammaryglandatx400froma Wnt-1TGp53+1+ female.Apoptoticcellsarestained darkbrown bytheTUNELassay. B, hyperplasticmammaryglandatx400froma Wnt-1TGp53-I- female.Apoptoticcellsarestained darkbrown.C, TUNELstainingforapoptosisinhyperplasticmammaryglandsof Wnt-1TG females.Thedataareexpressedastheaveragepercentageofepithelialcellsundergoingapoptosis(forover1000cellscountedperslide).Forp53+/+hyperplasticmammaryglands, n = 8;p53+1-, n = 10;p53-I-, n = 4. Bars, SE. terparts.Thetumorgrowthassaysindicatedahigherrateof growthintumorcellsmissingp53(p53-/-)orhavinglostp53(p53+/-LOH)comparedtotumorcellswithafullcom- plementofp53(p53+/+).Themaintenanceofnormaltissuehomeostasisisdepend-entonbalancingtherateofcelldivisionandtherateofcelldeath.Anincreaseincellproliferationratesoradecreaseincelldeathratescanupsetthebalancethatmaintainsthe normalcellularintegrityofthetissue,andtheendresultcan beatumor.Lossofnormalp53functioncouldplayaroleinaffectingtissuehomeostasiseitherbyincreasingthefractionofcellsprogressingthroughthecellcycleorbyreducingthelevelsofcellularapoptosis.Previousanimalmodelstudieshaveexaminedthemechanismsbywhichabsenceofp53mayconferagrowthadvantageduringtumorprogression (26-28).Ingeneral,thesestudieshavesupportedthecon- ceptthatp53lossacceleratestumorgrowthprimarily throughlossofnormalapoptoticfunction.Thedatapre- sentedhereindicatethatinthe Wnt-1/p53 model,theab- senceofp53haslittleornoeffectonapoptosis(thoughsubtledifferencesinapoptosisnotdetectablebyourassayscannotberuledout).However,thelossorabsenceofp53doesresultintumorcellswithsignificantlyhigherratesofproliferation.Whetherthisincreasedproliferationisadirecteffectofp53lossoranindirecteffectduetosuchfactorsasgenomicinstabilityremainsunclear.InarecentstudybyHundley etaL (51),theoffspringofMMTV-c-rasmicematedtop53-deficientmicealsodemonstratedincreasedtumor cellproliferationintheabsenceofp53.Moreover,thep53-deficient c-ras tumorsdidnotshowthep53-dependentdif-ferencesinapoptoticcelllevelsdescribedinothermodels(26-28).Theseresults,togetherwiththeobservationofin-creasedgenomicinstabilityinthep53-deficienttumors,closelyparalleledourownresultsandsuggestthatp53loss
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