TopoGEN/Topoisomerase In Vivo Link Kit/TG1022 (Topo II)/200 Assays

TopoisomeraseI/IIInVivoLinkKit(ICEBioassay)ProductDescription
TopoGENhasextensiveexperiencewithassaysfortopoisomeraseinhibitioninvivo. Theseexperimentsallowtheinvestigatortoascertainwhetheranovelagentisactiveagainstendogenoustopoinachromosomalsettinginnuclei. Animportantbenefittothisanalysisisthatonecanuseanytumorcellortissueinordertoestablishclinicalefficacyofatestdrugagainstaspecifictumorcellline. WeuseessentiallythesamebasicapproachforTopoIasforTopoII. Themethodsarebaseduponphysicallyseparatingthetopo/DNAadductsfromfreeDNAandusingantibodiestomeasureboundtopoIorII. Inthisanalysis,tissueculturecellsaretreatedwithatestcompoundalongwithnegativecontrols(nodrug)andpositivecontrols(withknowninhibitors). ThecellsaredrugtreatedandrapidlylysedwithSarkosylwhichtrapssomefractionoftheendogenoustopoonDNAinacovalentcleavagecomplex. Followingdetergentlysis,thelysateisdilutedtofullydissociatenon-covalentDNA/proteincomplexes. Covalenttopo/DNAcomplexesareresolvedonastepCsClgrADIent(ionicDNA/proteininteractionsarealsopreventedby5MCsCl). ThegradientresolvesDNA,chromatinaggregates,andprotein,respectively.Gradientsarecentrifugedovernightandfractionated. TheamountoftopocoincidentwiththeDNApeakisameasureofcovalentDNA/topocomplexes. TopoconcentrationintheDNApeakisdeterminedbyimunoblottingusingantibodytotopoIorIIasprobe. IntheabsenceofagentsthatstABIlizethecleavablecomplex(etoposide,camptothecin)onlylowlevelsoftopoarefoundintheDNApeak;thisisparticularlyobviouswithtopoIIsincethetypeIIenzymeisnottrappedbythismethodunlessaninhibitorisused. Incontrast,topoIistrappedtoalowextentevenintheabsenceofcamptothecin. TheratiooftopoattheDNAdensityandtheproteindensityreflectstherelativeefficiencyofstabilizationofthecleavablecomplexes.

KitContents:

-Camptothecin(suppliedlyophilizedwithTG1021Top1)
-Etoposide(suppliedlyophilizedwithTG1022Top2)
-Sarkosyl(20%)
-AntibodytoTopoisomeraseI(SuppliedwithTG1021Top1)
-AntibodytoTopoisomeraseII(170kDaform,suppliedwithTG1022Top2)
-CsClStockSolution
-DetailedInstructionManual

GeneralProcedures:
Anoutlineofthemethodisshownbelow. Cellstobetestedmaybeaparticularcellline,virusinfectedcellortumortissue. Cellsareincubatedunderconditionsthatfavorendogenoustopoactivity(definedasphysiologicalconditionsconducivetocellgrowth);thus,theendogenousenzymeisengagingtheDNAtemplateinaseriesofbreakingandresealingsteps. Concurrently,centralgeneticprocessessuchasDNAreplication,transcriptionandrepairareongoing. Thecellsarethenrapidlylysedwithadetergent(sarkosyl). Itisimportantthatlysisbecarriedoutwhilemaintainingthecellsat37°C;ifthecellsarecooledormanipulatedpriortolysis,thecleavagecomplexestendtore-ligateandyieldnegativeresults(seeTraskandMuller,1988). ThenextsteprequirespurificationofDNAawayfromfreeprotein;however,organicextractionsorproteinasedigestionsmustbeavoided. WeuseastepCsClgradientforthispurpose. ThedensitystepsaredesignedtoresolveDNAfromfreeproteinandthereisquantitativerecoveryofboth.CovalentcomplexescontainingtopoandDNAsedimenttothepositionofDNA. ItisknownthatcovalentlyboundproteincanshiftthedensityofDNAinCsClandthemagnitudeofthedensityshiftisproportionaltothetotalamountofprotein. Invitro,topoImayproduceadensityshiftofDNAunderconditionsofstoichiometricexcessofprotein(datanotshown);however,invivosignificantlylessproteiniscoupledtoDNA. Infact,DNAtopoisomerasedoesnotcauseanydensityshiftofgenomicDNAinthisanalysis(seebelow)fortworeasons. First,thenumberofboundtopomeculesperDNAmoleculeisverylowandinthesegradients,complexesbehavelikefreeDNA(vs.DNA/proteinadducts). Second,thegradientsareverysteepanditisnotpossIBLetoresolvesmalldensitydifferencesanyway. AfterCsClcentrifugation,thegradientsarefractionatedandtheamountofboundandfreetopoismeasuredbyWesternblottingusingslotordotblotsandantibodiesdevelopedbyTopoGEN. Theratioofbound/freeisadirectmeasureofthecleavablecomplexformationintheparticularcellsystem. Wehavefoundthattopoisomerasesaredifficult(ifnotimpossible)totrapascleavablecomplexeswithincellsintheabsenceofinhibitors;thus,anytestcompoundthatresultsindetectionoftopoIorIIintheDNApeak(seebelow)ismostlikelyatopo-activeagent.TheInVivoLink-KitthenallowstheinvestigatortoevaluateacompoundforactivityagainsttopoIandIIindifferenttissuesettingsorwithvirusinfectedcells. Finally,itispossibletocombinetheanalysisoftopoIandIIinthesameexperiment. Inthiscase,onecanevaluatetopoisomeraseIinhibitionusingtheantibodytotopoisomeraseIortopoisomeraseII. Somedrugsmayconceivablyactuponbothenzymes(TraskandMuller,1988).