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).

美国TopoGEN公司成立于1991年，是一家诊断公司，由一批在拓扑异构酶领域工作和在基础、临床、应用及相关方面研究经验丰富的研究者创建，为以药物开发为基础的拓扑异构酶和机制研发人员提供创新产品和服务。药物筛选试剂盒可以分析拓扑异构酶活性药物，用于癌症化疗和抗菌方面。TopoGEN公司还能提供原核和真核化验拓扑异构酶 (I型，II，IV和旋转酶)检测试剂盒，体外体内诊断均可应用。TopoGEN公司还提供DNA载体、DNA裂解目标、拓扑异构酶抑制剂、拓扑异构酶抗体和相关试剂盒等完整产品线。