3-76 Kinetics of DNA Repair Proteins 53BP1 and γ-H2AX in HeLa Cells Irradiated with X-rays

3-75FirstInterdisciplinaryExperimentUsingHigh

EnergyMicrobeam

DuGuanghua,GuoJinlong,LiuWenjing,ChenHao,WuRuqunandGuoNa

ThehighenergyionbeamoftenstohundredMeV/upossessesmm-to-cmpenetrationdepthinmaterialsandcanbeeasilyextractedintoairwithoutsignificantenergylossandbeamscattering.Combinationofhighenergyionsandmicrobeamtechnologyfacilitatesthemicroprobeapplicationtomanypracticalstudiesinlargescalesam-ples.TheIMPheavyionmicrobeamfacilityhasre-centlybeenintegratedwithmicroscopicpositioningandtargetingirradiationsystem.ThefirstinterdisciplinaryexperimentsperformedattheIMPmicrobeamfacilityusingthebeamof80.5MeV/ucarbonionsincludeby-standereffectstudy,irradiationeffectonnervesystem,securityalgorithmattackstudy.Bystandereffectin-ductionviamediumtransferringwasnotfoundinthemicro-irradiationstudyusingHeLacells.Themouseir-radiationexperimentdemonstratedthatcarbonirradia-tionof10Gydosetoitstuberomammillarynucleusdidnotimpairthesleepnervesystem.ThefaultinjectionattackonRSA(Rivest–Shamir–Adleman)decryptionprovedthatthecommercialfield-programmablegatear-raychipisvulnerableinsingleeventeffecttolowlinear-energy-transfercarbonirradiation,andtheattackcancausetheleakageofRSAprivatekey(Fig.1).These

Fig.1Rasterscanirradiationofmousenervenucleususingexperimentsdemonstratethepotentialofhighenergyhighenergymicrobeamof80.5MeV/ucarbonions.Themicrobeaminitsapplicationtobiology,biomedical,ra-upper-leftinsertshowsthephotoscanofEBT2filmraster-diationhardness,andinformationsecuritystudies.

scanirradiatedwithandwithoutenergydegrader.

3-76KineticsofDNARepairProteins53BP1andγ-H2AXin

HeLaCellsIrradiatedwithX-rays

ChenHao,WuRuqunandDuGuanghua

Double-strandedDNAbreaks(DSBs)arethemostlethaltypeofDNAdamage,therefore,theinefficientorinaccuraterepaircancreatemutationsandchromosomaltranslocationswhichinducegenomicinstability[1].DSBscanbeinducedbymanyeventssuchasionizingradiation,reactiveoxygenspeciesandradiomimeticdrugs.Non-homologousendjoining(NHEJ)andhomologousrecombination(HR)arethemainDSBrepairpathways[2].IthasbeenreportedthathistonevariantH2AXphosphorylation(γH2AXformation)atDSBsitesisrequiredfortheaccumulationofmanyDNAdamageresponseandrepairproteinssuchas53BP1[3].53BP1isanestablishedplayerinthecellularresponsetoDNAdamage[4].Thisworkinvestigatedthekineticsofionizingradiationinducedfoci(IRIF)ofbothproteinsinHeLacellsirradiatedwith0.2and1.0GyX-rays.

HeLacellswerekindlyprovidedbyDr.Zhou(InstituteofModernPhysics,CAS,China)routinelymaintainedinRPMI-10mediumsupplementedwith10%fetalbovineserum,100mg/mLstreptomycinand100U/mLpenicillin.Sampleswerefixedin4%paraformaldehydefor10minandinmethanolat-20˚Cfor20min,blockedwith5%blockingbufferfor2handstainedwithprimaryantibodies(mouseanti-γ-H2AX,#ab26350andrabbitanti-53BP1,#ab36823,Abcam)for2h.Theprimaryantibodywasvisualizedusinggoatanti-rabbitlabeledwithRhodamineorgoatanti-mouselabeledwithFITCsecondaryantibody.Then,allthesamplesweresubjectedtothecaptureof3Dpicturesandthefocinumberwascountedwithconfocalmicroscope.TheFociPicker3DpluginsofImageJwaswrittenandutilizedtoacquireinformationfromthe3DconfocalmicroscopicimageoftheIRIFinHeLacells.

Thefollowingfigureswereanalyzedforγ-H2Axand53BP1IRIFkineticsinHeLacellsafterapplicationof0.2and1.0GyX-rays.Withimmunofluorescence,theγ-H2AXIRIFnumberexhibitedthesimilarbehavioras53BP1in

2014IMP&HIRFLAnnualReport·169·

thelowerdosesamples,whilearapidassemblageofγ-H2AXcomparedwiththedelayedkineticsof53BP1in1.0Gyirradiatedcells(Fig.1).Wefoundmore53BP1IRIFsinthelowerdosesamplesatalmostallthetimepointsbutthecontrastmeasurementshowedintheotherone(Fig.1).AsillustratedinFig.1(b),theinitialIRIFsformationinthehigherdosesampleswasrapidcomparedwiththecontrolandlowerdosesamples(Fig.1).Thedifferencebetween53BP1andγ-H2AXIRIFdynamicswasdistinctatthetimepointrangeof5to35min(Fig.1(b)),afterthataslowdecayoccurredinbothsamples(Fig.1).TodeterminethefinerecruitmentkineticsofbothproteinsincelllineHeLa,WeanalyzedthetotalfluorescenceintensityofbothproteinsinHeLacells.Withtheimmunofluorescenceassayassatedabove,thetotalintensitywasutilizedasanindextoinvestigatethefinerbehaviorofIRIFsthanthenumberoffoci.Indeed,thetendencyofcurvesinFig.2(a)agreedwithkineticsofIRIFsinFig.1(a),althoughtheabsolutedifferenceissignificant.However,themaximumofproteinsintensitypresentedafterirradiationtime35min,especially53BP1(Fig.2(b)),whichwerenotcloselyinlinewiththenumberoffoci(Fig.1(b)).Attheendoftimecourse,therewereobviousdifferencesbetweenexperimentalsamplesandthecontrol(Fig.2(b)).TheresultsshowedthattherecruitmentkineticswerenotsynchronouswiththoseintheformationofIRIFsintheupperdosesamples.Theremightbesomedelayedactionintheassemblageprocess.TheseevidencescouldsuggesttheresistantDSBsinducedbyionizationweremorecomplexthanthemostone.

Fig.1Thekineticsofγ-H2AXand53BP1focinumberpercell.Morethan1000cellswereprocessedbyFociPicker3D.

Fig.2

Thekineticsofγ-H2AXand53BP1quantitypercell,totalintensitycanberegardedasprotein

quantityapproximatelyinimmunofluorescenceassay.

TheassemblyofproteinsatDSBssiteoccursinahighlyordered,strictlyhierarchicalandrapidlyfashion.Thequantificationofγ-H2AXand53BP1fociallowstheestimationofDNArepairandresponse.ThisworkhasbeenattributedtotheDNAdamageandrepairinducedbylowdoseX-rays.Theapplicationoffinerevaluationindexcontributstopreciseassessment.References

[1]JingsongYuan,RachelAdamski,JunjieChen,FEBSLetters,584(2010)3717.[2]J.SanFilippo,P.Sung,H.Klein,Annu.Rev,Biochem,77(2008)229.[3]SimonBekker-Jensen,NielsMailand,DNARepair,9(2010)1219.[4]AngelaT.Nam,AarowA.Goodarz,DNARepair,10(2010)1071.