Morphological Transformation, Focus Formation, and Anchorage

43558_75752_full.pdf [CANCERRESEARCH47,5752-5757,November1,1987] MorphologicalTransformation,FocusFormation,andAnchorageIndependence InducedinDiploidHumanFibroblastsbyExpressionofaTransfectedH-rasOncogene1 PeterJ.Hurlin,DennisG.Fry,VeronicaM.Mäher,andJ.JustinMcCormick2

CardnogenesisLaboratory,DepartmentsofMicrobiologyandBiochemistry,MichiganStateUniversity,EastLansing,Michigan48824-1316

ABSTRACT

InanattempttodeterminehownormalhumanfibroblastsrespondtohighexpressionoftheT24H-rasoncogene,wetransfectedsuchcells

withtheplasmidvectorpHO6Tl(D.A.SpandidosandN.M.Wilkie,

Nature(Lond.),310:469-475,1984),containingtheT24H-rasoncogenewith5'and3'enhancersequences,andtheaminoglycosidephosphotrans-

ferasegenewhichconfersresistancetothedrug,G418.Approximately

1.5%oftheG418-resistantcoloniesobtainedaftertransfertionand

selectionconsistedofcellsexhibitingobviousmorphologicaltransfor mation;i.e.,theywerehighlyrefractileandmoreroundedthannormal

fibroblasts.DNAhybridizationanalysisshowedthatthemorphologicallytransformedcellscontainedthetransfectedT24H-rasoncogene,and

radioimmunoprecipitationanalysisshowedthattheywereexpressingthe T24H-rasproteinproduct,M,21,000protein.Morphologicallytrans formedcellsformedcoloniesinsoftagaratafrequencyatleast60times higherthanthatofcellsthathadbeentransfectedwiththecontrol plasmidcontainingthenormalcellularII-raxgene.Cellstransfectedwith

plasmidpHO6Tlcouldalsobeidentifiedbytheirabilitytoformdistinctfociwhengrowntoconfluenceinnonselectivemediumfollowingtransfer-

tion.ThisstudydemonstratesthatnormaldiploidhumanfibroblastsinculturecanbetransformedbytransfectionwithaH-rasoncogene,and

thatsuchtransformationcorrelateswithexpressionofthemutantM,

21,000protein.

INTRODUCTION

Asignificantproportionofhumantumorsfromvarioussites

inthebodyhavebeenshowntocontainactivatedoncogenesfromtherasfamily(1-3).Thisfinding,whichsuggeststhatthe

rasoncogenesareinvolvedincausingsuchtumors,has promptedinvestigationsintotherolerasoncogenesplayin bringingabouttransformationofcellsinculture.Transfection studiesusingprimaryorearlypassagerodentfibroblastsas recipientsofrasoncogenesindicatethatthesegenesactina

dominantmannertocausetransformation(forreview,seeRef.4).Thetransformedphenotypesobservedinrasoncogene-

transfectedrodentfibroblastsincludemorphologicalalteration

(5,6),focusformation(6),anchorageindependence(5,8,9),and,insomeinstances,tumorigenicity(6-9).Severalofthese

studiesindicatethatthelevelofexpressionoftherasoncogene

canbeanimportantfactorgoverningthedegreeoftransformationbyrasoncogenes(7-9)andthathighexpressionofthe

transfectedrasoncogeneisrequiredfortheinductionofthetumorigenicphenotypeinsuchfibroblasts(6-9). Incontrasttotheresultsachievedusingrodentfibroblasts, theresultsofmostDNAtransfectionstudiesinwhichearly passagediploidhumanfibroblastswereusedastherecipients suggestedthatthesecellsaremoreresistanttotransformation

Received2/16/87;revised7/6/87;accepted7/29/87.

Thecostsofpublicationofthisarticleweredefrayedinpartbythepayment ofpagecharges.Thisarticlemustthereforebeherebymarkedadvertisementin

accordancewith18U.S.C.Section1734solelytoindicatethisfact.1ThisresearchwassupportedinpartbyDepartmentofEnergyGrantDE-

FG02-87-ER60524andbyDepartmentofHealthandHumanServicesGrant CA21289fromtheNationalCancerInstitute.D.G.F.isarecipientofaLeukemia

SocietyofAmericaSpecialFellowshipAward.2Towhomrequestsforreprintsshouldbeaddressed,atCarcinogenesisLab

oratory,FeeHall,MichiganStateUniversity,EastLansing,MI48824-1316.byraÃoncogenes.Forexample,severalgroupshavereported

that,followingrasoncogenetransfection,humanfibroblasts failedtoexhibitmorphologicaltransformation(9,10),focus

formation(10),anchorageindependence(9),ortumorigenicity(9-11).Sutherlandetal.(11)reportedthattransfectionof

humanfibroblastswiththeT24H-rasoncogeneinaplasmid devoidoftranscriptionalenhancersequencescausedthetarget populationtoexhibitaveryhighfrequencyofcellsabletoform coloniesinsoftagar.Sageretal.(10,12)reportedthat,follow ingtransfectionofnormalhumanfibroblastswiththesame

oncogene,butinaplasmidwhichcontainsSV40transcriptionalenhancersequences,expressionoftheH-rasp213couldbe

detected.However,thisdidnotresultinmorphologicaltrans formation,focusformation,ortumorigenicity.Theseinvesti gatorsdidnotassaythetransfectantsforanchorageindepend ence.

Anexplanationforthenegativeresultswithhumanfibro

blasts,suggestedbythetransfectionstudieswithanimalfibro blasts,isthatthetransfectedrasoncogenewasnotexpressed orthatthelevelofexpressionoftherasoncogenewasnothigh

enoughtocausemeasurableeffects.InanattempttoachievehighexpressionoftheT24H-rasoncogeneinearlypassage,

diploidhumanfibroblasts,weusedaplasmid,pHO6Tl,ob tainedfromSpandidosandWilkie(8),inwhichtheoncogene isinsertedbetweentwosetsoftranscriptionalenhancerse quences.Weherereportthatthetransfectantsexhibitedtrans formationtomorphologicalalterationandfocusformation, thatprogenyofmorphologicallyalteredcoloniesexhibitedan chorageindependence,andthatsuchtransformationwascorrelatedwithexpressionoftheT24H-rasoncogeneprotein productp21inthehumanfibroblasts.

MATERIALSANDMETHODS

CellsandCultureConditions.Diploidhumanfibroblastswereiniti atedfromforeskinsofclinicallynormalnewborninfantsasdescribed previously(13).ThehumanbladdercarcinomacelllineT24waspur chasedfromtheAmericanTypeCultureCollection(Rockville,MD). EMEM,supplementedwith0.2mMserine,0.2miviaspartate,1.0mM pyruvate,10%FBS(GrandIslandBiologicalCo.,GrandIsland,NY), penicillin(100units/ml),andstreptomycin(100Mg/ml)(complete

EMEM),wasusedforroutineculturingofcells.Cellsweregrownat37°C,in5%CO2,water-saturatedincubators.

Plasmids.Homer6plasmids(8)containingtheT24H-rasoncogene (pHO6Tl),thecellularH-rasgene(pHO6Nl),ornoadditionalse quences(pHO6)werekindlyprovidedbyDr.NeilWilkie.These plasmidscontaintheselectablemarkergene,aminoglycosidephospho- transferase,whichconfersresistancetothedrug,G418. DNATransfection.ThePolybrene/DMSOmethod,adaptedfor

transfectionofdiploidhumancellsbyMorganetal.(14),wasused.Briefly,1xIO5cells(passages7to10)wereplatedinto100-mm-

diameterdishesincompleteEMEMandincubatedat37°C.After16 'Theabbreviationsusedare:p21,M,21,000protein;DMSO,dimethylsulf- oxide;EMEM,Eagle'sminimalessentialmedium;FBS,fetalbovineserum; StaphAbuffer,0.2Mphosphatebuffer(pH7.4)-1%TritonX-100-0.1%sodium dodecylsulfate-0.5%sodiumdeoxycholate-0.1%NaNj-0.1MNaCl.

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TRANSFORMATIONOFHUMANCELLSBYT24H-ras

to24h,themediumwasremovedandreplacedwithtransfection medium,i.e.,2.5mlofcompleteEMEMcontaining7.5ngofPolybrene

perml(SigmaChemicalCo.,St.Louis,MO)and1.2to2.0u%ofplasmidperml,andthedisheswerereturnedto37°C.Thetransfection

mediumwasdistributedoverthecellsbyshakingonceeveryhour.After

6hthetransfectionmediumwasremoved,andthecellsweretreated

for4minatroomtemperaturewith5mlofcompleteEMEMcontaining30%freshlydistilledDMSO.FollowingDMSO"shock"thecells

wererinsed2timeswithEMEM,refedwithcompleteEMEM,andincubatedat37°C. SelectionofG418Resistance.Ifthetransfectedpopulationwereto beselectedfordrugresistance,24hafterDMSOshockthemedium wasexchangedforcompleteEMEMcontaining200ngofactiveG418 (GIBCO)perml.Colonieswereallowedtodevelopfor14days,with onerefeedingwiththisselectivemedium. AssayforFocusFormation.Ifthetransfectedcellsweretobeassayed fortheabilitytoformmacroscopicfocipiledupontopofamonolayer ofcontact-inhibitedcells,theywerenotselectedforG418resistance,

butinsteadwereallowedtogrowtoconfluenceincompleteEMEMfollowingDMSO"shock."Thecellswerefedonceperweekfor3wk

andthenstainedwithméthylèneblue,andthefociwerecounted.AssayforAnchorageIndependence.Cellsweretrypsinizedandresus-pendedinamodifiedversionofHam'sMCDB110(15)formulatedin

thislaboratoryforusewithserumreplacementsinstudiesrequiring absenceofserum(16).Forthepresentassay,1%FBSwassupplied. Thecellswerecountedelectronically(CoulterCorp.,Hialeah,FL)and platedintotopagaratadensityof4000cellsperml.Thetopagar consistedofthemodifiedMCDB110mediumsupplementedwith1% FBSand0.33%SeaPlaqueagar(FMCCorp.,Rockland,ME).The bottomagarwaspreparedwiththesamemedium,butsupplemented with2%agar.Onemloftopagarwasplatedper35-mm-diameterdish ontopof2mlofsolidifiedbottomagar.Thefollowingdayandweekly thereafter,1mlofmodifiedMCDB110mediumwith1%FBSwas addedtothetopagartocompensateforevaporation.After3wkthe numberofcolonieswithadiameterlargerthan60Mmwasdetermined microscopically. SouthernBlotAnalysis.GenomicDNAwasisolatedaspreviously described(17).DNAwasdigestedwithrestrictionenzymesunder conditionsrecommendedbythesupplier(NewEnglandBiolabs,Bev erly,MA).TenngofdigestedgenomicDNAor5.6^gofplasmidDNA wereelectrophoresedin0.8%agarosegelsandtransferredtonylon filters(GeneScreenPlus;NewEnglandNuclear,Boston,MA)using standardtechniques(18).Nylonfilterprehybridization,hybridization,

andhighstringencywashingconditionswerecarriedoutasrecommendedbythesupplier.The2.9-kilobaseSadfragmentoftheT24fi-

rasoncogene,nicktranslatedwith32P-labeleddeoxyribonucleoside

triphosphates(NewEnglandNuclear,Boston,MA;600Ci/mmol)toaspecificactivityof1to2x10scpm/Vg,wasusedforhybridization.

FilterswereexposedtoKodakXARX-rayfilm(Kodak,Rochester,

NY)withtheaidofintensifierscreens(CronexLightningPlus;Dupont,Inc.,Wilmington,DE)for72hat-70°C.

RNAAnalysis.CytoplasmicRNAdotblotswereperformedasde scribedbyWhiteandBancroft(19). Detectionofp21.Proteinlabelingandimmunoprecipitationwere carriedoutessentiallyasdescribedpreviously(20,21)withthefollow ingmodifications.Cellsgrowingexponentiallyinthemodifiedversion

ofMCDB110mediumsupplementedwith10%FBSwereplatedinto100-mm-diameterdishesatId"cells/dish.After24hthemediumwas

exchangedfor3mlofthismediumlackingmethionineandFBS,but

supplementedwithserumreplacements(16).After45min,1.0mCiof["Sjmethionine(1148Ci/mmol;NewEnglandNuclear,Boston,MA)

wasadded,andthecellswereincubatedfor24h.Cellswerelysedwith

1.0mlofSiapiiAbuffercontaining2.0HIMphenylmethanosulfonyl

fluoride(SigmaChemicalCo.,St.Louis,MO)and135kallikrein inactivatorunits/mlaprotinin(Sigma).Lysedcellswerecentrifugeaat

35,000rpminaBeckman50Tirotorfor35min.Thesupernatant,containing10*cpmoftrichloroaceticacid-precipitablecounts,was

immunoprecipitatedfor2hat4°CwiththemonoclonalantibodyY13-

259(22),kindlysuppliedbyDr.M.Anderson.ProteinA-Sepharose

(PharmaciaChemicalCo.,Piscataway,NJ)coatedwithgoatanti-ratTable1Frequencyoftransformationtoalteredcellularmorphology

andfocusformation

PlasmidpH06pHO6NlpHO6TlNo.ofdishesused

intrans

fection"2210119TotalG418-resistantcoloniesdetected5731752461G418-resistantcolonies/10Â"cellstransfected*260175201Morphologicallyalteredcolonies/IO6

cellstransfected003Foci/10*cellstransfected'0039 "Cells(10')per100-mm-diameterdishweretransfectedwith3/igofplasmid DNA.*Determinedafter2wkofG418selectionfollowingtransfection. cDeterminedafter2wkofgrowthincompleteEMEMfollowingtransfection.

IgG(CooperBiomedicai,Malvern,PA)wasadded,andthemixturewasincubatedfor30minat4°C.Themixturewascentrifuged,the

supernatantwasremoved,andtheSepharosefractionwaswashedtwice withStaphAbuffer,oncewithasolutionof50mMTris-Cl(pH7.5J-1

MMgClz,andonemoretimewithStaphAbuffer.Electrophoresissamplebufferwasadded,andthesampleswereheatedat90°Cfor5

min.Thesampleswerecentrifuged,andaliquotsofthesupernatant, alongwithprestainedproteinmolecularweightmarkers(Bethesda

ResearchLaboratories,Bethesda,MD),wereelectrophoresed(23)through12.5%sodiumdodecylsulfate-polyacrylamidegelsfor4hat

200V.Thegelswerefixed,treatedwithautoradiographicenhancer

(Enlightning;NewEnglandNuclear,Boston,MA),dried,andexposedtoX-rayfilmat-70°Cfor24to72h.

RESULTS

FrequencyofTransfectiontoG418Resistance.PlasmidpHO6Tl,whichcontainstheT24H-rasoncogene,waschosen fortransfectionofdiploidhumanfibroblastsbecauseofits demonstratedabilitytotransformearlypassagerodentfibro

blastsintomalignantcells(8)andbecauseinourpreliminaryexperiments,otherplasmidscontainingtheT24H-rasonco

geneprovedtobeineffectiveintransforminghumanfibroblasts.4PlasmidpHO6Nl,whichcontainsthenormal,endog enousH-rasgene,andplasmidpHO6,whichhasnorasgene, wereusedascontrolsinthetransfectionexperiments.Recom binantplasmidswereintroducedintoearlypassage(passages7 to10)diploidhumanfibroblastsusingamodifiedPolybrene/

DMSOtransfectiontechnique.ThistransfectiontechniquewaschosenbecausethefrequencyofG418-resistantclonesobtained

wasshowntobe10to40timesthatachievedusingcalcium phosphate(14).Toincreasetheprobabilitythatcoloniesse lectedforresistancetoG418representeduniquetransfection

events,transfectionandselectionofcellswereperformedinthesameculturedish.ThefrequencyofG418-resistantcolonies

obtainedaftertransfectionofhumanfibroblastswiththevar iousHomer6plasmidsisshowninTable1.EachplasmidconstructionyieldedapproximatelythesamenumberofG418- resistantcoloniesperpgoftransfectedplasmid,i.e.,200per106targetcells. TransformationtoAlteredMorphology.G418-resistantcolo niesweremicroscopicallyexaminedforevidenceofaltered cellularmorphology2wkfollowingtransfectionandselection inG418medium.OnlypHO6Tlinducedmorphologicaltrans formation,asindicatedinTable1.Arepresentativecolonyof morphologicallytransformedcellsisshowninFig.1.The morphologicallytransformedcellsdividedrapidly,werehighly retractile,andgrewinirregularpatterns,ratherthanthehighly orientedpatternofnormalhumanfibroblasts. TransformationtoFocusFormation.Cellstransformedfollow

4P.J.IInrlin.D.G.Fry,V.M.Mäher,andJ.J.McCormick,unpublished

data.

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Fig.1.Comparisonofnormalandtransformedmorphologies,a,normalhumanlibro blasts;b,fibroblaststransfecledwithpHO6Tl.

Confluentcultureswerefixedwithmethanol

andstainedwithméthylèneblue,x100.TRANSFORMATIONOFHUMANCELLSBYT24H-ras-'M

,,^/VK,||'r,'-*¿•'¿'•'•••rï.<'f*.•"''•'

'••••/•-••'-,'*,s'i'f'

'''•••:,<ÃOE'.W' 'rtÂ"'^W^.^'(Â"'A'^/ia'.,M r.';»

Fig.2.Thefocus-formingabilityofpHO6Tl-transfectedfibroblasts.FollowingtransfectionwithpHO6Tl,whichcontainstheT24H-rasoncogene,orpHOóNl,

whichcontainsthenormalcellularH-rasgene,cellswereallowedtogrowfor3wkundernonselectiveconditions.Confluentcellsweretrypsinized,replatedata1:3

dilution,andthenallowedtogrowforanadditional3-wkbeforebeingstainedwithméthylèneblue,a,focigeneratedinadishcontainingpHO6Tltransfretanis;b,

confluentmonolayerinadishcontainingpHO6Nltransfectants;c.x40magnificationofafocusfromthedishshownina.

ingtransfectionwithpHO6Tlcouldbealsoidentifiedbytheir abilitytogrowintoa3-dimensionalarrayofcells,afocus,on topofamonolayerofcontact-inhibitedcells.Thistypeof

aberrantgrowthbehaviorisacommoncharacteristicoftumorcells,includingthosederivedfromhumanfibrosarcomas.4Mac

roscopicfociandanindividualfocusofpHO6Tl-transfected fibroblastssurroundedbyadjacentnontransformedfibroblasts areshowninFig.2.Thenumberoffociobservedper100-mm- diameterdishfollowing3wkofgrowthundernonselective conditionswasapproximately10-foldgreaterthanthenumber ofmorphologicallytransformedcoloniesobservedafter2wk ofG418selection(seeTable1).Nofociweregeneratedfollow ingtransfectionofthecontrolplasmids,pHO6NlandpHO6, whichlacktheoncogene.Microscopicinspectionofthemor phologyofthecellsmakingupthefocusrevealedthatnotall cellscapableofformingfociexhibitedthetransformedmor phology.Thus,thenumberoffociisamoresensitivedetermi nationofthefrequencyoftransformedcellsfollowingtransfec tionwithpHO6Tlthanisthenumberofmorphologically transformedcolonies.

InductionofAnchorageIndependence.Morphologicallytransformed,G418-resistantcoloniesobtainedfollowingtransfec

tionwithpHO6TlandG418-resistantcoloniesobtainedfol lowingtransfectionwiththecontrolplasmid,pHO6Nl,were comparedfortheirabilitytoformcoloniesinsoftagar.The cellpopulationsassayedwerederivedfrompooledcoloniesthat hadbeenisolated24hearlier.Nocolonieswereobservedin

thecontroldishes,representingatotalof8000cellsassayed(frequency,<1.25x10~4).Incontrast,122colonieswitha

diameterequaltoorlargerthan60/¿mwereobservedwiththe oncogene-transformedcells,ofatotalof16,000cellsassayed(frequency,76xIO"4).

Aconcomitantcomparisonoftheabilityofthetwopopula

tionstoformcoloniesonplasticshowedthatthecloning efficiencyofthecontrolcellswastwicethatofthetransformed cells(datanotshown).Therefore,whenmeasuredunderthe stringentconditions(1%FBS)usedinthisassay,cellsexhibit ingthetransformedmorphologyformedcoloniesinsoftagar atafrequencyatleast60timesthatofthecontrolcells. AnalysisforthePresenceofTransfectedSequences.G418- resistantcoloniesthatexhibitedthetransformedmorphology wereisolated,pooledingroupsofseveralclones,andexpanded

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TRANSFORMATIONOFHUMANCELLSBYT24H-ras

forseveralpopulationdoublings.High-molecular-weightDNA wasextractedanddigestedwithrestrictionendonucleasesXbal andHindlll(doubledigestion)toliberatespecificsizefrag

ments(2.5and4.5kilobases)thatarediagnosticforanintactregionofplasmicipHOóTlcontainingtheT24H-rasoncogene,itsendogenouspromoter,andexogenous5'and3'enhancer

sequences(8).TheDNAwasanalyzedbythemethodofSouth ern(18).AsshowninFig.3,cellsexhibitingthetransformed morphologycontainthe4.5-and2.5-kilobasefragments.The intensityofthesetwofragmentswasapproximatelyhalfthatof theendogenousraxsequences,indicatingthatthecellscontain onlyonecopyofthetransfectedrasgene.Wefoundthat,ifthemorphologicallytransformedG418- resistantcellswerepassagedextensively,eveninthepresence ofselectivemedium,theyeventuallyrevertedtoanormalmor phology.Ofthe38morphologicallytransformedcoloniesana lyzed,38revertedtoanormalmorphologywithin25population doublingsfollowingtransfection.Theserevertedcellswereas

sayedfortheabilitytoformfoci,toformcoloniesinsoftagar,andforthepresenceoftheT24H-rasoncogeneanditsprotein

product.Theydidnotformfociorcoloniesinagar.However, Southernblotanalysisshowedthattherevenantsstillpossessed thetransfectedoncogene(Fig.40).ExpressionoftheT24H-rasOncogeneinTransformedCells. Substitutionofvalineforglycineatthe12thaminoacidposition oftheT24//-raj-encodedp21altersitselectrophoreticmobil

ity,makingitdistinguishablefromnormal,endogenousp21(24).SevenindependentG418-resistant,morphologicallytrans

formedclonalpopulationsobtainedafterpHO6Tltransfection and5clonalpopulationsthatrevertedtothenormalfibroblast morphologywereanalyzedforthepresenceofthemutated

formofp21byradioimmunoprecipitationanalysisusingmonoclonalantibodyY13-259(22).Fig.4showsacomparisonof

p21expressioninpHO6Tltransfectantsexhibitingatrans formedmorphology,andincellswhichrevertedtoanormal morphology.Incellstransfectedwiththecontrolvector(Lanes

1and7)andinpHO6Tltransfectantsthatreverted(Lanes3to5),thecharacteristicp21doublet(20)isexpressed(see

dashes).ThepHO6Tltransfectantsexhibitingatransformed morphologyexpressedanadditionalp21speciesthatÂ"»mi gratedwithap21speciesexpressedinthehumanbladder carcinomacelllineT24.Themutantspeciesmigratedimme diatelybelowtheslowermigrating,normallypresentp21spe cies(seearrow).Asimilarpatternofexpressionhasbeen observedinallsevenofthemorphologicallytransformedpop

ulationsanalyzed.Theadditionalp21specieswasnotobservedinanyoftherevertedpopulations,orincontrol,vector(pHO6)-

transfectedpopulationstested. RNAdotblotanalysisofcytoplasmicRNAsamplesindicated

thattransfectantsmaintainingthetransformedmorphologyexpressedhigherthannormallevelsof//-raj-specificRNA.

Fig.5showsthatsamplesfromsuchcells(Lanes2and3)expressedapproximately4timestheamountofH-rasRNA

expressedincontrolcells(vectortransfected;Lane1).ThishigherlevelofexpressionofH-rasRNAcorrelateswithexpres

sionoftheT24-H-rasp21speciesinpHO6Tl-transfected, morphologicallytransformedcells.AnalysisforTumorigenicity.ThreeG418-resistant,morpho logicallytransformedcoloniesobtainedfollowingtransfection

withpHO6Tlwereexpandedintolargepopulationsinselectivemediumandassayedfortumor-formingabilityinathymicmiceasdescribedpreviously(25),exceptthat2xIO6testcellsplus

8XIO6normalhumanfibroblastswereinjectedpermouse.

Threemicewereusedpertest.Notumorsorindicationsof

growthwereobservedoveraperiodofgreaterthan1yr. However,progenyofthemorphologicallytransformedcells revertedtothenormalfibroblastmorphologyduringtheexpan

sionrequiredtoobtainenoughcellsforthisassay.Whentheserevenantcellswerecontinuallypassagedaspartofalife-span

assay,theysenescedatapopulationdoublinglevelcomparable tothatofnormalhumanfibroblastpopulations.

DISCUSSION

Resultsofthepresentstudyshowthatnormaldiploidhuman fibroblastscanbetransformedintomorphologicallyaltered, 1245

Fig.3.Southernblotanalysisofcellular

DNAfrommorphologicallytransformedcells

obtainedfollowingtransfectionwithpHO6Tl.

FollowingtransfectionofpHO6TlandG418

selection,coloniesofmorphologicallytrans formedcellswereisolatedandpooledsothat eachpoolrepresented3to4separateclonal populations.Theclonalpopulationswereexpanded,andhigh-molecular-weightDNAwas extractedfromthemandfromtheparental strainofnormaldiploidhumanfibroblasts usedfortransfection.Ten^gofDNAfromeachsampleweredigestedwithA'Aoland ///'mllll(doubledigest),electrophoresed througha0.8%agarosegel,andtransferredto nylonfilters.Thefilterswerehybridizedwiththe2.9-kilobaseSacIfragmentoftheT24ti rosoncogeneradioactivelylabeledbynick translation,a.LaneI,nontransfectednormal diploidhumanfibroblasts;Lanes2to5,pooled populationsofmorphologicallytransformed cells.Hybridizingfragmentsof4.5and2.5 kilobasesarediagnosticforthepresenceoftransfectedT24H-rasoncogenesequences,b,

Southernblotanalysisofhigh-molecular-

weightDNAextractedfromprogenycellsof thoseanalyzedinFig.4a.butwhichhadre vertedtothenormalfibroblastmorphology.DNAswereanalyzedasabove.Lanes2'to5' aretheprogenycellsfromtherespectivelanesinFig.4Â":kt>.kilobases.kb 4.5-

2.5-2'3'415'

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123456TRANSFORMATIONOFHUMANCELLSBYT24H-ras

789123

Fig.4.Analysisofrasgeneexpression.[3*S]Methionine-labeledcellularex tractswereimmunoprecipitatedwithmonoclonalantibodyY13-259,theimmu- noprecipitateswereelectrophoresedon12.5%polyacrylamidegels,andthegels wereanalyzedbyfluorography.Lanes1and7,cellstransfectedwiththecontrol vector.pHO6;Lanes2and8,thehumanbladdercarcinomacellline,T24;Lanes

3to5,independentclonalpopulationsthatrevertedfromatransformedmor

phologytoanormalmorphologyfollowingtransfectionwithpHO6T1;Lanes6and9,pHO6T1-transfected.morphologicallytransformedcells;Lanes7to9,a

secondanalysisofSamples1,2,and6,respectively.Dashes,endogenousp21doublet;arrows,mutant(T24)H-rasp21. focus-forming,andanchorage-independentcellsfollowing transfectionoftheT24H-rasoncogene.Ourresults,showing thatthetransformedcellsexhibitedanchorageindependence, supporttheresultsobtainedbySutherlandetal.(11)andextend thatstudybyshowingthatthetransformedcellsexpressedthe T24H-rasprotein.However,unlikeSutherlandetal.(11)we didnotuseanchorageindependencetoidentifytransformed populationsofcellsdirectlyfollowingtransfection.Therefore, itisnotpossibletodirectlycompareourfrequencyofanchorage-independentcellswiththeirs.

IftheanchorageindependenceexhibitedbycellstransfectedwiththeT24H-rasoncogeneintheenhancer-freeplasmidused

bySutherlandetal.(11)isanindicationthatthetransfectants wereexpressingthemutantp21,thenitisdifficulttounder standwhySageretal.(10,12),didnotobserveanyevidenceof transformation.Theyusedthesameoncogene,butinaplasmid whichcontainsasetoftranscriptionalenhancersequences,and theyfoundexpressionofmutantp21.Anobviousexplanation forthedifferencebetweentheresultsofSageretal.(10,13) andthosewepresenthereisadifferenceinthelevelofexpres sionofp21resultingfromtheuseofdifferentplasmidconstructionscontainingtheT24H-rasoncogene.Whenwebegan transfectionstudiesusingnormaldiploidhumanfibroblastsas

recipients,likeSageretal.(10,12)weconstructedandusedapSV2-derivedplasmidcontainingtheT24H-rasoncogeneand

found,astheydid,noevidenceofmorphologicaltransformation*•2x105 IxlO5

2.5xl04

6.25xl03

B123 2xl05

Fig.5.H-rasRNAexpression.Cytoplasmicsampleswereextractedfrom8x10scells,denatured,dilutedtotheequivalentcellnumberindicated,andspotted

ontonitrocellulosefilters.I.hybridizationwiththeSad3-kilobaseT24H-rasfragment,nicktranslatedwith"P-labeleddeoxyribonucleosidetriphosphates.

Lane1,cellstransfectedwiththecontrolvector,pHO6;Lanes2and3,cellstransfectedwithpiKHiI1andexhibitingatransformedmorphology.B,hybridizationofthesamesamplesasinAwithaA-rnicroglobulinprobetocontrolfor

variabilityintheamountofcytoplasmicextractloaded. orfocusformation.4Whatismore,therewasnoinductionof anchorageindependence.4Thepositiveresultsreportedinthe presentstudyweregeneratedusingaplasmid(pHO6Tl)that

wasspecificallydesignedbySpandidosandWilkie(8)toelicithighexpressionoftheT24H-rasoncogeneinmammaliancells

byinsertingtheoncogenebetweentwosetsoftranscriptional enhancersequences.Therefore,weinterpretourresultstoin dicatethatasufficientlyhighlevelofexpressionoftheT24ti rasoncogenewasachievedtocausethetransformationof normalhumanfibroblasts,andthatthepSV2-T24plasmidof Sageretal.(12)wasnotcapableofgeneratingsuchlevels.This interpretationdoesnotexplainthenegativeresultsofSpandi dos(9),whorecentlyreportedthattransfectionofnormal humanfibroblastswithpHOoTIdidnotcauseanchoragein dependenceoranyobviousmorphologicaltransformation. However,Spandidosusedadifferentmethodoftransfection (calciumphosphate)thenwedid(DMSO/PoIybrene)andex aminedarelativelysmallnumberofindependentG418-resist- antcolonies(~200)comparedtothetotalnumberweexamined (>2461). Theresultsofnumerousrasoncogenetransfectionstudies carriedoutwithrodentfibroblastssuggestthathighexpression oftheT24H-rasoncogenecanbeattained,andthatwithsuch expressionthetumorigenicphenotypeisinduced(6-9).Our studieswithhumanfibroblastsindicatethatthetransfected oncogeneisexpressedatlowlevels,comparedtowhathasbeen observedinrodentfibroblasts,andthatsuchexpressionislost asthecellsarepassaged.Becauseoftheeventuallossof expression,andtheaccompanyinglossofthetransformed phenotypeduringtheextensivepropagationoftheclonaliso latestosufficientsizefortestingtumorigenicity,wecouldnot properlyassessthisphenotypeinpHO6Tltransfectants.How ever,thereisevidencethatrasoncogenesareinvolvedinneo- plastictransformationofhumanfibroblasts.Forexample.

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TRANSFORMATIONOFHUMANCELLSBYT24H-ras

transfectionofdiploidfibroblastsderivedfromBloom'ssyn dromepatientswithDNAfromNIH3T3cellsthathadbeen transformedbyHarveymurinesarcomavirusDNAresultedin cellsabletoformnodulesinathymicmice(26).Thesenodules subsequentlyregressed(26).Also,Harvey(27)orKirstenmu rinesarcomavirus(28)infectionofhumanfibroblaststhathad acquiredanindefinitelifespaninculturefollowingSV40virus transformationorrepeatedexposuretoyradiationresultedin cellsabletoformtumors.Onegroup(27)indicatedthatthe tumorsregressed;theother(28)foundtheydidnot.Three groups(29-31)havesucceededincausingneoplastictransfor mationofhumanepithelialcellsbyrasoncogenetransfection. Furtherstudiestodetermineifdiploidhumanfibroblaststhat continuetoexpresstheT24H-rasoncogenearetumorigenic andwhetherthisgenecooperateswithotheroncogenes(e.g., myc)incausingmalignanttransformationofhumanfibroblasts areinprogress.

ACKNOWLEDGMENTS

WethankDr.NeilWilkieforkindlysupplyinguswiththepHO6seriesofplasmids,Dr.MarshallAndersonfortheY13-259antibody

toH-rasp21,andCarolHowlandfortypingthemanuscript.

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