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AxinC61,1,1,1,1,1,2BiologicaleffectsofAxingeneexpressioningliomaC6cellsZHANGLi-ying1,LIQing2,CHENGuang-sheng1,YEJing1,ZHANGFeng1,LIFan-fan1,LINSheng-cai21DepartmentofPathology,XiJingHospital,FourthMilitaryMedicalUniversity,Xi’an710032,China;2SchoolofLifeScience,XiamenUniversity,Xiamen361000,China.AbstractObjective:ToobservetheeffectsofAxingeneexpressiononthebiologicalcharacteristicsofgliomacells.Methods:TheproliferationabilityoftransfectedcellswasexaminedbyMTTassay,cellgrowthcurveandcolo2nyformationassay.Thechangeofcellcyclewasanalysesbyflowcytometry(FCM).TheapoptosiswasmeasuredbyTUNELstaining.ImmunostainingwasusedtodetecttheexpressionofKi67,CyclinD1andp53inC6cellsandtrans2fectedcells.Results:TheprogressionofthecellcyclewasarrestedinG1phaseaftertransfectedwithAxinplasmids;thecellviability,growingspeedandcolonyformationabilityweredecreasedsignificantlyatthesametime;theapop2toticrateincreased,theexpressionsofKi67andCyclinD1decreased,butthep53levelincreased.Conclusion:AxingenecouldinhibitC6cellproliferationandarrestthetransfectedcellsinG1phase,andthisbiologicaleffectmightbedependedontheincreasedexpressionofp53andthedecreasedexpressionofCyclinD1.ThemolecularmechanismofAxinincellproliferationneedsmoreresearch.KeywordsAxingene;gliomacells;growthcurve;cellcycle;apoptosis;p53(ModernOncology2006,14(5):05210524):AxinC6:(MTT),(FCM),TdT(TUNEL)Ki67CyclinD1p53:AxinG1;;;Ki67CyclinD1,p53:Axin,,Axinp53Cy2clinD1Axin;;;;;p53R730.264A1672-4992-(2006)05-0521-04Axin(axisinhibition)1997ZengL[1]Axin[2],[3],Axin[4]Axin,,-cateninWnt,MEKK1/4MEKK4/7JNK[5]Axin2005-11-09(30125012);(02ma04)1,,7100322,361000(1977-),,,,(1955-),,,,,p53,HIPK2(homeodomain-interactingproteinkinase-2)p5346,p53[6]Axin[7],21%Axin1036,[8]Axin,AxinC6[9]Axin,11.1p53CyclinD1(SantaCruz),Ki67(NeoMarkers),SABC(),TUNEL(),SP(),MTT(AMRESCO),DAB(12520065145)1.21.2.1[9]1.2.2(MTT)0.25%Axin,60096,375%CO2,96MTT(5mg/ml)20ul,37,4h,,150ulDMSO,10min,;490nmOD,1.2.3(FCM)0.25%Axin,PI,1.2.40.25%,,100,375%CO223W,Giemsa30min,1mm1.2.5TUNELTUNELAxin,PBSTUNEL:,,,(%)=(500/500)100%1.2.6Ki67cyclinD1p53,4%20min;3%H2O210min,2,PBS5min;100,20min,;Tri2tonX-10010min,PBS3,5min;15min;Ki67CyclinD1p53,PBS,,4;40min,()IgG,3740min,PBS3,5min;,3740min,PBS3,5min;DAB,10min;,10,1000,1.3t2SPSS11.522.1MTTAxinAxin(1)1AxinFig1EffectsofAxinexpressionongliomacellgrowth2.2AxinAxin,G1,S(1)1AxinC6()(%)G1G2SG2/G1C6-Vector-Axin74.86.318.91.981C6-Vector65.49.824.81.896C668.68.023.41.8642.3AxinAxin70.2%(P0.01),22.4Axin,10.79%7.10%,Axin,33.20%,(P0.01),32.5Ki67cyclinD1p53Ki67cyclinD1p53(4),Ki67Axin(2=131.75,P0.01),CyclinD1Axin(2=11.75,P0.01),p53Axin(2=133.83,P0.01)2AxinA:C6-Vector-AxinB:C6-VectorC:C6Fig2EffectsofAxinexpressiononcellcloningefficiencyA:C6-Vector-AxinB:C6-VectorC:C6225MODERNONCOLOGY,May.2006,VoI114,NO153AxinA:C6-Vector-AxinB:C6-VectorC:C6Fig3EffectsofAxinexpressiononapoptosisA:C6-Vector-AxinB:C6-VectorC:C64Ki67cyclinD1p53(SP200)3AxinFused(Fu),Wnt[1],Wnt,AxinAPC/GSK-3/CKI-catenin[10],[11][12]Axin,Axin[13],Axin[14]Axin[7]Axin[8],AxinC6,C6,,,AxinC6,C6G1,S,Ki67,,Axin,Ki67,AxinCyclinD1,CDK4/6,G1SDNACyclinD1,[15]p53,,[16][6]Axinp53HIPK2p53AxinHIPK2p5346,p53,p21,CDK,AxinC6,p53,CyclinD1,G1,,p53CyclinD1Axinp53p21,p21CDK,CyclinD1,CDK4/6,RB,E2F,G1,Axin,p53,CyclinD1[1]ZengL,FagottoF,ZhangT,etal.ThemouseFusedlocusen2codesAxin,aninhibitoroftheWntsignalingpathwaythatregu2latesembryonicaxisformation[J].Cell,1997,90:181192.[2]SSalahshor,JRWoodgett.Thelinksbetweenaxinandcarcino2genesis[J].JClinPathol,2005,58:225236.[3]CowanCA,HenkemeyerM.TheSH2/SH3adaptorGrb4transduc2esB-ephrinreversesignals[J].Nature,2001,413:174178.[4]ChristianJL,MoonRT.InteractionsbetweenXwnt-8andspe2mannorganizersignalingpathwaysgeneratedorsoventralpatternintheembryonicmesodermofXenopus[J].GenesDev,1993,7:1328.[5]LuoW,NgWW,LinSC,etal.AxinutilizesdistinctregionsforcompetitiveMEKK1andMEKK4bindingandJNKactivation[J].JBiolChem,2003,278:3745137458.[6]RuiYN,XuZ,LinSC,etal.Axinstimulatesp53functionsbyactivationofHIPK2kinasethroughmultimericcomplexformation[J].EMBOJ,2004,23:45834594.[7]PeiferM,PolakisP.Wntsignalinginoncogenesisandembryogen2esis-alookoutsidethenucleus[J].Science,2000,287:1606325200651451609.[8],,,.Axin[J].,2003,2(4):334337.[9],,,.pIRES2-EGFP-Ax2in[J].,2005,21(4):408410.[10]LiuC,LiY,SemenovM,etal.Controlofbeta-cateninphos2phorylation/degradationbyadual-kinasemechanism[J].Cell,2002,108:837847.[11]IshizakiY,IkedaS,FujimoriM,etal.Immunohistochemicala2nalysisandmutationalanalysesofbeta-catenin,AxinfamilyandAPCgenesinhepatocellularcarcimomas[J].IntJOncol,2004,2:10771083.[12]JinLH,ShaoQJ,LuoW,etal.DetectionofpointmutationsoftheAxin1geneincolorectalcancers[J].IntJCancer,2003,107:696699.[13]SatohS,NakamuraY,KatoT,etal.AXIN1mutationsinhepa2tocellularcarcinomas,andgrowthsuppressionincancercellsbyvirus-mediatedtransferofAXIN1[J].NatGenet,2000,24:245250.[14]HsuW,ShakyaR,CostantiniF,etal.ImpairedmammaryglandandlymphoiddevelopmentcausedbyinducibleexpressionofAxinintransgenicmice[J].JCellBiol,2001,155:10551064.[15]YoussefEM,HasumaT,MorishimaY,etal.Overexpressio