糖原合酶激酶3β以cyclin D1依赖性方式引发人肺腺癌细讲解.docx

糖原合酶激酶3β以cyclin D1依赖性方式引发人肺腺癌细讲解.docx

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糖原合酶激酶3β以cyclinD1依赖性方式引发人肺腺癌细讲解

204ActaPhysiologicaSinica,April25,2007,59

（2）:

204-209

ResearchPaper

Glycogensynthasekinase3βinducescellcyclearrestinacyclinD1-dependentmannerinhumanlungadenocarcinomacelllineA549

LIJian-Sha,ZHUMin,TIANDan,WANGMan-Xiang,WANGFang,LINa-Ping,WURen-Liang*

DepartmentofPathology,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology,andKeyLaboratoryofPulmonaryDiseaseofMinistryofHealthofChina,Wuhan430030,China

Abstract:

Theeffectofglycogensynthasekinase3β（GSK3β）hasbeenrepeatedlyimplicatedincellproliferation,butstudiesontheeffectofGSK3βindifferentcelllineswithdifferentstimulihavedrawndifferentconclusions.ToinvestigatethedirecteffectofGSK3βoncellgrowthinhumanlungadenocarcinomacelllineA549,wechangeditsactivitybytransienttransfectionwithtwokindsofGSK3βmutantplasmids,constitutivelyactiveformS9A-GSK3βanddominantnegativeformKM-GSK3β.Twenty-fourhourslater,cellcounting,flowcytometryandWesternblotdetectionweremaderespectively.TheresultsshowedthatenhancingGSK3βactivitycausedadecreaseincellnumber,aswellasahigherpercentageofcellsatG1phase.Further,theexpressionofcyclinD1wasdown-

regulatedbyGSK3β.Takentogether,ourobservationssuggestthatGSK3βmayinduceG1cellcyclearrestinacyclinD1-dependent

fashionandthereforepossiblyplaysagrowth-inhibitoryroleinA549cells.

Keywords:

glycogensynthasekinase3β;cellproliferation;cellcycle;cyclinD1

糖原合酶激酶3β以细胞周期蛋白D1依赖性方式引发人肺腺癌细胞A549细胞周期阻滞

李建莎，朱敏，田丹，王满香，王芳，李娜萍，吴人亮*

华中科技大学同济医学院病理学系，卫生部呼吸系统疾病重点实验室，武汉430030

摘要：

对糖原合酶激酶3β（glycogensynthasekinase3β,GSK3β）在细胞增殖中的作用研究，在不同细胞系和不同刺激因素作用下得出了不同结论，本文旨在探讨GSK3β在人肺腺癌细胞系A549细胞生长中的直接作用。

A549细胞瞬时转染持续激活型S9A-GSK3β以及显性负突变型KM-GSK3β两种GSK3β突变型质粒，改变GSK3β活性。

24h后，分别进行细胞计数，流式细胞术及Westernblot检测。

结果显示，增强GSK3β活性可导致细胞数量下降，G1期细胞百分比升高。

细胞周期蛋白D1

表达水平被GSK3β下调。

结果提示，GSK3β可能以细胞周期蛋白D1依赖性方式引发A549细胞的G1期阻滞，从而发挥生

长抑制效应。

关键词：

糖原合酶激酶3β；细胞增殖；细胞周期；细胞周期蛋白D1

中图分类号：

R322.3

Glycogensynthasekinase3β（GSK3β）isaserine/threo-

nineproteinkinasethatwasfirstdescribedinametabolic

pathwayforglycogensynthaseregulation[1].Incontrast

tomanyotherkinases,GSK3βisconstitutivelyactivein

un-stimulated,restingcellsandbecomesinactivethrough

phosphorylationatserine9byitsupstreamproteinkinases.

Ithasavarietyofputativesubstrates,playingimportantrolesinmetabolism,cellproliferation,differentiationandsurvival[2].GSK3βattractsmoreandmoreattentionforitsrolesinadiverserangeofcellularprocessesanditskeypositionatseveralsignalingpathwaysthatarecrucialincancerandotherhumandiseases.Duringembryonichairfollicledevelopment,GSK3βmayserveasacentralsig-nalinghub,allowingfortightlyintegratedandspatiallycon-Received2006-11-23Accepted2006-12-29

ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina（No.30470757）.

*Correspondingauthor.Tel:

+86-27-83692619;Fax:

+86-27-83650729;E-mail:

renliangwu@

LIJian-Shaetal:

GSK3βInducesG1CellCycleArrestinA549Cells

205

trolledproliferativeresponses[3].RecentdocumentsabouttumorspresentopposingeffectsofGSK3βoncellproliferation.Inastudyofcoloncancercells,inhibitionofGSK3βactivitybychemicalinhibitorsanditsexpressionbyRNAinterferencetargetingGSK3βinducedapoptosisandattenuatedproliferation,suggestingapossibleroleofGSK3βinpromotingtumorcellsurvivalandproliferation[4].Conversely,inmostinvestigationsoftumorssuchasMCF-7breastcancercellsandhepatocellularcarcinoma,GSK3βshowedgrowth-inhibitoryeffectandthereforewaspre-dictedatumorsuppressor[5,6].

Lungcanceristheleadingcauseofcancer-relatedmor-talityintheworld[7].ItsdevelopmentiscloselyassociatedwithdysregulationofWntsignaling[8],inwhichGSK3βhasbeenillustratedasagenuineswitchthatdictatesbothonandoffstatesofapivotalregulatorypathway[9].However,littleisknownabouttheroleofGSK3βinlungcancer.OurpreviousworkhasshownthatGSK3βisrichinlungtissueandculturedpigbronchialepithelialcells[10].Furthermore,wehavedemonstratedthatthisenzymeisinvolvedinairwayrepairaswellassquamousdifferentia-tionofairwayepithelialcells[11-13].Inthepresentstudy,thedirecteffectofGSK3βoncellproliferationinthehumanlungadenocarcinomacelllineA549wasassessed.FirstwechangedtheactivityofGSK3βbytransienttransfec-tionwithtwoGSK3βmutantplasmids,constitutivelyac-tiveform（S9A-GSK3β）[14]anddominantnegativeform（KM-GSK3β）[15].ThenweinvestigatedtheeffectsofchangedGSK3βoncellgrowthaswellascellcyclepro-gressioninA549cells.Ourresultssupportedagrowth-inhibitoryroleofGSK3βinA549cells.

1MATERIALSANDMETHODS

1.1Cellculture

A549celllinewasobtainedfromtheCellBankofChineseAcademyofSciences（Shanghai,China）.A549cellsweremaintainedinDulbecco’smodifiedEagle’smedium（DMEM）supplementedwith10%newcalfserum（ZhejiangSanliBiotechnologyCompany,China）andantibiotics（100IU/mLpenicillinand100µg/mLstreptomycin）at37oCin5%CO2.Experimentswereperformedinthesamepas-sageofA549cellsandrepeatedatleast3times.1.2Transienttransfection

TheconstitutivelyactiveGSK3βmutant（S9A-GSK3β）anddominantnegativeGSK3βmutant（KM-GSK3β）weregenerouslyprovidedbyProfessorJamesR.Woodgett（OntarioCancerInstitute,Canada）.S9A-GSK3βwascon-structedbymutationofserine9toalanineusingthepAlter-1

method[14],andthemutationrendersthekinaseinsensitivetoinhibitoryphosphorylationandhenceconstitutivelyactive.WhileKM-GSK3β,whichwasgeneratedbyalteringthetwoconsecutivelysineresiduesintheATP-bindingsitetoamethionineandanisoleucine,hadnokinaseactivityandspecificallyinterferedwithendogenousGSK3βactivitypresumablybyblockingaccesstosubstrates[15].Therefore,itcanbeutilizedasadominantinhibitorofendogenousGSK3β.TransienttransfectionwascarriedoutusingLipofectamine2000（Invitrogen,CA,USA）accordingtotherecommendationfrommanufacturerandthemethoddescribedbyTuckeretal.[16]withminormodification.Thecellswereplatedonto6-wellplateonedaypriortotransfection.Followingconfirmationof70%-80%confluence,thecellsweretransfectedwiththeGSK3βmutantplasmids（1µgS9A-GSK3βand1µgKM-GSK3β,respectively）aswellasemptyvectorcontrolplasmid（1µgpcDNA3）.Twenty-fourhourslater,amorphologicalobservationwasmade,andthenthecellswereharvestedforcellcounting,flowcytometry,orthewholecellpro-teinwasextractedforafurtherWesternblotdetection.1.3Cellcounting

Thecellswereallowedtoattachfor24hatadensityofabout2×105cells/wellin6-wellcultureplate.Aftertran-sienttransfectionandafurtherculturefor24h,thecellswerewashedwithphosphate-bufferedsaline（PBS）,incu-batedwith0.25%trypsinfor15minat37oC.Thediges-tionsolutionwasmixedwith1.5mLPBS,andaspiratedrepeatedlytomakesinglecellsuspension.Cellcountingwasperformedusingahematocytecountingchamber.1.4Flowcytometry

Twenty-fourhoursaftertransfection,thecellswerewashedwithcoldPBS,trypsinizeddownandcollectedbycentrifugation,andthenfixedwith80%coldethanolat−20oCovernight.Beforemeasurement,thecellswerewashed,collectedandre-suspendedinPBScontaining10µg/mLpropidiumiodideand100µg/mLDNase-freeRNaseA（Sigma,USA）,andthenincubatedat4oCforatleast30min.ThepercentageofcellsatG1,SandG2phaseswasdeterminedbyFACSCalibur（BectonDickinson,USA）.1.5Westernblotanalysis

Afterthesamedurationoftransfection,thecellswererinsedtwicewithcoldPBS,collectedbytrpsinizationandcentrifugation,thenlysedinbuffer（50mmol/LTris-HCl,pH8.0,100mmol/LNaCl,1mmol/LEDTA,1mmol/Ldithiothreitol,1%TritonX-100,0.1%SDS,50mmol/Lsodiumfluorideand1mmol/Lsodiumvanadate）contai-ningaproteaseinhibitorcocktailtoobtainthewholecell

206ActaPhysiologicaSinica,April25,2007,59

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protein.ProteinconcentrationwasdeterminedbyBCAkit（PierceChemicalCompany,USA）.Equalamountofpro-teinswerefractionatedbySDS-polyacrylamidegelelectrophoresis,andtransferredontonitrocellulosemembrane.Themembraneswereblockedwith5%non-fatmilkinTBSTandincubatedwithanti-GSK3β（1:

1000,SantaCruz,CA,USA）,anti-phosphorylatedGSK3β（1:

500,CellSignaling,USA）,anti-cyclinD1（1:

500,SantaCruz,CA,USA）andanti-β-actin（1:

1000,SantaCruz,CA,USA）antibodiesovernightat4oC.Thesignalwasdetectedbyusingahorseradishperoxidase-conjugatedse-condaryantibody（SantaCruz,CA,USA）andenhancedchemiluminescence（ECL,PierceChemicalCompany,USA）,andthenexposedtoX-rayfilms（Fuji,Japan）.1.6Statisticalanalysis

Datawerepresentedasmean±SD.One-wayANOVAtestwasusedtodeterminesignificanceformultiplegroupcom-parison（SPSS12.0software）.Differenceswereconsi-deredtobestatisticallysignificantatP<0.05.

KM-GSK3βtransfectionmightpromotecellgrowth（P<0.05comparedwiththecontrolgroup）（Table1）.

WithWesternblotanalysis,itwasconfirmedthatthe

Table1.EffectofGSK3βonproliferationofA549cells

ControlS9A-GSK3βKM-GSK3β

Cellnumber（×104）

39.3±5.2

21.6±3.4*

50.8±8.7*

n=3independentexperimentsdoneinduplicate.*P<0.05ascom-paredwiththecontrolgroup.

2RESULTS

2.1GSK3βinhibitedproliferationofA549cellsToinvestigatewhetherGSK3βhadadirecteffectonA549cellproliferation,wechangeditsactivitybytransienttrans-fectionwithtwokindsofGSK3βmutantplasmidsfor24h.Subsequently,wemadeamorphologicalobservationofA549cellgrowth.Underphasecontrastmicroscope,theemptyvect