NF2κB信号通路在食管鳞癌细胞系中的激活

:2005203209;:2005207208:211([2002]2):1.450052;2.:,E2mail:[e-mail]2B1,1,1,1,2,1TheActivationofNF2BSignalingTransductionPathwayinEsophagealSquamousCellCarcinomaCellLinesTIANFang1,XUPei2rong1,HOUWei2hong1,LIUHong2tao1,CHENKui2sheng2,XUELe2xun11.LaboratoryforCellBiology,ZhengzhouUniversity,Zhengzhou450052,China;2.Departmentofpathology,TheFirstAffiliatedHospital,ZhengzhouUniversityCorrespondingAuthor:XUELe2xun,E2mail:[e-mail]:ObjectiveMoreandmoreevidenceshaveshownthattranscriptionfactorNuclearFactor2kap2paB(NF2B)playsacriticalroleintheinitiationandprogressionofcarcinogenesis.Inthepresentstudy,weinvestigatedwhethertheNF2BsignalingtransductionpathwaywasconstitutivelyactivatedinESCCcelllines.MethodsImmunocytochemistryandWesternblottingwereusedtodeterminetheproteinlevelsofthesubunitsofNF2B,p50andp65,inhibitor2BandIKKintwoESCCcelllines.NuclearproteinsfromtheESCCcelllineswereextractedtoevaluatetheDNA2bindingactivitybyelectrophoreticmobilitygelshiftassay(EMSA).ResultsImmunocytochemicalanalysisshowedthatp50andp65,tworepresent2ativesubunitsofNF2B,IB,anaturalinhibitorofNF2B,anditsupstreamkinaseIKKweremainlyexpressedandlocalizedinthecytoplasm.Cytoplasmproteinextractsfromthecelllineseachwereana2lyzedbyWesternblotting.TheresultsrevealedthattherewasthesamehighNF2Bactivityinthecyto2plasmintwoESCCcelllinescomparedtothatofthepositivecontrolcellline.Inaddition,theresultsofEMSAanalysisfornuclearproteinextractshowedhighDNA2bindingactivityofp50andp65.ConclusionThefindingsdemonstratethatNF2Bsignalingtransductionpathwayisconstitutivelyactivatedinhu2manESCCcelllines,suggestingthatNF2Bmayplayacriticalroleincarcinogenesisoftheesophagus.Keywords:Esophagealcancer;NF2B;Signalingtransductionpathway:NF2kappaB(NF2B),NF2B,NF2BWesternblottingNF2Bp50p65,IBIKKEMSANF2BDNANF2B,p50p65IBIKK,p50/p65DNANF2B,NF2B:;NF2B;:R735.1:A:100028578(2006)01200112040,[1],,,,,,,NF2B(nuclearfactor2kappaB)1986B,k[2],NF2B5:p50/p105(NF2B1)p65(RelA)C2Relp52/p100(NF2B)RelB,Rel,NF2B,NF2Bp50p65112006331,NF2BIBs,,NF2B,IB,NF2B,NF2B,,IBNF2B,IBIKKNF2B,,,,,NF2B[325],NF2B,,NF2BIBIKK,NF2B11.11.1.1Eca109,EC9706,HeLa22910%,100u/ml,100g/ml1640(Gibco),37,5%CO2,1.1.2p65(sc28008)IKK(sc28014)p50(sc2114)IB(sc2371)SantaCruzBiotechnology3(89818)EMSA(78833)PierceSP1.21.2.180%(Eca109EC9706),6,375%CO224h,SPp65p50IBIKK,11001.2.2,,Bradford,-701.2.3WesternBlotting50g,2SDS11,10min,,SDS2PAGE,5%TBST2h,p50p65IBIKK11004,TBST10min3,(150000)1h,TBST10min3,DAB1.2.4(electrophoreticmobilityshiftassay,EMSA)NF2BDNA3NF2B52AGTTGAGGGGACTTTCCCAGGC23,32TCAACTCCCCTGAAAGGGTCCG25()(10g)20l(10MTris2HCl,pH8.5,0.5mMEDTA,5mMMgCl2,1MKCl,0.05%NP240,2.5%Glycerol,1g/lPoly(dIdC)and0.2g/lBSA),6%,,120mJ5min,DNAHRP,,X,,22.1NF2Bp50,p65EC9706Eca109,NF2Bp50,p65EC9706Eca109,1WesternBlottingp50,p65Eca109EC9706,HeLa229,EC9706Eca109p50,p65,21p50p65EC9706Eca109(400)2.2NF2BIBIKK,IB2120063312EC9706Eca109HeLa229NF2Bp50,p653IBandIKKEC9706Eca109(400)4EC9706Eca109HeLa229NF2BIBIKK5EC9706Eca109HeLa229NF2BIKKEC9706Eca109,3,EC9706Eca109HeLa229IBIKK,42.3NF2BDNA,EC9706Eca109HeLa229NF2BDNA,53,NF2B,[6]NF2B,[7,8]NF2B,p50/p65NF2B,Yang[9],,p50/p65WesternBlot,NF2Bp50p65,EMSAp50p65DNANF2B30min,NF2B,TNF2IL21,NF2B,,,NF2BNF2B,IBNF2B,IBIB,,IBIKKIKK,IKKIKKIKK,IKKNF2B,WesternBlot,IBIKK,IBIKKNF2BIKK2IB2p65/p50Wang[10],(52),NF2BNF2B,,NF2B,,,,,NF2B,NF2BNF2B,NF2BNF2B:[1]PisaniP,ParkinDM,BrayF.Estimatesoftheworldwidemortalityfrom25cancersin1990[J].IntJCancer,1999,83(6):18229.[2]SenR,BaltimoreD.Mutiplenuclearfactorsinteractwiththe312006331immunoglobulinenhancersequences[J].Cell,1986,46(5):7052716.[3]WangJH,HuangQK,ChenMX.TheroleofNF2Binhepa2tocellularcarcinomacell[J].ChinMedJ,2003,116(5):7472752.[4]YuHG,YuLL,YangY.IncreasedexpressionofRelA/nucle2arfactor2kappaBproteincorrelateswithcolorectaltumorigen2esis[J].Oncology,2003,65(1):37245.[5]NairA,VenkatramanM,MaliekalTT.NF2Bisconstitutive2lyactivatedinhigh2gradesquamousintraepitheliallesionsandsquamouscellcarcinomasofthehumanuterinecervix[J].Oncogene,2003,22(1):50258.[6]GreenDR.DeathandNF2kappaBinTcellactivation:lifeattheedge[J].MolCell,2003,11(3):5512552.[7]PhamLV,TamyoAT,YoshimuraLC.Inhibitionofconstitu2tiveNF2BactivationinmantlecelllymphomaBcellsleadstoinductionofcellcyclearrestandapoptosis[J].JImmunol,2003,171(1):88295.[8]TamataniT,AzumaM,AotaK.EnhancedIBkinaseactivi2tyisresponsiblefortheaugmentedactivityofNF2Binhu2manheadandneckcarcinomacells[J].CancerLetters,2001,171(2):1652172.[9]YangJ,RichmondA.ConstitutiveIkappaBkinaseactivitycorrelateswithnuclearfactor2kappaBactivationinhumanmelanomacells[J].CancerRes,2001,61(12):490124909.[10]WangW,CassidyJ.Constitutivenuclearfactor2kappaBmR2NA,proteinoverexpressionandenhancedDNA2bindingactivi2tyinthymidylatesynthaseinhibitor2resistanttumorcells[J].BrJCancer,2003,88(4):6242629.[:]3:;:R736.2:D:100028578(2006)0120014201:2005202201;:2005203230:312000,3:1:,453,,:5cm4cm3.5cm,,,,,T3T4TSHB:()5cm4cm3.5cm,,2:,3122,,,:3cm3cm,,,,B:T3T4TSH3cm3cm3.5cm,,,,,3:,5244,,,,:5cm5cm4cm,,,,B:44cm42cm49cm:T3T4FT3FT4TSH5cm5cm4cm,,,,,:1.5%3.5%,,1.53.51,[1],[2],,1.010cm,4cm,[2]:,,,PTH