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ARTICLESCellResearch(2004);14(1):8l85h

    ;ttp://www.cellresearch.com

    ;Thecellcyclerelatedapoptoticsusceptibilitytoarsenictrioxideisassociated ;withthelevelofreactiveoxygenspecies

    ;FeiGAO,JingYI,JingQiYUAN,GuiYingSHI,XueMingTANG,

    ;.DepartmentofCellBiology,ShanghaiSecondMedicalUniversity,Shanghai200025,China ;DepartmentofAutomation,ShanghaiJiaotongUniversity,Shanghai20003l,China. ;ABSTRACT

    ;Doublestainingflowcytometrywasperformedusing7aminoactinomycinDand6carboxy2‟.7‟

    ;dichlorodihydrofluoresceindiacetate.todetectthelevelfluctuationofreactiveoxygenspecies(ROS)d

    uringthecell

    ;cycleofnormalNB4cells.OurresultsshowedthatNB4cellspossessedhigherlevelofROSinG2/MphasethaninG1

    ;andSphases.Doublestainingflowcytometry,withTdTmediateddUTPnickendlabeling(Tune1)andpropidiumiodide

    ;(PI),indicatedthatAs,O(2LI

    )couldinduceapoptosisinNB4cellsprevailinglyfromG2/Mphase,andthisemcacy ;wasenhanceduponcoadministrationof2,3.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[161.Althoughit

    ;hasbeenwidelyacceptedthat,likeAs,O.manychemo.

    ;therapeuticdrugsfunctionasproapoptoticsignalswhich

    ;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 ;itslevelrelativelystableunderphysiologicalconditions14

    ;161.Recentdatashowedthatmanyproapoptoticsignals

    ;couldraisethecellularROSleve1.andcelluarinherentROS ;levelmaybeassociatedwiththesusceptibilityoftumor ;cellstoproapoptoticsignals1013,l729].

    ;SomeresearchersreportedthatlOWconcentrationf12

    ;mM)ofAs,OpreferentiallyinducedapoptosisinNB4cells ;-Correspondenceauthor:XueMingTANG

    ;Te1:+862l63846590(ext.776480),Fax:+862153065329

    ;Email:xmtang@shsmu.edu.ca

    ;ofG2/Mphase301.0urrecentworkindicatedthatdi

    ;ferentsusceptibilityofcelllinestoAs,O3-inducedapoptosis ;maypossessdifferentROSlevels.andthussuggestedthat ;inherentROSlevelmightbedeterminativeincellular ;apoptoticsusceptibilitytoAs2O3lII.Wthereforeques

    ;tionedwhethertherewasaROSlevelfluctuationincell ;cycleduration.andcellcyclerelatedapoptosisofNB4

    ;cellsinducedbyAs,OwasassociatedwithsuchaROS ;levelvariationincellcyclephases.Weherein.first.using ;aflowcytometricdoublestainingmethod,detectedthe

    ;ROSlevelsintherespectivephasesofthenormalcellcycle. ;andtheninvestigatedtherelationshipbetweenthevaria. ;tionofROSlevelduringcellcycleandtheapoptosisin. ;ducedbyAs,OinNB4cells.

    ;MATERIALSANDMETHoDS

    ;Reagents

    ;As,O.6.carboxy2‟.7‟dichlorodihvdrof1uoresceindiacetate ;(DCFHDA),7-aminoactinomycinD(7AAD),propidumiodide ;(PI)andNAcetylcystein(NAC)werepurchasedfromSigmaChemi ;calCo.(StLouis,M0,USA).2,3dimethoxyl,4naphthoquinone

    ;(DMNQ)waspurchasedfromCalbiochem(SanDiego,CA,USA). ;CatalasewaspurchasedfromHuameiCompany(Luoyang,Henan. ;China).AsOwasdissolvedinsmallamountsof1.0MNaOH,then ;dilutedtol0.0mMwithphosphatebufferedsaline(PBS)asstock

    ;solution

    ;Cellculture

    ;AcutepromylocyticleukemiacelllineNB4,kindlysuppliedby ;Shanghaiinstituteofhematology,wasculturedinRPMI1640me

    ;

    ;Cellularreactiveoxygenspeciesleveldeterminesapoptoticsusceptibilityhttp://www.cellresearch.

com

    ;dium(GibcoBRL,Gaitherburg,MD,USA)supplementedwith100 ;IU/mLpennicillin,100mg/mlstreptomycin,and10%fetalbovine ;serum(FBS;GibcoBRL,inafullyhumidifiedatmospherewith5% ;CO2at37.C.Cellculturewasdailysplitandmaintainedatadensity ;lessthan5x10cells/m1.

    ;RoSdetection

    ;DCFHDAwasusedforROSdetection.DCFHDAjscleaved

    ;intracellularlybynonspecificesterasestoformDCFH.whichis ;furtheroxidizedbyROStoformthefluorescentcompoundDCF ;l3l1.DCFHDAworkingsolutionwasaddeddirectlytothemedium ;toreachl0uM.andthenincubatedat37.Cforl5min.Cellswere ;thenwashedonce.resuspendedinPBSandkeptoniceforanimme

    ;diatedetectionbvFACScan(BectonDickinson.USA.Whendeter

    ;miningtheeffectsofdrugsandantioxidantsonROSlevel,cellswere ;exposedtoantioxidantscatalaseorNACfor2hbeforedrugtreatment. ;andDCFHDAstainingwasappliedsimultaneouslywiththedrug ;exposureforl5min.

    ;Doublestainingflowcytometrywasperformed,using7AADto ;displayDNAcontent,andDCFHDAtodetectROS,thusshowing

    ;thelevelfluctuationofROSduringanormalcellcycleofNB4cells ;freeofproapoptotictreatment..7AADwasappliedtomeasure ;DNAcontentinordertoidentifytheeel1cycledistributionI32}. ;NB4cellsweretreatedwithl0DCFHDAforlOmininnormal

    ;culturedconditions.AtierwashinginPBS.cellswereresuspendedin ;7AADlabelingbufferf0.1Mphosphatecitratebuffcrcontaining0.

    ;15NaC1.5mMEDTA.0.5%BSAand0.004%saponinpH6.01 ;before7AADwasaddedinafinalconcentrationof20f321.Cells ;wereanalyzedbyFACScan(BectonDickinson,USA)following ;7AADincubationfor57min.

    ;Apoptosisandcellcycleanalysis

    ;Inordertovisualizetheapoptosis.NB4cellswereexposedto ;either2AsOaloneorincombinationwithsomeROSproducer ;andinhibitorsforl6hours.

    ;TdTmediateddUTPnickendlabeling(Tunel1/propidiumiodide ;(PI)doublestainingflowcytometrywasperformed,toassessthe ;apoptoticrateandtoknowfromwhichphasetheseapoptoticcells ;came.Cellswerefixedwith4%paraformaldehydefor30minat ;roomtemperature.AfterwashingtwicewithPBS,cellswereresus

    ;pendedwith70%precoldethanolandimmediatelytransferredinto ;freezer.Thesampleswerestoredat20.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,OinducedapoptosiswasROSdependant,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/MphasewasROSdependant.

;DlSCUSSION

    ;ThepreviousstudiesindicatedthatthechangesofROS ;levelmightplayaroleinapoptosisinducedbyA2O3[12, ;13].Ourrecentworkindicatedthatcelllinesdifferently ;susceptibletoAs,O3-inducedapoptosispossesseddiffe

    ;entROSlevelswhichmightbedetermitiveintheirapoptotic ;susceptibilitytoAs,O1.Forinstance,wemanifestedthat, ;therewasainherentlyexisteddifferenceinwholeROS ;quantityamongfourkindsofleukemiacelllinesNB4.HL60. ;K562andU937,andapositivecorrelationbetweenthe ;inherentROSlevelandtheirapoptoticsensitivitytoAs,O1. ;Furthermore,bVinterferenceusingaR0SDroducer ;DMNQ,wedemonstratedthatanelevationofROSlevel ;wouldsensitizethecellstoAs,Oinducedapoptosisf11].

    ;Inthepresentstudyourresultsshowedthatalowdose ;(2UofAs,OinducedtheapoptosisofNB4ce1lspre

    ;

    ;Cellresearch,14(1),Feb2004FeiGA0.f

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    ;Fig1.7AAD/DCFHDAdoublelabelingflowcytometrydisplay ;ingROSlevelduringanormalcellcycle.A.DNAhistogram.B ;7AAD/DCFHDAbivariatedotblots,R1:G1phase,R2:Sphase

    ;R3:G2/Mphase.

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    ;200%

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    ;o15O%

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    ;50%

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    ;DMNQ25laM

    ;CataIase600U,mI

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    ;Fig3.EffectofDMNQonelevatingROSleveldeterminedbyflow

    ;cytometryandrepresentedbyDCFfluorescenceintensity(mean~ ;SD.n=3).NB4cellsweretreatedwithDMNQaloneortogether ;withAs203,catalaseorNACfor15min.(CatalaseandNACwere ;preincubatedwiththecellsfor2h.)

    ;vailinglyfromG2/Mphase.Thiswascoincidentwithother ;researcher‟sresults301.

    ;Toourknowledge,methodologyapplying7AADand ;DCFHDAdoublestainingflowcytometryhadnotbeen ;documented.evenif7AADusedinDNAmeasurement ;andDCF‟H—DAinROSdetectionwereseparatelyestab

    ;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/ ;threoninekinaseinhibitorH7orwithgammairradiation

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    ;Fig2.Tunel/PIbivariatedotblotsdisplayingprevaillingapoptotic ;cellsfromG2/MphaseofNB4cellsuponadministrationofAs2O3 ;at2pMaloneorincombinationwith2.5uDMNQfor16h.A: ;control;B:As2O3alone:C:As2O3incombinationwithDMNQ. ;25

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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 ;modificationofcyclindependentkinase2andothercell

    ;cycleproteins.Inthepresentstudywefoundthelinkof ;ROSlevelvarianceinthecellcyclewiththecycle.related

;apoptosisinducedbyAs,O.SinceROShasbeenconsid

    ;eredasoneofmediatorsofcellapoptosisinducedbvUV ;exposureandgammairradiation[371,andalsobyarsenic ;trioxideI10131invariedtypesofcell,thehigherapoptosis ;susceptibilityinG2/Mphasetogammairradiationmight

    ;bepresumedtoreflectamechanismsimilartoonede

    ;scribedhere.inwhichROSplayedacrucialroleindeter. ;miningthecellcyclerelatedapoptosissusceptibility. ;ThelinkofROSvarianceduringthecellcyclewiththe ;83

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    ;Cellularreactiveoxygenspeciesleveldeterminesapoptoticsusceptibilityhttp://www.cellresearch.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

    ;ecutionofapoptosisthroughROSdependentpathway

    ;34].SinceDMNQproducedROSwhereascatalaseand ;NACremovedROSfromcells.ourresultsfurtherargued ;thatROSleveldifferenceintherespectivecellcyclephases ;0fNB4cellsshouldbepivotalincyclerelatedcellularsen

    ;sitivitytoAs,O3-inducedapoptosis.

    ;Assomeotherinvestigatorsdemonstratedthatahigh ;doseofAs,O(8raM)inducedapoptosisofNB4cellsfrom ;eachphaseofcellcycle[30],andaverylowdoseofAs,O ;(1essis.ROSlevelsineachphaseofNB4cellcycle. ;upon8As,Oadministration,mightsurpassthethresh

    ;oldandelicitanintensiveapoptosisofcellsinallphases. ;whileROSlevelskeepsafeforthecellsinallphasesafter ;administrationwithlessthan0.5IMAs,O1

    ;TheresultsinthepresentstudyandtheROSthreshold ;hypothesisweproposedbasedonthemseemedtoshed ;morelightsonthemechanismsbywhichAs,Oinduce ;apoptosisincertaintypesoftumorcells.NotonlyAs,O ;inducedapoptosisbeROSdependent,alsoROSlikelybe ;ofdeterminationincellularsuceptibilitytoapoptosisin

    ;ductionbyAs,O.Asithasbeenclearthatmitochondriais ;acentralexecutorinapoptosis391,itisconceivableto

    ;recognizetheuniqueandpivotalroleofROS.Identifica

    ;tionindetailhowROSmediatesandsignalsAs,Oinduce ;aDoptosiswillcertainlyhelptoelucidatethemechanisms ;ofapoptosisinducedbyAs,O1andotherchemotherapeutic ;84

    ;agents

    ;ACKNoWLEDGMENTS

    ;Thisprojectwassupportedbyresearchgrantsfrom ;NationalNaturalScienceFoundationofChina(No. ;30170475).

    ;ReceiveDec12.2002

    ;,ised,July10.2003

    ;Accepted,Sep7,2003

    ;REFERENCES

    ;ShenZX,ChenGQ,NiJH,LiXS,XiongSM,QiuQY,eta1.Use ;ofarsenictrioxide(As,O)inthetreatmentofacutepromyelocytic ;leukemia(APL1:II.Clinica

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