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ExperimentalHematology32(63Anovelmechanismforimatinibmesylate(STI571)resistanceinCMLcelllineKT-1:RoleofTC-PTPinmodulatingsignalsdownstreamfromtheBCR-ABLfusionproteinTakatsuneShimizua,YoshitakaMiyakawaa,SatoshiIwatab,AkikoKuribarab,TonyTiganisc,ChikaoMorimotob,d,YasuoIkedaa,andMasahiroKizakiaDepartmentofInternalMedicine,KeioUniversitySchoolofMedicine,Tokyo,JbDivisionofClinicalImmunology,AdvancedClinicalResearchCenter,InstituteofMedicalScience,UniversityofTokyo,Tokyo,JcDepartmentofBiochemistryandMolecularBiology,MonashUniversity,Victoria,AdDepartmentofLymphoma/Myeloma,MDAndersonCancerCenter,Houston,Tex.,USA(Received28January2004;revised8July2004;accepted21July2004)aObjective.Acquiredresistancetoimatinibmesylate(STI571)inchronicmyelogenousleukemia(CML)patientshasbecomeaseriousclinicalproblem.WepreviouslyestablishedSTI571-resistantsublines(designatedKTRcells)fromtheCMLcelllineKT-1.Tcellproteintyrosinephosphatase(TC-PTP)wasmarkedlydownregulatedinallKTRcellscomparedtoparentalKT-1cells.Therefore,weexaminedwhetherthesuppressionofTC-PTPexpressionmightcontributetoimatinibmesylate-resistanceinKTRcells.MaterialsandMethods.WetransducedthenuclearisoformofTC-PTP(TC45)andcatalyticallyinactiveTC45-D182AcDNAsintoKTRcellsbyretroviralgenetransfer.Subsequently,weanalyzedthesensitivitytoimatinibmesylateandthestatusofsignalingpathwaysinthetransducedcells.Results.TheoveralllevelsofSTAT5phosphorylationweresigni?cantlyhigherinKTRcellsascomparedtoKT-1cells,butreconstitutionofTC-PTPinKTRcellsresultedinadramaticdecreaseofSTAT5phosphorylation.Furthermore,STAT5phosphorylationwasablatedbyimatinibmesylateinKT-1cellsbutremainedelevatedinKTRcells.Incontrast,weobservednodifferenceinBCR-ABLorJAK2phosphorylationandnodifferenceinactivationofothersignalingpathways.Importantly,reconstitutionofTC-PTPinKTRcellstolevelsfoundinparentalKT-1cellsrestoredtheirsensitivitytoimatinibmesylateasmonitoredbyreducedproliferationandincreasedapoptosis.Conclusions.WehavedemonstratedthatforcedexpressionofTC-PTPinimatinibmesylate-resistantKTRcellscanrestoresensitivitytoimatinibmesylate.OurstudiesindicatethatlossofTC-PTPmayrepresentanovelmechanismbywhichCMLcellscanacquireimatinibmesylate-resistance.?2004InternationalSocietyforExperimentalHematology.PublishedbyElsevierInc.TheABLselectivetyrosinekinaseinhibitorSTI571(Gleevec?,INovartisAG,Basel,Switzer-land)isthemajortherapeuticagentfortreatmentofpatientswithchronicmyelogenousleukemia(CML)[1,2].However,imatinibmesylatedoesnotprovidegoodclinicalresultsinpatientswithblastcrisisandacquiredresistancetoimatinibmesylatehasbecomeaseriousproblem[3,4].Severalmech-anismsofimatinibmesylateresistancehavebeenreported,Offprintrequeststo:MasahiroKizaki,M.D.,DivisionofHematology,DepartmentofInternalMedicine,KeioUniversitySchoolofMedicine,35Shinanomachi,Shinjuku-ku,Tokyo160-8582,JE-mail:makizaki@sc.itc.keio.ac.jpincludinggeneampli?cationofbcr-abl[5,6],point-muta-tionsinthekinasedomainofbcr-ablgene[5,7C9],multidrugresistantgeneproductexpression[10],andinactivationofimatinibmesylatebybindingofα-1-acidglycoprotein[11].Inaddition,BCR-ABLindependentmechanismsforima-tinibmesylate-resistancesuchasactivationofalternativepathwayshavebeenreportedrecently[12C15].WepreviouslyestablishedSTI571(imatinibmesylate)-resistantCMLsublines,designatedKTRcells,fromKT-1cells[16].KTRcellsexpressedhigherlevelsofp-glycopro-teinthanparentalKT-1cellsandshowedcross-resistancetodoxorubicin.Verapamil,ap-glycoproteinblocker,over-cameresistancetodoxorubicinbutnottoimatinibmesylate,$Cseefrontmatter.Copyrightdoi:10.1016/j.exphem.?2004InternationalSocietyforExperimentalHematology.PublishedbyElsevierInc.1058T.Shimizuetal./ExperimentalHematology32(63indicatingthatp-glycoproteinwasnotinvolvedinimatinibmesylateresistanceinKTRcells.Noampli?cationsofthebcr-ablgeneandnomutationsresultinginamino-acidssub-stitutionsintheadenosinetriphosphate(ATP)bindingsiteofABLkinasedomainweredetectedinKTRcells.Interest-ingly,expressionofthetyrosinephosphataseTC-PTPwasmarkedlydownregulatedinallKTRcells[16].Inthisstudy,wereportthatlossofTC-PTPcontributestoimatinibmes-ylateresistanceinKTRcells.Ourstudiesindicatethatde-creasedtyrosinephosphataseexpressionmayallowforenhancedsignalingdownstreamofBCR-ABLandpromoteimatinibmesylateresistanceinCML.MaterialsandmethodsReagentsImatinibmesylatewaskindlyprovidedbyNovartisPharmaceuti-cals(Basel,Switzerland).AmousemonoclonalantibodyagainstTC-PTPwasobtainedfromOncogeneResearchProducts(SanDiego,CA,USA).Rabbitanti-humanSTAT5,anti-humanJAK2,andgoatanti-humanactinantibodieswerefromSantaCruzBio-technologies(SantaCruz,CA,USA).Mousemonoclonalantibod-iesagainstphosphotyrosine(clonePY20and4G10)wereobtainedfromBDBiosciences(SanJose,CA,USA)andUpstate(Charlottesville,VA,USA),respectively.Amonoclonalanti-c-AblantibodywasfromBDBiosciences.Polyclonalanti-phospho-AKT,AKT,phospho-p44/42MAPK(ERK1/2)andp44/42MAPK(ERK1/2)antibodieswerepurchasedfromCellSignaling(Beverly,MA,USA).Monoclonalanti-phospho-SAPK/JNKandpolyclonalSAPK/JNKantibodieswerefromNewEnglandBioLabs(Beverly,MA,USA).Ahorseradishperoxidase-conjugatedsecondaryanti-bodywaspurchasedfromAmershamBiosciences(Piscataway,NJ,USA).CellcultureThehumanCMLcelllineKT-1(kindlyprovidedbyDr.Sakai,EhimeUniversity,Japan)[17]and0.5μMSTI571(imatinibmesy-late)-resistantclones(KTR2and3)weremaintainedasdescribedpreviously[16].Plat-EpackagingcellswerekindlyprovidedbyDr.Kitamura(UniversityofTokyo,Japan)[18].PT67amphotropicpackagingcellswerepurchasedfromBDBiosciences.K562,MOLT4,andJurkatcellswereobtainedfromtheJapanCancerResearchResources(Tokyo,Japan).PlasmidandretroviralgenetransferThenuclearisoformofTC-PTP(TC45)anditscatalyticallyinactivesubstrate-trappingmutantTC45-D182AcDNA[19]wereclonedintotheretroviralplasmidpMX-IRES-GFP[20](kindlyprovidedbyDr.Kitamura).Retroviralgenetransferwasperformedusingtheping-ponginfectionmethod.Brie?y,Plat-EcellsweretransfectedwitheachplasmidusingtheFuGene6reagent(Roche,Mannheim,Germany).PT-67cellswereinfectedwiththeviralsupernatant.AfterestablishingPT67-transfectantsstablyproducingeachretro-virus,thesupernatantwasusedtoinfectKTRcellswith5μg/mLofPolybrene(Sigma,St.Louis,MO,USA).Sevendaysafterinfection,green?uorescentprotein(GFP)-positivecellsweresortedbyEPICSELITEESP?owcytometer(Coulter,Hialeah,FL,USA).CellproliferationassayCellproliferationwasmeasuredusingaMTTproliferationassaykit(Roche)asdescribedpreviously[16].FlowcytometryCellswereculturedintheabsenceorpresenceof0.5μMimatinibmesylatefor48hours.CollectedcellswerestainedwithannexinV-PEand7-amino-actinomycinDstaining(BDPharmingen,Mis-sissauga,ON,Canada).AnalysiswasperformedusingFACSCali-ber?owcytometer(BDBiosciences).WesternblottingandimmunoprecipitationWesternblottingwasperformedasdescribedpreviously[16],withminormodi?cationstothelysisbuffer(1%NonidetP40,40mMTris-HCl[pH8.0],150mMNaCl,0.1%sodiumdodecylsulfate[SDS],1mMsodiumorthovanadate,1μLofaprotinin,0.5%sodiumdeoxycholate,10μg/mLleupeptin,1μg/mLpepstatinA,5mMethylenediaminetetraaceticacid,and1mMphenylmethyl-sulphonyl?uoride).TyrosinephosphorylationofBCR-ABLwasdetectedwithananti-phosphotyrosineantibody4G10.Forimmu-noprecipitations,wholecelllysateswereincubatedfor2hoursat4?Cwiththespeci?edantibodiesandproteinA/GPLUS-Agarosebeads(SantaCruz)addedandincubationscontinuedovernightat4?C.ImmunoprecipitateswereresolvedbySDS-polyacrylamidegelelectrophoresis,transferredtopolyvinylidenedi?uoridemembranesandimmunoblottedwithamonoclonalphosphotyrosineantibody(PY20),andthenstrippedandre-probedasindicated.Semi-quantitativeRT-PCRofTC-PTPTotalRNAwasisolatedusingRNeasyMiniKit(Qiagen,Hilden,Germany).Reversetranscriptase-polymerasechainreaction(RT-PCR)wasperformedusingReverTraAcereversetranscriptaseandKODPLUSTaqpolymerase(Toyobo,Osaka,Japan).TheprimersusedinthePCRforTC-PTPandβ-actinwereasfollows:forward:5′-cctcatagagtggccaagt-3′andreverse:5′-caagtgtctaccagagag-3′,forward:5′-caagagatggccacggctgct-3′andreverse:5′-tccttctgcatcc-tgtcggca-3′,respectively.Semi-quantitativePCRwasperformedasfollows:1cycleof94?Cfor2minutesand23cyclesof94?Cfor1minute,50?Cfor1minute,and72?Cfor1minute.PCRproductsweremeasuredbyQuantityOne(BioRad,Hercules,CA,USA)densitometrysoftware.Datawereexpressedasaratiorelativetoβ-actininarbitraryunits.StatisticalanalysisDataareexpressedasmean±SD.TheunpairedStudent’st-testwasusedtoevaluatestatisticalsigni?cance.ResultsEffectsofTC-PTPexpressiononthesensitivityofKTRcellstoimatinibmesylateAswereportedpreviously[16],TC-PTPwasdownregulatedinSTI571(imatinibmesylate)-resistantKT-1sublines(KTR2,KTR3)(Fig.1).WeutilizedretrovirusestotransducecDNAsofTC45(thenuclearisoformofTC-PTP)orthecatalyticallyinactivesubstrate-trappingTC45-D182AasanegativecontrolintoKTRimatinibmesylate-resistantcells.ExpressionleveloftransducedTC-PTPwasassessedbyWesternblottingandKTR2andKTR3cellssuccessfullyT.Shimizuetal./ExperimentalHematology32(63
1059Figure1.TC-PTPexpressioninKT-1cells,STI571(imatinibmesylate)-resistantKTRcellsandTC-PTPtransducedKTRcells.ProteinexpressionlevelsofTC45(thenuclearisoformofTC-PTP)anditscatalyticallyinactivesubstrate-trappingmutant(TC45-D182A)inKTRcellswereexaminedbyWesternblotting.KT-1:parentalcells,KTR2andKTR3:STI571(imatinibmesylate)-resistantsublinesofKT-1.expressedTC45andTC45-D182Aprotein(Fig.1).UtilizingMTTproliferationassays,wefoundthatproliferationofKTR2-TC45cellswassuppressedby37.0%,74.2%,and89.3%upontreatmentwith0.2,0.5,and1μMimatinibmesylate,respectively(Fig.2A),similartothesuppressionachievedinparentalKT-1cells[16].Incontrast,prolifera-tionofKTR2-mockcellswassuppressedby5.8%,39.9%,and75.6%with0.2,0.5,and1μMimatinibmesylate,respec-tively.ProliferationofKTR2-D182AcellswassimilartothatofKTR2-mockcells.TheseresultsindicatethatexpressionofcatalyticallyactiveTC45overcametheimatinibmesylateresistanceinKTR2cells.Similarresultswereobservedinanotherimatinibmesylate-resistantsubline(KTR3)uponrestorationofTC45(KTR3-TC45)(Fig.2A).NextweexaminedwhethertherewasanycorrelationbetweenthelevelofTC-PTPexpressionandtheresistancetoimatinibmesylate-inducedapoptosisinKTRcells.Afterstimulationwith0.5μMimatinibmesylatefor48hours,annexinV-PEincombinationwith?owcytometrywasuti-lizedtomonitorapoptoticcells.InKTR2-mockcells,thepercentageofannexinVpositiveapoptoticcellswas8%inthecontrolandwasincreasedto25%uponimatinibmesylatetreatment(Fig.2B).InKTR-TC45cells,thepercentageofapoptoticcellswasincreasedfrom12%to56%,suggestingthatTC45expressioninKTRcellsrestoredthesusceptibilitytoapoptosisbyimatinibmesylate.Percentageofimatinibmesylate-inducedapoptoticcellsinKTR2-D182Awassimi-lartothatofKTR-mockcells(Fig.2B).Takentogether,theseresultsindicatethatsensitivitytoimatinibmesylateinKTRcellscanbemodulatedbyTC-PTPexpression.EffectsofTC-PTPonSTAT5phosphorylationinKTRcellsToclarifythemechanismsunderlyingthedifferentialsensi-tivitytoimatinibmesylate,weexaminedthetyrosinephos-phorylationstateofproteinsintheBCR-ABLsignalingpathwayinTC-PTPtransducedKTR2cells.STAT5isre-portedtobeanimportantmoleculeinBCR-ABLsignaling,anditsconstitutivephosphorylationiscriticalfortheprolif-erationofCMLcells[21].Wethusexaminedthephosphory-lationstateofSTAT5inparentalKT-1cellsandKTR2cells.InparentalKT-1cells,phosphorylationofSTAT5wasabolishedwiththetreatmentof0.5μMimatinibmesylatefor1hour.Incontrast,inKTR2cells,STAT5phosphoryla-tionwasstrongerthanthatofKT-1cellsandremainedelevateduponexposureto0.5μMimatinibmesylate(Fig.3A).WenextcomparedtheSTAT5phosphorylationlevelsinKTR2transfectants.InKTR2-mockandKTR2-D182Atransducedcells,STAT5phosphorylationwasaugmentedcomparedtoKTR-TC45transducedcells.Upontreatmentwith0.5μMimatinibmesylatefor1hour,phosphorylationofSTAT5wasabolishedinKTR2-TC45cells,whereasitremainedelevatedinKTR2-mockandKTR2-D182Acells(Fig.3B
).<parisonofimatinibmesylatesensitivityinTC-PTPtrans-ducedKTR2andKTR3cells.(A)Cellswereculturedwith0.2,0.5,or1μMimatinibmesylatefor48hoursandsubjectedtoMTTproliferationassay.Resultsareexpressedaspercentagesrelativetoeachcontrolculture.Valuesaremeans±SDoftriplicateexperiments.TheunpairedStudent’st-testwasusedtoevaluatestatisticalsigni?cance(**p?0.01).(B)Detec-tionofapoptosisinKTR2transfectantsupon0.5μMimatinibmesylatetreatmentfor48hours.CellswerestainedwithannexinV-PEand7-amino-actinomycinDandthenumbersinthe?gureshowthepercentageofannexinVpositivecells(apoptoticcells).Similarresultswereobtainedinthreeindependentexperiments.1060T.Shimizuetal./ExperimentalHematology32(63
Figure3.TyrosinephosphorylationofSTAT5inKT-1,KTR2andTC-PTPtransducedKTR2cells.(A)KT-1,KTR2and(B)TC-PTPtransducedKTR2cellsweretreatedinthepresenceorabsenceof0.5μMimatinibmesylatefor1hourandSTAT5wasimmunoprecipitatedanditsphosphotyr-osinecontentwasassessedusingananti-phosphotyrosineantibody(PY20).Membraneswerere-probedwithantibodiesspeci?ctoSTAT5.ExpressionofTC-PTPdoesnotalterJAK2orBCR-ABLphosphorylationinKTRcellsWenextexaminedthephosphorylationstateofJAK2,aproteintyrosinekinaseinvolvedinBCR-ABLtransfor-mationinCMLcells[22].JAK2phosphorylationwasequallysuppressed1hourafter0.5μMimatinibmesylateexposureinKTR2cellsandtherespectiveTC-PTPtransfec-tants(Fig.4A).Next,weexaminedthephosphorylationstatusoftheBCR-ABLprotein.BCR-ABLwasalmostequallydephosphorylatedinKTR2andTC-PTPtransducedKTR2cellsafterstimulationwith0.5μMimatinibmesylatefor15minutes(Fig.4B).Importantly,expressionlevelsofTC-PTPwerenotalteredbythetreatmentof0.5μMimatinibmesylateforupto1hourinKT-1,KTR2,KTR2-mock,KTR-TC45,andKTR-D182Acells(Fig.4C).ExpressionofTC-PTPdoesnotalterproteinkinasePKB/AKTormitogen-activatedproteinkinasesignalinginKTRcellsWenextexaminedwhetherTC-PTPexpressioninKTRcellsaffectedtheactivationstatusofproteinkinasePKB/AKT,orthemitogen-activatedproteinkinaseJNKandERK1/2thathavepreviouslybeenreportedtobeactivatedinCMLcells[23,24].PKB/AKT,JNK,andERK1/2activationweremonitoredbyWesternblotanalysisutilizingantibodiesspeci?cforthephosphorylatedandactivatedformsoftheseproteins.WedidnotdetectanyappreciableactivationofPKB/AKTinKTR-transducedcells(Fig.5).Inaddition,JNKactivatedwasdetectedweakly,however,notsuppressedupontreatmentwith0.5μMimatinibmesylatefor1hour,indicatingthatJNKactivationmightbeindependentofBCR-ABLinKTRcells.Incontrast,ERK1/2wasequallysup-presseduponimatinibmesylatetreatmentinallKTRtransfectants(Fig.5).Theseresultsindicatethat
TC-PTPFigure4.TyrosinephosphorylationofJAK2andBCR-ABLinKTR2andTC-PTPtransducedKTR2cells.(A)Theindicatedcellsweretreatedwith0.5μMimatinibmesylate.JAK2wasimmunoprecipitatedanditsphospho-tyrosinecontentwasassessedusingananti-phosphotyrosineantibody(PY20).Thesamemembranewasre-probedwithantibodiesspeci?ctoJAK2.(B)TyrosinephosphorylationofBCR-ABLinwholecelllysatesofeachcelllinewasevaluatedwithananti-phosphotyrosineantibody(4G10).Thesamemembranewasre-probedwithananti-c-ABLantibody.K562andMOLT4cellswereusedasapositiveandnegativecontrol,respectively.(C)TheTC-PTPproteinexpressionlevelinwholecelllysatesofeachcelllinewasdetectedbyWesternblotting.Thesamemembranewasre-probedwithananti-actinantibodytoensureequalproteinloading.expressiondoesnotmodulatePKB/AKTormitogen-acti-vatedproteinkinaseactivationinKTRcellsandthattheeffectsofTC-PTPonimatinibmesylatesensitivityarein-dependentofthesesignalingpathways.KTRcellshavereducedTC-PTPmRNAlevelsTodeterminewhetherdownregulationofTC-PTPproteininKTRcellsmightbeattributedtosuppressionofmRNAorotherresultsfromposttranslationalmechanisms,weun-dertookasemi-quantitativeRT-PCRanalysisofTC-PTPmRNAlevelsinKT-1andKTRcells.TC-PTPexpressionwassigni?cantlyreducedinKTR2andKTR3cellswhencomparedtotheparentalKT-1cells(morethanfourfold,p?0.05)(Fig.6),indicatingthatsuppressionofTC-PTPT.Shimizuetal./ExperimentalHematology32(63
1061Figure5.TC-PTPdoesnotalterPKB/AKTormitogen-activatedproteinkinasesignalinginKTRcells.Theindicatedcellsweretreatedwithorwithout0.5μMimatinibmesylatefor1hour.ThephosphorylationstateofproteinkinasePKB/AKTandtheMAPKsJNKandERK1/2wereexaminedbyWesternblottingusingtheindicatedantibodies.AtotalcellextractfromJurkatcellswasusedasapositivecontrolforphospho-PKB/AKT.proteininKTRcellswasattributable,atleastinpart,toreducedmRNAlevels.DiscussionInthisstudywehaveexaminedtheroleofTC-PTPinimatinibmesylateresistanceinKTRcells,whichwasde-scribedinourpreviousinvestigation[16].Initially,weana-lyzedp-glycoproteinexpression,thenumberofbcr-ablfusiongenes,andthesequenceoftheATPbindingsiteofABLkinasedomain.However,thesewerenotresponsibleforimatinibmesylateresistanceinKTRcells.Interestingly,TC-PTPproteinlevelsweremarkedlydownregulatedinKTRcellsascomparedtoparentalKT-1cells[16].TC-PTPisexpressedpredominantlyinhematopoieticcellsandplaysimportantrolesinhematopoiesisandcytokinesignaling[25C28].Accordingly,weexaminedwhetherTC-PTPlossmayaccountfortheimatinibmesylateresistanceinKTRcells.Tooursurprise,sensitivitytoimatinibmesylateinKTRcellsasmonitoredbyproliferationandsurvivalwasrestoredbytheforcedexpressionofTC-PTPwithretroviralgenetransfer.Notably,onlyTC45-transducedcellswereaffectedbytreatmentofimatinibmesylateat0.2μM(Fig.2A),indicatingthatTC45activitywasessentialforincreasedsensitivitytoimatinibmesylate.AmongmembersoftheSTATfamilyoftranscriptionfactors,STAT5isactivatedbytheBCR-ABLkinaseandisimplicatedinpathogenesisofCML[21].WeobservedthatSTAT5phosphorylationinKTR2-mockandKTR2-D182Atransducedcellswasele-vatedwhencomparedtoKTR2-TC45cells.Wedid
notFigure6.Semi-QuantitativeRT-PCRanalysisofTC-PTPexpressioninKT-1andKTRcells.(A)TC-PTPmRNAexpressionwasdown-regulatedinKTR2andKTR3cellscomparedtoKT-1cells.Datawereexpressedasaratiorelativetoβ-actininarbitraryunits.Valuesaregivenasmeans±SDoftriplicateexperiments.TheunpairedStudent’st-testwasusedtoevaluatestatisticalsigni?cance(*p?0.05).(B)Arepresentativeexperi-mentofthreeindependentexperimentsisshown.observeanydifferenceinBCR-ABLorJAK2phosphoryla-tion,indicatingthatsuppressionofSTAT5phosphorylationbyTC45expressionwasnotattributabletoTC-PTPactingupstreamonBCR-ABLorJAK2.OurresultsareinlinewiththoseofSimoncicetal.[27]andLaMontagneetal.[29],whoreportedthatTC-PTPdoesnotdephosphorylateJAK2orBCR-ABL,respectively.Inaddition,wedidnotobserveanydifferenceinactivationofothersignalingpath-waysreportedtobeimportantforproliferationandsurvivalofCMLcells(Fig.5).AlthoughsomecontroversyexistsastowhetherTC-PTPmightactdirectlyonSTAT5inothersystems[26C28],itremainsonewaybywhichTC-PTPmightmodulateSTAT5phosphorylationandsensitivitytoimatinibmesylate.ConsistentwiththiswefoundthatTC-PTPandSTAT5couldbeco-immunoprecipitatedinKT-1cells(Shimizuetal.,unpublishedobservation).AlthoughwedidnotobserveenhancedphosphorylationofSTAT5inKTRcellsexpressingtheTC45substrate-trappingmutant(KTR2-D182A),previousstudieshaveshownthat,whenexpressedatlowlevels,theTC45-D182Amutantdoesnotnecessarilysuppressallpathwaysaffectedbywild-typeTC45[30],suggestingthatthesubstrate-trappingabilityofTC45-D182Amaybelesseffectivethanthedephosphoryla-tionmediatedbywild-typeenzymes.Inthisstudy,wedemonstrateforthe?rsttimethataug-mentationofsignalingpathwaysdownstreamofBCR-ABLbylossofatyrosinephosphatasecouldpromoterelativeresistancetoimatinibmesylateinCMLcells.InadditiontoTC-PTP,itispossiblethatlevelsofotherPTPsmightalsoaffectaugmentationofsignaling.ConsistentwiththiswefoundthatSH-PTP1wasdownregulatedinKTR2cells1062T.Shimizuetal./ExperimentalHematology32(63comparedtoparentalKT-1cells,butwedidnot?ndanydifferenceinlevelsofSH-PTP2andPTP1B([16]anddatanotshown].AlthoughwecannotexcludethecontributionofotherPTPstotheenhancedimatinibmesylatesensitivityofKTRcells,ourresultsnonethelessindicatethatTC-PTPsuppressionplaysaprimaryroleinthisprocessandindeedthatlossofTC-PTPisthemainreasonforhyperactivationofSTAT5inKTRcells.ConcerningthemechanismbywhichTC-PTPproteinlevelsaresuppressedinKTRcells,wecon?rmeddownregu-lationofTC-PTPmRNAinKTRcellsrelativetoparentalKT-1cellsbysemi-quantitativeRT-PCR(Fig.6).KTRcellshadi(18)(q10)abnormalfusionisochromosomes[16],whichlacks18p11.2-11.3;thelocationofTC-PTPgene.Althoughtheprecisemechanismsstillremaintobeelucidated,wespeculatethatformationoftheisochromosomemighthavecausedthedownregulationofTC-PTPmRNAinKTRcells.Inconclusion,wehavedemonstratedthatdownregulationofTC-PTPaugmentedSTAT5phosphorylationaccompaniedbyrelativeresistancetoimatinibmesylateinKT-1CMLderivedcells.Althoughourresultsarelimitedtooneparti-cularCMLcelllineanditwillbenecessarytoascertaintherelevanceofthismechanismforimatinibmesylate-resist-anceinprimarysamples,wewouldliketoproposethatthismayrepresentanewmolecularmechanismforimatinibmesylateresistanceinCML.AcknowledgmentsTheauthorsthankDr.ToshioKitamuraforprovidingtheretrovirusvectorandpackagingcells,Dr.IkuyaSakaiforKT-1cells,andNovartisPharmaceuticalsforprovidingimatinibmesylate.TheauthorsalsothankDr.AtsushiOdaforcriticalcommentsandMs.KaoriSaitofortechnicalassistance.References1.DruckerBJ,TalpazM,RestaDJ,etal.Ef?cacyandsafetyofaspeci?cinhibitoroftheBCR-ABLtyrosinekinaseinchronicmyeloidleukemia.NEnglJMed.1C1037.2.KantarjianH,SawyersC,HochhausA,etal.Hematologicandcytoge-neticresponsestoImatinibmesylateinchronicmyelogenousleukemia.NEnglJMed.C652.3.DrukerBJ,TalpazM,RestaDJ,etal.Activityofaspeci?cinhibitoroftheBCR-ABLtyrosinkinaseintheblastcrisisofchronicmyeloidleukemiaandacutelymphoblasticleukemiawiththePhiladelphiachro-mosome.NEnglJMed.8C1042.4.SawyersCL,HochhausA,FeldmanE,etal.Imatinibinduceshemato-logicandcytogeneticresponsesinpatientswithchronicmyelogenousleukemiainmyeloidblastcrisis:resultsofaphaseIIstudy.Blood.0C3539.5.GorreME,MohammedM,EllwoodK,etal.ClinicalresistancetoSTI571cancertherapycausedbyBCR-ABLgenemutationorampli?-cation.Science.C880.6.leCoutreP,TassiE,Varella-GarciaM,etal.InductionofresistancetotheAbelsoninhibitorSTI571inhumanleukemiccellsthroughgeneampli?cation.Blood.8C1766.7.BubnoffN,SchnellerF,PeschelC,DuysterJ.BCR-ABLgenemuta-tionsinrelationtoclinicalresistanceofPhiladelphia-chromosome-positiveleukemiatoSTI571:aprospectivestudy.Lancet.C491.8.BranfordS,RudzkiZ,WalshS,etal.Highfrequencyofpointmutationsclusteredwithintheadenosinetriphosphate-bindingregionofBCR/ABLinpatientswithchronicmyeloidleukemiaorPh-positiveacutelymphoblasticleukemiawhodevelopimatinib(STI571)resistance.Blood.2C3475.9.Roche-LestienneC,Soenen-CornuV,Grardel-Du?osN,etal.Sev-eraltypesofmutationsoftheAblgenecanbefoundinchronicmyeloidleukemiapatientsresistanttoSTI571,andtheycanpre-existtotheonsetoftreatment.Blood.4C1018.10.MahonFX,DeiningerMWN,SchultheisB,etal.Sele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