Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES) : impact of experimental conditions

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sul fi de (CEES): Impact of experimental conditions

Malgorzata Debiak

, Kirsten Lex

, Viviane Ponath

, Waltraud Burckhardt-Boer

, Horst Thiermann

, Dirk Steinritz

, Annette Schmidt

, Aswin Mangerich

, Alexander Bürkle

*

aUniversityofKonstanz,MolecularToxicologyGroup,DepartmentofBiology,78457Konstanz,Germany

bBundeswehrInstituteofPharmacologyandToxicology,80937Munich,Germany

cWaltherStraubInstituteofPharmacologyandToxicology,80336Munich,Germany

BiologicaleffectsofCEES treatedcellsaresignificantlyinfluencedbytreatmentprotocol.

OptimizedprotocolforthetreatmentofHaCaTcellswithCEES.

CEESinducesadose andtime dependentPARylationresponseinHaCaTcells.

CEES inducedPARformationispredominantlyduetotheactivationofPARP1.

Keywords:

Poly(ADP-ribose)

Poly(ADP-ribose)polymerases Sulfurmustard

CEES

HaCaTkeratinocytes Solvent

ABSTRACT

Sulfurmustard(SM)isabifunctionalalkylatingagentwithalonghistoryofuseasachemicalweapon.

Althoughitslastmilitaryuseisdatedfortheeightiesofthelastcentury,apotentialuseinterroristic attacksagainstcivilians remainsa significant threat.Thus, improving medical therapyofmustard exposedindividualsisstillofparticularinterest.PARPinhibitorswererecentlybroughtintothefocusasa potentialcountermeasureformustard inducedpathologies,supportedbytheavailabilityofefficient compoundssuccessfullytestedincancertherapy.

PARPactivationafterSM treatmentwasreportedinseveralcelltypesand tissuesundervarious conditions;however,adetailedcharacterizationofthisphenomenonisstillmissing.Thisstudyprovides thebasisforsuchstudiesbydevelopingandoptimizingexperimentalconditionstoinvestigatepoly(ADP ribosyl)ation(PARylation)inHaCaTkeratinocytesupontreatmentwiththemonofunctionalalkylating agent 2 chloroethyl ethyl sulfide (“half mustard”, CEES). By using an immunofluorescence based approach,weshowthatoptimizationofexperimentalconditionswithregardstothetypeofsolvent, dilutionfactorsandtreatmentprocedureisessentialtoobtainahomogenousPARstaininginHaCaTcell cultures.Furthermore,wedemonstratethatdifferentCEEStreatmentprotocolssignificantlyinfluence thecytotoxicityprofilesoftreatedcells.Usinganoptimizedtreatmentprotocol,ourdatarevealsthat CEESinducesadose andtime dependentdynamicPARylationresponseinHaCaTcellsthatcouldbe completelyblockedbytreatingcellswiththeclinicallyrelevantpharmacologicalPARPinhibitorABT888 (also known as veliparib). Finally, siRNA experiments show that CEES induced PAR formation is predominantlyduetotheactivationofPARP1.Inconclusion,thisstudyprovidesadetailedanalysisofthe CEES inducedPARylationresponseinHaCaTkeratinocytes,whichformsanexperimentalbasistostudy themolecularmechanismofPARP1activationanditsfunctionalconsequencesaftermustardtreatment ingeneral.

*Correspondingauthorat:MolecularToxicologyGroup,DepartmentofBiology,UniversityofKonstanz,D-78457Konstanz,Germany.

E-mailaddress:alexander.buerkle@uni-konstanz.de(A.Bürkle).

1Currentaddress:FederalEnvironmentAgency,06844Dessau-Rosslau,Germany.

Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-300780

1.Introduction

Chemical weapons have been repeatedly used in military conflicts,since theirfirstreleaseduringWorldWarIahundred years ago (Debiak et al., 2009; Mangerich and Esser, 2014).

Although, due to considerable international efforts chemical warfarebecamealmostcompletelybannedbytheinternational community, the risk of potential use of chemical weapons by terroristsremainsa constantthreat.Astheproductionofsulfur mustard(SM)doesnotrequirespecializedtechnologyandlarge stockpilesmightbeavailableintheregionsaffected,itsuseagainst civiliansisarealisticdanger(Keheetal.,2009;KeheandSzinicz, 2005).ThelatesteventsduringtheSyriancivilwarleadingtothe releaseofsarin,chlorinegas,andpotentiallyalsoSMprovedthat suchathreatismorerealthanever.

ThefirstsynthesisofSMfallsintothemiddleof19thcentury.

Alongwithphosgeneandchlorine,SMenteredthebattlefieldsof WorldWarI.While,theexposureisrarelylethal,SMcausessevere skinand eyelesionsand acute respiratory pathologies. Since a specificantidoteisstillmissing,patienttreatmentissymptomatic and requires intensive and costly medical care and long term hospitalization(Keheetal.,2009).Theneedforthedevelopmentof effectivetherapyhasdrawntheattentiontothemechanism of actionofSM inducedtoxicity.Despiteintensiveresearchendeav orsthroughoutthelastdecades,thedetailedmechanismofSM inducedtoxicityremainsunclear.Itiscommonlyacceptedthatthe exposurecausesrapidformationofSM proteinandDNAadducts, resultingin massivecelldeathand inflammationof localtissue (Debiaketal.,2009;Keheetal.,2009).Earlyon,aninvolvementof poly(ADP ribose)polymerase1(PARP1)wasreported[reviewedin (Debiak et al., 2009)]. It was postulated that PARP1 becomes activated upon SM treatment in an apurinic endonuclease dependent manner (Papirmeister et al., 1985). Moreover, the treatment of cells with the first generation PARP inhibitor 3 aminobenzamide(3AB) preservedcellularNAD⁺ pools.Interest ingly,PARPinhibitiondidnotinfluencecellsurvivalingeneral,but shiftedthemodeofcelldeath,fromnecrotictoapoptoticcelldeath (Keheetal.,2008;Meieretal.,1987;Papirmeisteretal.,1985).It hasbeenhypothesizedthatthis shiftisresponsibleforreduced mustard inducedpathologiesunderconditionsofPARPinhibition inarodentanimalmodel(Cowanetal.,2003).

PARP1isaubiquitouslyexpressedproteinplayingkeyrolesin genomicmaintenance,chromatinorganization,transcription,and theregulationofcelldeath(MangerichandBürkle,2012;Rouleau etal.,2010).PARP1bindstoDNAstrand breaksandbyusingNAD⁺ asasubstrate,catalyzestheformationofhomopolymericbranched chains of poly(ADP ribose) (PAR) of different length that are covalentlyboundto“acceptor”proteinsatglutamate,aspartateor lysineresidues.StrongactivatorsofPARP1areionizingradiation, alkylatingagentsinducingsmallbasemodifications,andreactive oxygenspecies;leadingtotheinductionofdirectorindirectDNA strandbreaksgeneratedintheprocessofbase excisionrepair.SM isabifunctionalalkylatingagent.ThereactionwithDNAleadsto rapid formation of bulky monoadducts and DNA cross links (Debiaketal.,2011).ThemajorityofDNAalkylationconsistsof adductsattheN7positionofguanineandN3positionofadenine, accounting for 61% and 16% of total alkylations, respectively (Ludlumetal.,1994).DNAdi adducts,ontheotherhand,represent about15%ofSM inducedDNAlesionsandareformedbyreaction ofoneSMmoleculewithN7positionsof twoguaninesinclose proximity(LudlumandPapirmeister,1986).SManditsderivative CEESarenotexpectedtoinduceDNAstrandbreaksdirectly,thus

potentialPARP1activationbysuchcompoundsmaybemediated byunusualDNAstructuresexhibitingPARP1 activatingpotential orbyDNAstrandbreaksthatariseasDNArepairintermediatesby enzymaticprocessingoftheoriginalDNAlesion.

TheobjectiveofthisstudywastoinvestigatePARP1activation inimmortalizedhumankeratinocytes[i.e.,HaCaTcells(Boukamp etal.,1988)]aftertreatmentwiththeSManalogueCEES.CEESisa monoalkylatingagentthathasbeenwidelyusedasaSMsurrogate substance(Bennettetal.,2014;Inturietal.,2011;Keheetal.,2013;

Wang et al., 2012), because its use is not subject to specific restrictions.It exhibitssimilarchemical propertiescomparedto SM, however, in contrast to SM, CEES is a monofunctional alkylating agent, and therefore lacks any crosslinking activity.

PresumablyduetolackofcapabilitytoproduceDNAcrosslinks, CEESisabout10timeslesstoxicthanSMafteroralapplicationin rodents (Wang et al., 2012). The profile of cellular and tissue damagefor CEESiscomparable tothat ofSM(Jainetal.,2011;

Tewari Singhet al., 2009).Based onpreviouslydescribed CEES treatment protocols, an optimized treatment protocol was developed to study PARP1 activation by immunofluorescence microscopyona single celllevel. Our results revealedthat the experimentalprotocolofCEEStreatmentsignificantlyaffectsthe qualityandquantityofPARformationandcytotoxicityinHaCaT cells.Moreover,wedefineoptimalconcentrationsandincubation timesforpharmacologicalPARPinhibitionandsiRNAexperiments demonstrate that PARP1 is responsible for the bulk of PAR formationuponCEEStreatment.

Thisstudyprovidesthebasisforabroadercharacterizationof sulfur and nitrogen mustard induced PARylation response and cellularconsequencesthereof,whichisdescribedinanaccompa nyingarticle(Mangerichetal.,2016).

2.Materialandmethods

2.1.Celllinesandcultureconditions

ThehumankeratinocytecelllineHaCaTwaskindlyprovidedby Dr.PetraBoukamp,GermanCancerResearchCenter,Heidelberg, Germany (Boukamp et al.,1988).HaCaTcells werecultured in Dulbecco’sModifiedEagleMedium(Invitrogen)containing4.5g/l glucose,0.58g/lL glutamineand0.11g/lsodiumpyruvatesupple mented with10%fetal calf serum(PAA Laboratories), penicillin (100U/ml),streptomycin(100U/ml)(Invitrogen)inahumidified atmosphereat37Cand5%CO2.Cellswerepassagedtwiceaweek ina1:6or1:10ratio.Fordetachment,cellswereincubatedwith 0.25%trypsin EDTA(Invitrogen)for5minat37C.

2.2.CEEStreatment

2 Chloroethylethylsulfide(CEES)(Sigma)wasdilutedin100%

DMSO(protocol#1) or95%ethanol/0.5%HCl (v/v)(protocols

m

12#3).SerialdilutionsofCEESwerepreparedatconcentrationsof 100 of the final concentrations (protocols #1 and #3). Equal distributionof CEESin PBSwas ensuredby shortbutintensive vortexingorinvertingthetube.Thesolventwaskeptconstantata finalconcentrationof 1%(v/v).The cellswerewashedcarefully withPBSpre warmedto37CandimmediatelytreatedwithCEES dilutedinpre warmedPBStothefinalconcentration.ThepHofthe mediumwasmonitoredafterCEESadditionandprovedtoremain stable.Thetreatmentoccurredfor10 60minat37Cand5%CO2. Thereafter,cellswerewashedwithPBSandsupplementedwith freshcellculturemedium.InpreliminaryexperimentsCEESina

Fig.1.CEEStreatmentprotocolaffectsdistributionofcellularPARstaininginHaCaTcellcultures.

MonolayersofHaCaTcellsweretreatedfor10minwith2mMCEESpredisolvedindifferentsolventsanddilutionfactorsasindicated(protocols#1-3).CEESwaseither predilutedinETOH/HClata1000concentratedstocksolutionandaddeddirectlytothecellculturemedium(protocol#2)orinDMSOorEtOH/HClin100concentrated stocksolutionswhichwerepremixedinpre-warmedPBS,vortexed,andimmediatelyappliedtocells(protocols#1and#3,respectively).PARsynthesiswasanalyzedby immuno-chemicalstainingusingthePARspecificantibody10Hfollowedbyepifluorescenceanalysis.H2O2treatmentfor5minservedasapositivecontrol.Dashedlinesin

1000concentratedstocksolution[95%ethanol/0.5%HCl (v/v)]

wasaddeddirectlyintothemediumorPBStothecellcultureplates atafinalconcentrationofthesolventof0.1%(v/v)(protocol#2).In ordertodistribute CEESequally inthe culturemedium, dishes wereswiveledseveraltimes.

2.3.ImmunochemicalanalysisofcellularPARformation

Cellsweregrownonsterileglasscoverslipsandtreatedwith CEESasdescribedabove.Fordetectionofpoly(ADP ribose),cells werefixedandpermeabilizedwithice coldmethanolfor7min.

The cover slips werewashed 3 for 5min withPBS and then blockedin5%milkpowderinPBSwith0.1%Tween 20for30minat 30C. Specimens were incubated with the primary mAB 10H (Kawamitsuetal.,1984)overnightat4Cinahumidifiedchamber, washedwithPBSandincubatedwithsecondaryantibody(anti mouse Alexa488) for 1hat 37C in the darkin a humidified chamber. Nuclei were visualized by Höchst 33342 (Molecular Probes, 1:20000 in PBS) staining for 5min. Specimens were washedwithPBS,andslides weremountedinAquaPolymount (Polysciences).SampleswereexaminedonLeicaDMIRBorZeiss Axiovert 200M microscopes using Axiovision software. For quantification,atleast100nucleiwerecountedpersampleand analyzedusingImageJsoftware.

2.4.Viabilityassay

Cells were grown to 80% confluency, trypsinized, pelleted at300gfor5minatroomtemperature(RT)andresuspended in fresh medium to a concentration of 110⁶cells/ml. Cell suspensions of 1ml each were transferred to 1.5 ml reaction tubes and pelleted at 300g for 5min at RT. Cells were resuspendedin1mlofPBScontainingCEESfinalconcentrations as indicated. The treatment with CEES was performed in a heatingblockat 37Cfor 1h.Cells werepelletedat300g for 5minat RT, washed withPBS and resuspendedin cellculture medium.Sampleswereseededinto96 well platesinquadrupli cates(6000cells/well)andincubatedunderstandardcellculture conditionsfor72h.Afterdiscardingthemedium,100

m

medium containing 10% Alamar Blue reagent (Invitrogen) was addedtoeachwellandafter2 3hofincubation(37C,5%CO2)the fluorescence signal was measured at 550nm excitation wave lengthand590nmemissionwavelength.Resultswereexpressed as relative fluorescence of the sample to the fluorescence of control[in%].

2.5.FluorimetricalkalineDNAunwindingassay(FADUassay)

DNAstrandbreakswereassessedbytheautomatedFADUassay asdescribedpreviously(Debiaketal.,2011).Briefly,HaCaTcells weretreatedwithdifferentdosesofCEESaccordingtotheprotocol

#3, washed with ice cold PBS and resuspended in suspension buffer(10mMNa phosphatebuffer,pH7.4;0.25Mmeso inositol;

1mMMgCI2)atthefinalconcentrationof210⁵cells/ml.70

m

thecellsuspensionwastransferredoniceinto96 wellplatesand lysedwith70

m

cyclohexanedinitrilotetraacetic acid, 0.1 % SDS). Samples were incubatedfor12minat0C.Pre chilledalkalinebuffer(42.5%lysis buffer,0.2MNaOH)wasoverlaidontopofthecelllysate.Alkaline unwindingwas performedat 30C for 60min. Unwinding was stopped with140

m

b

m

Germany)wasadded.Samplesweremixedbyautomaticpipetting up and down. Fluorescence was measured in a 96 well plate readerbyexcitationat492nmandemissionat520nm.Foreach sampleaninternalcontroloftheDNAcontentwasincorporated without alkaline unwinding process. Here the unwinding was preventedbyaddingofneutralizationbuffertosamplespriorto theadditionofalkalinebuffer.

The amount of double stranded DNA was calculated as (fluorescence of sample/fluorescence of sample without unwinding)100%.

2.6.PARP1knock down

RNAinterferenceexperimentswereperformedbytransfecting cellswithON TargetplussiRNAtargetingPARP1orcontrolsiRNA (ThermoScientific). After48h, cellswereharvested forWestern blotanalysisortreatedwithCEESforPARanalysisbyimmunoflu orescencemicroscopy.

2.7.SDS PAGEandWesternblotting

Cells(110⁶cells/ml)wereresuspendedinhot1RotiLoad1 buffer(Roth)andsonicatedtwicefor30swith50%pulsefor30 cycles.ProteinswereseparatedbySDS PAGEandweretransferred on ECL nitrocellulose membrane (GE Healthcare). Membranes wereblockedin5%milkpowderinTBS(15mMNaCl,1mMTris Base,pHadjustedto8.0)for 1hatRTandincubatedwithanti PARP1antibody(CII 10;1:300,4C,overnight).Then,membranes werewashedandincubatedwithsecondaryantibodygoat anti mouse HRP coupled (1:2000; DakoCytomation) for 1h at RT.

DetectionwasperformedbymeansofECLAdvance/PrimeWestern Blotting DetectionKit (GE Healthcare) accordingto the manu facturer’sprotocol.ImageswereacquiredwithImageQuantLAS 4000mini(GEHealthcare).

2.8.Statisticalanalysis

StatisticalanalysisofdatawasperformedwithGraphPadPrism (GraphPadSoftware)software.Theresultswereanalyzed using Student’st testor2 wayANOVAasappropriate.

3.Resultsanddiscussion

3.1.OptimizationoftheCEEStreatmentprotocolforcellulardetection ofpoly(ADP ribose)

Since CEESundergoesrapid hydrolysis inaqueoussolutions, varioustreatmentprotocols for CEES havebeenreported using non aqueoussolventssuchasDMSO,ethanol,oracetone[e.g.,(Jain etal.,2011;Paromovetal.,2011a;Tewari Singhetal.,2010,2014)].

Furthermore, conditions reported in the literature vary by treatment temperature, time, pre dilution factors and medium inwhichthetreatmentisperformed.Inordertodefineoptimal CEEStreatmentconditionsforthedetectionofPARformationin HaCaTcells wesetout totest differentexperimental protocols.

Therefore, wetreated cells for 10minwithCEES pre dilutedin differentstocksolutionsandanalyzedPARformationviaimmuno fluorescence microscopy based on a previously established protocol(Küpperetal.,1996).AsitisevidentfromFig.1,applying CEESinDMSOina100concentratedstock solution(protocol#1) didresultinPARformationonlyinaminorsubfractionofcells, potentially due to DMSO hygroscopicity and related CEES hydrolysis. Based on available literature information, we imagedatafromprotocol#2indicateareas,wherecellsdidnotshowanysignificantPARylationresponse,illustratingtheheterogeneityinPARylationresponseunderthese treatmentconditionsonasinglecelllevel.Scalebarindicates200m^m.

expectedtostabilizeCEESbyshiftingthehydrolysisequilibrium from 2 hydroxyethyl ethyl sulfide towards CEES (Wang et al., 2012).Ina first approach, wepreparedCEES asa 1000stock solution,whichwasthenaddeddirectlytothecellculturemedium (protocol#2).AsitisevidentfromFig.1,considerablymorecells displayeddetectablelevelsofPARformation,howeverresponding cellswereunevenlydistributedthroughouttheculturedish.While cellsinsomeregionsoftheculturedishshowedastrongstaining for PAR, the signal intensity in other regions did not differ significantlyfromcontrolcells.Incontrast,PARPactivationincells treatedwithhydrogenperoxide(H₂O₂)wasregularlydistributed.

Interestingly, CEES treated cells showing high PAR levels were localizedin defined groups/regions.Sincea detailed analysisof microscopic slides excluded any clonal effects of cells growth, whichmighthaveaccountedforthisphenomenon,weconcluded that these results were presumably caused by an unequal distributionofCEESintheculturedishes(Fig.1).Tothebestof ourknowledge,suchdifficultiesintreatingcellswithCEEShave notbeenreportedbefore.Firsttheuniquecombinationofdifferent factorssuchasveryshortassessment timeanda verysensitive endpointanalyzedatasinglecelllevelusedinourstudy(i.e.,PAR formationanalyzedbyimmunofluorescencemicroscopy)resulted intheappearanceofthiscomplication,whichpotentiallyalsoleads toresearchartefactswithregardstoothercellularend points.

Inthefollowingthetreatmentprotocolwasoptimizedtobetter take into account the physico chemical properties of CEES.

Therefore, CEES was pre diluted in EtOH/HCl in a 100 stock solution.Furthermore,aCEESworkingsolutionwaspreparedby addingtheCEESstock solutiontopre warmedPBS(37C)shortly beforecellswereexposed.Aftershortbutintensivemixing,the CEES solution was immediately added to the adherent cells (protocol#3).AsitisevidentfromFig.1,usingthisprotocol,PAR formation occurred in all cells exposed to CEES. Furthermore, signalintensitieswereconsiderablehigherasincellstreatedwith CEES diluted in DMSO, thereby significantly improving the sensitivity ofthe method.Importantly,solventcontrolsdidnot showanyPARformationabovebackgroundlevels(Fig.1).

3.2.ImpactoftreatmentprotocolonCEEStoxicity

Following the observation that individual CEES treatment protocols significantly affect the intensity and homogeneity of cellular PAR formation, individual treatment protocols were analyzedwithregardstooverallcytotoxicity.Previousstudiesof CEES induced cytotoxicity in HaCaTcells reportedLC50 values differingbyatleastoneorderofmagnitudewithinarangeof0.25 2mM depending on treatment time and conditions applied (Paromov et al., 2011a,b; Tewari Singh et al., 2010, 2014). One mayassumethatcellculturesdisplayingheterogeneousareasof PARformationthroughouttheculturedish(Fig.1),wereunequally exposed to CEES, which may lead toan overall lower toxicity compared to cultures showing uniform PAR formation, which indicateshomogenousexposureofCEEStoallcellswithinaculture dish.ThethreedifferentCEEStreatmentprotocolsasmentioned above(protocols#1 3)wereanalyzedfortheirabilitytoinduce cytotoxicityinHaCaTcellcultures.Tothisend,cellsweretreated with different doses of CEES for 60min and cell viability was measured 72h after treatment (Fig. 2). As supposed, solvent conditionshadasubstantialimpactonCEES inducedcytotoxicity.

TheLC50ofthe1000concentratedEtOH/HClCEESstocksolution (protocol#2)wasintherangeof1mMandthereforealmostfour times as high as compared to the 100 concentrated stock solutionswithCEESdissolvedinEtOH/HClorDMSO(LC500.25 mM, protocols #1and #3). It canbe assumed that in cultures

treatedbyprotocol#2,individualcellswereabletosurvivethe treatmentwithdosesashighas3mMofCEES,whereasthelower pre dilution(i.e., protocols#1and #3)resulted inalmost 100%

toxicityat a concentrationof 1mM. In contrast tothedilution factor,thechoiceofthesolventbetweenDMSO(protocol#1)and EtOH/HCl(protocol#3)didnotsignificantlyinfluencecytotoxicity, indicating that in both solvents CEES has been distributed uniformlythroughouttheculturedish.However,thedifferential resultswithregardstothePARylationresponse(c.f.Fig.1)suggest that dependingonthesolventused, CEESexhibitssignificantly differenttoxicokineticsintheshort term,whichcouldsignificant lyinfluencemeasurementsofmolecularandcellularend pointsof toxicity.

3.3.ApplicationoftheoptimizedCEEStreatmentprototocolforthe analysisofPARPactivationinHaCaTcells

Afterdefiningoptimaltreatmentconditions,wecharacterized theCEES inducedPARylationresponseinHaCaTindetail.Several previous studies reported PAR formation after SM treatment, however at present those remain incidental and lack any systematic time and dose dependent analysis (Debiak et al., 2009;Hinshawet al.,1999;Keheet al.,2008;Rosenthal etal., 1998). In terms of CEES induced PAR formation, Paromov analyzedPARPactivityinHaCaTcellextractsusingbiotinylated NAD⁺,leadingtoamaximumofa10 foldinsignalintensitiesat 5mM CEEStreatment (Paromovet al., 2011a).However, tothe bestofourknowledge,intracellularPARformationinintactcells withendogenousNAD⁺assubstratehassofarnotbeenreported uponCEEStreatment.SincecellularPARylationisaverydynamic process,duetoseveralPARdegradingenzymessuchaspoly(ADP ribose) glycohydrolase (PARG), we performed a time course analysis of PAR formation in intact HaCaT cells upon CEES treatment. As it is evident from Fig. 3A, PAR activation was detectedassoonas5minafterCEEStreatment.PARlevelspeaked at10minposttreatmentandalmostcompletePARdegradation wasobserved30min aftertreatment. Next we focusedonthe time point of maximum PAR formation (i.e., 10min post treatment) to analyze CEES PARylation dose response relation ships.Wedemonstratedadose dependentincreaseincellularPAR formation(Fig.3B).TheincreaseincellularPARwasobservedat 1mMCEESand3mMCEESresultedincircaeight foldincreaseof PAR concentration in comparison to the untreated control.

However, although a highly toxic dose was applied the PARP activation was not as high as the activation upon hydrogen peroxidetreatment.

Fig.2.TreatmentprotocolaffectsCEES-inducedcytotoxicityinHaCaTcells.

HaCaTcellsweretreatedinsuspensionwithdifferentconcentrationsofCEESusing differenttreatmentprotocolsasindicatedanddescribedinthetextbody.Cellswere incubatedwithCEESfor60min,washedwithPBS,andcellviabilitywasanalyzed 72haftertreatmentusinganAlamarBlueassay.n=3SD.Anon-linearcurvefit modelwasapplied.

Inordertoprovethespecificityofthe10H derivedPARsignalin fluorescencemicroscopy,wesuppressedPARformationinHaCaT cells by using the clinically relevant pharmacological PARP

inhibitor ABT888 (also knownas veliparib). These experiments revealedthatPARformationwasalmostcompletelyinhibitedafter genotoxictreatmentwithCEESorH2O2,whenHaCaTcellswere Fig.3.Timeanddose-dependentanalysisofcellularPARylationresponseuponCEEStreatment.

(A)Time-dependentcellularPARylationinHaCaTcellsaftertreatmentwithCEESasanalyzedbyepi-immunofluorescencemicroscopy.Cellswerefixedattimepointsas indicated,andstainedforcellularPARusingtheanti-PAR-specificmAB(10H).H2O2(1mM)treatmentfor5minservedasapositivecontrol.(B)Doseresponserelationshipof CEEStreatmentandPARformationanditsinhibitionbythepharmacologicalPARPinhibitorABT888.CellsweretreatedwithCEES(for10min)orH2O2(for5min)at concentrationsasindicated.Scalebarindicates50m^m.(C)DensitometricanalysisofimmunofluorescencedatashowninB.PBSreferstoPBScontrol,EtOHtoEtOH/HCl solventcontrol(1%(v/v)finalconcentration.(D)Determinationofconcentrationsandpre-incubationtimesofABT888treatmentforeffectiveinhibitionofCEES-andH2O2- inducedPARylation.

pre incubatedfor30minwith1

m

respect,itisimportanttonotethatPARPinhibitorsarediscussedas apotentialtreatmentoptiontoreducemustard relatedpatholo gies in exposed victims (Debiak et al., 2009). However, the moderatePARPactivationinducedbyCEESpresumablyleadsonly to minor changes in intracellular NAD⁺ concentrations, which speaks against the hypothesis that SM causes massive PARP activation resulting in ATP depletion, necrotic cell death, and subsequenttissueinflammation.Foradetailedcellculturestudy characterizing the PARylation responseinduced by bifunctional mustardsaswellasa discussionof thetherapeuticpotential of PARPinhibitorsinSM relatedpathologiesthereaderisreferredto anaccompanyingpaper(Mangerichetal.,2016)

Itis interestingtonotethatPARformationafterH2O2 peaks alreadyat5minposttreatment(Martelloetal.,2013),whereas maximumPARylationwasobserved10minpostCEEStreatment.

One possibility for the different PARylation dynamics are substance specific toxicokinetics. Alternatively, different PARy lationdynamics maybea directconsequences ofthe different typesofDNAlesionsformed,i.e.,H2O2inducesmainlyoxidative damage and strand breaks, while CEES induces mainly DNA alkylation, withoutbeingthoughttoinduceDNAstrand breaks directly.PreviousreportsshowedthatCEES inducedDNAmono adducts arepredominantly repairedby BER andNER pathways (Jowseyetal.,2009;Matijasevicetal.,2001).PARylationplaysan activeroleinbothrepairpathway,potentiallybybeingactivated

Pineset al.,2013,2012;Robertet al.,2013).Thus, eventhough mustardsdonotinduceDNAstrandbreaksdirectly,strandbreaks can occur in response to mustard treatment by enzymatic processes that actively introduce them in the course of repair processes, thereby triggering PARP activation. Potential CEES inducedDNAstrandbreakformationinHaCaTcellswasassessed bytheautomatedFADUassay(Fig.4).Theassaymakesuseofthe directrelationshipbetweentheextentofDNAunwindingunder alkalineconditions and the number ofDNA strandbreaks.The acquisitionis basedondetectionof remainingdouble stranded DNAbymeansofa fluorescent DNAintercalating dye.Thus the relativeamountofdoublestrandedDNAisinverselyrelatedwith thenumberofDNAstrandbreaks.HaCaTcellsweretreatedfor 60minwith differentdoses of CEESand analyzed immediately thereafter. During the processing, cells were kept on ice to suppressDNArepair.CEEStreatmentledtoadose dependentDNA strand break induction, which was evident already the lowest applieddoseof0.5mMCEES.DNAstrandbreakformationbyCEES was analyzed previously also in human PBMCs (Debiak et al., 2011).Innon proliferatingcellstheunwindingtakesplaceonlyat chromosomeends.ThustheamountofdoublestrandedDNAin controlPBMCisabout90%,whereasinproliferatingHaCaTcells, wherereplicationdependentbreaksactasadditionalunwinding initiators,only 50 60% DNAremains double stranded. The low backgroundincontrolPBMCsinfluenced substantiallytheassay sensitivityandDNAbreaksinPBMCweredetectedalreadyafter treatmentwith50

m

damage observed after CEES treatment in PBMC suggests a replication independent mechanism of DNA break induction such as direct reaction of CEES, ROS, BER or NER activity (Debiaketal.,2011).

InadditiontoPARP1,PARP2andPARP3canalsobeactivated uponcertaingenotoxicstimuli.WhilePARP1isinvolvedinmost DNArepairprocesses,suchasbaseandnucleotideexcisionrepair, aswellasDNAstrandbreakrepair,PARP2appearstocontribute mainlytoBERandsinglestrandbreakrepairandPARP3todouble strandbreakrepair(Becketal.,2014;DeVosetal.,2012).Totest thehypothesisthatthePARP1homologueisresponsibleforPAR formationupon CEEStreatment,presumablythroughitsrolein BERandNER,wesilencedPARP1expressionbytransfectingHaCaT cellswithsiRNAtargetingPARP1transcripts.Theefficiencyofthe PARP1knockdownwasover80%(Fig.5A).Ofnote,PARformation afterCEEStreatmentcorrelateddirectlywiththePARP1 protein level,indicatingthatofthethreeknownDNA damagedependent Fig. 4.HaCaT cells exposed to CEES incorporate DNA strand breaks in dose

dependentmanner.

HaCaTcellsweretreatedwithCEESfor60min,washedwithPBSandpreceded immediatelybytheautomatedFADUassay.ThedegreeofDNAunwindingisrelated withtheamountofDNAstrandbreaks.DatarepresentsmeanvaluesSDofthree independentexperiments(n=3).

Fig.5.PARP1isresponsibleforPARsynthesisafterCEEStreatment.

(A)and(B)PARP1isthemaincontributortoCEES-inducedPARylationinHaCaTcells.CellsweretransfectedwithsiRNAtargetingPARP1(PARP1siRNA)orcontrolsiRNA(scr siRNA).(A)Left.WesternBlotanalysisofPARP1proteinlevels48huponsiRNAtransfection.DetectionofERK2servedasloadingcontrol.Right.Densitometricanalysisof3 independentexperiments.(B)AnalysisofPARylationresponse48haftersiRNAtransfectionandtreatmentofcellswith3mMCEESfor10min.DatarepresentmeansSD from3independentexperiments.StatisticalanalysiswasperformedusingaStudent’sttest.***P<0.001.

PARPs(i.e.,PARP1 3),PARP1activationisresponsibleforthebulkof PARproductionuponCEEStreatment.

4.Conclusions

In conclusion, this study provides a detailed analysis of the cellularPARylationresponseinHaCaTcellsupontreatmentwith the sulfur mustard derivative CEES. By comparing different treatmentprotocolswecouldoptimizeexperimentalconditions, which allow the analysis of a homogenous, time and dose dependent PARylation response on a single cell level using immuno chemical based epifluorescence microscopy. Using siRNAdirected againstPARP1, we provideevidence that PARP1 isthemaincontributortoCEES inducedPARylation.Furthermore, wedefinetreatmentconditionstoeffectivelyinhibitCEES induced PARylationbyusingtheclinically relevantPARPinhibitorABT888 (veliparib).Takentogether,thisstudyformsanexperimentalbasis tostudythemolecularmechanismsof mustard induced PARP1 activationanditscellularconsequences.Itwillbeinterestingto applytheoptimizedmethodologyasestablishedinthisworkto studythePARylationresponseincells treatedwithbifunctional sulfurandnitrogenmustards.Thiswillhavesignificantimplica tionstowards thequestionifPARP inhibitorsmaybeused asa therapeutic option to mitigate SM induced pathologies, as suggestedbyseveral animal studies(Cowanet al.,2003;Gross etal.,1985;Moletal.,1991;Youricketal.,1991,1993;Zhangetal., 1995)ortosensitizecancercellstonitrogenmustardtreatment,as testedinpreclinicalandclinicalsettings(Donawhoetal.,2007;

Norrisetal.,2014).Severaloftheseaspectsareaddressedininan accompanyingarticle(c.f.Mangerichetal.,2016).

Acknowledgements

WethankMatthiasBirtelforhiscontributionstoexperiments includedinFig.3.ThisworkwassupportedbytheGermanFederal MinistryofDefense(GrantE/UR3G/AG001/94804).

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