[PDF] Morphological Transformation of Established Rodent Cell Lines by

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MOLECULARANDCELLULARBIOLOGY,Apr.1986,p.1253-1260Vol.6,No.40270-7306/86/041253-08$02.00/0CopyrightC1986,AmericanSocietyforMicrobiologyMorphologicalTransformationofEstablishedRodentCellLinesbyHigh-LevelExpressionoftheAdenovirusType2ElaGeneALLENW.SENEARANDJAMESB.LEWIS*BasicSciencesDivision,FredHutchinsonCancerResearchCenter,Seattle,Washington98104Received16September1985/Accepted30December1985Whenastrongpromoterderivedfromthemousemetallothioneingenewassubstitutedforthehomologousadenovirustype2Elapromoter,leadingtoenhancedlevelsofElaRNAsandproteinsincellstransfectedwiththechimericgene,theElagenealonewasabletoinduceinestablishedcellinesalterationsinceBularmorphologyandgrowthpropertiessimilartothoseproducedbythecombinedactionofElaandElbgenes.ThequalitativeeffectsofElageneexpressionuponcellularpropertiesthusdependonthelevelofexpressionoftheElagene.Furthermore,Elamaybetheprimarytransforminggeneofadenoviruses,sinceitproducedmanyofthecharacteristicsoftransformedcellsthathadpreviouslybeenattributedtoElb.Thetransformationofcellsinvitrobyoncogeneshasbeenconsideredatwo-stepprocessinwhichprimarycellsarefirstimmortalized(bygenessuchasmycorpolyomalargeT)toproduceestablishedcelllinesofindefiniteproliferativeca-pacityandthenfurthertransformed(bygenessuchasrasorpolyomamiddleT)toproducecellsofalteredmorphologythatarelesssusceptibletoarrestofgrowthbycontactinhibitionandotherfactors(10,13,19).Additionalfactorsdeterminethedegreetowhichthesefullytransformedcellsaretumorigenicinvivo.Applicationofthismodeltothetransformationofrodentcellsinculturebyhumanadenovi-ruses(Ad)identifiesElageneproductsasnecessaryandsufficientfortheimmortalizationstep(10,19).Elbgeneproductswerethoughttobenecessaryforfulltransforma-tionalthoughtheresultsofmorerecentexperimentsinwhichElbgeneswereexpressedinestablishedcelllinesintheabsenceofElaimplythatElbisnotsufficientforthesecondstep(23).Moreover,themorphologyandotherpropertiesofcellstransformedbytheentireElregiondependinpartonwhichoftwodifferentAdserotypesisusedasasourceofElagenes(24).ThustheassignmentofimmortalizationandmorphologicaltransformationfunctionstotheElaandElbregions,respectively,maynotbeassimpleassupposed.ThesituationisfurthercomplicatedbythemultiplicityofElproteinsproducedbytheuseofalternativesplicesitesinbothElaandElbgenesandtheuseoftwodifferentinitiationcodonsinElb(seeFig.1fordetails).Wehaveconstructedaseriesofplasmidsasameansofstudyingtheeffectsupontransformingfunctionoftheex-pressionincellsofvariousamountsofparticularAdtype2(Ad2)Elproteins.Theseplasmidsweretransfectedintoestablishedlinesofcontact-inhibitedrodentcellstodeter-minethepotentialofthesegeneproductstomorphologicallytransformalreadyimmortalizedcells.WereportherethatthelevelofexpressionofElageneproductshasprofoundeffectsoncellularproperties:whenexpressedathighlevels,Elaproteinsaloneareabletoinducemanyoftheattributesofmorphologicaltransformationthatwerepreviouslyas-signedtoElbproteins.*Correspondingauthor.MATERIALSANDMETHODSConstructionofplasmids.AllplasmidsusedarederivativesofpBR322andwereconstructedbystandardmethods.TheAdandmetallothionein(MT)sequencesinsertedintoeachplasmidareshowninFig.1anddescribedinitslegend.Transformationassays.TransfectionofplasmidDNAsintoNIH3T3cellswasperformedbythecalciumphosphate-DNAcoprecipitationmethodofvanderEbandGraham(25).Toeach60-mmdishcontaining3x105cellsseededthepreviousdaywasaddedaprecipitateformedfromapproxi-mately1to1.5pLgofplasmidDNA(amountswereadjustedforsizesothatequimolaramountsofeachplasmidwereused)and10p.gofcalfthymuscarrierDNA.Toassayforfocusformation,culturesweremaintainedfor3to4weeksinDulbeccomodifiedEaglemedium(DME)plus10%ofetalbovineserumbeforefixingandstaining.InsomeexperimentsG418-resistanttransfectedcellswereisolatedaftertransfectionasabovewithadditionof50ngoftheplasmidpko.neo(26)toeachtransfectionmixtureandsubsequentselectionbytheadditionof400,ugofG418(GIBCOLaboratories,GrandIsland,N.Y.)permltotheculturemedium.EachG418-resistantpopulationconsistedofthecellspooledfromtwosuchtransfecteddishesandcontainedafewdozenclones.Tomeasureanchorage-independentgrowth,5x103cellsfromeachpopulationwereseededin0.37%agarovera0.90%agarbase;bothlayerscontainedDMEplus10%fetalbovineserum.Liquidmedium(DMEplus10%fetalbovineserum)overlayingtheagarwaschangedweekly.AnalysisofRNAfromtransfectedcells.TotalcellularRNAwasisolatedfrompopulationsofG418-resistantcellstransfectedwithvariousregionElplasmids,essentiallybythemethodofFavaloroetal.(6),withbufferscontaining10mMvanadylribonucleosidecomplex.Ahybridizationprobetodetectthealternative5'exonsofElamRNAswaslabeledbydigestingtheplasmidpMTEla(Fig.1)atthe5'endoftheElagenewithBamHI,removingapproximately40nucleo-tidesfromthe3'endofthecomplementarystrandwithexonucleaseIII,andfillingintheendswiththeKlenowfragmentofDNApolymerasebyusinglow-specific-activity[32P]dCTP(300Ci/mmol),followedbydigestionwithEcoRIandagarosegelelectrophoresistoisolatethelabeledDNAfragment.Hybridizations(6)wereperformedat54°C,fol-1253

1254SENEARANDLEWISEIA1000II2000EIB30001a4000.IIIIII1IIIt1LJ,LeSst1ll4,CIaIXbaIHpal4,tIBstEllHindNIBgl11BclI289R_243R_i-I1745R*495R..----*_----->155RpLeHpa-ipMTEla-'pMTEla7144pClaBclApMTBstBclIpLeBclIpMTElob1pMTElab714FIG.1.MapofadenovirusregionElDNA,mRNAs,proteinsandplasmids.Thelineatthetoprepresentsapproximately4.5kilobasesofDNAfromtheleftend(Le)oftheAdgenome.Mapcoordinatesinnucleotides(8)areindicatedabovethelineforpositionsmarkedbysmallhatchmarks.Down-pointingarrowsindicatesitesofrestrictionenzymecleavageforenzymesidentifiedbelowtheline.mRNAsareindicatedbythinlines;intronsequencesremovedbysplicingfromeachofthemRNAsareindicatedbydottedlines.AllElmRNAsaretranscribedfromlefttorightonthismap.ThetranslatedregionsforeachofthefiveElproteinsareshownbythicklines.Eachproteinisdesignatedaccordingtoitsnumberofaminoacidresidues(R),assuggestedinreference1.AlthoughtheElb175RproteinisshownassociatedwiththesmallestoftheElbmRNAs,itcanbetranslatedfromallElbmRNAsshown.Onlythemajor,well-characterizedmRNAsandproteinsareshown;otherElaandElbRNAandproteinspeciesarebelievedtoexistinvivo.ThebottomportionofthefigureshowstheextentofAdDNAsequencesfoundintheplasmidsusedintheseexperiments.MTgenepromoter-regulatorysequencesareindicatedbythethickarrowpointingtotheright;thissegmentisnotdrawntoscalewiththeviralsequences.pLeHpacontainsviralDNAsequencesderivedfromAd5(byusingpLA1[22])betweentheleftendoftheviralgenomeandtheSstIIsite(nucleotide353oftheAd2sequence)andfromAd2betweenN353andtheHpaIsite(N1596)andthuscodesforAd2ElaproteinsunderAdcontrol.pMTElacontainsapproximately1.7kilobasesofmousemetallothionein-IDNA(21)locatedfrom-1,700to-12relativetotheMTcapsitejoinedbyaBamHIlinkertoAd2DNAextendingfromaHaeIIIsite(N494)locatedjust5'totheElacapsitetotheHpasite(N1569)andthuscodesforElaproteinsunderMTpromotercontrol.pMTEla7l4hasthesamesegmentofMTDNAjoinedtoElaDNAextendingfromaHaeIIIsiteatN714totheHpaIsite(N1569);thisplasmidmaycodeforN-terminallytruncatedElaproteinslacking70aminoacidresidues.pClaBclcontainsAd2DNAlocatedbetweenClaI(N916)andBclI(N4036)andcodesforallElbproteinsunderElbpromotercontrol.pMTBstBclcontainsthe1.7-kilobasesMT-IDNAsegmentjoinedthroughaBamHIlinkertoAd2DNAextendingfromaBstEIIsite(N1912)totheBclsite(N4036)andshouldcodefortheElb155Rand495Rproteins,butnottheElb175Rprotein,underMTpromotercontrol.pLeBclcontainstheviralsequencescontainedinpLeHpaandextendstotheBcllsite(N4036)andthuscodesforbothElaandElbproteinsunderAdcontrol.pMTElabandpMTElab7l4containthesameMTandElasequencesaspMTElaandpMTEla7l4,respectively,butextendtoN4036andthusalsocodeforElbproteins.lowedbySinucleasedigestionat37°C.Eachassaycon-tained30jxgofcellularRNAor2jxgofRNAfromHeLacellsinfectedwithAd2and5ng(5,000cpm)oftheDNAprobes.S1nuclease-digestedhybridswereanalyzedbyelectrophoresison6%polyacrylamide-8Mureagelsfollow-ingdenaturingbyboilingin90%formamide.Analysisofproteinfromtransfectedcells.AnantiserumtotheEla289Rand243RproteinswaspreparedessentiallyasdescribedbyYeeetal.(27);forimmunizationapeptidehomologoustotheC-terminalfiveresiduesoftheElaproteinswasused.Rabbitantiserumwasaffinitypurifiedasdescribedpreviously(1).Amoredetailedcharacterizationofthisantiserumwillbepresentedelsewhere(R.C.Schmitt,M.L.Fahnestock,andJ.B.Lewis,manuscriptinprepara-tion).Whole-cellextractswerepreparedandusedforim-muneprecipitationaspreviouslydescribed(14).SouthernblotanalysisofDNAfromtransfectedcells.A15-,ugportionofDNAfromeachpopulationoftransfectedwwbroMOL.CELL.BIOL.IIII

ADENOVIRUSElaGENEEXPRESSIONANDTRANSFORMATION1255cellswasdigestedwithEcoRIandHindIll.Forcomparison,amountsoftransfectedplasmidDNAequivalentto10mol-eculespercell,assuminganaverageDNAcontentof10pgperNIH3T3cell,weremixedwith15,ugofcalfthymusDNAbeforedigestionwithEcoRlandHindIll.Agarosegelelectrophoresis,transfertonitrocellulose,nicktranslation,hybridization,andwashingconditionswereasdescribedbyBrinsteretal.(4).RESULTSFocusformationwithNIH3T3cells.Variousplasmids(Fig.1)containingElaorElbgenesorbothweretransfectedintoNIH3T3cells,andthefocioftransformedcellswhichovergrewthemonolayerwerecountedandjudgedforsizeanddensity(Table1;Fig.2).Asexpected,aplasmidcontainingonlytheElagenewithitsownpromoter(pLeHpa)gaverisetoafewsmall,poorlystainedfoci.TransfectionoftheElbgene(pClaBcl)alonegaverisetofew,ifany,transformants.However,transfectionofbothElaandElbgenestogether,eitheronseparateplasmids(pLeHpapluspClaBcl)oronasingleplasmid(pLeBcl),yieldedmanymorefoci,whichwerealsolargerandmoredenselystainedthanthefewobservedwithElaalone.ThusElaandElbgenescancooperatetogiveefficienttransfor-mation.Theefficiencyofthiscomplementationislowerwhenthetwogenesareonseparateplasmids.Thislowerefficiencymaybeduetothelossofinteractionsbetweenthetwogenesortheirassociatedsequenceswhentheyarephysicallyseparated(forexample,apossibleeffectofen-hancerelementslocatedinElauponexpressionofElb),oritmayconceivablybeduetoalowerprobabilityofgettingbothgenesintoasingletransfectedcell.TheresultsweredramaticallydifferentwhentheEla(Ad)upstreamregulatorysequenceswerereplacedbythemouseTABLE1.TransformationassaysFocusformation'GrowthinsoftagarbPlasmidExpt1Expt2No.ofNo.ofmicro-macro-No.ofSizeNo.ofSizescopicscopicfocifocicoloniescoloniespBR3222+0-610pLeHpa15+16+430pClaBcl9+0-520pLeHpa+79++63++16376pClaBclpLeBcl196++++65+++431171pMTEla192+++52+++309215pMTEla+190+++NDND321135pClaBclpMTElab237++++77++++297123pMTE1a714NDND2+pMTElab7142+NDNDpLeHpa+17+NDNDpMTBstBclaNIH3T3cellmonolayersweretransfectedasdescribedinMaterialsandMethods.Theextentoftransformationwasmeasuredbothbycountingthenumberoffociandbyjudgingthetypicalsizeanddensityofstainingofthefocioneachdish.Thenumbersshownforeachexperimentarethesumofduplicatedishes;allduplicateswereverysimilar.ND,Notdone.bPopulationsofG418-resistantNIH3T3cellsthathadbeencotransfectedwiththeplasmidsindicatedwereassayedforgrowthinsoftagarasdescribedinMaterialsandMethods.After5weeksinculture,allvisiblecoloniesofanysizewerecounted(microscopicagarcolonies).Afteranadditional4weeks,coloniesofapproximately1mmorgreaterwerecounted(macroscopicagarcolonies);theother(microscopic)colonieswerestillonlymarginallyvisible.pLeHpapCIaBcipLeBclpMTElapMTElabFIG.2.FocusformationbyAd2ElplasmidsonNIH3T3cells.TransformationassayswereperformedasdescribedinMaterialsandMethods.(MT)upstreamregulatorysequences.TheseexperimentsdidnotinvolvetheinductionofMT-regulatedexpressionbytheadditionofmetalions,butinsteadreliedonthelevelofexpressioninducedbythenormalcomponentsofserumandmedium.TheMTpromoterseemstobecomparativelystrongevenwithoutinduction(4,15),sothatitmightpromotehigherlevelsofsynthesisofElproductsthanisfoundwiththeAdpromoter.TransfectionwitheitherpMTElaborpMTElagavemanyfoci.pMTElawasapprox-imatelyasefficientasAdElab(pLeBcl),andthepresenceofElbintheMTplasmidhadonlyamarginaleffect.Transfor-mationrequiredintactElaprotein,sinceconstructions(pMTEla714orpMTElab7l4)inwhichtheMTpromoterhasbeenfusedtotheElagenewithinthecodingregionandthuscanonlyproduceElaproteinslacking70amino-terminalresidues,areinactiveinthetransformationassay.Formally,pMTElamightbeactiveintransformationbe-causethe1.7-kilobase(kb)regionofMTDNAcontainsanundescribedoncogene.ThispossibilitywasruledoutbytheinabilityofplasmidscontainingtheMTpromoterlinkedtofragmentsofElb(pMTBstBcl)ortofragmentsofothergenes(Elaorthymidinekinase,datanotshown)tocomple-mentpLeHpainthefocusassay.MorphologicalandgrowthcharacteristicsofElatrans-formedcells.TheaboveresultssuggestthattheElageneispotentiallysufficientforfullmorphologicaltransformationofestablishedlinesofrodentcellsandthattheMTpromoterisformallyequivalenttothepresenceofElbgenes.Ifso,cellstransformedeitherbyElaplusElborbypMTElashouldhavesimilarproperties.Tofurthercharacterizesuchcells,variousElplasmidsweretransfectedintoNIH3T3cellsalongwithaplasmidforresistancetotheneomycinderiva-tiveG418(or,notshown,intoRat2cellsalongwithathymidinekinaseplasmid).TransformationfrequencieswerejudgedasthepercentageofG418-resistantorthymidinekinase-positivecoloniesthatshowedalteredmorphology,asdescribedbelow,andincreaseddensity,andforfullycom-petentplasmidsthesefrequenciesrangedfrom30to90%.pBR322P.eHpaVOL.6,1986

1256SENEARANDLEWIS.**.si-r:,S.4.tgA..FIG.3.MorphologyofNIH3T3cellstransfectedwithAd2Elgenes.RepresentativeregionsofG418-resistantcoloniesphotographedaftertransfectionofNIH3T3cellswithpko.neopluspBR322(panelsAandB),pLeHpa(panelsCandD),pMTEla(panelsEandF),orpLeBcl(panelsGandH).Suchfrequenciesaretypicalforcotransfectionofanytwomarkers,sothattransformationbytheAd2Elregionisintrinsicallyefficient,confirmingtheresultsofvandenElsenetal.(24).Theresultsofthesecolonyexperiments(trans-formationfrequenciesnotshown)areverysimilartothoseofthefocusformationexperimentsinTable1.TheAdElaplasmidaloneisnotabletoefficientlyinducetransformedcolonies,buttheElaandElbgenestogether,orElaunderMTcontrol,dotransformefficiently.Visualanalysis(Fig.3)ofsuchcoloniesshowsthatcellstransfectedwithpBR322(panelsAandB)orwithElbalone(notshown)maintaintheextremelyflattenedappearanceoftheparentalcellsandceasegrowingwhentheybecomeaconfluentmonolayer.CellstransfectedwithpLeHpa(AdEla;panelsCandD)maintainsomeofthemorphologicalcharacteristicsoftheirparents.Thesecellscontinuetogrowasaflat,tightlyadherentmonolayer;however,theyaregenerallysomewhatsmaller,havealargernuclear-to-cytoplasmicratio,andarefrequentlyorganizedintofairlyregulararraysofpolygonalorelongatedcells.Occasionalcoloniescontainsomecellsthatresemblethecellsdescribedbelow.CellstransfectedwithpLeBcl(AdElab;panelsGandH),pMTEla(panelsEandF),andpMTElab(notshown)formathirdmorphologicalclass.Thesecellsaremuchsmaller,adoptalooselyattachedroundedorcylindricalshape,andreadilyovergrowoneanotherinahighlydisor-ganizedmanner(panelsFandH).Focicontaininglargenumbersofverysmallcellspiledupseverallayersdeeparefoundscatteredthroughoutdensecolonies.Whenatlowdensityorontheedgesofisolatedcolonies(panelsEandG),thesecellsmaytakeonamoreflattenedpolygonalorelongatedshapesimilartothatseenwiththeAdElacells,butthismorphologyislostinthepresenceofanycrowding.Forfurtherstudies,weanalyzedpopulationsofcells,containingseveraldozenindependentcolonieseach,gener-atedbytransfectionperformedbyusingalowratioofG418resistancetoElplasmids,sothatnearlyallG418-resistantcoloniescotransfectedwithappropriateplasmids(pLeBcl,pMTEla,pMTElab)weretransformed.Weusedpoolsofcellsselectedfordrugresistanceratherthanclonalcelllinestoavoidanybiasesintheselectionofparticularcellsforstudyandbecausewehopedthatpooledpopulationswouldgiveresultsclosetotheaverageofmanylines,avoidingtheconsiderablevariationinthelevelofexpressionoftransfectedgenesthatisoftenobservedamongclones.However,asjudgedbyvisualmorphology,thesepopula-tionsdidappeartobefairlyhomogeneous.Aswiththemorphologyofsinglecolonies,thegrowthpropertiesofthesepopulationsfellintothreeclasses.ThegrowthrateofthesecellswasnotsignificantlyaffectedbythepresenceofElDNA;in10%serumallcelltypesgrewwithadoublingtimeofapproximately20h,whilein1%serumtheyallgrewverypoorly.However,asexpectedfromourvisualobservation,theydifferedgreatlyintheirmaxi-mumcelldensity.ThepBR322contransfectedcellsgrewtoafinaldensityof3x106to4x106cellsper60-cmdish,asdidtheparentalNIH3T3cells.ThepopulationtransfectedwithAdEla(pLeHpa)grewtoasomewhathigherdensity,10x106.TheAdElab(pLeBcl),pMTEla,andpMTElabpopulationsallbehavedsimilarly,growingrapidlytoadensityof30x106,withslowerpatchyovergrowthlateryieldingprogressivelyhigherdensitiessothatnofinalsatu-rationdensitycouldbeclearlydefined.Anchorage-independentgrowthinsoftagaryieldedonlytwoclassesofcells(Table1).At1monthafterplating,thepBR322andAdEla(pLeHpa)populationsgaverisetoafewmicroscopiccolonies(afewdozencellseach)whichfailedtogrowfurther.TheAdElab(pLeBcl),AdElaplusAdElb,pMTEla,andpMTElabpopulationsallhada5-to10-fold-highercloningefficiency;furthermore,evenat1monthMOL.CELL.BIOL.

ADENOVIRUSElaGENEEXPRESSIONANDTRANSFORMATION1257manyofthesecoloniesweresignificantlylargerthanthoseofthefirstclass,andtheycontinuedtogrowuntiltheywereseveralmillimetersindiameter.Wethusfoundthatthemorphologyandbothanchorage-dependentandanchorage-independentgrowthpropertiesoftheNIH3T3cellstransformedwiththepMTElagenewereverysimilartothoseofcellstransformedwiththeElaandElbgenestogether,whilethecellstransfectedwiththeAdElaplasmidaloneweregenerallyaffectedtoamuchlesserextent,ifatall.ThatthesestatesmaybedifferentpointsalongasinglecontinuumwassuggestedbyobservationsofrareAdElacoloniescontainingsomewhatmoretrans-formedproperties,andofsomecoloniesinthepMTElaandAdElabpopulationswiththelowerdensityandflatpoly-gonalmorphologycharacteristicofAdElacells.ExpressionofElamRNAsandproteins.WeinvestigatedthepossiblerelationshipbetweentransformationpotentialandlevelofElageneexpressionbyanalyzingbothRNAand[35S]methionine-labeledproteinfromourtransfectedcellpopulations.TomeasureElamRNAlevelstotalcellularRNAwasisolatedandhybridizedtoaDNAfragmentlabeledatthe5'endoftheElageneonthecomplementarystrand;hybridswerethendigestedwithSinucleaseanddenatured,andtheproductswereanalyzedbypolyacryl-amidegelelectrophoresis(Fig.4).Thisprotocolallowedustospecificallymeasurethealternative5'exonsthatcanbeproducedfromtheElagene.ThemajorElamRNAsof13Sand12S,whichcodeforthe289Rand243Rproteins,respectively,generateprotectedfragmentsof615and475nucleotides,respectively,andthe9Sspeciesthatisob-servedininfectedcellsatlatetimesgivesa120-nucleotideI~~~~~1--QProbe"MM_to-13sf-12s-9sobcdef5'3-3S12sProbeFIG.4.AnalysisofRNAfromtransfectedcells.RNAextractedfrompopulationsofNIH3T3cellstransfectedwithpko.neoplusplasmidscontainingAd2Elgeneswashybridizedtoa3'end-labeledcomplentarystrandDNAprobe(structuresofthetwomajorElaRNAspeciesandoftheprobeareshownabove);hybridsweredigestedwithS1nucleaseandanalyzedbypolyacrylamidegelelectrophoresis.289RyAwl,243Rd--obcdefghijkFIG.5.Analysisofproteinsfromtransfectedcells.Transfectedcells(lanesathroughi),Ad2-infectedHeLacells(lanesjandk),ormock-infectedHeLacells(lane1)werelabeledbyincubationwith250,uCiof[35S]methioninein3mlofmethionine-freemediumcontaining5%fetalbovineserumper100-mmdishfor3h.Tocontrolfornonspecificprecipitationofcellularproteins,theanti-bodiesusedinthelanesmarked(+)werefirstincubatedwithanexcess(20jig)ofthepeptideusedforimmunization.Fluorographyofthesodiumdodecylsulfate-17.5%acrylamideelectropherogramwasfor7days(lanesathroughi)or1day(lanesjthrough1).protectedfragment.Wedetectedthemajor13Sand12SmRNAs,butnotthe9SmRNA,inallofourcellpopulationsthatcontaintheElagene.However,thelevelsoftheElamRNAvariedmarkedly;thepLeHpa(AdEla)cellshadlowlevelsofElamRNA,whilepLeBcl(AdElab),pMTEla,andpMTElabcellshadprogressivelyhigherlevels.Eventhehighestlevels,however,weremuchlowerthanthosefoundinAd2-infectedHeLacells(trackfhad15-foldlessRNA).Asexpected,theMTpromoterwassignificantlystrongerthantheviralElapromoter,inducingabout10-to15-fold-higherlevelsofElageneexpression.TheformalequivalenceinfocusformationofthepresenceoftheMTpromotertothepresenceofregionElbwasalsoreflectedinthisassay,sincethepresenceoftheElbgeneresultsinfour-tofivefold-higherlevelsofElamRNA.TheeffectsoftheMTpromoterandtheElbgeneonElaexpressionappeartobeindependentandadditive.Weanalyzed[35S]methionine-pulse-labeledproteinsfromthesecellsbyimmunoprecipitationwithanantibodyraisedagainstasyntheticpeptidehomologoustotheCterminusoftheEla289Rand243Rproteins(Fig.5).ThisantiserumspecificallyimmunoprecipitatesfourpolypeptidesfromAd2-infectedHeLacellsthatmigratewiththefourpolypeptidessynthesizedinvitrofrompurifiedElaRNA(datanotshown).EachofthetwomajorElamRNAsgivesrisetotwopolypeptideforms,presumablyowingtoposttranslationalmodification(27).Inadditiontononspecificprecipitationofseveralcellularpolypeptides,theanti-Elaserumspecifically(thereactionisblockedbypreincubationoftheantiserumwiththesyntheticpeptide)precipitatesthefourexpectedpolypeptidesfromtheEla-transfectedcells.AlthoughthepresenceofhostcellpolypeptidesintheElaregionofthegelmadequantitationdifficult,theresultswerequalitativelysimilartothosefoundwithmRNA.CellstransfectedbyVOL.6,1986

1258SENEARANDLEWISC-,......fabcdefghiFIG.6.AnalysisofDNAfromtransfectedcells.DNAfromtransfectedcellpopulations(lanesathroughf)orplasmidDNAs(lanesgthroughj)weredigestedwithEcoRIandHindIII,analyzedbyagarosegelelectrophoresis,transferedtonitrocellulose,andhybridizedtoanick-translatedprobecontainingtheentireElacodingregion(N494toN1569).AutoradiographyofcellularDNAsamples(lanesathroughf)wasfor22h;autoradiographyofplasmidDNAsamples(lanesgthroughi),whichcontainedapproximately10moleculesofplasmidDNApercellDNAequivalent,wasfor110min.pMTElabsynthesizedthehighestlevelofElaprotein,withlessincellstransfectedwithpMTElaandstilllower(notmeasurableowingtothebackgroundofnonspecificallyprecipitatedcellularproteins)amountsincellstransfectedbypLeBcl(AdElab)andpLeHpa(AdEla).ThehigherlevelsofElaRNAandproteinseenintrans-formedcellsthatcontaineithertheMTpromoterorregionElbaremostprobablyduetohigherlevelsofexpressionofthecotransfectedElagene(s)inducedbyeitherofthesetwoelements.Toeliminateanalternativepossibility,thatthetransfectionfrequencyofElagenesishigherinthepresenceofElbortheMTelements,SouthernblotanalysisforElaandElbsequenceswasperformedonseveralpopulationsoftransfectedcells(Fig.6).AnaverageofonetotwocopiesofElsequencespercellwerecontainedineachpopulation,withnomoreElaDNAinMTElaorAdElabthaninAdElacellpopulations.Thus,increasedgenedosagecannotaccountforincreasedlevelsofElageneproductsinthefirsttwocasescomparedwiththelast.DISCUSSIONEfficiencyoftransformationbyadenoviraloncogenes.Wehaveexaminedthetwo-stepmodelofadenoviraltransfor-mationbyaskingwhichAd2Elgenesaresufficienttoproducefociofcellsalteredinmorphologyandgrowthpropertieswhentransfectedintoestablishedrodentcells.Insomeexperimentstheappearanceoffocithatescapedthegrowthrestrictionsimposedbythemonolayerwasassayed.Inothers,cellswerecotransfectedwithElplasmidsandaplasmidcontainingaselectablemarker,andthepropertiesofthoseG418-resistantorTK+coloniesthatalsoappearedmorphologicallytransformedwereassayed.Althoughtrans-formationfollowinginfectionbyAdortransfectionofAdDNAisinefficient,thehighpercentage(30to90%)ormorphologicallytransformedcoloniesseenwithappropriateElconstructionsintheexperimentsinvolvingcotransfectionwithadominantselectablemarkerdemonstratesthatAdElgenesarecapableofefficienttransformationwhentakenupbycellscapableofexpressingforeigngenesandarethusdominanttransformingoncogenes.Consequently,thelimit-ingfactorintransformationbyAdislikelytobeviralfunctionsinvolvedininsertingviralDNAintothecellinawayinwhichitcanbeefficientlyexpressed,ratherthanthefunctioningoftheEloncogenesonceexpressed.EvidencefortheroleoftheformerfunctionsinviraltransformationisprovidedbygroupIIhostrangemutants,whicharedefec-tivefortransformationbyvirusbutwildtypefortransfor-mationviatransfection(18).ThepolyomamiddleTantigenhassimilarlybeenshowntobeamuchmoreefficientoncogenewhenintroducedviameansotherthanpolyomavirusinfection(5).IncreasedtransformingcapacityoftheElageneexpressedathighlevels.Thelimitedtransformationcapabilityprevi-ouslyascribedtoEla(10)onthebasisofitsinabilitytofullytransformprimaryrodentcellsisreflectedintheseresultsbytheinabilityofthesameElasequencestoefficientlytrans-formestablishedrodentcelllines.AcomparisonofthecapacitytotransformcellsofplasmidsinwhichAdElagenesarecontrolledbyhomologous(Ad)upstreamse-quencesandbyplasmidscontrolledbyheterologous(MT)upstreamsequencesdemonstratesthatthislimitedcapabilityoriginatesfromtheregulationofElaexpression,notfromintrinsiclimitationsofElageneproducts.ThustheAdElaconstructionexhibitedverylimitedcapacityformorpholog-icaltransformation,whiletheMTElaconstructionwasessentiallyascapableaseithertheAdElaborMTElabconstructions.WehavenotprovedthattheMTElagenewillsubstituteforElabinallaspectsofmorphologicaltransformation;however,thepropertiesthatwehaveinves-tigatedaddressamajorfractionofthecharacteristicsthatwereattributedtoAdtransformation.ThecellstransformedbyMTElaandthecellstransformedbytheentireElregionshareatleastfourmajorcharacteristicsofadenovirustrans-formedcells:(i)theyproducecellsthatovergrowthemonolayer,(ii)theyhavesimilarlyalteredmorphology(muchsmallerandrounded),(iii)theyhaveagreatlyin-creasedsaturationdensity,and(iv)theyhaveagreatlyincreasedcapacityforgrowthinsoftagar.NoneofthesegenesappearedtoappreciablyaffectthegrowthrateofNIH3T3cells,sothattheproductionoffocicannotbeeasilyattributed.toasimpleincreaseingrowthrate.Characteris-ticsofAd-transformedcellsthatwehavenotexaminedincludealteredserumrequirements,growthatlowcalciumlevels,changesinfibronectinorothercellsurfacemolecules,andtumorigenicityofcellsinvivo.Manyofthesecharac-teristics,however,includingtumorigenicity,arenotuniver-sallyattributesofAd-transformedcells,evenincludinglinesofepitheloidmorphologyderivedfromvirusinfectionofprimaryratcells(7).Furtherstudiesareclearlyneededtodeterminewhetherappropriatecellsmorphologicallytrans-formedbyElaalonehavethesamespectrumofthesecharacteristicsasdocellstransformedbybothElaandElb.OursuppositionthattheMTElachimerasexhibitgreatercapabilityfortransformationbecauseofhigherlevelsofElaexpressionwasconfirmedbySinucleaseanalysisofElamRNAandbyradioimmuneprecipitationofElaproteinsinpopulationsoftransformedcells.Althoughtheimmunopre-cipitationexperimentsarenotassensitiveastheRNAdeterminations,bothapproachesdemonstratethattransfor-mationefficiencycorrelateswithincreasedlevelsofexpres-MOL.CELL.BIOL.

ADENOVIRUSElaGENEEXPRESSIONANDTRANSFORMATION1259sionuptothelevelseenwitheitherMTElaorAdElab.ThefurtherincreaseinexpressionseenwithMTElabisnotcorrelatedwithafurtherincreaseintheefficiencyoftrans-formation,presumablyreflectingaplateauofmaximumeffectofElageneproductsupontheprocessoftransforma-tion.TheseexperimentsalsoindicatethatatleastoneeffectofthepresenceofElbgenesistoincreasethelevelofElageneexpression,sothatbothincreasedtransformationandincreasedElaexpressioncanbeachievedeitherbythepresenceofElborbyregulationbyMTsequences.ThemodeloftransformationfavoredbyourresultsisthatalthoughElamaybecapableofimmortalizingprimarycellsatlowlevelsofexpression,itisalsocapableofmorpholog-icallytransformingestablishedcelllinesatahigherlevelofexpression.WehavenotyetdoneexperimentstomeasurethelevelsofElarequiredforimmortalization,butvandenElsenetal.(24)havereportedthatsomecelllinespartiallytransformed(immortalized)byElaalonehaveunusuallylowlevelsofElaRNA.Montelletal.(16)recentlydemonstratedthatbothoftheElaproteins(289Rand243R)areinvolvedintheprocessoffulltransformationbyAd.AlthoughexpressionofbothtypesofElaproteinisincreasedbytheMTpromoter,wedonotyetknowwhetherincreasedexpressionofonlyonewouldbesufficient.ThehigherlevelofElageneexpressionthatoccurredwiththeMTpromotercouldreflecteitherahigherintrinsicstrengthoftheMTpromoterthantheElapromoterordifferencesinthemannerinwhichthetwopromotersrespondtolevelsofthetwoElaproteins,whichhavebeenshowntoregulatetranscriptionbothpositivelyandnega-tivelyfromavarietyofpromoters,includingtheElapro-moter(3,16).AnissuethatWehavenotyetaddressediswhethertheimportantfactorinenhancedtransformationis(i)theoverallincreaseinthelevelofElageneexpression,(ii)achangeinthelevelofElaexpressionwhenthecellisataparticularpointofthecellcycle,(iii)changesinElaexpressionwhencellgrowthisinhibitedbyculturecondi-tions.Implicationsforthetwo-stepmodeloftransformation.Theresultsdescribedaboveareinconflictwiththesimplestformofthetwo-stepmodeloftransformation,whichsuggestthatseparateclassesofgenesareresponsibleforinductionofimmortalizationandofmorphologicaltransformation.Al-thoughElageneproductsmaybeintrinsicallymoreactiveasimmortalizingagents,theyarealsoactiveinmorpholog-icaltransformation.Similarcrossoverinactivitybetweenthetwoclassesofoncogeneshasbeenobservedforotherprototypicaloncogenes.SpandidosandWilkie(20)andYoakumetal.(28)reportedthatappropriaterasgenecontainingplasmidscan,inatleastsomecelltypes,producebothimmortalizationandmalignanttransformation.Simi-larly,Keathetal.(12)demonstratedthattheintroductionintoNIH3T3orRat2cellsofthec-myconcogenelinkedtoviralpromoterscouldinducetumorigeniccelllines.Theseresults,aswellasoursandthoseofLandetal.(13)andRuley(19),arenotallnecessarilydirectlycomparable,sincetheremaybeimportantdifferencesbetweendifferentcelltypeswithrespecttogenefunctionsnecessaryformorpho-logicaltransformation.Forexample,itwillbeofespecialinteresttodeterminewhetherhighlevelsofexpressionofElawillalsomorphologicallytransformprimarycells.OnecouldimaginethatNIH3T3cellsarenotonlyimmortalizedbutalsopartiallytransformed,sothatenhancedlevelsofElamightprovidethefunctionsneededtomorphologicallytransformthesecells,butnotprimarycells.Takentogether,theseresultssuggestthatthedivisionofthetransformationprocessintoimmortalizationandmorphologicalchangesisartificialandatleastpartiallymisleading.Rather,thesetwotypesofphenotypicchangemayactuallyrepresentdifferentpartialdisruptionsofasinglemechanismregulatingcellulargrowthandstructure.ModulationofElaexpressionbyElb.AmajorimplicationoftheresultsdescribedaboveisthatthepresenceoftheElbgenesignificantlyincreasesthelevelofexpressionofElageneproducts,thereverseofthewell-establishedroleofElainenhancingtheexpressionofElb(2,11).ThiseffectofElbonElaexpressioncorrelateswithanenhancementoftrans-formingactivityandmayrepresentaroleofElbintrans-formation,ashasalsobeensuggestedbyvandenElsenetal.(24).ThepresenceoftheElbgenecouldincreaseElageneexpression,eitherbecauseofthepresenceofcis-actingregulatorysignalscontainedwithintheElbregion,orbe-causeofatrans-actinginteractionofanElbproteinwithElaregulatorysignals,locatedeitherupstreamof(9)orwithinthetranscribedportionoftheElagene(17).Prelim-inaryresultsfromtransientexpressionexperimentsper-formedwithbothNIH3T3andHeLacellsdemonstratethatatleastalargefractionofthestimulationofElageneexpressionbycotransfectionofElboccursevenifElaandElbgenesareonseparateplasmids,andisthusduetoatrans-actingElbfunction(unpublisheddata).However,cis-actingsequencesmayalsocontributetotheeffectofElbuponEla.Wecurrentlydonotknowwhetherthiseffectisattheleveloftranscription,RNAprocessing,ormRNAstabi-lization.RoleofElbintransformation.Finally,wedonotwishtoimplythattheElbgenenecessarilyplaysnoroleintrans-formationbeyondregulatingElaexpression.Resultsofpreliminaryexperimentswithplasmidsinwhicheitherthe175orthe495codonreadingframeofElbhasbeeninterruptedsuggestthatdifferentElbgeneproductsarerequiredforthecomplementationofElainthetransforma-tion(focusformation)assayandforthestimulationofElaexpressioninthetransientexpressionassay(unpublishedresults).Furthermore,althoughthecellstransformedbythecooperationofElaandElbgenesandthosetransformedbytheMTElagenearesimilarwithrespecttofourmajorfeaturesofmorphologicaltransformation,moredetailedstudiesmayrevealsomedifferencesbetweenthetwocelltypes.Alternatively,thetwotypesofcellsmaybetrans-formedbyverydifferentmolecularmechanismseveniftheirphenotypesaresimilar.TheElbgenemayhaveactivities(otherthantheregulationofElaexpression)thatarenor-mallyimportantfortransformation,butwhichbecomedis-pensableifElaproteinsareexpressedatsufficientlevels,muchasmycandrasoncogenescancooperatetoinducefulltranformationwhenexpressedatlowlevels,butcanappar-entlyactindependentlywhenexpressedathighlevels.Therelativeimportanceofthetwosetsofgenesmayalsodependonthecellularenvironmentinwhichtheyfunction.ElaandElbgenesmaywellhaveevolvedasasinglefunctionalunitandcooperatenotonlybyregulatingtheexpressionofeachotherbutalsobyworkingtogetherinotherways.ACKNOWLEDGMENTSWethankNiallGlanvilleforprovidingNIH3T3cellsandforinstructioninthecalciumphosphatetransformationmethod,WilliamToppforprovidingRat2cells,GaryStuartandRichardPalmiterforprovidingtheMTpromoterfragment,BruceStillmanforprovidingpLA1,TeresaMilloyforperformingtheimmunoprecipitationVOL.6,1986

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