Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

jo u r n al ho me p a g e :w w w . e l s e v i e r . c o m / lo c a t e / m o l m u t C o m mu n i ty a dd r e s s :w w w . e l s e v i e r . c o m / l o c a t e / m u t r e s

Suppression of DNA-dependent protein kinase sensitize cells to radiation without affecting DSB repair

Ann-Soﬁe Gustafsson

^∗

, Andris Abramenkovs, Bo Stenerlöw

SectionofBiomedicalRadiationSciences,DepartmentofRadiology,OncologyandRadiationScience,RudbeckLaboratory,UppsalaUniversity, DagHammarskjöldsVäg20,SE-75185Uppsala,Sweden

a r t i c l e i n f o

Articlehistory:

Received9April2014

Receivedinrevisedform4June2014 Accepted16June2014

Availableonline22June2014

Keywords:

DNArepair DNA-PKcs Ionizingradiation DNA-PKdeﬁciency NU7441

a b s t r a c t

EfﬁcientandcorrectrepairofDNAdouble-strandbreak(DSB)iscriticalforcellsurvival.Defectsinthe DNArepairmayleadtocelldeath,genomicinstabilityanddevelopmentofcancer.Thecatalyticsubunitof DNA-dependentproteinkinase(DNA-PKcs)isanessentialcomponentofthenon-homologousendjoining (NHEJ)whichisthemajorDSBrepairpathwayinmammaliancells.Inthepresentstudy,byusingsiRNA againstDNA-PKcsinfourhumancelllines,weexaminedhowlowlevelsofDNA-PKcsaffectedcellular responsetoionizingradiation.DecreaseofDNA-PKcslevelsby80–95%,inducedbysiRNAtreatment,lead toextremeradiosensitivity,similartothatseenincellscompletelylackingDNA-PKcsandlowlevelsof DNA-PKcspromotedcellaccumulationinG2/Mphaseafterirradiationandblockedprogressionofmitosis.

Surprisingly,lowlevelsofDNA-PKcsdidnotaffecttherepaircapacityandtheremovalof53BP1or␥- H2AXfociandrejoiningofDSBappearednormal.Thiswasinstrongcontrasttocellscompletelylacking DNA-PKcsandcellstreatedwiththeDNA-PKcsinhibitorNU7441,inwhichDSBrepairwereseverely compromised.ThissuggeststhattherearedifferentmechanismsbywhichlossofDNA-PKcsfunctions cansensitizecellstoionizingradiation.Further,fociofphosphorylatedDNA-PKcs(T2609andS2056)co- localizedwithDSBandthiswasindependentoftheamountofDNA-PKcsbutfociofDNA-PKcswasonly seeninsiRNA-treatedcells.OurstudyemphasizesonthecriticalroleofDNA-PKcsformaintainingsurvival afterradiationexposurewhichisuncoupledfromitsessentialfunctioninDSBrepair.Thiscouldhave implicationsforthedevelopmentoftherapeuticstrategiesaimingtoradiosensitizetumorsbyaffecting theDNA-PKcsfunction.

1. Introduction

Ionizingradiationandendogenousprocesses,e.g.freeradicals formedinnormal cellularmetabolismor collapseofreplication forks during replication of DNA, may result in complex damage to the DNA. Among the different types of DNA damage, DNA double-strand break (DSB) is the most unfavorable type of DNA lesion, and unrepaired or misrepaired DSB can leadto genemutations, chromosomalaberrations,permanentcellcycle arrest, apoptosis, mitotic cell death and cancer development [1–3].

InDSBrepairtwopathwayshavebeenimplicatedtoprocess thebreak,non-homologousendjoining(NHEJ)andhomologous recombination (HR) [4]. Some of the key proteins involved in NHEJareDNA-dependentproteinkinasecatalyticsubunit(DNA- PKcs), Ku70 and Ku80 that together form the DNA-dependent

∗ Correspondingauthor.Tel.:+46184713887;fax:+46184713432.

E-mailaddress:ann-soﬁe.gustafsson@bms.uu.se(A.-S.Gustafsson).

proteinkinase(DNA-PK)complex[5].DNA-PKcsisamemberofthe phosphatidylinositol3(PI-3)-likekinasefamilythatincludesATM (ataxia-telangiectasiamutated)andATR(ATMandRad3-related) [6]. Besidesfunctioning in NHEJ, DNA-PKcsis alsorequired for V(D)Jrecombinationofimmune-globulingenesandT-cellrecep- torgenes,andtelomerelengthmaintenance[7].NHEJisbelieved toplayamoreimportantrolethenHRinmammaliancells,andis presentatalltimeduringthecellcycle,HRislimitedtolateS,G2 andMphase[8–10].

ProteinsinvolvedintherepairandsignalingofDSBplayavital roleincellsurvivalandhealthyhumanlife[11–13].Manytumors typescharacterizedbygenomicinstabilityarecausedbymutations in DSB-responsivegenes.The expressionofDNA-PKcsin tumor biopsiescanbeboth up-and down-regulatedin speciﬁctumor types[14].SeveralcancertypeswithdecreasedlevelsofbothDNA- PKcsandKu70/80havebeenfound[15–17]whichcouldindicate thatpartialdeﬁciencyofproteinsinvolvedinDSBrepairmaycon- tributetoincreasedriskofdevelopingcancer[14,18].Incontrast, elevatedlevelsofDNA-PKcshavebeenfoundinvarioustumortypes andlinkedtopoorsurvival[19].

http://dx.doi.org/10.1016/j.mrfmmm.2014.06.004

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2 A.-S.Gustafssonetal./MutationResearch769(2014)1–10

Besidesitsdirectroleinrepair,itisbecomingevidentthatDNA- PKcsmayhaveotherfunctionsthatpromotecellsurvival.Arlander etal.(2008)suggestedthatDNA-PKcscontributestotheG2check- pointsandcellcycleregulation[20].Recentlyitwasalsoshown thatDNA-PKcsisinvolvedinstabilizationofthemitoticspindle formationinawaythatreducedlevelsofDNA-PKcsmaydirect thecelltowardmitoticcatastrophe[21–23].Furtherdataindicates thatDNA-PKcsisacentralplayerinG2progressionandlowlev- elsofDNA-PKcspromotesafastprogressionfromSphasetoG2, butinterfereswithprogressionthroughmitosisandleadstocell accumulationinG2[22].HumancellsdeﬁcientinDNA-PKshowa severesensitivitytoradiationandreducedcapacitytorepairDSBs [11,24,25]anditispossiblethatthisinabilitytoprogressthrough thecell-cycleisaffectedbythepresenceofunrepairedDNA[26].

Inthepresentinvestigation,westudiedDNArepairincellswith reducedlevelsofDNA-PKcs.HumancellstransfectedwithsiRNA targetingDNA-PKcs,resultingin5–20%oftheoriginalDNA-PKcs content,exhibitedsimilarextremeradiosensitivityascellscom- pletelylackingDNA-PKcs.Surprisingly,thelowDNA-PKcs-levels hadnosigniﬁcanteffect onDSBrepair capacity, butlow levels of DNA-PKcs affected thecell cycle, resulting in siRNA treated cellaccumulationinG2afterirradiationandinabilitytoproceed throughmitosis.Thesedatasupportrecentﬁndingssuggestingthat DNA-PKcsisanimportantregulatorofthecell-cyclewhilewehere showthatthisfunctionseemstobeuncoupledfromthecellular capacitytorepairDSBs.

2. Materialsandmethods 2.1. Celllinesandcultureconditions

HumanepithelialcancercelllineA431(ATCC,Manassas,VA, USA) was grown in Ham’s F-10. Human oral squamous carci- nomacell line H314 (ATCC) was grown in DMEM/Ham’s F-12.

HumancolorectalcarcinomacelllineHCT116(ATCC)wasgrownin McCoy’s.NormalhumanskinfibroblastGM5758cellline(Human GeneticMutantCellRepository,Camden,NJ,USA)wasgrownin MEM, supplemented with 2× concentration of MEM vitamins, essentialandnon-essentialamino acids.Thehumangliomacell lines M059J deficient in DNA-PKcs (ATCC) and M059K (ATCC) withwild-typeDNA-PKcswereculturedinDMEM/Ham’sF12sup- plementedwithnon-essential amino acids.Culturemedium for allthecells was supplemented with10%FCS (Sigma, St. Louis, MO),2mMl-glutamine,100IU/mlpenicillinand10␮g/mlstrep- tomycin(BiochromKg,BerlinGermany).Allcellswereculturedin ahumidifiedincubatorwith5%CO2at37^◦Candtrypsinizedwith trypsin–EDTA(0.25%trypsin,0.02%EDTA,BiochromKg).

2.2. Irradiationandinhibitortreatment

Cellswereirradiatedwith¹³⁷Cs␥-rayphotons(Gammacell40 Exactor,MDSNordion,Kanata,Canada)atadoserateof∼1Gy/min.

NU7441wasobtainedfromSelleckchem(Houston,TX).NU7441 wasdissolvedindimethylsulfoxide.Fortreatment,cellswerepre- treatedin37^◦Cfor1hwith2–10␮MNU7441beforeirradiation.

ClonogenicsurvivalassayNU7441wasremoved24hafterirradi- ationwithfreshmedium.Dimethylsulfoxidewasusedin mock treatedcells.

2.3. Antibodies

Primarymouse-monoclonalantibodiesusedwere,DNA-PKcs (ab1832,Abcam), p-DNA-PKcs (Thr2609)(ab18356, Abcam), ␤- actin(Sigma)and ␥-H2AX(Millipore).Primaryrabbit-polyclonal antibodyusedwere53BP1(BethylLab,Montgomery,TX),p-DNA- PKcs(Ser2056)(ab18192,Abcam),p-DNA-PKcs(Thr2609)(Santa

Cruz),p-AKT(Ser437)(CellSignaling)andp-HistoneH3(Millipore).

NucleuswasstainedwithDAPI(Invitrogen).Secondaryantibod- iesAlexaFluor488goatanti-mouseandAlexaFluor555donkey anti-rabbit(Invitrogen).

2.4. siRNAtransfection

ForthesiRNAtransfectionofDNA-PKcsaSMARTpoolprovided byDharmacon(ThermoScientific)wasusedwithfoursiRNAspe- cificforPRKDC(catalogueno.L-005030-00-0020)andaSMARTpool asnon-specific control(mock) (catalogueno.D-001810-10-20).

TransfectionswereperformedwithDharmaFECT1 Transfection Reagent(cataloguenoT-2001-01)according totheinstructions ofthesupplier.TheconcentrationofsiRNAwas100nMandwas addedtothecellstogetherwithDharmaFECTI.Cellswereseeded onedaypriortransfection,andcellswereanalyzedthreedaysafter transfection.

2.5. SDS-PAGEandWesternblotanalysis

Cellswerelysedinicecoldlysisbuffercontaining1%Tween-20, 20mMTris(pH8.0)137mMNaCl,10%glycerol,2nMEDTA,1mM activatedsodiumorthovanadate(Sigma) and protease inhibitor cocktail (P8340, Sigma). Cell extracts were loaded and elec- trophoresedonNuPAGENovex3–8%Tris–acetategels(Invitrogen).

Wettransfertonitrocellulosemembraneswasdoneovernightand thenthemembraneswereblockedfor1hin5%BSA,PBSandthen incubatedwiththeprimaryantibodyovernightat4^◦C.Secondary antibodies(626520and656120)(Invitrogen)labeledwithHorse RadishPeroxidase wereincubatedfor1hatroomtemperature.

ImmunoreactivebandswerevisualizedinaCCDcamera(SuperCCD HR,Fujiﬁlm,Japan)aftertreatmentwithelectrochemiluminescent solution(Immobilon). The intensity ofthe bands wasanalyzed usingImageJsoftware(NIH,US).

2.6. Cellsurvival

Cellswerecountedandanappropriatenumberwasseededinto T-25cellﬂaskintriplicateforeachdose.Cellswereirradiatedwith 1–4Gy,4hafterseeding.After1–2weekscellswereﬁxedin95%

EtOHandstainedwithMayer’shematoxylinandcolonieswith>50 cellswerescored.

2.7. Mitoticanalysis

ThreedaysaftertransfectionwithsiRNAcellswereirradiated with5Gy,andlettorepairfor24hinfreshmediumbeforeﬁxation andimmunostainingwithp-HistoneH3 (p-H3)antibody.Atthe microscopep-H3positivecellswerescored,atleast>250cellsper datapoint.

Fornocodazoletreatment,cellsweretreatedwith1␮l/mgnoco- dazole(Sigma),3daysaftertransfectionwithsiRNA,andincubated for6h.After6h,nocodazolewasremovedandcellswereirradiated with2Gyandlettorepairfor18hinfreshmediumbeforeﬁxation andimmunostainingwithp-H3antibody.Atthemicroscopep-H3 positivecellswerescored,atleast>400cellsperdatapoint.

2.8. Immunoﬂuorescencestainingandquantiﬁcationoffoci

Cellsweregrownonmicroscopeslidesforthreedaysaftertrans- fectionandwereirradiatedwith1–5Gy.AfterrepairofDNAfor varioustimesat37^◦C,thecellswereﬁxedinmethanolfor20min at−20^◦Cfollowedbypermeabilizationinicecoldacetonefor10s.

Blockingwasperformedin10%FCS-PBSfor1hatroomtemperature andthenincubationovernightat4^◦Cwithprimaryantibodies.Sec- ondaryantibodieswereincubatedfor1hat37^◦C(dilution1:400).

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Fig.1. ReductionofDNA-PKcsleadstoextremeradiosensitivity.(A)WB:expressionofDNA-PKcsaftersiDNA-PKcstransfectionfor3daysinA431,HCT116,H314,GM5758 cellsandinDNA-PKcsdeﬁcientM059Jcells.QuantiﬁcationofbandintensitywithImageJ,fractionofresidualDNA-PKcsisshownforeachcellline,±SDfromthreeexperiments.

(B)RepresentativeimagesofA431cellmock-transfectedandsiDNA-PKcstransfectedfor3days,ﬁxedandimmunostainedwithantibodiesagainstDNA-PKcs.Nucleiwere visualizedwithDAPIstaining.(C)Clongenicsurvivalinthreecelllines,A431,HCT116andH314,transfectedwithsiRNAagainstDNA-PKcs(siDNA-PKcs)for3daysortreated withnon-speciﬁcsiRNA(mock).Incomparison,survivalforM059Jcells(lackingDNA-PKcs)andM059K(wtDNA-PKcs)areshown.Colonieswith>50cellswerescored10–15 daysafterirradiation.SF=survivingfraction.Datarepresentmean±SDofatleastthreeindependentexperimentsforeachcellline.(D)Radiationsensitivity,summaryof meansurvivingfractionat2Gy.

Slides werecapturedwithZeiss LSM510Meta confocalmicro- scope.ImageswereprocessedandfociwerecountedusingImage Jsoftware(NIH,US).

2.9. DSBRejoiningandpulsed-ﬁeldgelelectrophoresis

Cells were labeled with 2kBq/ml [methyl-¹⁴C] thymidine (PerkinElmer),togetherwithsiRNAtransfection,threedaysprior toirradiation.Cellswereirradiatedwith40Gyandembeddedin 90␮lagaroseplugs(0.6%w/v,InCert,Cambrex).Cellplugswere lysedusingatwo-stepcoldlysisprotocolinESPlysisbuffer(2%N- lauroylsarcosine(Sigma),1mg/mlProteinaseK(Roche),alldiluted in0.5MEDTA(Na₃)atpH8.0[27].After>20hthecellplugswere movedtoHS-bufferandincubatedovernightat4^◦C(HS:HighSalt;

1.85MNaCl,0.15MKCl,5mMMgCl2,2mMEDTA,4mMTris,0.5%

TritonX-100,pH7.5,TritonX-100isaddedjustbeforeuse).Plugs werewashedtwicein0.1MEDTAandoncein0.5×TBEat4^◦C prior toelectrophoresis.Theplugswerethen loadedintowells inachilled(4^◦C)agarosegel(0.8%SeaKemGold,Lonza).Thegel wasplacedintoaPFGEunit(GeneNavigator,AmershamPharmacia Biotech,Uppsala,Sweden)with120^◦betweentheﬁelds.Following electrophoresisin0.5×TBEat10^◦C,thegelswereslicedattheposi- tionofthe5.7MbpchromosomefromSchizosaccharomycespombe (BioRad),and¹⁴Cinthegelsegmentsweremeasuredbyliquidscin- tillation.ThefractionofradioactivitycorrespondingtoDNAofsize lessthan5.7Mbpwasdividedbythetotalradioactivityinthelane, givingtheFractionActivityReleased(FAR)whichisameasureof

DNAdouble-strandbreaks.Datawasnormalized,setto100%at t=0.

3. Results

3.1. ReductionofDNA-PKcsleadstoextremeradiosensitivity

ThereductionofDNA-PKcswasconﬁrmedonWesternblotper- formedinparallelwitheachexperiment(Fig.1A).Proteinlevels weremarkedlydecreasedtwodaysfollowinginitialtransfection andwerestableforatleastfourdays(Supplementary Fig.S1A).

Thetransfection efficiencyvariedslightly betweenexperiments andcelllines,buttherewasalarge80–95%reductioninDNA-PKcs levelsinallofthetestedhumancelllines,A431epithelialcancer cells,H314oralsquamouscarcinomacells,HCT116colorectalcar- cinomacellsandGM5758normalfibroblasts.Thedepletionwas alsoevidentonimageswithimmunofluorescentstainingagainst DNA-PKcs(Fig.1B).ATMlevelswerealsoanalyzedforpotential lossasaconsequenceofDNA-PKcsknock-down,howeverATMlev- elswerenotaltered(SupplementaryFig.S1B).TheM059Jglioma celllinewhichcompletelylacksDNA-PKcsissensitivetoionizing radiationandisshownforcomparison.Totestwhetheran80–95%

depletionofDNA-PKcsinﬂuencethesurvivalandproliferationof cells,aclonogenicsurvivalassaywasperformedonthreediffer- entcelllines,A431,H314andHCT116(Fig.1C).siRNAmediated depletionofDNA-PKcsmarkedlyreducedthesurvivingfractionin allcelllinesandatadoseof2Gytherewasa3–10foldreduction

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Fig.2. CellswithlowlevelsofDNA-PKcsaredelayedinthemitoticprogression.(A)AccumulationofmitoticcellsinasynchronousA431cellswithlowlevelsofDNA-PKcs.

Cellswereirradiatedwith5Gyandscoredforp-H3-positivecells24hafterirradiation.Datarepresentmean±SDoftwoindependentexperiments.Atleast250cells/time pointwerescoredperexperiment.(B)SuppressionofDNA-PKcsleadstoaccumulationofmitoticcellsfollowingirradiationandtreatmentwithnocodazole.Cellswere synchronizedfor6hwithnocodazoleandirradiated,andafter18hincubationinmediumwithoutnocodazole,cellswereﬁxedandp-H3-positivecellswerescored.Ctr cellswereunirradiated.Datarepresentmean±SDoftwoindependentexperiments.Atleast400cells/timepointwerescoredperexperiment.(C)PhosphorylatedDNA-PKcs (Thr2609)isdislocatedfromchromatininmitoticcellsandlocatedatthemidbody(whitearrows).Thr2609(green)ismergedwithp-H3-positive(red).Incomparison,total DNA-PKcs(green)ininterphaseandmitoticcellsareshown(right).NucleiwerestainedwithDAPI(blue).

inthesurvival(Fig.1CandD).Incomparison,M059Jcells,which completelylacksDNA-PKcs,areshown.Thus,partialreductionin DNA-PKcsmayleadtosimilarextremeradiosensitivityasseenin cellscompletelylackingDNA-PKcs.

3.2. CellswithlowlevelsofDNA-PKcsaredelayedinthemitotic progression

Recentdata suggest that DNA-PKcs, beyonditsrole in DNA repair,mayhaveothercellularfunctionsandcouldbeanimpor- tantregulatorofthecell-cycle[21–23].Totestthehypothesisthat lowDNA-PKcslevelscouldleadtoaberrantmitosis,asynchronous A431cellswerescoredforphosphorylatedHistoneH3(p-H3)24h afterirradiation.p-H3isrelatedtoseveralphasesofmitosisand

usedasamarkerhereinforpositiveG2/Mcells.Cellstreatedwith siRNAagainstDNA-PKcsexhibitedtwo-foldincreaseofp-H3posi- tivecellswhencomparedtomocktreatedcells(Fig.2A).Further,in siRNAtreatedcellsasubstantialnumberofthemitoticcellswere inlatemitosis(metaphase,anaphaseortelophase),whichwasin strongcontrasttomocktreatedcells(datanotshown).Toenrich thefractionofcellsinmitosis,cellsweretreatedwithnocodazole, anagentusedtoarrestandsynchronizecellsinG2/M-phase.Cells wereincubatedfor6hwithnocodazole,irradiatedandreleased for18hbyremovingthedrugbeforeﬁxationandscoringofp-H3- positivecells.Afterirradiationwith2Gytherewasa2-foldincrease in the fractionof p-H3-positive cells when treated with siRNA againstDNA-PKcscomparedtomocktreatedcells(Fig.2B).Thus, theseresultssuggestthatlowamountsofDNA-PKcscouldleadto

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accumulationofcellsinG2/Mphaseafterirradiation.Theresults indicatethatirradiatedcellswithlow levelsof DNA-PKcsenter mitosisascellswithnormalproteinlevels,butarethendelayed inthemitoticprogression.Thisfurtherstrengthensthehypothesis that DNA-PKcs hasa role in regulation of the mitotic progression.Indeed,inthemitoticA431cells DNA-PKcswasdislocated fromchromatinandmitosis-speciﬁcDNA-PKcs phosphorylation (Thr2609)appearedtoconcentrateattwositesonoppositeside ofthenucleusimplyingadifferentrolethaninDSBrepair(Fig.2C), although there was no evident difference in the protein dis- tribution between siDNA-PKcs or mock treated cells (data not shown).

3.3. CellswithlowresiduallevelsofDNA-PKcshaveapparently normalDSBrepair

SincecellswithdefectiveDNA-PKhaveareducedrepaircapac- ity,itisreasonabletoassumethattheextremeradiosensitivityand themitoticfailure,seeninDNA-PKcsdepletedcellsabove,canbe explainedbyinabilitytorepairDSB.Totestthishypothesis,the formationanddisappearanceof53BP1(p53bindingprotein1)foci and␥-H2AXwasanalyzedinsinglecellsafterirradiation.GM5758 andA431cellsweretransfectedwithsiRNAagainstDNA-PKcsor mocktreatedandafter3dayscellswereirradiatedandincubated forrepair.53BP1fociwererapidlyformedinbothcelllinesafter irradiationand theinitial numberof 53BP1 fociwas similarin cellstransfectedwithsiRNAagainstDNA-PKcsandmocktreated (Fig.3A).However,somewhatsurprising,thereducedamountof DNA-PKcsdidnotsigniﬁcantlyaffectthekineticpropertiesofeither 53BP1or␥-H2AXineithercellline(Fig.3BandD)and1–24hafter irradiationthereweresimilarnumbersoffociincellstransfected withsiRNAagainstDNA-PKcsand mocktreatedcells. Similarly, therewasnodifferencebetweensiDNA-PKcs andmock treated cellswhentheresidualnumberof53BP1fociwasscored24hafter irradiationwithdifferentdoses(Fig.3C).Theseresultsshowthat siDNA-PKcsdoesnotaffecttheformation anddisappearanceof boththeDSBmarker53BP1and␥-H2AXafterirradiation.

TofurtherinvestigateiflowDNA-PKcslevelsaffectthecapacity torepairradiation-inducedDSBs,DSBrejoiningwasanalyzedusing pulsed-ﬁeldgelelectrophoresis(PFGE).Despitethelargereduction inthelevelsofDNA-PKcs,therewasnoeffectonDSBrejoiningin anyofthefourtestedcelllines(Fig.3E),notevenforradiationdoses upto200Gy,whichinducesatleast5000–6000DSBpercell(Sup- plementaryFig.S2). Incontrast,M059Jcells,completelylacking DNA-PKcs,showedonlymarginalrejoiningupto24hpostirra- diation(Fig.3E).Insummary,thesedatasuggestthatdespitelow residuallevelsofDNA-PKcs,i.e.5–20%oftheoriginalproteinlevels, cellshaveapparentlynormalDSBrepair.

3.4. InhibitionoftheDNA-PKcskinaseactivitysensitizecellsto radiationandreduceDSBrepair

BecausesiDNA-PKcstreatmentmarkedlyreducedcellsurvival afterirradiation,despiteanefﬁcientDNArepair,wewantedtocom- paresiDNA-PKcstreatedcellswithcellstreatedwiththespeciﬁc DNA-PKcsinhibitorNU7441.Cellsurvivalwasmarkedlyreduced inA431cellstreatedwithNU7441(Fig.4A),andthedose-response wassimilartothatinM059Jcells,lackingDNA-PKcs(Fig.1C).We nextinvestigatedwhethertreatmentwithNU7441affectedthefor- mationanddisappearance53BP1inA431andGM5758cells.The initialnumberof53BP1fociafter1hwassimilarinuntreatedcells andcells treatedwithNU7441,however,after4–24hofrepair, theresidualnumbersof53BP1fociwerehigherin theNU7441 treatedcells(Fig.4B).Further,inGM5758cellsDSBrejoiningwas almostcompletelyinhibitedbyNU7441(Fig.4C).EvidentlyDSB repairisdifferentiallyaffectedwhethertheactivityofDNA-PKcs

is inhibited, or ifthere are low amountsof DNA-PKcs present.

OurdatashowthatinhibitionofDNA-PKcsactivityreduceboth DSBrepairandcellsurvival afterexposuretoionizingradiation (Fig.4).Incontrast,loweringofDNA-PKcslevelsviasiRNAtreat- mentmarkedlyreducedcellsurvivalwithoutanycleareffectson repair(Figs.1and3).Thus,thepresentdataimplicatethatthereare differentmechanismsbywhichlossofDNA-PKcsfunctionsensitize cellstoionizingradiationandsuggestthatDNA-PKcshasacritical roleinmaintenanceofsurvivalafterradiationexposure,besidesits importantfunctioninDSBrepair.

AnumberofstudiessuggestacloseinteractionbetweenAKTand DNA-PKcs[28,29].TreatmentwithNU7441orsiDNA-PKcsabol- ishedtheactivityofAKT(Ser473)afterirradiation(Supplementary Fig.S3A),supportingtheroleofDNA-PKcsinactivationofAKTand thatlowamountsofDNA-PKcs,viadownregulationofAKT,may promoteapoptosis.Totestthis,cellswereirradiatedandscoredfor apoptosisbymeasuringcleavedPARP.Comparedtounirradiated cellstherewasaclearincreaseinPARPcleavage72hafterirradi- ationwith8Gy,buttherewasnodifferencebetweensiDNA-PKcs andmocktreatedcells(SupplementaryFig.S3B).

3.5. PhosphorylationofDNA-PKcsatThr2609andSer2056

ATMandDNA-PKcsarethemajorkinasesactivatedfollowing radiation.DNA-PKcsisrapidlyactivatedbybothphosphorylation andautophosphorylationuponirradiationandseveralphosphor- ylation sites have been identiﬁed so far. ATM is essential for phosphorylation of Thr2609 [30] and DNA-PKcs is responsible forSer2056phosphorylation[31].Westernblotanalysiswasper- formedonA431cellstotestifradiation-inducedphosphorylation ofThr2609andSer2056areaffectedbyadepletionofDNA-PKcs.

The overall phosphorylationof both Thr2609 and Ser2056 was almostcompletelyabolishedincellstargetedwithsiRNAagainst DNA-PKcs(Fig.5A),mostlikelyduetodecreasedlevelsofDNA- PKcs.However,cellsinhibitedwiththeDNA-PKcsinhibitorNU7441 showednodifferenceinDNA-PKcsphosphorylation.

Despite the reduced amount of phosphorylated DNA-PKcs in Western blot,formation and disappearance of Thr2609 and Ser2056fociafterirradiationweresimilarinmock-andsiRNA- treated cells, and thesefoci co-localizedwiththe DSBmarkers 53BP1and␥-H2AX,indicatingthatthephosphorylationofDNA- PKcsatDSBsiteswerenotaffectedbytheamountofDNA-PKcs (Fig.5BandC).Further,noapparentdifferencecouldbeseeninfoci intensitybetweensiDNA-PKcsandmocktreatedcells.Theseresults suggestthattheproteinsleftinthecellsaregatheringaroundDSB andarefullyactivatedatthesesites.CellstreatedwiththeDNA- PKcsinhibitorNU7441exhibitedanapparentlynormalformation ofThr2609andSer2056fociatearlytime-pointsafterirradiation.

However, 4–24h post-irradiation,thenumbers of Thr2609 and Ser2056focishowedonlyasmalldecrease(Fig.5C),whichcoin- cidedwiththe53BP1analysisandisinlinewiththehypothesisthat ATMactivationpromotesDNA-PKcsbindingtoDNAbutinhibition byNU7441preventsDNA-PKcstoparticipateinDSBrepair.

Interestingly,fociofDNA-PKcswereclearlyvisible60minafter irradiationofcellstreatedwithtargetsiRNA(Fig.6).TheDNA-PKcs focioverlappedwiththeDSBmarker53BP1andthesefociwere notseenincellswithnormallevelsofDNA-PKcs(mock-treated).

ThiscouldbeexplainedbythehighlevelsofDNA-PKcsinnormally presentinhumancellsbutwhentheproteinlevelisdecreasedby 80–95%theallocationaroundDSBofactiveproteinscanbeseen.

Notably,theDNA-PKcsfocibecamevisible60minafterirradiation andafter4–24htheybecamemoredistinct,whereascellsanalyzed atshorterrepairtimesjustdisplayedaweakersignalofDNA-PKcs overthewholenucleus.Thus,lowlevelsofDNA-PKcsmaycausea delayintheredistributiontotheDSBsites.

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Fig.3.CellswithlowresiduallevelsofDNA-PKcshaveapparentlynormalDSBrepair.(A)representativeimagesofA431andGM5758cellsmocktreatedorsiDNA-PKcs transfected,irradiatedwith2Gyor1Gy,respectively,andallowedtorecoverfortheindicatedtimes.0Gywasusedascontrol.Cellswereﬁxedandimmunostainedwith 53BP1(red)andnucleiwerestainedwithDAPI(blue).(B)Kineticsof53BP1fociincellstreatedasin(A).Atleast100cells/timepointswerescoredforfoci.Datarepresent mean±SDofthreeindependentexperiments.(C)Doseresponsebyaveragenumberof53BP1focipercell.CellswereeithermocktransfectedorsiDNA-PKcstransfectedand thenirradiatedatindicateddosesandallowedtorecoverfor24hbeforeﬁxationandimmunostaining.Countingandstatisticsasin(B).(D)Timeresponseof␥-H2AXfociin cellstreatedasin(A).Countingandstatisticsasin(B).(E)RejoiningofDSBsinA431,HCT116andH314cellsmock-transfectedorsiDNA-PKcstransfectedbeforeirradiation.

CellswereallowedtorecoverfortheindicatedtimespriortoPFGEanalysis.M059KandM059Jdataareshownascomparison.DatawerenormalizedtotheDSBlevelat t=0handeachdatapointrepresentsmean±SDofthreeindependentexperiments.

4. Discussion

ItiswellknownthatdecreasedcapacitytorepairDNAdamage byradiationorotherDNAdamagingagentsisacancerriskfactor andDNA-PKhasbeenimpliedtobedownregulatedinmanydiffer- enttumortypes.Theexpressionseemstobetissuedependentand ithasbeendemonstratedthattissuefromuterine,breastandlung

cancerhaveatendencyoflowDNA-PKlevelswhereasheadand neckcancershownormallevels[15,16,32].Humancellsmaintain ahighreservoirofproteinsinvolvedinNHEJofDSBrepairandhigh levelofproteinsallowsthecelltoprotectthegenomebytheimme- diatepresenceofrepairproteinsatthebreaks,minimizingthetime forassemblyandactivationatthebreaksite.Therearenearlyhalfa millionKumoleculesandaroundonehundredthousandDNA-PKcs

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Fig.4.InhibitionoftheDNA-PKcskinaseactivitysensitizecellstoradiationandreduceDSBrepair.(A)ClonogenicsurvivalinA431cellstreatedwiththeDNA-PKcsinhibitor NU7441(2␮M).CellswerepreincubatedwithNU74411hbeforeirradiationatindicateddoses.NU7441wasremoved24hafterirradiationandcolonieswith>50cellswere scoredafter10days.(B)53BP1fociformationandremovalinA431andGM5758cells.Cellswereincubatedwith10␮MNU74411hbeforeirradiationatindicateddosesand allowedtorecoveratindicatedtimesbeforeﬁxationandimmunostaining.Fortimeresponsecellswereirradiatedwith2Gy,andfordoseresponsefociwerecountedafter 24h.Atleast100cells/timepointswerescoredforfocianddatarepresentmean±SDofthreeindependentexperiments.(C)RejoiningofDSBsinGM5758cellstreatedwith 5␮MNU7441.CellswereincubatedwithNU74411hpriortoirradiationwith40GyandallowedtorecoverfortheindicatedtimesbeforepreparationandanalysisbyPFGE.

Datawasnormalized(t=0h)andeachdatapointrepresentsmean±SDofthreeindependentexperiments.

moleculespercell[33].Thedatapresentedhereprovidefurther indicationsoftheimportanceofDNA-PKcsforpromotingcellular survival.Irrespectiveofthestrongradiosensitizingeffect,weshow thatwithlevelsofDNA-PKcsbelow20%comparedtonormalcells, cellsretaintheirabilitytorejoinDNAfragmentsandremove53BP1 and␥-H2AXfociwithnormalspeed.Toourknowledge,thepresent studyistheﬁrsttoshowthatknock-downofcriticalDSBrepairpro- teinsleadstoextremeradiosensitivitywithoutanyapparenteffect ontherepair.Thus,thehighlevelsofDNA-PKcsinnormalcells mayindicateimportantrolesinothercellularfunctionsandthatan excessofmoleculesneedtobepresentinthestressresponseupon severeDNAdamage.Ourobservationsshowthatafterirradiation, cellswithlimitedlevelsofDNA-PKcshaveatwo-foldincreasein thefractionofcellsarrestinginG2/M,indicatingthatlowlevelsof DNA-PKcsmaystallmitosis.Thiswasbothshowninsynchronized andasynchronouscells(Fig.2).Theseobservationsareinlinewith astudybyShangetal.,showingthatDNA-PKcscontributestothe normalspindleformationandcentrosomestabilityaswellasinac- tivationofDNA-PKcscouldcausemultipolarspindleandmitotic catastropheafterDNAdamage[21].WeherealsoshowthatDNA- PKcsisdislocatedfromthechromatininthemitoticnucleusand aDSB-independentThr2609phosphorylationwasconcentratedat twodistinctsitesoneachsideofthechromatin(Fig.2C),indicat- ingaroleofDNA-PKatcentrosomesinthemitoticcell.Thisisin linewithrecent datashowingco-localizationofphosphorylated DNA-PKcs(Thr2609)andPlk1inmitoticcells[21–23,34].Deple- tionofthismitoticphosphorylationofDNA-PKcscouldresultin delayanddysfunctioninthemitotictransition.Thiscouldleadto alossorgain ofawholechromosome,which isthemostcom- monchromosomalinstabilityassociatedwithhumancancers[23].

ItwouldberationaltoassumethatthepresenceofunrepairedDSB inDNA-PKcsdeficient/suppressedcellsshouldcausedelayincell- cycleprogressionandmitoticfailure.Importantly,ourdatasuggest that DNADSB repair capacity isnot thelimiting factor in cells withlowDNA-PKcslevels.Thisinformationcouldbecrucialforthe understandingofthecomplexityofDNAdamagestressresponse, and specifically,themultifunctional roles ofDNA-PK. ATM and ATRhavebeenfirmlyestablishedascentralplayersintheactiva- tionofthecheckpointpathwaysthatrelayontheDNAdamage

signaling to downstream effectors through phosphorylation [35,36] and perhaps DNA-PK plays a part in this role as well.

InhibitionofPI-3kinasesincreasemitoticarrestandmitoticcell death[37]and ATM,ATRandDNA-PK haveall beenconﬁrmed tolocalizetocentrosomesduringmitosis[34].Thissuggeststhat thesePI3-likekinasescomplementeachotheroncentrosomesand thefollowingdownstreamphosphorylationsarenecessaryforcell cycleprogressionandsynthesisofmicrotubules[34].Wecannot excludethepossibilityofalternativepathways(e.g.homologous recombination)mayrejointheDSBs,howeverthisdoesnotexplain thearrestofcellsinG2/Mphase.

CellswithtotaldeficiencyofDNA-PKcs,liketheM059Jcellline, displayahypersensitivitytoionizingradiationthatreflectsacrucial roleofthisenzymeinmaintainingrepaircapacity[24,25,38,39]and chromosomestability[40].PhosphorylationofDNA-PKcsiscritical forthebindingandreleasefromtheDNAend andinhibitionof DNA-PKcswillresultinblockedDNAends,whichinhibitfurther processingandefficientligation.Themajorityofphosphorylation sitesarelocatedattheABCDE-andPQRclusterandmutationsat eitherofthesesitesimpairsDNArepair[30,35,39,41–44].There- fore,targetingandinhibitingDNA-PKactivityisanattractivecancer therapystrategyandthemostsuccessfulapproachhasbeenwith theuseofsmallmoleculestargetingtheATPbindingsiteofthe kinasedomain[45].A promisingdrugis theNU7441,based on theLY294002backbone,whichshowsastronginhibitionofDNA- PKcsandDNArepairincells[46].InhibitionbyNU7441doesnot resultinthesameradiosensitivityascellstotallylackingDNA-PKcs [47],howeverweobservedanalmostcompleteinhibitionofDNA repairincellstreatedwithNU7441,whichwasinstrongcontrastto cellswithdecreasedlevelsofDNA-PKcs.Eventhoughtheinhibitors holdpromisingresultsforimprovingcancertherapy,themolecules knownsofararelimitedbytheirpoorpharmacokinetics[45,48].

Further,ourdataimplicatesthatsuppressionofDNA-PKcscouldbe apreferabletherapeuticstrategy;whereasinhibitionofthekinase activitybyNU7441blockstheDSBrepairinbothdividingandnon- dividingcells (Fig.4), whilesuppressionof DNA-PKcsbysiRNA mainlyaffectsthedividingcells.

ThehighsensitivitytoionizingradiationincellswithlowDNA- PKcslevelscouldbeduetodysregulationofdownstreammolecules.

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Fig.5.PhosphorylationofDNA-PKcsatThr2609andSer2056.(A)WB:relativelevelsofDNA-PKcs,p-Thr2609(DNA-PKcs),p-Ser2056(DNA-PKcs)inA431cells,mock- transfected,siDNA-PKcstransfectedortreatedwith10␮MNU74411hbeforeirradiationwith2Gy.Cellswereallowedtorepairfor1hbeforepreparationforWB.Equal loadingwasconﬁrmedby␤-actinimmunoblot.(B)RepresentativeimagesofA431cells,mock-transfected,siDNA-PKcstransfectedortreatedwith10␮MNU74411hprior toirradiationwith2Gy.Cellswereallowedtorepairfor1hor24hbeforeﬁxationandimmunostainingwitheitherp-Thr2609(DNA-PKcs)(green)andco-stainedwiththe DSBmarker53BP1(red),orp-Ser2056(DNA-PKcs)(red)andco-stainedwiththeDSBmarker␥-H2AX(green).Co-localizationbetweenthedifferentantibodiesisdepicted inthemergedimagesasyellow,nucleiwerestainedwithDAPI(blue).(C)Averagenumberofp-Thr2609(DNA-PKcs)andp-Ser2056(DNA-PKcs)fociinA431andGM5758 cellsmock-transfected,siDNA-PKcstransfectedortreatedwith10␮MNU7441followedbyirradiationatindicateddosesandallowedtorecovertheindicatedtimes.Data representmean±SDofthreeindependentexperiments.Atleast100cells/timepointwerescoredperdatapoint.

Indeed,AKTphosphorylation(Ser473)decreasedinsiRNAtreated cellsafterirradiation.However,increasedapoptosiswasobserved in both, DNA-PKcs silenced cells and mock treated cells when cleavedPARPpathwaywasstudied.(SupplementaryFig.S3).Fur- ther,wecouldnotdetectanysignsofacceleratedsenescence(data notshown), which wasrecently reportedwhen DNA-PKcswas blockedwiththePI3-inhibitorNVP-BEZ235incombinationwith radiation[49].

InourstudyweshowthatincellswithlowlevelsofDNA-PKcs therewasclearvisualizationofDNA-PKcsfocithatoverlappedwith 53BP1,whichwasnot seeninmocktreatedcells. However,the formationofDNA-PKcsfociwasrelativelyslowandthiscouldindi- catea delayinthere-localizationof DNA-PKcstotheDSBsites

insiRNA-treatedcells. Wecouldnotidentifyadifferenceinthe numbersofThr2609andSer2056focibetweencellswithlowDNA- PKcslevelsandcellswithnormalDNA-PKcscontent,howeverin NU7441treatedcellsthemajorityoffociretainedevenafter24h repair.Thisimpliesthattheproteinsleftareactiveandrepairing DSBasinnormalcells,butinhibitionofDNA-PKcskinaseactivity leavesthecellunabletorejoinDNAends.Ourobservationsalso showare-localizationofp-DNA-PKcsduringmitosis,indicatinga phosphorylationeventthatmaybecriticalformitosis.

Insummary,ourdatashowthatDNA-PKcsisnecessaryforreg- ulationof mitoticprogressionafterirradiation,and thisprocess seemstobeindependentofitsroleinDSBrepair.Thisincreaseof radiationsensitivity,comparedtonon-dividingcells,couldbean

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Fig.6.LowlevelsofDNA-PKcsdisplayfociformation.Representativeimagesof A431cellsmock-transfectedorsiDNA-PKcstransfected.Cellswereirradiatedwith 2Gy,ﬁxedandimmunostainedafter15minor4hwithDNA-PKcs(green)andthe DSBmarker53BP1(red).Thetwoimagesweremergedandco-localizationofDNA- PKcsand53BP1aredepictedasyellow,nucleiwerestainedwithDAPI(blue).

advantageinradiotherapyoftumors.Identiﬁcationofadditional functionsofDNA-PKcs,beyonditsstrongroleinDSBrepair,could giveimportantknowledgeaboutregulationofgenomicstability andthiscomplexitywouldbeimportanttokeepinmindforthe developmentofnewtherapeutic approachestargeting theDNA damagesignalingandrepairpathways.

Conﬂictofintereststatement

Theauthorsdeclarethattherearenoconﬂictsofinterest.

Acknowledgements

Microscopic imaging wasperformed with equipment main- tainedby theSciencefor LifeLabBioVisPlatform,Uppsala.We sincerelythankK.KarlssonforhelpwithdatafortheM059cells.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, in the online version, at http://dx.doi.org/10.1016/j.mrfmmm.

2014.06.004.

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