Inhibition of ATM blocks the etoposide-induced DNA damage response and apoptosis of resting human T cells

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Inhibition of ATM blocks the etoposide-induced DNA damage response and apoptosis of resting human T cells

Z. Korwek

^a

, T. Sewastianik

^a

, A. Bielak-Zmijewska

^a

, G. Mosieniak

^a

, O. Alster

^a

, M. Moreno-Villaneuva

^b

, A. Burkle

^b

, E. Sikora

^a,∗

aLaboratoryoftheMolecularBasesofAgeing,NenckiInstituteofExperimentalBiology,PolishAcademyofSciences,02-093Warsaw,Poland

bMolecularToxicologyGroup,BoxX911UniversityofKonstanz,Universitaetsstrasse10D-78457Konstanz,Germany

Keywords:

FADU

␥H2AX DSBs Caspases KU55933

a b s t r a c t

ItisbelievedthatnormalcellswithanunaffectedDNAdamageresponse(DDR)andDNAdamagerepair machinery,couldbelesspronetoDNAdamagingtreatmentthancancercells.However,theanticancer drug,etoposide,whichisatopoisomeraseIIinhibitor,cangenerateDNAdoublestrandbreaksaffecting notonlyreplicationbutalsotranscriptionandthereforecaninduceDNAdamageinnon-replicating cells.Indeed,weshowedthatetoposidecouldinfluencetranscriptionandwasabletoactivateDDRin restinghumanTcellsbyinducingphosphorylationofATManditssubstrates,H2AXandp53.Thisledto activationofPUMA,caspasesandtoapoptoticcelldeath.LymphoblastoidleukemicJurkatcells,ascycling cells,weremoresensitivetoetoposideconsideringthelevelofDNAdamage,DDRandapoptosis.Next, weusedATMinhibitor,KU55933,whichhasbeenshownpreviouslytobearadio/chemo-sensitizing agent.PretreatmentofrestingTcellswithKU55933blockedphosphorylationofATM,H2AXandp53, which,inturn,preventedPUMAexpression,caspaseactivationandapoptosis.Ontheotherhand,KU 55933incrementedapoptosisofJurkatcells.However,etoposide-inducedDNAdamageinrestingTcells wasnotinfluencedbyKU55933asrevealedbytheFADUassay.AltogetherourresultsshowthatKU 55933blocksDDRandapoptosisinducedbyetoposideinnormalrestingTcells,butincreasedcytotoxic effectonproliferatingleukemicJurkatcells.Wediscussthepossiblebeneficialandadverseeffectsof drugsaffectingtheDDRincancercellsthatarecurrentlyinpreclinicalanticancertrials.

1. Introduction

The cell cycle of normal somatic cells is regulated with extremelyhighprecision.Thisisachievedbyanumberofsignal transductionpathways,knownascheckpoints,whichcontrolcell cycleprogressionensuringaninterdependencyoftheS-phaseand mitosis,theintegrityofthegenomeandproperchromosomeseg- regation[1].Thecellcyclecheckpointsarecriticalforprotection fromuncontrolledcelldivisionwhichisthemainfeatureofcancer development.

DNA damage checkpoints are activatedwhen cells undergo DNAreplication(S-phase)orifDNA(G1andG2)isdamagedby reactiveoxygenspecies orgenotoxicandotherinsults.Thesig- nalsof double-strandDNAbreaks(DSBs)aretransducedbythe socalledDNA damageresponse (DDR)pathwayand determine cellfateasoneofthethreeresponses:transientcellcyclearrest

∗Corresponding authorat: NenckiInstituteof ExperimentalBiology, Polish AcademyofSciences,3PasteurSt,02-093Warsaw,Poland.Tel.:+48225892436;

fax:+48228225342.

E-mailaddress:e.sikora@nencki.gov.pl(E.Sikora).

(repair), stablecellcyclearrest(senescence) orcelldeath(apoptosis).DDRismediatedbyDNAdamageproteinsensors,suchas theMRN(Mre11–Rad50–Nbs1)complex,which triggertheacti- vationofasignaltransductionsystemwhichincludestheprotein kinases:ATM(ataxiatelangiectasiamutated),ATR(ATMandrad3- related),Chk1andChk2.Ultimately,theDDRactivatesp53,which contributes to either an apoptotic or senescence response via transactivationof pro-apoptoticproteinsbelongingtotheBcl-2 proteinfamilyorcyclindependentkinaseinhibitorp21,respec- tively(reviewedby[2,3]).

InductionofDSBstriggersphosphorylationofoneofthevari- antsofthenucleosomecorehistone,namelyH2AX,onSer-139.This phosphorylationismediatedbyATM,whichitselfisactivatedby autophosphorylationonSer-1981.Thepresenceofphosphorylated H2AX,named␥H2AX,canbedetectedimmunocytochemicallyin theformofdistinctnuclearfociwhereeachfocusisassumedtocor- respondtoasingleDSB[4].Co-localizedwith␥H2AXareproteins suchasRad50,Rad51,Brca1andthep53bindingprotein1(53BP1), recruitedtotheDSBsite.ConcomitantactivationofATMandH2AX phosphorylationisconsideredtobeareliablehallmarkofDSBs[4].

Recentlyalso53BP1hasbeenrecognizedasaconvenientmarker ofDSBs,formingnuclearfocitogetherwith␥H2AX[5].

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-217198

https://dx.doi.org/10.1016/j.dnarep.2012.08.006

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Thereareanumberofdocumentedgeneticlesionsincheck- pointgenes,orincellcyclegenes,whichresulteitherdirectlyin cancerdevelopmentorinapredispositiontocertaincancertypes andgenomicinstability[6].Ontheotherhand,radio/chemotherapy inducesDNAdamageincancercellswhichthenswitchonDDRthat leadstocellsenescenceorcelldemiseviaapoptosisorthemitotic catastrophe[7].TherearemanyagentsinducingDNAdamagein cancercellsandetoposideisoneofthem.

Etoposidehasbeenusedinthetreatmentofawidevarietyof neoplasms,includingsmall-celllungcancer,Kaposi’ssarcoma,tes- ticularcancer,acuteleukemiaandlymphoma[8].Etoposideisa poisonof topoisomerasetypeII(Top2)[9],which stabilizesthe cleavage complex leading toTop2 mediated chromosome DNA breakage[10].Inmammals,therearetwoisozymesofDNAtopo- isomeraseII,Top2␣andTop2␤bothofwhich,seemtobeinvolved notonlyinreplicationbutalsointranscription[11–13].Thus,it couldbeexpectedthatetoposidecanexertadverseeffectonslowly ornon-proliferatingnormalcellsbyinﬂuencingbothTop2␣and Top2␤duringtranscription.

The majorside effect of radio/chemotherapy, including that elicitedwiththeuseofetoposide,isleucopeniacausedbydrug cytotoxicitytomyeloidcellsandmaturelymphocytes.Themain mechanism of thecytotoxic effect of etoposide might be apo- ptosisoftheimmune cells[14].Veryrecently,theinductionof

␥H2AXhasbeenobservedinperipheralbloodlymphocytesirra- diatedinvitro[15]andtherelationbetweenDNAdamagefociand withapoptosisofrestinglymphocytesfromirradiatedpatientswas revealed[16].However,toourknowledge,therearenopublica- tionsshowingarelationbetweenetposide-inducedDNAdamage, DDRandapoptosisofrestinglymphocytes.Weexpectedthatthe DNAdamageresponseandsubsequentapoptosismighttakeplace inprimary non-proliferatinghuman Tcells treatedwithetoposide.Indeed,weshowinthispaperthatthetreatmentofTcells withetoposidecausedDNAdamageandinducedactivationofthe DNAdamage signalingpathwayfollowed byp53-and caspase- dependentapoptosis.PretreatmentofcellswiththeATMinhibitor, KU55933,successfully blockedDDR,butdidnotinﬂuenceDNA damage level measured by the FADU technique. In a seminal paperdescribingKU55933itwasshownthattheATMinhibitor sensitizedHeLacellstothecytotoxiceffectsofetoposideasmea- suredbytheclonogenicityassay[17].Weshowsurprisingly,that KU55933protectsTcellsagainstapoptosisindicatingitsoppo- site action onnormal resting cells and on proliferatingcancer ones.

2. Materialsandmethods 2.1. Cellculture

HumanTcellswereisolatedfrombuffycoatsofbloodsamples obtainedfrominformedhealthyvolunteerdonors,inaccordance withlocalethicalregulations,andprovidedbyDomesticBloodCen- ter,Warsaw,Poland.IsolationwasperformedusingtheRosetteSep HumanTCellIsolationCocktail(StemCellTechnologies,Vancou- ver,Canada),accordingtothemanufacturer’sinstruction.Thecell purity wasusually more than 95%(estimated byﬂow cytome- try).Cells wereseededat adensity of1×10⁶ cells/mlin RPMI 1640mediumsupplementedwith10%FBS,2mMl-glutamineand antibioticsandkeptinhumidiﬁedatmosphere(37^◦Cand5%CO₂ intheair).

JurkatE6.1cellsobtainedfromECACC(EuropeanCollectionof CellCulture)wereculturedinRPMI1640mediumsupplemented with10%FBS,2mMl-glutamineandantibioticsandkeptinhumid- iﬁedatmosphere(37^◦Cand5%CO₂intheair).Thecellswereseeded 24hbeforetreatmentatadensityof4×10⁵cells/ml.

Etoposide (Sigma–Aldrich, Poznan, Poland) and KU 55933 (TocrisBioscience,Bristol,UK)weredissolvedinDMSOandadded tothemediumtoagivenﬁnalconcentration.KU55933wasadded tothemediumfor2hbeforeetoposidewithoutmediumexchange.

TheDMSOconcentrationincellculturedidnotexceed0.1%,which didnotinﬂuencecellsurvival.

2.2. Transcriptiondetection

DetectionofnewlysynthesizedRNAwasestimatedusingthe Click-iT^® RNA HCS Assays (Invitrogen). T cells (1×10⁶) were treated with transcription inhibitors either 10␮M ␣-amanitin (Sigma–Aldrich) for 17h or 40␮M 1-␤-d-ribofuranoside (DRB) (Sigma–Aldrich) for 1h before theaddition of 1mM 5-ethynyl uridine (EU) for 1h at 37^◦C. Afterwards cells were ﬁxedwith 3.7%formaldehydeinPBSfor15minandpermeabilizedwith0.5%

Triton-X100inPBSfor15min.EUincorporationwasdetectedusing theClick-iT^®reactioncocktailcontaininggreen-ﬂuorescentAlexa Fluor^®488azide.Afterthewashingstep,meanﬂuorescenceofcells wasmeasuredusingFACSCalibur(BDBiosciences,Warsaw,Poland) andCellQuestProsoftware(BDBiosciences,Warsaw,Poland).

2.3. Apoptosisdetection

Externalizationof phosphatidylserine (PS)totheouterlayer ofcellmembranewasexaminedbybindingofAnnexinVinthe presenceof7-AAD,adyewhichstaineddeadcells.Theassaywas performedusingthePEAnnexinVApoptosisDetectionKitI(BD Biosciences, Warsaw,Poland).Cellswerewashed,suspended in theAnnexinVbindingbufferandstainedwithPE(Phycoerythrin) conjugatedwith Annexin V and 7-AADfor 15min at RT.Flow cytometricanalyseswereperformedusingFACSCaliburandthe CellQuestProanalysissoftware.

2.4. Immunocytochemistry

Cellswerewashedandﬁxedwith2%PFAfor20min,atRT.Cells werewashedtwiceandattachedtotheSuperfrost^® PlusMicro- scopeSlides (Thermo Scientiﬁc, Braunschweig, Germany) using thecytospincentrifuge.Afterwardstheywerepermeabilizedwith 70%ethanolovernightat−20^◦C.Next,cellswereblockedwith5%

bovineserumalbumin(BSA)inPBScontaining0.5%Tween-20and 0.1%TritonX-100for30min.Afterwashingcellswereincubated withprimaryanti-p-ATMSer1981(Abcam,Cambridge,UK),anti-

␥H2AX,(Abcam),anti-53BP1(NovusBiologicals,Cambridge,UK) andanti-Ki-67(Dako,Gdynia,Poland)antibodiesdiluted1:500in 1%BSA/PBS(0.5%Tween-20and0.1%TritonX-100)for2handthen withtheanti-mouseAlexa488/anti-rabbitAlexa555secondary antibodies(Invitrogen,Warsaw,Poland),1:500in1%BSA/PBS(5%

Tween-20and0.1%TritonX-100)for1h.DNAwasstainedwith DRAQ5(BiostatusLimited,Leicestershire,UK)diluted1:400inPBS for10minandthecoverslipsweremounted.Stainingswerevisu- alizedwithaLeicaTCSSP5laserscanningconfocalmicroscopewith a63×(1.4NA)PlanApoobjective.Forﬂuorescenceintensityeval- uationatleast50cellsfromeachexperimentwereanalyzedusing theLASAFsoftware(LeicaMicrosystems).

2.5. DNAcontentmeasurement

ForDNAcontentanalysiscellswerewashedinPBS,ﬁxedwith 70%ethanolandkeptovernightin−20^◦C.Afterwashingcellswere stainedwithPIsolution(3.8mMsodiumcitrate,50␮g/mlRNAseA, 500␮g/mlPI,inPBS)for30min.Flowcytometryanalysisof10,000 cellswasperformedusingFACSCaliburandtheCellQuestProsoft- ware.

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2.6. Westernblottinganalysis

WholecellproteinextractswerepreparedaccordingtoLaemmli [18].Equalamountsofproteinwereseparatedelectrophorectically in8or12%SDS-polyacrylamidegelsandtransferredontonitrocel- lulosemembranes.Membraneswereblockedwith5%non-fatdry milkdissolvedinTBScontaining0.1%Tween-20for1hatRTand incubatedovernightat4^◦Cwithoneoftheprimaryantibodies:

anti-ATM(1:500)andanti-H2AX(1:500)(Millipore);anti-p-ATM Ser1981(1:1000)andanti-␥H2AXSer139(1:1000)(Abcam);anti- p53(1:500)andanti-p21(1:250)(SantaCruzBiotechnology,Santa Cruz,USA);anti-p-p53Ser15(1:500),anti-Puma(1:1000), anti- caspase-3(1:500),anti-caspase-9(1:500);anti-caspase-8(1:500) (Cell Signaling, Boston, USA); anti-Poly(ADP-ribose)polymerase (PARP) (1:1000) (Enzo Life Sciences, Exeter, UK); anti ␤-actin (1:50,000)(Sigma–Aldrich).Speciﬁcproteinsweredetectedafter 1h incubation at RT with one of the horseradish peroxidase- conjugatedsecondary antibodies(1:2000) (Dako), using anECL system (GE Helthcare, Buckinghamshire, UK), according to the manufacturer’sinstructions.

2.7. Flowcytometrymeasurementofcaspase-2

Caspase-2activationwasmeasured24hand48haftertreat- ment with etoposide and/or KU 55933 by the CaspGLOW^TM Fluorescein Active Caspase-2 Staining Kit (BioVision, Milpitas, USA).3×10⁵ofcellsweresuspendedin300␮lofmediumand1␮l ofFITC–VDVAD–FMKwasadded.Thencellswereincubatedfor1h at37^◦Cwith5%CO₂.Aftertwowashesﬂuorescencewasanalysed byFACSCaliburwiththeCellQuestProsoftware.

2.8. FluorimetricdetectionofalkalineDNAunwinding(FADU) method

Amodifiedandautomatedversionofthe‘fluorimetricdetec- tionofalkalineDNAunwinding’methodwasemployedtomeasure thelevelofDNAdamageandrepairincellstreatedwithetoposide and/orKU55933.ThelevelofDNAstrandlesionswasanalyzed 30minaftercelltreatmentasdescribedpreviously[19].Themea- surementofDNAstrandbreaksbyFADUisbasedonthepartial denaturation(“unwinding”)ofdouble-strandedDNAundercon- trolledalkalineandtemperatureconditions.DNAstrandbreaksare siteswheretheunwindingofDNAcanstart.Briefly,afterinfliction ofDNAdamage,celllysiswasperformed.Unwindingwastermi- natedbyaddinganeutralizingsolution.Toquantifytheamountof DNAremainingdouble-stranded,acommerciallyavailablefluores- cencedye(SybrGreen^®)wasusedasamarkerfordoublestranded DNA.

2.9. Statisticalanalysis

DatawereevaluatedusingtheMann–Whitneytest.

3. Results

3.1. EtoposideinducesapoptosisofrestingandproliferatingT cells

Previously it was shown by Tanaka et al. [20] that human lymphoblastoidcells,whichareintheG1phaseofthecellcycle, preferentially underwent apoptosis following treatment with etoposide(ETO).Wewereinterestedwhethercellswhichremain outof thecellcycle(G0phase)arealsosensitivetoETOtreat- ment.Tothisend,weperformedexperimentsonhumanTcells, whicharerestingcellsand,forcomparison,weusedproliferating lymphoblastoidleukemicJurkatcells.Wedecidedtoperformour

studiesusinganisolated pure(morethan95%)population ofT cells,insteadofperipheralbloodlymphocytescommonlyusedfor dosimetry,whicharethemixtureofcellsofdifferentfunctions, lifespanandpropensitytoundergoapoptosisinvivoandunder culturecondition.Moreover,Tcellsderivedfromhealthypeople aretrulyintheG0phase.

Toshowthisweperformedfollowinganalyses.Firstwechecked DNAcontentin restingTcells andJurkat cellsbyﬂow cytome- try.TheresultspresentedinFig.1Ashowthatthevastmajority ofrestingT cellswerein G0/G1phase (96%),whilstwithinthe population of Jurkat cells only abouthalf of them werein the G0/G1phase.Previously,weshowedthatPHAstimulationinduced proliferationofrestingTcells[21].However,DNAcontentmea- surementdoesnotdiscriminatebetweencellsintheG0andG1 phase.Thus,weperformedadditionalanalysis,namelytheKi67 expressionwasmeasuredbyimmunocytochemistryinrestingand PHA(0.5␮g/ml)stimulatedTcells.Ki67isacommonmarkerof proliferatingcells.AscanbeseeninFig.1B,beforestimulationall cellswereKi67-negative,whilstafterPHAstimulationsomecells wereKi67-positive.

WemeasuredtheapoptoticindexofnormalTcellsandJurkat cells treatedfor 24hwithetoposideatdifferentconcentrations rangingfrom1to20␮M.Apoptosiswasdetectedbyﬂowcytometry usingtheAnnexinV/7-AADassay.Theapoptoticindexwasdeﬁned asthesumofthepercentageofcellswhichwereAnnexinV-pos- itiveand7-AAD-negative(earlyapoptosis,membraneintegrityis preserved)andthosewhichwereAnnexinV-and7-AADpositive (endstageofapoptosisandcelldeath).Fig.2Ashowscumulative valuesoftheapoptoticindexforrestingTcells.Asexpected,the highestapoptosislevelwasobservedincellstreatedwith20␮M ETO,howevera10␮Mdrughasalreadyinduceddeathinasub- stantialamountofrestingTcells(25%). Accordingly,for further experimentsweused10␮METO(ifnotstatedotherwise)asithas beensuggestedpreviouslythatthiscelltreatment(10␮M,24h) mimicsoneofthetherapeuticregimes[22].

When we measured the apoptotic index in Jurkat cells it appearedthattheyweremuchmoresensitivetoETOtreatment.

Namely,already5␮METOinducedapoptosisin40%ofcellsand 10␮METOwastwicemorecytotoxic.Thetimecourseof10␮M ETOcytotoxicityalsoindicatedhighersensitivityofleukemicthan normalnon-proliferatingTcellstoETOtreatment(Fig.2AandB).

3.2. EtoposideinducesDNAdamageinrestingandproliferating (Jurkat)Tcells

We were interested whether ETO induced apoptosis by introducingDNAbreaksleadingtoDDRinnormalrestinghuman TcellsandproliferatingJurkatcells.First,wecheckedDNAlesions byusingtwodifferentmethods,namely“ﬂuorimetricdetectionof alkalineDNAunwinding”andimmunocytochemicaldetectionof DNAdamagefoci.

TheFADUmethodservestoquantifytheformationandrepair ofbothsingleanddoubleDNAstrandbreaks.Thisisaverysensi- tiveandquantitativemethod[19,23].Sincethismethoddoesnot discriminatebetweenprimaryandapoptoticDNAlesions,weonly analysedcellsaftertreatmentwithetoposideforashortperiodof time(30min).Thismethodwasusedjusttoshowwhetheretopo- sidewasabletoinduceconcentration-dependentDNAdamagein restingTcellsandcyclingJurkatcells.Lowﬂuorescenceintensities indicatedalargenumberofDNAstrandbreaks.Indeed,thismethod revealedthatETOaffectedDNAinbothnormalandleukemiccells.

HoweverlowerﬂuorescencecouldbeobservedinJurkatcellsafter treatmentwithallofthetestedconcentrations(Fig.3).Inthecase of10␮METOitwasabout30%oftheinitialﬂuorescencevaluein comparisonwithabout90%innormalrestingTcellsprovingthat restingTcellswerelesssensitivetotheDNAdamagingagentthan

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Fig.1. NormalTcellsarerestingones.

(A)DNAcontentofTcellsandJurkatcellswereanalyzedbyﬂowcytometry.Percentageofcellsindifferentphasesofthecellcycleareindicated.(B)RestingTcellsafterPHA stimulationenteredthecellcycleaswasevidencedbyKi67staining.Theresultsarerepresentativeofatleast10measurements.Barscorrespondto20␮M.

Fig.2. ApoptoticindexofETO-treatedTcellsandJurkatcells.

(A)Dose-dependenceofapoptoticindexmeasured24hafterETOtreatmentandtimecourseofapoptosisinTcellstreatedwith10␮METO.(B)Dose-dependenceofapoptotic indexmeasured24hafterETOtreatmentandtimecourseofapoptosisinleukemicJurkatcellstreatedwith10␮METO.ApoptoticindexwasestimatedbytheAnnexinV/7- AADﬂowcytometryassay.Thebarsshowmeans±SDvalues.ApoptoticindexwasobtainedfromfourindependentexperimentsinthecaseofJurkatcellsandfromfour differentdonorsinthecaseofTcells.

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Fig.3. DNAlesionsinducedbyETOinTcellsandJurkatcellsmeasuredbyFADUassay.

TcellsandJurkatcellsweretreatedwithETOatdifferentconcentrations.DNAlesionsweremeasuredafter30min.Thevaluesaremeans(±SDobtainedfromsixdonorsand sixmeasurementsinJurkatcells).

proliferatingJurkatcells.Toconﬁrmtheseresultsweusedanother methodwhichdetectsonlyDNAdoublestrandbreaks(DSBs)typi- calforETOaction,thatisphosphorylationofH2AXonSer-139.Fig.4 shows␥H2AXfociobservedunderaconfocalmicroscope.Asitcan beseenETOinducedformationof␥H2AXfocivisibleinJurkatcells already1haftertreatment.ContrarytoJurkat,restingTcellshad muchlessDSBsvisualizedas␥H2AXfociinducedbyETO.How- ever,24haftertreatmentwithETOmanycellsstainedfor␥H2AX wereintensivelygreen,butnofociwereobserved.Thiseffectisvery spectacularespeciallyinrestingTcellsthenucleiofwhichwerenot asfragmentedasthoseofJurkatcells.Asitwasreportedpreviously [24,25],thiseffectischaracteristicforDNAdamageinapoptotic cells,whichdisplaymuchstrongerphosphorylationofH2AXand moreintenseﬂuorescencethantheoneobservedinthecaseofpri- marylesions.Altogether,ourresultsevidencedthatproliferating JurkatcellsweremoresensitivetoETOthannormalrestingTcells.

Moreover,inbothtypesofcellsDNAdamageinducedbyETOtrig- geredtheDDRfollowedbyapoptoticcaspasesactivation(chapter belowandsupplementaryFigure1).

3.3. KU55933inhibitsATMandDNAdamageresponseinresting Tcells

UpontheoccurrenceofDSBs ATMisactivatedbyautophos- phorylation. Recently, an ATP-competitive inhibitor, KU 55933 (KU), that inhibits ATM was identiﬁed and its speciﬁcity was

demonstratedbytheablationofphosphorylationofarangeofATM targets,includingp53,H2AXandothersinducedbyDNAdamage.

[17].

WewereinterestedwhetherATMinhibitionwouldaffectthe propensity of resting T cells to undergo DNA damage-induced apoptosis.Accordingly,wepretreatedTcellswith10␮MKUfor 2handthen10␮METOwasaddedtothemedium.First,using theconfocalmicroscopywecheckedthepresenceofphosphory- latedATMinETO-treatedcells,includingthosepretreatedwithKU (KU+ETO). ResultspresentedinFig.5revealedthatindeedETO inducedaccumulationofp-ATMSer1981whichwaspreventedby KU.

Next,wecheckedbyWesternblottingthelevelofATMandsome otherkeyproteinsoftheDDRpathwayuponETOand/orKUtreat- mentofrestingTcells.Asitisshown inFig.6A,ETOincreased thelevelofp-ATMSer1981already1haftertreatmentfollowed byanincreaseinitssubstrates,namely␥H2AXandp-p53Ser15.

Inductionoftotalp53anditsphosphorylationinETO-treatedcells wasfollowedbyincreasedlevelsofitsdirecttarget,namelythe proapoptoticPUMA.Asexpectedtheotherp53target,p21,which is a cellcycleinhibitor wasnotdetectedin non-proliferatingT cells.KUeffectively preventedtheinductionofp-ATMSer1981, p-p53 Ser15 and PUMA for at least 48h after ETO treatment.

Alsothe␥H2AXlevelinKU+ETOtreatedcellswassubstantially lowerfor aslong as12hafterKU+ETOtreatment. Collectively, we can assume that activationof ATM and phosphorylationof

Fig.4. ␥H2AXstaininginnormalrestingTcellsandleukemicJurkatcellstreatedwithETO.CellsweretreatedwithDMSOor10␮METOandstainedfor␥H2AX(green)and DNA(red).Representativeconfocalimagesareshown.Barscorrespondto10␮m.

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Fig.5. KUpreventsphosphorylationofATMinducedbyETOinrestingTcells.

Tcellsweretreatedwith10␮METOfor6h.KUwasaddedtotheculturefor2hbeforeETO.(A)ATMﬂuorescencerevealedbyconfocalmicroscopyofcellsimmunostainedby antibodyagainstp-ATMSer-1981.DNAwasstainedwithDRAQ5.Theresultsarerepresentativeof3independentexperimentsperformedonTcellsisolatedfrom3donors.

Atleast50cellsfromeachgroupwereanalyzed.Barscorrespondto10␮m.(B)QuantiﬁcationofATMﬂuorescence(means±SD;***p<0.001).

Fig.6. InhibitionofATMbyKUleadstoattenuationofDDR(A)andprotectionfromapoptosisinrestingTcells(B).

TcellswerepretreatedwithKUfor2handthencultivatedwithorwithout10␮METOupto48h.Theblotisrepresentativeof3independentexperimentsperformedonT cellsderivedfrom3donors.

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thedownstreamproteinsweresuccessfullyreducedbyKUtreat- ment.

However, KU had no inﬂuence on the DNA damage level introducedbyETOasmeasuredbyFADUassay(Supplementary Figure2A).

3.4. KU55933diminishedapoptosisofrestingTcellstreatedwith etoposide

AsPUMAisamediatorofapoptosiswecouldassumethatKU protectscellsalsoagainstETO-inducedapoptosis.Thusweveri- ﬁedthisbyothermarkers.Fig.6BshowsthatthePARPproteolysis detectedinETO-treatedcells 24hand48hafterETO-treatment wasdiminishedinKU+ETO-treatedcellsandhardlyvisibleinKU- treatedcellssuggesting,atleast,areducedlevelofapoptosisin KU+ETOtreatedcellsincomparisonwithETO-treatedcells.The samecouldbeconcludedfromthecomparisonofthe␥H2AXlevel.

PhosphorylatedH2AXisamarkerofDNAdamagewhichappears withinsecondsafterDNAbreak[4,26].However,itcanalsoreﬂect DNAfragmentationoccurringduringapoptosis[25,27],whichis ATM-independent[28,29].Actually,already after24hand later, concomitantlywiththeincreasedlevelof␥H2AX,weobserveda dropinp-ATMSer1981andtypicalATM“ladder”forapoptosisin ETO-treatedcellssuggestingthat␥H2AXcanbeaverysensitive markerofapoptoticDNAdegradationwhichoccursindependently ofearlyDDRactivation.Thesamesuggestionhasbeenmadepre- viouslybyotherresearchers[29].

Collectively, ETO induced symptoms of apoptosis such as:

increasedlevelofPUMA,cleavageofPARPandATM,andH2AX phosphorylationinrestingTcells.Allthesesymptomswerealmost completelysuppressedbyKUwhenchecked24hand48hafter KU+ETO-treatment.

TofurtherverifywhetherKUblocksapoptosiswecheckedthe apoptotic index(Fig.7A)andkey apoptoticcaspases uponnor- malTcelltreatmentwithETOandKU+ETO(Fig.7BandC).Asit canbeseen(Fig.7A)theapoptoticindexincreasedabout4times 48h aftercell-treatmentwith ETO.In cells pretreated withKU followedbyETOtreatmenta substantialreductionoftheapop- toticindexwasobservedincomparisonwithjustETOtreatedcells (p<0.001).Wealsocheckedthekeycaspasesinvolvedinapoptosis, namelycaspases-2,3,8and9.ResultsobtainedbyWesternblot- tingrevealedthatthelevelsofcleavedcaspases-3,8and9(Fig.7B) werehigherinETO-thaninKU-orKU+ETO-treatedcells.KUalso loweredthenumberofcellswithactivecaspase-2asmeasuredby ﬂowcytometry(Fig.7C).

Thus,wecansummarizethatKUattenuatesactivationofATM andDDRsignaltransduction,whichinturnsubstantiallydimin- ishescaspase-dependentapoptosisinETO-treatedrestingTcells.

AsithasbeenshownpreviouslythatKUdidnotinhibitapo- ptosis,butquitetothereverse,itincrementedtheapoptoticeffect ofDNAdamagingagentsinmanycancercells[17],wepretreated Jurkatcells withKUand checkedtheapoptoticindex24hafter ETO-treatment.TreatmentwithKUaloneinducedapoptosisin40%

ofJurkatcellsandtheapoptoticindexwasincreasedseveraltimes incellstreatedwithKU+ETO(SupplementaryFigure1).

3.5. InhibitionoftranscriptionattenuatesDDRresponseinTcells treatedwithetoposide

It couldbeexpectedthat ETOexertsitscytotoxicactivityin restingTcells byinﬂuencing transcription.Toverifythis,inthe followingexperimentsweusedtranscriptioninhibitors,namely␣- amanitinandDRB,whichdonotinduceDNAdamagebythemselves [30]. Bothofthem inhibitedtranscription,although ␣-amanitin wasmoreeffective.Cellspretreatedwitheither␣-amanitin(17h) or DRB (1h)displayed lower level of DNA damageinduced by

ETO (measured as53BP1 foci) and had substantially decreased DDRresponse consideredas thelevelsof p-ATM Ser 1981and p-p53Ser15,measuredafter3hofETOtreatment(Fig.8).Accord- ingly, it can be assumed that ETO activity is associated with transcription.However,theinhibitorsdidnotprotectcellsagainst ETO-inducedapoptosismeasuredatlongertimes(24hafterETO- treatment).Moreoverlongerincubationwiththeinhibitors(41h for␣-Amanitinand25hforDRBinducedapoptosisbythemselves (SupplementaryFigure2B).

4. Discussion

Theaimofourstudywastoanswerthefollowingquestions:

(i)whether theDNA damagingagent,etoposidewould beable toevokeDDRandDDR-dependentapoptosisinnon-proliferating normalhumanTlymphocytes,and(ii)whetherinhibitionofATM wouldaffectthepropensityofnormalcellstoundergocelldeath.

Previouslyithasbeenshownthattheinhibitoroftopoisomerase I,caphotectin,activatesATManddownstreamproteinsinnormal humanperipheralbloodlymphocytesbyinhibitionoftranscription [30].WeshowedthatETO,thewellrecognizedinhibitoroftopo- isomeraseII,alsoaffectedtranscription,andthuswehypothesized thatitwouldactivateDDRinrestinghumanTcells.Indeed,weshow inthispapertheactivationofATMandofp53inTcellsupontreat- mentwithETO,followedbyapoptosis.AsexpectedKUsubstantially reducedthelevelofp-ATMSer1981andp-p53Ser15.Sordetetal.

[30]alsoreportedthatblockingATMautophosphorylationbyKU reducedthelevelofdownstreamproteinphosphorylationinnor- malhumanperipheralbloodlymphocytes.Howevertheydidnot addressthequestionofthepropensityofcellspretreatedwiththe ATMinhibitortoundergoapoptosis.

Our results revealed that KU protected T cells against ETO- inducedcaspasesactivationandapoptosis.Toourknowledgethis istheﬁrstsuchreport.Eventhoughitisratherunlikelythatres- tingTcellscanundergosenescenceasweshowednop21induction, wecheckedSA-␤-galactosidaseactivity,whichisawellrecognized markerofcellularsenescence[31].Theresults,asexpected,were negative(notshown).Instead,weshowedthatKUblockedallcru- cialcaspases,andmoreimportantly,weobservedanincreasedlevel ofPUMAinETO-treatedcellsbutnotinKU+ETO-treatedcells.Asit hasbeenshownpreviously,“noPUMAnodeath”,asthisproteinis necessaryforbothp53-dependentandp53-independentcelldeath [32].AlltheseresultsprovedthatKUreducedthelevelofETO- induceddeathofrestingTcells.Thisisquiteoppositetowhatis observedincancercells.Indeed,weshowedthatKUinducedapo- ptosisandincrementedtheapoptoticindexinJurkatcellstreated withetoposide.TherearealsootherreportsshowingthatKUsen- sitizescancercellstoradio-andchemotherapytreatment[33–35]

and to various DDR-inhibitory drugs, including those targeting ATM,whichareinpreclinicalandclinicaldevelopment[3].More- over,aswassuggestedbyJackson andBartek[3]this approach couldselectivelytargetcancercells.Firstly,differentDNA-repair pathwayscanoverlapinfunction,andsometimessubstitutefor eachother.Inhibitionofagivenpathwayshouldinsomecaseshave agreaterimpactoncancercellsthanonnormalcells,whichcon- trarytocancercells,haveallpathwaysunaffected.Secondly,cancer cellsareproliferatingmorerapidlythanmostnormalcellsandthe SphaseisaparticularlyvulnerabletimeforDNA-damagetooccur.

IndeedweshowedthatJurkatcellsweremuchmoresensitiveto ETO-inducedDNAdamageandthefollowingapoptosisthannor- malrestingTcells.Thus,theantiapoptoticactivityofKUinnormal cellswithinducedDNAdamagesupportstheideaofdeveloping abranchofATMinhibitorswhichcouldactselectivelyoncancer cells.However,itisvery wellknownthatATMdeﬁciency leads toataxia-telangiectasia(A-T),agenomicinstabilitywithhallmarks

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Fig.7. InhibitionofATMprotectsrestingTcellsagainstDNAdamage-inducedapoptosis.

TcellswerepretreatedwithKUfor2handthencultivatedwithorwithout10␮METOfor24hand48h.(A)ApoptoticindexestimatedbyAnnexinV/7-AADﬂowcytometry assay.Upperpanelshowsarepresentativedensityplot.Bargraphshowsdataanalyzedfrom10independentexperimentsonTcellsfrom10donors(means±SD;***p<0.001).

(B)Levelsofcaspase-3,8,9measuredbyWesternblotting.Representativeblotsareshownfrom3experimentsperformedonTcellsfrom3donors.Arrowsindicatethefull lengthprocaspasesandtheircleavedforms.Molecular-massmarkersareshownontheleft.(C)Cellspositivefortheactiveformofcaspase-2asmeasuredbyﬂowcytometry.

Theresultscomefrom3independentexperimentsperformedonTcellsisolatedfrom3donors(means±SD).

ofneurodegeneration,immunodeficiencyandradiationsensitivity [36]suggestinghigherpropensityofA-Tcellstoundergoapopto- sis.Interestingly,othersshowedthatATMdeficiencyresultedina significantresistanceoflymphoidcellsderivedfromA-Tpatients

toFas-inducedapoptosisandthesameeffectcouldbeachievedby ATMinhibition(KU)inestablishedcelllines[37]advocatingthat thepropensitytoapoptosisofnormalcellswithATMdeﬁciencyis stillawaitingelucidation.

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Fig.8. InhibitionoftranscriptionreducesDDRinETO-treatedrestingTcells.

(A)TranscriptionlevelafterTcelltreatmentwithinhibitors.Tcellswereuntreatedortreatedwith1-␤-d-ribofuranoside(DRB;1h,40␮M)or␣-amanitin(17h,10␮M) beforetheadditionof5-ethynyluridine,theincorporationofwhichwasdetectedandisexpressedasﬂuorescenceintensity.(B)DNAdamageinTcellstreatedwithETO reducedbytranscriptioninhibitors.CellswerepretreatedwiththeinhibitorsasindicatedinAandfollowedby3htreatmentwithETO.Then,cellswerestainedforp53BP1.

The53BP1fociinatleast60cellspereachtreatmentwerecountedunderconfocalmicroscope.(C)InhibitionofDDRbytranscriptioninhibitorsinTcellstreatedwithETO.

TheleveloftotalandphosphorylatedATMandp53proteinsweremeasuredincellstreatedasin(B).Representativeresultsfortwoindependentexperimentsareshown.

BlockingapoptosisincellstreatedwithanagentinducingDNA damageraisesthequestionwhetherthecellswhichsurvivedcould have unrepaired DNA damage. Actually, we showed using the FADUassay,thatKUdidnotinfluenceDNAprimarylesionsinT cells, althoughthis wasmeasuredonly ina shorttime, namely after30minofETOtreatment.However,onecannotexcludethat cellswhichsurvivedtheKU+ETO-treatmentcouldhaveunrepaired DNAduetoattenuationoftheDNArepairmachinery.Thustheben- eficialactionofKUindiminishingapoptosisinnormalTcellsmay beweakenedbypossibleadverseeffectssuchasdelayedapopto- sisorincreasedgenomicinstabilityduetothepersistenceofDNA damage.ItwasdocumentedthatATM[38]andH2AX[39]arecrit- icalforfacilitatingtheassemblyofspecificDNA-repaircomplexes ondamagedDNA.Ontheotherhand,itcanbeimaginedthatinan organism,duetothesupportivesurveillance,thecellscouldsur- vivelongerandhaveenoughtimeforDNArepair,especiallythat KUcompeteswithATPand itsinhibitoryactiononATMshould bereversible[17].Recently,ithasbeenshown thatallproteins neededfortherepairof␥-irradiationinducedDNA-damage,that canbedetectedbythealkalinecometassay,arealreadypresentin G0cellsatsufficientamountsanddonotneedtobeinducedonce lymphocytesarestimulatedtostartcycling[40].

5. Conclusions

ItiscommonlyacceptedthatDNAdamageresponseoperates atthecell cyclecheckpointsofproliferatingcells and itcanbe thetargetforchemotherapy.Ontheotherhanddataconcerning DDRinnormalnon-proliferatingcellsareveryscarce,althoughthe harmfuleffectelicitedbyradio/chemotherapyonrestingTcellshas beenreported.Accordingly,theaimofourstudywastoanswerthe followingquestions:(i)whethertheDNAdamagingagent,etopo- sideisabletoevokeDDR-dependentapoptosisinnon-proliferating normalhumanTlymphocytes,and(ii)whetherinhibitionofATM, whichisthekeyenzymeinDDRaffectsthepropensityofnormal cellstoundergocelldeath.

Weshowfortheﬁrsttimethatetoposide,whichisatopoiso- meraseIIinhibitorinducedDNAdamageresponseviainﬂuencing transcription andthe subsequentapoptosisinnormal restingT cells. Both DDRand apoptosiswere blocked byATM inhibitor, KU 55933. The resultis intriguingin the light of the fact that thisinhibitorsensitizescancercellstoanticancerdrugtreatment.

Nonetheless,itcouldnotbeexcludedthatblockingDDRinnormal cellsdoesnotprotectagainstDNAdamagewhichmayeitherper- sistinnon-proliferatingcellsorinducedelayedapoptosis.Thus,to

judgewhetherATMinhibitorsdonotcausesideeffectsadditional studiesonclinicalmaterialareneeded.

Acknowledgements

Thiswork wassupported bythe NationalCenter ofScience (grant0727/B/P01/2011/40).

AppendixA. Supplementarydata

Supplementary data associated with this article can be found,intheonlineversion,athttp://dx.doi.org/10.1016/j.dnarep.

2012.08.006.

References

[1]L.H.Hartwell,T.A.Weinert,Checkpoints:controlsthatensuretheorderofcell cycleevents,Science246(1989)629–634.

[2]F.d’AddadiFagagna,Livingonabreak:cellularsenescenceasaDNA-damage response,Nat.Rev.Cancer8(2008)512–522.

[3]S.P.Jackson,J.Bartek,TheDNA-damageresponseinhumanbiologyanddisease, Nature461(2009)1071–1078.

[4]W.M.Bonner,C.E.Redon,J.S.Dickey,A.J.Nakamura,O.A.Sedelnikova,S.Solier, Y.Pommier,GammaH2AXandcancer,Nat.Rev.Cancer8(2008)957–967.

[5]L.B.Schultz,N.H.Chehab,A.T.D.Malikzay,Halazonetis,p53bindingprotein1 (53BP1)isanearlyparticipantinthecellularresponsetoDNAdouble-strand breaks,J.CellBiol.151(2000)1381–1390.

[6]Z.Storchova,D.Pellman,Frompolyploidytoaneuploidy,genomeinstability andcancer,Nat.Rev.Mol.CellBiol.5(2004)45–54.

[7]I.B.Roninson,E.V.Broude,B.D.Chang,Ifnotapoptosis,thenwhat?Treatment- inducedsenescenceandmitoticcatastropheintumorcells,DrugResist.Updat.

4(2001)303–313.

[8]K.Kobayashi,M.J.Ratain,Pharmacodynamicsandlong-termtoxicityofetopo- side,CancerChemother.Pharmacol.34(Suppl.)(1994)S64–S68.

[9]J.L.Nitiss,TargetingDNAtopoisomeraseIIincancerchemotherapy,Nat.Rev.

Cancer9(2009)338–350.

[10]C.C.Wu,T.K.Li,L.Farh,L.Y.Lin,T.S.Lin,Y.J.Yu,T.J.Yen,C.W.Chiang,N.L.

Chan,StructuralbasisoftypeIItopoisomeraseinhibitionbytheanticancer drugetoposide,Science333(2011)459–462.

[11]N.Mondal,J.D.Parvin,DNAtopoisomeraseIIalphaisrequiredforRNApoly- meraseIItranscriptiononchromatintemplates,Nature413(2001)435–438.

[12]B.G.Ju,V.V.Lunyak,V.Perissi,I.Garcia-Bassets,D.W.Rose,C.K.Glass,M.G.

Rosenfeld,AtopoisomeraseIIbeta-mediateddsDNAbreakrequiredforregu- latedtranscription,Science312(2006)1798–1802.

[13]J.L.Nitiss,DNAtopoisomeraseIIanditsgrowingrepertoireofbiologicalfunc- tions,Nat.Rev.Cancer9(2009)327–337.

[14] K.Nagami,Y.Kawashima,H.Kuno,M.Kemi,H.Matsumoto,Invitrocytotoxicity assaytoscreencompoundsforapoptosis-inducingpotentialonlymphocytes andneutrophils,J.Toxicol.Sci.27(2002)191–203.

[15]E. Markova, J. Torudd, I. Belyaev, Long time persistence of residual 53BP1/gamma-H2AXfociinhumanlymphocytesinrelationshiptoapoptosis, chromatincondensationandbiologicaldosimetry,Int.J.Radiat.Biol.87(2011) 736–745.

[16] E.C.Bourton,P.N.Plowman,D.Smith,C.F.Arlett,C.N.Parris,Prolongedexpres- sionofthegamma-H2AXDNArepairbiomarkercorrelateswithexcessacute

(10)

andchronictoxicityfromradiotherapytreatment,Int.J.Cancer129(12)(2011) 2928–2934.

[17]I.Hickson,Y.Zhao,C.J.Richardson,S.J.Green,N.M.Martin,A.I.Orr,P.M.Reaper, S.P.Jackson,N.J.Curtin,G.C.Smith,Identiﬁcationandcharacterizationofanovel andspeciﬁcinhibitoroftheataxia-telangiectasiamutatedkinaseATM,Cancer Res.64(2004)9152–9159.

[18]U.K.Laemmli,Cleavageofstructuralproteinsduringtheassemblyofthehead ofbacteriophageT4,Nature227(1970)680–685.

[19]M.Moreno-Villanueva,R. Pfeiffer, T.Sindlinger, A.Leake, M.Muller,T.B.

Kirkwood,A.Burkle,Amodiﬁedandautomatedversionofthe‘Fluorimetric DetectionofAlkalineDNAUnwinding’methodtoquantifyformationandrepair ofDNAstrandbreaks,BMCBiotechnol.9(2009)39.

[20]T.Tanaka,H.D.Halicka,F.Traganos,K.Seiter,Z.Darzynkiewicz,Inductionof ATMactivation,histoneH2AXphosphorylationandapoptosisbyetoposide:

relationtocellcyclephase,CellCycle6(2007)371–376.

[21] A.Brzezinska,A.Magalska,E.Sikora,ProliferationofCD8+incultureofhumanT cellsderivedfromperipheralbloodofadultdonorsandcordbloodofnewborns, Mech.AgeingDev.124(2003)379–387.

[22]ISchonn,JHennesen,D.C.Dartsch,Cellularresponsestoetoposide:celldeath despitecellcyclearrestandrepairofDNAdamage,Apoptosis15(2)(2010) 162–172.

[23]M.Moreno-Villanueva,T.Eltze,D.Dressler,J.Bernhardt,C.Hirsch,P.Wick,G.

vonScheven,K.Lex,A.Burkle,TheautomatedFADU-assay,apotentialhigh- throughputinvitromethodforearlyscreeningofDNAbreakage,Altex28(4) (2011)295–303.

[24]H.D.Halicka,X.Huang,F.Traganos,M.A.King,W.Dai,Z.Darzynkiewicz,His- toneH2AXphosphorylationaftercellirradiationwithUV-B:relationshipto cellcyclephaseandinductionofapoptosis,CellCycle4(2005)339–345.

[25]T.Tanaka,A.Kurose,X.Huang,W.Dai,Z.Darzynkiewicz,ATMactivationand histoneH2AXphosphorylationasindicatorsofDNAdamagebyDNAtopo- isomeraseIinhibitortopotecanandduringapoptosis,CellProlif.39(2006) 49–60.

[26]T.Tanaka,D.Halicka,F.Traganos,Z.Darzynkiewicz,CytometricanalysisofDNA damage:phosphorylationofhistoneH2AXasamarkerofDNAdouble-strand breaks(DSBs),MethodsMol.Biol.523(2009)161–168.

[27] G.Mosieniak,M.Sliwinska,K.Piwocka,E.Sikora,Curcuminabolishesapo- ptosisresistanceofcalcitriol-differentiatedHL-60cells,FEBSLett.580(2006) 4653–4660.

[28]C.Lu,F.Zhu,Y.Y.Cho,F.Tang,T.Zykova,W.Y.Ma,A.M.Bode,Z.Dong,Cellapo- ptosis:requirementofH2AXinDNAladderformation,butnotfortheactivation ofcaspase-3,Mol.Cell23(2006)121–132.

[29]B.Mukherjee,C.Kessinger,J.Kobayashi,B.P.Chen,D.J.Chen,A.Chatterjee,S.

Burma,DNA-PKphosphorylateshistoneH2AXduringapoptoticDNAfragmen- tationinmammaliancells,DNARepair(Amst.)5(2006)575–590.

[30]O.Sordet,C.E.Redon,J.Guirouilh-Barbat,S.Smith,S.Solier,C.Douarre,C.

Conti,A.J.Nakamura,B.B.Das,E.Nicolas,K.W.Kohn,W.M.Bonner,Y.Pommier, Ataxiatelangiectasiamutatedactivationbytranscription-andtopoisomerase I-inducedDNAdouble-strandbreaks,EMBORep.10(2009)887–893.

[31] E.Sikora,T.Arendt,M.Bennett,M.Narita,Impactofcellularsenescencesigna- tureonageingresearch,AgeingRes.Rev.10(2010)146–152.

[32] J.Yu,L.Zhang,PUMA,apotentkillerwithorwithoutp53,Oncogene27(Suppl.

1)(2008)S71–S83.

[33] I.G.Cowell,B.W.Durkacz,M.J.Tilby,Sensitizationofbreastcarcinomacells toionizingradiationbysmallmoleculeinhibitorsofDNA-dependentpro- teinkinaseandataxiatelangiectsiamutated,Biochem.Pharmacol.71(2005) 13–20.

[34] S.E.Golding,E.Rosenberg,N.Valerie,I.Hussaini,M.Frigerio,X.F.Cockcroft, W.Y.Chong,M.Hummersone,L.Rigoreau,K.A.Menear,M.J.O’Connor,L.F.

Povirk,T.vanMeter,K.Valerie,ImprovedATMkinaseinhibitorKU-60019 radiosensitizesgliomacells,compromisesinsulin,AKTandERKprosurvival signaling,andinhibitsmigrationandinvasion,Mol.CancerTher.8(2009) 2894–2902.

[35]F.S.Shaheen,P.Znojek,A.Fisher,M.Webster,R.Plummer,L.Gaughan,G.C.

Smith,H.Y.Leung,N.J.Curtin,C.N.Robson,TargetingtheDNAdoublestrand breakrepairmachineryinprostatecancer,PLoSONE6(2011)e20311.

[36]Y.Shiloh,ATMandrelatedproteinkinases:safeguardinggenomeintegrity,Nat.

Rev.Cancer3(2003)155–168.

[37]V.Stagni,M.G.diBari,S.Cursi,I.Condo,M.T.Cencioni,R.Testi,Y.Lerenthal,E.

Cundari,D.Barila,ATMkinaseactivitymodulatesFassensitivitythroughthe regulationofFLIPinlymphoidcells,Blood111(2008)829–837.

[38]A.A.Goodarzi,A.T.Noon,D.Deckbar,Y.Ziv,Y.Shiloh,M.Lobrich,P.A.Jeggo, ATMsignalingfacilitatesrepairofDNAdouble-strandbreaksassociatedwith heterochromatin,Mol.Cell31(2008)167–177.

[39]A.Celeste,S.Petersen,P.J.Romanienko,O.Fernandez-Capetillo,H.T.Chen,O.A.

Sedelnikova,B.Reina-San-Martin,V.Coppola,E.Meffre,M.J.Diﬁlippantonio, C.Redon,D.R.Pilch,A.Olaru,M.Eckhaus,R.D.Camerini-Otero,L.Tessarollo,F.

Livak,K.Manova,W.M.Bonner,M.C.Nussenzweig,A.Nussenzweig,Genomic instabilityinmicelackinghistoneH2AX,Science296(2002)922–927.

[40]C.Mayer,O.Popanda,O.Zelezny,M.C.vonBrevern,A.Bach,H.Bartsch,P.

Schmezer,DNArepaircapacityaftergamma-irradiationandexpressionpro- ﬁlesofDNArepairgenesinrestingandproliferatinghumanperipheralblood lymphocytes,DNARepair(Amst.)1(2002)237–250.