The
ARTICLESCellResearch(2004);14(1):8l一85h
;ttp://www.cell—research.com
;Thecellcyclerelatedapoptoticsusceptibilitytoarsenictrioxideisassociated ;withthelevelofreactiveoxygenspecies
;FeiGAO,JingYI,JingQiYUAN,GuiYingSHI,XueMingTANG,
;.DepartmentofCellBiology,ShanghaiSecondMedicalUniversity,Shanghai200025,China ;DepartmentofAutomation,ShanghaiJiaotongUniversity,Shanghai20003l,China. ;ABSTRACT
;Doublestainingflowcytometrywasperformedusing7aminoactinomycinDand6carboxy一2‟.7‟
;dichlorodihydrofluoresceindiacetate.todetectthelevelfluctuationofreactiveoxygenspecies(ROS)d
uringthecell
;cycleofnormalNB4cells.OurresultsshowedthatNB4cellspossessedhigherlevelofROSinG2/MphasethaninG1
;andSphases.Doublestainingflowcytometry,withTdTmediateddUTPnickendlabeling(Tune1)andpropidiumiodide
;(PI),indicatedthatAs,O(2LI
朋)couldinduceapoptosisinNB4cellsprevailinglyfromG2/Mphase,andthisemcacy ;wasenhanceduponco—administrationof2,3.dimethoxy1,4.naphthoquinone(DMNQ)(2.5JaM)whichcouldpro
;ducetheendogenousROS.TheseresultssuggestedthatdifferentROSlevelindifferentcellcyclephaseso
fNB4cells
;mightdetermintheselectiveinductionofG2/Mapoptosisandthecells‟susceptibilitytoapoptosisbyAs,
O.
;Keywords:arsenictrioxide,apoptosis,cellcycle,reactiveoxygenspecies(ROS) ;INTRoDUCTIoN
;Arsenictrioxide(As,O1)hasbeensuccessfullyintro—
;ducedintoclinictrialandthusapprovedbyUSFoodand
;DrugAdministration(FDA)fortreatmentofacute
;promyelocyticleukemia(APL)recently[1—61.Althoughit
;hasbeenwidelyacceptedthat,likeAs,O.manychemo.
;therapeuticdrugsfunctionaspro—apoptoticsignalswhich
;cantriggerapoptosisintumorcells.theheterogeneityof
;cellularsusceptibilitytothesecytotoxicagentslimitedtheir
;usageonmoretypesoftumorsinclinic[7.131.
;Reactiveoxygenspecies(ROS),includingsuperoxide
;radical(O2.‟),hydrogenperoxide(H2O2),hydroxylradical
;(OH)andsingletoxygen(O2),continuouslygeneratedfrom
;mitochondrialrespiratorychain.havepowerfuloxidative
;potential,andareknowntobecapableofresultinginthe
;oxidativedamages.Cellspossessanefficientantioxidant ;defensesystem,composedofmainlytheenzymessuch ;assuperoxidedismutase,glutathioneperoxidase.and ;catalase,whichcanscavengetheexcessiveROSandkeep ;itslevelrelativelystableunderphysiologicalconditions『14
;161.Recentdatashowedthatmanypro—apoptoticsignals
;couldraisethecellularROSleve1.andcelluarinherentROS ;levelmaybeassociatedwiththesusceptibilityoftumor ;cellstopro—apoptoticsignals【10—13,l7—29].
;SomeresearchersreportedthatlOWconcentrationf1—2
;mM)ofAs,OpreferentiallyinducedapoptosisinNB4cells ;-Correspondenceauthor:XueMingTANG
;Te1:+86—2l一63846590(ext.776480),Fax:+86—21—53065329
;E—mail:xmtang@shsmu.edu.ca
;ofG2/Mphase『301.0urrecentworkindicatedthatdi
;ferentsusceptibilityofcelllinestoAs,O3-inducedapoptosis ;maypossessdifferentROSlevels.andthussuggestedthat ;inherentROSlevelmightbedeterminativeincellular ;apoptoticsusceptibilitytoAs2O3【lII.Wthereforeques—
;tionedwhethertherewasaROSlevelfluctuationincell ;cycleduration.andcellcycle—relatedapoptosisofNB4
;cellsinducedbyAs,OwasassociatedwithsuchaROS ;levelvariationincellcyclephases.Weherein.first.using ;aflowcytometricdouble—stainingmethod,detectedthe
;ROSlevelsintherespectivephasesofthenormalcellcycle. ;andtheninvestigatedtherelationshipbetweenthevaria. ;tionofROSlevelduringcellcycleandtheapoptosisin. ;ducedbyAs,OinNB4cells.
;MATERIALSANDMETHoDS
;Reagents
;As,O.6.carboxy一2‟.7‟一dichlorodihvdrof1uoresceindiacetate ;(DCFH—DA),7-aminoactinomycinD(7AAD),propidumiodide ;(PI)andN—Acetyl—cystein(NAC)werepurchasedfromSigmaChemi ;calCo.(StLouis,M0,USA).2,3一dimethoxy—l,4一naphthoquinone
;(DMNQ)waspurchasedfromCalbiochem(SanDiego,CA,USA). ;CatalasewaspurchasedfromHuameiCompany(Luoyang,Henan. ;China).AsOwasdissolvedinsmallamountsof1.0MNaOH,then ;dilutedtol0.0mMwithphosphate—bufferedsaline(PBS)asstock
;solution
;Cellculture
;AcutepromylocyticleukemiacelllineNB4,kindlysuppliedby ;Shanghaiinstituteofhematology,wasculturedinRPMI一1640me—
;
;Cellularreactiveoxygenspeciesleveldeterminesapoptoticsusceptibilityhttp://www.cell—research.
com
;dium(GibcoBRL,Gaitherburg,MD,USA)supplementedwith100 ;IU/mLpennicillin,100mg/mlstreptomycin,and10%fetalbovine ;serum(FBS;GibcoBRL,inafullyhumidifiedatmospherewith5% ;CO2at37.C.Cellculturewasdailysplitandmaintainedatadensity ;lessthan5x10cells/m1.
;RoSdetection
;DCFH—DAwasusedforROSdetection.DCFH—DAjscleaved
;intracellularlybynonspecificesterasestoformDCFH.whichis ;furtheroxidizedbyROStoformthefluorescentcompoundDCF ;l3l1.DCFH—DAworkingsolutionwasaddeddirectlytothemedium ;toreachl0uM.andthenincubatedat37.Cforl5min.Cellswere ;thenwashedonce.resuspendedinPBSandkeptoniceforanimme—
;diatedetectionbvFACScan(BectonDickinson.USA.Whendeter—
;miningtheeffectsofdrugsandantioxidantsonROSlevel,cellswere ;exposedtoantioxidantscatalaseorNACfor2hbeforedrugtreatment. ;andDCFH—DAstainingwasappliedsimultaneouslywiththedrug ;exposureforl5min.
;Doublestainingflowcytometrywasperformed,using7AADto ;displayDNAcontent,andDCFH—DAtodetectROS,thusshowing
;thelevelfluctuationofROSduringanormalcellcycleofNB4cells ;freeofpro—apoptotictreatment..7AADwasappliedtomeasure ;DNAcontentinordertoidentifytheeel1cycledistributionI32}. ;NB4cellsweretreatedwithl0DCFH—DAforlOmininnormal
;culturedconditions.AtierwashinginPBS.cellswereresuspendedin ;7AADlabelingbufferf0.1Mphosphate—citratebuffcrcontaining0.
;15NaC1.5mMEDTA.0.5%BSAand0.004%saponinpH6.01 ;before7AADwasaddedinafinalconcentrationof20f321.Cells ;wereanalyzedbyFACScan(BectonDickinson,USA)following ;7AADincubationfor5—7min.
;Apoptosisandcellcycleanalysis
;Inordertovisualizetheapoptosis.NB4cellswereexposedto ;either2AsOaloneorincombinationwithsomeROSproducer ;andinhibitorsforl6hours.
;TdT—mediateddUTPnick—endlabeling(Tunel1/propidiumiodide ;(PI)doublestainingflowcytometrywasperformed,toassessthe ;apoptoticrateandtoknowfromwhichphasetheseapoptoticcells ;came.Cellswerefixedwith4%paraformaldehydefor30minat ;roomtemperature.AfterwashingtwicewithPBS,cellswereresus—
;pendedwith70%pre—coldethanolandimmediatelytransferredinto ;freezer.Thesampleswerestoredat一20.Cforovernight.Cellswere
;thenrehydratedbywashingtwicewithPBSbeforeresuspendedin ;50mLTunelreactionmixture(InStuCelldeathDetectionKit. ;Roche,Germany)andincubatedfor60minat37.Cinahumidified
;atmosphereindark.AtierwashedtwicewithPBS,cellsweretreated ;withl00U/mlRNAsecontaining0.002%TritonXl00forl5minutes. ;andstainedwithPI(thefinalconcentration50Bg/ml1for20minutes ;beforeanalyzedbvFACScan[30,331.
;RESUrS
;TheROSlevelvariedduringthenormalcellcycle ;OfNB4cells
;Duringthesinglecellcycle,intracellularROSlevelsof ;theintactNB4cellsincreasedsignificantlyfromG1,S ;phase,toG2/Mphase(Fig1).
;82
;As2O3selectivelyinducedapoptosisofNB4cells ;phasesfromG2/Mphase
;Upon2/aMAs2O3administrtionfor16h,NB4cells ;wereselectivelyinducedapoptosisfromG2/Mphase(Fig ;2A,B).
;DMNQenhancedtheefficacyofAs,OinROS-de- ;pendantmanner
;2uMAs,Oincombinationwith2.5uMDMNO
;(endogenousROSproducingreagent[34])inducedmore ;cellstoapoptosisfromG2/MphasethanAs,O1alone(Fig ;2C1.
;ToknowwhethertheeflfectofDMNOonpromoting ;As,O一inducedapoptosiswasROS—dependant,weinves—
;tigatedcellularROSleveluponDMNOtreatmentaloneor ;togetherwithtwotypesofantioxidants.orROSinhibitors ;andscanvangers,catalaseandNAC.Thedatashowedthat ;DMNOdidelevatethecellularROSlevelandthetreat—
;mentofcatalaseorNACcouldreversittotally.Exposure ;toAs,O1alonealsoresultedinaROSelevation,andthis ;efficacywasstrikinglyenhancedbvDMNOcotreatment. ;CatalaseorNAChadthecapacitytoreverseROSlevel ;elevatedbyAs2O3/DMNOcombination(Fig3). ;Correspondingly,theproportionofapoptoticcellswas ;closelyassociatedwiththecellularROSleve1.DMNQ ;dramasticllyenhancedtheefficacyofAs,Oonapoptosis ;inductionofNB4cells.Uponadministrationof2mMAs,O ;togetherwithDMNQandcatalase(orNA0treatment.the ;propotionofapoptoticcellswassimilartothatwithAs,O ;treatmentalone(Fig4).Thisshowedthattheapoptosis—
;enhancingefficacyofDMNOwasreversedbycatalase ;orNAC.Thuswecameintoaconclusionthattheeffect ;ofDMNOonpromotingAs2O3-inducedapoptosisfrom ;G2/MphasewasROS—dependant.
;DlSCUSSION
;ThepreviousstudiesindicatedthatthechangesofROS ;levelmightplayaroleinapoptosisinducedbyA2O3[12, ;13].Ourrecentworkindicatedthatcelllinesdifferently ;susceptibletoAs,O3-inducedapoptosispossesseddiffe卜
;entROSlevelswhichmightbedetermitiveintheirapoptotic ;susceptibilitytoAs,O1.Forinstance,wemanifestedthat, ;therewasainherentlyexisteddifferenceinwholeROS ;quantityamongfourkindsofleukemiacelllinesNB4.HL60. ;K562andU937,andapositivecorrelationbetweenthe ;inherentROSlevelandtheirapoptoticsensitivitytoAs,O1. ;Furthermore,bVinterferenceusingaR0SDroducer ;DMNQ,wedemonstratedthatanelevationofROSlevel ;wouldsensitizethecellstoAs,O一inducedapoptosisf11].
;Inthepresentstudyourresultsshowedthatalowdose ;(2UofAs,OinducedtheapoptosisofNB4ce1lspre—
;
;Cellresearch,14(1),Feb2004FeiGA0.日f
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;050100150200250
;7AAD
;B
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;《
;《
;卜
;10010‟1001010一
;DCF
;Fig1.7AAD/DCFHDAdoublelabelingflowcytometrydisplay ;ingROSlevelduringanormalcellcycle.A.DNAhistogram.B ;7AAD/DCFHDAbivariatedot—blots,R1:G1phase,R2:Sphase
;R3:G2/Mphase.
;.250%
;200%
;(,)
;o15O%
;100%
;50%
;O%
;As2O32laM
;DMNQ25laM
;CataIase600U,mI
;NAC25riM
;Fig3.EffectofDMNQonelevatingROSleveldeterminedbyflow
;cytometryandrepresentedbyDCFfluorescenceintensity(mean~ ;SD.n=3).NB4cellsweretreatedwithDMNQaloneortogether ;withAs203,catalaseorNACfor15min.(CatalaseandNACwere ;pre—incubatedwiththecellsfor2h.)
;vailinglyfromG2/Mphase.Thiswascoincidentwithother ;researcher‟sresults『301.
;Toourknowledge,methodologyapplying7AADand ;DCFH—DAdoublestainingflowcytometryhadnotbeen ;documented.evenif7AADusedinDNAmeasurement ;andDCF‟H—DAinROSdetectionwereseparatelyestab一
;1ished[3l.321.Thefeasibilityofthisnovelapproachwas ;demonstratedinthepresentwork.Ourdatashowedthat ;duringanormalcellcycleinNB4cells.ROSlevelsgradu—
;allyincreasedfromG1.StoG2/Mphases.oralternatively ;speaking,G2/Mphasecellspossessedinherentlygreater ;contentofROSthanGlandSphasecells.Thehigher ;levelofROSinG2/Mphasemightbethuslogicallyas—
;sumedtoaccountforthatG2/Mcellsweremorepredis—
;posedtoapoptosisinductionbV2UMAs,O
;Thecellcyclerelateddifferencesinsusceptibilityto ;apoptosisinducedbyvariousantitumoragentshavebeen ;noticedforlonginHL.60cells[35].inwhichcellsinG2/ ;Mphasewerelargelyapoptoticwhentreatedwithaserine/ ;threoninekinaseinhibitorH7orwithgamma—irradiation
;AP0178%
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;Fig2.Tunel/PIbivariatedot—blotsdisplayingprevaillingapoptotic ;cellsfromG2/MphaseofNB4cellsuponadministrationofAs2O3 ;at2pMaloneorincombinationwith2.5uDMNQfor16h.A: ;control;B:As2O3alone:C:As2O3incombinationwithDMNQ. ;25
;2O
;15
;1O
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;DMNQ25uM?
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;Fig4.ApoptoticratesanalyzedbyTunel/PIdoublelabelingflow ;cytometry(mean~SD,n:3).NB4cellsweretreatedwithAs2O3 ;aloneortogetherwithDMNQ,catalaseorNACfor16h. ;whereassomeotheragentsinducedGlorSphasecell ;apoptosisselectively.Thoughtheclinicrelevanceofthis ;modeofcelldeathwasdiscussed.themechanismsun. ;derlyingcellcyclerelateddifferencesinapoptosisSUS. ;ceptibilitywerevirtuallyobscure[351.B1ymphocytes‟
;growtharrestandapoptosisinducedbyactivationdisplayed ;Glphasespecificity[36].andthiswaspartiallyexplained ;bythedelicatelyregulatedexpressionorDosttranslation ;modificationofcyclin—dependentkinase2andothercell
;cycleproteins.Inthepresentstudywefoundthelinkof ;ROSlevelvarianceinthecellcyclewiththecycle.related
;apoptosisinducedbyAs,O.SinceROShasbeenconsid—
;eredasoneofmediatorsofcellapoptosisinducedbvUV ;exposureandgamma—irradiation[371,andalsobyarsenic ;trioxideI10—131invariedtypesofcell,thehigherapoptosis ;susceptibilityinG2/Mphasetogamma—irradiationmight
;bepresumedtoreflectamechanismsimilartoonede—
;scribedhere.inwhichROSplayedacrucialroleindeter. ;miningthecellcyclerelatedapoptosissusceptibility. ;ThelinkofROSvarianceduringthecellcyclewiththe ;83
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;Cellularreactiveoxygenspeciesleveldeterminesapoptoticsusceptibilityhttp://www.cell—research.com
;apoptosissusceptibilitymightimplythatthereexisteda ;“thresholdlevel”forROS.underwhichcellcouldsurvive
;itsoxidativedamagesandapoptosistriggering.Thinking ;ofthatAs,OcouldelevatetheROSlevelofNB4cells,we ;deducethat,upontreatmentwithAs,O,theROSlevelof ;G2/Mphasecellsexceededthatcellcouldendure.Hence ;thecellsinG2/Mphasewereinducedtoapoptosisaftera ;transitoryreplicationarrest.TheROSlevelsofGlandS ;phasescellswerestillunderthe”thresholdlevel”after
;treatmentwithAs,O1.Thesecellscouldprogresstonext ;stageofcellcycle.butultimatelybearrestedinG2/Mphase ;andtriggeredtoapoptosisaswel1.
;ToaffirmtheassociationofROSvarianceinthecell ;cyclewithapoptosissusceptibility.weuseDMNQincom—
;binationwithAs,Oforapoptosisjnduction.DMNOfacili—
;tatedtheefficacYofAs,Oonselectiveinductionof
;apoptosisofNB4cellsfromG2/Mphase.Thisemcacy ;0fDMNQcouldbereversedbycatalaseandNAC.DMNO, ;onetypeofquinonesthatunderwentredoxcyclingand ;waswidelyusedtoinvestigateoxidativestress,hasbeen ;provedtogenerateendogenousROSandcarriedintoex—
;ecutionofapoptosisthroughROS—dependentpathway
;『34].SinceDMNQproducedROSwhereascatalaseand ;NACremovedROSfromcells.ourresultsfurtherargued ;thatROSleveldifferenceintherespectivecellcyclephases ;0fNB4cellsshouldbepivotalincycle—relatedcellularsen—
;sitivitytoAs,O3-inducedapoptosis.
;Assomeotherinvestigatorsdemonstratedthatahigh ;doseofAs,O(8raM)inducedapoptosisofNB4cellsfrom ;eachphaseofcellcycle[30],andaverylowdoseofAs,O ;(1essis.ROSlevelsineachphaseofNB4cellcycle. ;upon8As,Oadministration,mightsurpassthethresh—
;oldandelicitanintensiveapoptosisofcellsinallphases. ;whileROSlevelskeepsafeforthecellsinallphasesafter ;administrationwithlessthan0.5I工MAs,O1
;TheresultsinthepresentstudyandtheROSthreshold ;hypothesisweproposedbasedonthemseemedtoshed ;morelightsonthemechanismsbywhichAs,Oinduce ;apoptosisincertaintypesoftumorcells.NotonlyAs,O ;inducedapoptosisbeROSdependent,alsoROSlikelybe ;ofdeterminationincellularsuceptibilitytoapoptosisin—
;ductionbyAs,O.Asithasbeenclearthatmitochondriais ;acentralexecutorinapoptosis【391,itisconceivableto
;recognizetheuniqueandpivotalroleofROS.Identifica—
;tionindetailhowROSmediatesandsignalsAs,Oinduce ;aDoptosiswillcertainlyhelptoelucidatethemechanisms ;ofapoptosisinducedbyAs,O1andotherchemotherapeutic ;84
;agents
;ACKNoWLEDGMENTS
;Thisprojectwassupportedbyresearchgrantsfrom ;NationalNaturalScienceFoundationofChina(No. ;30170475).
;ReceiveDec12.2002
;尺,ised,July10.2003
;Accepted,Sep7,2003
;REFERENCES
;ShenZX,ChenGQ,NiJH,LiXS,XiongSM,QiuQY,eta1.Use ;ofarsenictrioxide(As,O)inthetreatmentofacutepromyelocytic ;leukemia(APL1:II.Clinica