Mechanism of l-^-D-Arabinofuranosylcytosine-induced Cell Lethalityl

MyronKaron,2WilliamF.Benedict,andNatalieRuckerDivisionofHematology,DepartmentofPediatrics,Children'sHospitalofLosAngeles,andtheUniversityofSouthernCaliforniaSchoolofMedicine,LosAngeles,California90054SUMMARY1-0-D-Arabinofuranosylcytosine-induced celllethalityasmeasuredbysurvivalfractioninaplatingefficiencyassaywiththeuseofculturesofhamsterfibroblastscorrelateswiththeproductionoffiveormorechromatidbreakspermetaphase.BycontrastneitherthedegreeofinhibitionofDNAsynthesisnorthemagnitudeofunbalancedgrowthasdeterminedbycellsizingproducedbyavarietyof1-0-D-arabinofuranosylcytosinedosagescorrelatedwithcelldeath.Chromatidbreakagecanthereforebeusedasasensitivemeansofassessingcellcytotoxicity.INTRODUCTIONara-C3inhibitsDNAsynthesisinseveraltissueculturesystems(3,14,17,22).Atleast3molecularmechanismshavebeendescribedthatcouldexplaintheara-Ceffect:inhibitionofthereductionofCDPtodCDP(4);incorporationintoDNAandRNApolynucleotides(5,11,22);andinhibitionofDNApolymerase(9,11,18).Althoughdeoxycytidinecanpreventthetoxiceffectsofara-Cinvivowhenaddedsimultaneously withara-C,deoxycytidinedoesnotreversetheseeffects(8).MooreandCohen(20)demonstrated that5'-l-0-D-arabinosylcytosinediphosphatewasnomoreeffectiveininhibitingribonucleotidereductioninaninvitrosystemfromarattumorthanwasdCDP.ThisevidenceindicatesthattheinhibitionoftheribonucleotidereducA­asewasprobablynotthemajorsiteofactionofara-C.Ontheotherhand,ara-CTPwasacompetitiveinhibitorofdCTPforDNApolymerase,andconsequentlyitisthisenzymethatisprobablytheprimetargetfortheactivityofara-C(18,19).Similarconclusionshavebeendrawnfromworkinhumantissueculturecelllines(13).ThreedifferentDNApolymeraseactivitieshavebeendescribed(12,21).Studiesinbacterialsystemsindicatethatara-CdoesnotinhibitDNApolymeraseI(12),aDNArepairenzyme,butdoesinhibitDNApolymeraseII(21),anenzymeinvolvedinDNAreplication.Inspiteofthefactthattheprimeenzymatictargetofara-CisaDNApolymerase,celllethalitydoesnotbeara1-to-lrelationshipwiththeinhibitionofDNA'SupportedbyNIHGrantsCA11050-05fromtheNationalCancerInstituteandT-493fromtheAmericanCancerSociety.2ScholaroftheLeukemiaSocietyofAmerica,Inc.3Theabbreviationusedis:ara-C,l-/3-D-arabinofuranosylcytosine.ReceivedJune20,1972;acceptedAugust22,1972.synthesis(11,16).Suchinhibition,aconsequenceofthetreatmentofalmostanycellculturesystemwithacytotoxicagent,canthereforerarelybeusedpersetoexplainthemechanismofcelllethality.ToexplorehowacompoundthatinhibitsDNAsynthesissuchasara-Cmightcausecelllethality,westudiedtherelationshipofchromatidbreaksinducedbythisdrugtothesurvivalfraction.LossofproliferativecapacitycouldbecorrelatedwithinductionofchromatidbreakagebutnotwithDNAinhibitionorunbalancedgrowth.MATERIALSANDMETHODSTissueCultureCells.ThetissueculturecellsemployedintheseexperimentswereoftheDon-Clineofhamsterfibroblastsdescribedpreviously(15).CellsweregrowninmonolayerculturesinMcCoy'sMedium5Aand20%fetalcalfserum.ThephasesoftheDon-Ccellcyclehavebeendeterminedbyradioautographicmeans:GAŒ,3.9hr;S,6.2hr;G2,2.2hr;M,0.7hr;andTc,13hr(15).TheEffectofara-ConDNASynthesis.Duplicateculturesgrowingin30-mmPetridishesweretreatedwithara-C,1,10,or100jug/ml.Atvariousintervalsfollowingtheadditionofdrug,thecellswereexposedtoa30-minpulseoftritiatedthymidine, 1juCi/ml(specificactivity,6.7Ci/mmole).Followingincubationthereactionwasstoppedby3washeswith0.9%NaClsolutioncontainingthymidine,10/ug/ml,and0.008Msodiumazide.ThecellsweredislodgedfromthedishesbyuseofarubberpolicemanandwashedontoWhatmanGF/CFiberglasfilterswith20ml0.9%NaClsolution.Thefilterswerethentreatedwith20mlofcold5%trichloroaceticacid,dried,andcountedforradioactivityinaPackardliquidscintillationspectrometer,withtheuseofatoluene-containing phosphor.Theuptakeofradioactivethymidinewascomparedtothatobtainedincontrolculturesnottreatedwithara-C.SurvivalFractionFollowingExposuretoara-C.Thesurvivalfractionwasdeterminedbycomparingplatingefficiencybetweenculturesderivedfromuntreatedcellsandthosederivedfromtreatedcellsbymethodsdescribedpreviously(15).Inbrief,cultureswereexposedtoara-C,1,10,and100/Lfg/ml,for1,4,and8hr.Thesecultureswerewashedtwicewith rinsemedia, trypsinized with0.075% trypsin(WorthingtonBiochemicalCorp.,Freehold,N.J.),dilutedwithfreshmedium(1:1000to1:10,000),andplatedinplasticT-15flasks(FalconPlasticsCo.,Oxnard,Calif).Eachflaskcontains200to1,000cellsin5mlofmedium.Fivereplicate2612 CANCERRESEARCHVOL.32

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