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A rapid, quantitative and inexpensive method for detecting...
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Wedescribearapidandquantitativeflowcytometricmethodfordeterminingtheapoptoticoranti-apoptoticpotentialofageneinvariouscelltypes.Aplasmidcarryinggreenfluorescentprotein(GFP)isco-transfectedwithanexpressionvectorencodingthegeneofinterest.Subsequentlycellsarestainedwithpropidiumiodideand,utilisingflowcytometry,transfected,GFP-expressingsinglecellsaredetectedandapoptoticcellsinthispopulationareidentifiedbytheirDNAcontentof<2N.Themethoddetectsapoptosisasreliablyasestablishedmethodsusinginsitunick-endlabellingbutisfaster,easierandlessexpensive.

Apoptosisorprogrammedcelldeath(PCD)isageneticallycontrolledcellularsuicidemechanismforselectivelyeliminatingunwantedcells(forreviewssee1-4).PCDisanobligatoryprocessinavarietyofBIOLOGicalprocessesincludingembryonicandneuronaldevelopment,immunesystemregulation,organogenesis,tissuehomeostasisandinthepreventionofmalignanciessuchastumorgrowthandviralinfection.Apoptosisischaracterisedbyplasmamembraneblebbing,cellshrinkage,nuclearcondensation,endonucleolyticcleavageofgenomicDNAintointernucleosomallengthfragmentsandtheformationofapoptoticbodies.

Currentmethodsforstudyingapoptosisincludetheassessmentofmorphologicalchangesatthecellularlevelbylight-,electron-ortime-lapsemicroscopyincombinationwithvitalfluorescentdyes(reviewedin5,6),theuseofAnnexinVtomonitorthelossofmembranephospholipidasymmetryduringapoptosis(7)orassaystodetectDNAfragmentationbyagarosegelelectrophoresis(8-10),byinsitunick-endlabelling(TUNEL)(11),orbyusinganenzyme-linkedimmunoabsorbentassay(12).Mostofthesemethods,however,arecumbersome,inappropriateor,inthecaseoftheTUNELmethod,expensiveforstudyingtheeffectofputativecelldeathgenesintransienttransfectionassays.

Wehavedevelopedamethodsuitablefortheflowcytometricdeterminationofapoptosisinalargenumberofsamplesbypropidiumiodide(PI)inapopulationofcellstransientlyco-transfectedwithaplasmidencodingenhancedgreenfluorescentprotein(eGFP)(13)andaplasmidcarryingageneofinterest.ThisassaycombinestheFACS-optimisedGFPmutant(eGFP)(13)asaMarkertodetecttransientlytransfectedcellsandthereducedfluorescenceoftheDNA-bindingdyePIintheapoptoticsubpopulationasamarkerforapoptosis(14-17).ThereducedfluorescenceofPIinapoptosingcellsresultsintheappearanceofacharacteristicsub-2NfluorescencepeakwithrespecttotheG0/G1cellcycleregion.

Inordertovalidatethismethod,weinitiallyusedtheestablishedmurinetumorcelllines([beta]TCand[beta]HC)whichoriginatefrom[beta]celltumors(18)oftransgenicmiceinwhichtheinsulingeneregulatoryregion(Rip)targetstheexpressionofthesimianvirus40largeT-antigen(Tag)tothe[beta]cellsofthepancreaticislets(Rip1Tag2)(19).

Approximately80000cellswereseededina3cmdiameterwellofasix-welldishandgrowninDMEMsupplementedwith10%FCS(v/v),2mMglutamine,100IUpenicillinand100µg/mlstreptomycinto70%confluency.Cellsweretransfectedwith1µgofaplasmidencodingenhancedGFP(pEGFP-C1;Clontech)togetherwith1µgofeitheracontrolplasmid(pMEX),orapCMVplasmidcarryingeitherthepro-apoptoticadenovirusE1Aortheanti-apoptoticadenovirusE1B-19Kgene(20,21)using10µlLipofectAMINEReagent(Gibco-BRL)accordingtothemanufacturer"srecommendations.Followingtransfection,thecellswereallowedtorecoverincompletemediumfor16handweretheneitherleftuntreatedoranapoptoticstimulus(800ng/mlstaurosporine;Sigma)(22,23)wasaddedforanadditional16h.At32haftertransfection,floatingcellswerecollectedandcombinedwithtrypsinised,adherentcells,washedtwicewith4mlPBSandfixed[2%paraformaldehyde(PFA),100mMNaCl,300mMsucrose,3mMMgCl2,1mMEGTA,10mMPIPESpH6.8]atroomtemperaturefor30min,washedtwicewith4mlPBSandpost-fixedinice-cold70%EtOHfor10-14h.

Followingfixation,cellswerewashedtwicewith4mlPBSanddividedtoallowcomparisonofourmethodtothecommonlyusedmodifiedTUNELmethodusingfluorescentCy5-CTP.OnehalfofthesamplewastreatedwithRNaseA(50µg/ml)inPBSfor30min,washedtwicewith4mlPBSandstainedwithPI(50µg/ml)inPBSfor30minbeforeFACSanalysis.Theotherhalfofthesamplewasincubatedin50µlofterminaldeoxynucleotidyltransferase(TdT)reactionmixture(BoehringerMannheim;200mMpotassiumcacodylate,25mMTris-HClpH6.6,0.25mg/mlbovineserumalbumin,1mMCoCl2,0.25nmolofFluorOlinkCy5AP3-dCTP[Amersham],12.5UTdT)for1hat37°C.Thesamplewasthenwashedtwicewith4mlPBS,treatedwithRNaseA(50µg/ml)inPBSfor30min,washedtwicewith4mlHBS(Note:HBSisusedfromthissteponwardssinceDAPItendstocausemicro-precipitatesinPBS),stainedwithDAPI(10µg/ml;BoehringerMannheim)inHBSfor20minandanalysedonaBectonDickinsonFACSVantagemachine.FlowcytometricanalysisofthePI-stainedcellswascarriedoutonaBectonDickinsonFACScanequippedwithadoubletdiscriminationmodule.

AsshowninFigure1A,ourmethodreliablydetectstheexpectedapoptoticoranti-apoptoticeffectoftheadenovirusE1AorE1B-19Kgeneproducts,respectively(20,21).Theadditionofstaurosporinefollowingtransfectionfurtherexacerbatestheobservedeffects.SimilarresultsareobtainedusingfluorescentCy5-CTPandtheTdTreactiontomeasureapoptosis.AlthoughthesensitivityoftheGFP/PImethodisbelowthatoftheCy5-CTP/TdTmethod(Fig.1A),thenormalisedamountofapoptosis,expressedasaratiooftheapoptosismaximaobtainedwiththeE1Ageneproductwithineachdetectionmethod,isnearlyidentical(Fig.1B).

Figure1.Determinationofapoptosisintransientlytransfectedmurine[beta]tumorcells.(A)Thenumberofapoptotic[beta]HC13Ttumorcells(%apoptosis)inthetotaltransfectedeGFP-positivepopulationisshown.Co-transfectionsofpEGFPandtheplasmidsasindicatedwerecarriedoutusingLipofectAMINEasdescribedinthetext.ApoptoticcellsweremeasuredbyflowcytometricanalysiseitherusingPIstaininganddeterminationofsub-2NDNAcontent(GFP/PI;blackbars)orbytheincorporationoffluorescentCy5-CTPduringtheTdTreaction(GFP/TUNEL;whitebars).Staurosporine(800ng/ml)wasaddedfollowingtransfectionasindicated(+staurosporine).Excitationwavelengthof488nmwasusedforeGFPandPI,647nmforCy5andmultilineUVforDAPI.Emmisionfluorescencewascollectedusinga530/20nmbandpassfilterforeGFP,610nmlongpassfilterforPI,675/20nmbandpassfilterforCy5anda424/44nmbandpassfilterforDAPI.Doubletswereexcludedbypulseprocessing.CellsexpressingeGFPweregatedandanalysedforeitherCy5-orPI-fluorescence.DatawereanalysedusingCELLQuestsoftware(BectonDickinson).Eachbarrepresentstheaverageofthreetransfections,standarddeviationsareindicatedbyerrorbars.Eachmeasurementincluded40000totaleventsgatedforsizeandsinglecells.Transfectionefficiencywas20-30%.(B)Thenormalisedpercentageofapoptosisisshownforthevariousconstructs.Theapoptoticindexwasnormalisedusingthefunction(%apopotosisinX/%apoptosisineGFP-C1/E1A)×100.Theapoptoticindexwasnormalisedforeachdetectionmethodusedandforeachpost-transfectiontreatmentemployed(i.e.+/-staurosporine).Notethepro-apoptoticandanti-apoptoticeffectofE1AandE1B-19K,respectively.

Todemonstratethegeneralusefulnessofourassay,theGFP/PImethodwastestedonanon-transformedratfibroblastcellline(Rat1A)whichislesssensitivetoapoptoticstimuli.Rat1AcellsweretransientlytransfectedusingeitherLipofectAMINEasdescribedforthe[beta]tumorcellsabove,orpolyethylenimine-(PEI2000)-DNA-adenoviruscomplexesasdescribed(24).AcomparisonofthedifferenttransfectionandapoptoticdetectionmethodsusingRat1AcellsispresentedinTable1.Asobservedwiththe[beta]tumorcelllines,theGFP/PImethodisareliableapoptoticassayfortransientlytransfectedRat1Acells(Table1)aswellasforahumanlungcarcinomacelllineA549(ATCCCCL-185)(24).

Table1.DeterminationofapoptosisinRat1Acells

TransfectedPropidiumIodidebTUNELc
Constructa%Apoptosis(s.d.)%ApoptosisNormalisedd%Apoptosis(s.d.)%ApoptosisNormalised
LipofectAMINEReagent
pMEX(Ctr)3.3(0.7)68.86.5(0.6)71.4
pE1B-19K1.4(0.3)29.22.2(0.5)24.2
pE1A4.8(0.4)1009.1(2.6)100
PEI/Adeno
pMEX(Ctr)1.2(0.2)52.27.5(3.0)52.1
pE1B-19K0.7(0.1)30.45.2(0.8)36.1
pE1A2.3(0.6)10014.4(2.7)100
aTransfectionofRat1AcellswithpEGFP-C1andtheconstructsasindicatedusingeitherLipofectAMINEReagentorPEI(2000)-DNA-adenoviruscomplexes.Followingtransfectionthecellsweretreatedwithstaurosporine(800ng/ml)for16h.TheapoptoticindexofthetransfectedcellswasanalysedbyflowcytometryasdescribedinthetextandinthelegendtoFigure1.Eachvaluerepresentstheaverageofthreetransfections,standarddeviationisindicated(s.d.).Transfectionefficiencywas25-30%bothusingLipofectAMINEandPEI/Adeno.bTheapoptoticindexofeGFP-positivecellswasdeterminedbyPIstainingandsub-2NDNAcontent.cTheapoptoticindexofeGFP-positivecellswasdeterminedbytheincorporationoffluorescentCy5-CTPduringtheTUNELreaction.dThenormalisedapoptoticindexwascalculatedasdescribedinthelegendtoFigure1.

ThePFA/EtOHfixationprocedureusedinourprotocolisessential.OmissionofthemildPFAfixationstepleadstotherapidleakageofeGFPfromthecellandimpairsdetectionoftransientlytransfectedcells.TheEtOHfixation/permeABIlisationstepallowsdiffusionoflowmolecularweightDNAproductsfromapoptoticcellsandisrequiredtodetectthecharacteristicsub-2NDNApeakuponPIstaining(6,17).

Inconclusion,wepresentarapid,efficientandreproducIBLemethodfordeterminingtheapoptoticpotentialofagivengeneofinterestinavarietyofcelllines.Ourmethodisapplicabletoandcompatiblewithavarietyofcelltypesandtransfectionmethods.ItcombinestheeaseofusingGFPasamarkerfortransfectionandsub-2NPIstainingasasimple,inexpensiveassayforapoptosis.FurThermore,thisassaycanbecarriedoutonarelativelysimpleFACScananalyseranddoesnotrequireelaborateFACSsortertechnologyorexpertise.

ACKNOWLEDGEMENTS

TheauthorswishtothankJohannesHoffmanforprovidingtheTdT/Cy5-CTPprotocolforFACSanalyses,EileenWhiteforpCMV-E1AandpCMV-E1B-19KandThomasBaderandKanagaSabapathyforcriticalreADIngofthemanuscript.ThisworkwassupportedinpartbytheAustrianIndustrialPromotionFund.

REFERENCES

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