ResearchPaperInductionofFADDExpressionandCaspaseCascadesInvolvedinRC-RNase-InducedApoptosisinHumanCervicalCancerCellsHuey-ChungHuang,You-DiLiao,Li-LiChen,andTze-SingHuang1InstituteofBiochemistryandMolecularBiology,CollegeofMedicine,NationalTaiwanUniversity,Taipei,Taiwan[H.-C.Huang];InstituteofBiomedicalSciences,AcademiaSinica,Taipei,Taiwan[Y.-D.Liao];NationalInstituteofCancerResearch,NationalHealthResearchInstitutes,Taipei,Taiwan[L.-L.Chen,T.-S.Huang]AIM:RC-RNaseisanovelmemberofRNaseAsuperfamilyfromRanacatesbeiana(bullfrog)oocytes.ThisstudyistoinvestigatethecytotoxiceffectofRC-RNaseoncancercells.Thecytotoxicmechanismswereinvestigatedanddiscussed.METHODS:CytotoxicityofRC-RNasetohumancervicalcanerHeLaS3cellswasdeterminedbymicroculturetetrazoliumtestandtrypanblueexclusionassay.Apop-toticcharacteristics,suchaschromatincondensation,blebbingmorphology,DNAladder,phosphatidylserinetranslocation,caspaseactivation,andpoly(ADP-ribose)polymerase(PARP)cleavage,wereanalyzedbygenerallydescribedmethods.Cell-cyclephaseswereanalyzedbyflowcytometry,andcellularcyclinB1/CDC2activitywasdetectedbyinvitrohistoneH1kinaseassay.Westernblotanalyseswereper-formedtoexaminethelevelsofFas,FADD,TNFR1,TRADD,cyclinB1,CDC2,Wee-1,andthecleavedformsofBid,PARPandcaspases-3,8and9.RESULTS:RC-RNaseinducedcelldeathwithapoptoticcharacteristicsinHeLaS3cells.Invitroactivityassayssuggestedthatcaspases-3,8,and9activitieswereallincreasedinRC-RNase-treatedcells.TheresultsofWesternblotanalyses,includingKeywords:inductionofFADDexpressionandincreasesofthecleavedformsofBid,PARPandRC-RNasecaspases-3,8and9,confirmedthatRC-RNaseindeedinducedtheactivationofcaspases-3,8,and9.AlthoughRC-RNasedidnotsignificantlyaffectthecell-cycleFADDdistribution,cyclinB1/CDC2kinaseactivitywasincreasedinRC-RNase-inducedcaspaseapoptoticcells.CellularWee-1levelwasreducedbyRC-RNasetreatment,andCDC2waskeptinTyr-15-dephosphorylated(active)form.Wee-1CONCLUSION:RC-RNasewaspotenttoinduceFADDexpression,caspasecas-cyc