Cyclin-E2和Survivin在急性白血病的表達及其相關(guān)性

CyclinE2和Survivin在急性白血病的表達及其相關(guān)性

作者：王穎，徐世榮，林鳳茹，郭曉楠，任金海

【摘要】為了解細胞周期蛋白E2和存活蛋白在急性白血病中基因表達及兩者間的相關(guān)性，采用逆轉(zhuǎn)錄－聚合酶鏈反應(yīng)的方法檢測了84例成人急性白血病患者和20例正常人的cyclinE2和survivinmRNA的表達。結(jié)果表明：①cyclinE2在急性白血病中表達的陽性率高于對照組；survivin在急性白血病中表達的陽性率高于對照組；②在急性白血病中cyclinE2的表達與survivin的表達呈正相關(guān)性，r＝；③cyclinE2陽性組緩解率低于cyclinE2陰性組，cyclinE2陽性組復(fù)發(fā)率高于陰性組；復(fù)發(fā)組cyclinE2表達率在三組中最高，持續(xù)緩解組cyclinE2表達率在三組中最低，④cyclinE2在急性髓系白血病患者表達率低于急性淋巴細胞白血病患者的表達率；與發(fā)病時白細胞計數(shù)未見有統(tǒng)計學意義的相關(guān)。結(jié)論：首次證實cyclinE2在急性白血病中有異常表達，且對臨床預(yù)后有不良影響；cyclinE2在急性白血病中的表達與survivin有正相關(guān)，提示cyclinE2在急性白血病中有可能作為一種微小殘留病變的檢測指標。

【關(guān)鍵詞】存活蛋白

Asamalignanthemophaty，acuteleukemia(AL)，especially，acutelymphocyticleukemiaunderwentahighrelapserateandashortdisease-freesurvival(DFS)，inspiteoftheimprovementinclinicaloutcomebenefitedfromthedevelopmentofhematopoieticstemcelltransplantation(HSCT)andchemicalrelapseandprolongingDFSstillisthefocusofALresidualdisease(MRD)becameaneffectiveindicationforthefoundationofpost-remissiontherapyregulationsincetheroutinemorphologicalexaminationoftheperipheralbloodandbonemarrowsamplefromtheremissionpatientwasthefusiongenesuchasAML1/MTG8andPML/RARαasMRDmarker，wasonlyusedinM2，M3，soitisvaluabletosearchanothernewMRDmarkerfortheotherhand，RNAinterference(RNAi)asanewandprospectivegenetherapyforcancerhasbecomeanewfocusofthestudyofALtherapy［1］.Searchingforaneffectivenewtargetforatumor-specificRNAiisworthdevelopingforleukemia，asatypeofG1cyclin，cyclinE2wasfoundouttobethepotentialmarkeroftumorssinceitpresentedapositiveexpressioninthecelllinesofsolidtumorsandanegativeexpressioninproliferatingnormalcells［2］.However，therehasbeennoreportonitsexpressionandroleinordertofindouttheroleofcyclinE2intheprogressionandclinicaloutcomeofAlpatients，reversetranscriptionpolymerasechainreaction(RT-PCR)isadoptedtotesttheexpressionofcyclinE2andsurvivinmRNAin84adultpatientswithAL，20normalpersonsascontrolsandleukemiacellline，weinvestigatedtherelationshipbetweentheexpressionofcyclinE2andtheclinicalprogressionofALstudyofcyclinE2canprovideapreliminarytheoreticalbasisforsearchinganewtargetofALgenetherapyandanewMRDmarker.

MaterialsandMethods

Patientsenrolled

84adultinpatientswithALfromApril2002toMarch2003intheSecondAffiliatedHospitalofHebeiMedicalUniversitywereenrolledinthisdiagnosesofthesepatients[50malesand34females，14-62yearsold(meanofyears)］wereconfirmedbymorphological，cytochemicalandimmunologicmarkertotheFrench-American-British(FAB)classification，59patientswerediagnosedasacutemyelocyticleukemia(AML)，including2casesofM1，12casesofM2，21casesofM3，11casesofM4，11casesofM5，1casesofM6，1casesofpatientswerediagnosedasacutelymphocyticleukemia(ALL)including4casesofL1，19casesofL2，2casesofL3;60patientswithdenovoAL，16relapsecases，8casesofcontinuouslycompleteremission(CCR)(timeofdisease-freesurvival≥3years).20normalpersonsaged15-60years(meanof35yearsold)containing11malesand9femalesservedas:allthesepatientswithAML，exceptM3，receivedstandardADEregimen(cytarabinearabinoside150mg/m2for7days，daunorubicin40mg/m2for3daysandetoposide75mg/m2for3days-7days).PatientswithM3receivedall-trans-retinoicacid(20-40mg/day)andarsenictrioxide(10mg/day)asremissioninductionwithALLreceivedVITPregimen(vincristinemg/m2for1dayofeachweek，iphosphamideg/m2for3daysofeachweek，pirarabicin20mg/m2for3daysofeach2weeks，prednisone1mg/kgfor14daysandthengraduallydecrementuntiltheendofregimenthatlastsforabout28days).

Cellsandcellculture

Bonemarrowsamplesfrom84patientswithALand20controlswereusedformononuclearcells(MNC)mlofthisfreshheparinizedbonemarrowsampleswereseparatedbyFicoll-Hypaque(PharmaciaBiotech，Uppsala，Sweden)andwashedtwicewithphosphate-bufferedsaline(PBS)，followedbyextractionofwholecelllysates，andtheMNCconcentrationwas106cells/cellsamountedtoover80%inbonemarrowsamplesfrom84ALpatientsassesedbyWright′sstaining，andthentheMNCswerefrozenaftercentrifugationandstoredat-80℃untilfurthercelllinewasobtainedfromtheInstituteofHematology，ChineseAcademyofMedicalweremaintainedat37℃inahumidifiedatmospherecontaining5%growingsuspensionculturesofleukemiacellswerepropagatedbyreseedingat5×105cells/mlevery3-5days，inRPMI1640mediumsupplementedwith10%fetalbovineserum，50U/mlpenicillinG，and50μg/mlstreptomycincellswereharvestedatlogarithmicgrowthphase，separatedbyFicoll-HypaqueandwashedtwicewithPBS，thenfrozenaftercentrifugationandstoredat-80℃untiluse.

RNAextraction

TotalRNAwasextractedfromMNCandK562cellbyTRIZOLreagent(Gibco)，accordingtotherecommendationsofthemanufacturer.TheconcentrationandpurityoftotalRNAwasdeterminedbyUVspectrophotometry，TheratioofA260/A280reachedTheelectrophoresispatternon%agarosegelstainedbyethidiumbromidewasusedfordeterminingtheintegrityoftotalRNAshowingtwobandsof18Sand28SintheRNAwasfrozenat-80℃untilfurtheruse.

Semi-quantitativeRT-PCR

Reversetranscriptionreaction20μlreversetranscriptionreactionsystemincludingtotalRNA1μg，M-MLV200U，dNTPs2mmol/L4μl，RNasin(Promega)20U，randomprimerμg，5×RTbuffer4μlwaswarmedforreactionat37℃，60min，thenstopat95℃for5productswasfrozenat-20℃untilfurtheruse.

Polymerasechainreaction25μlPCRsystemincludingreversetranscriptionproducts2μl，oligonucleotideprimer25pmol，TaqDNApolymerase(Huamei）1U，dNTPsmmol/L，10×PCRbufferμl(MgCl215mmol/L，pH，Tris-HCl100mmol/L，KCl500mmol/L，BAS20μg/ml），reactionconditionasfollows:94℃40sec，55℃40sec，72℃60sec，totally29cyclesforcyclinE2andβ-actin;94℃30sec，55℃30sec，72℃60sec，totally35cycles，72℃10minforsurvivintostoptheeachsetofPCRreactions，parallelreactionusingdouble-distilledwaterinsteadofthecDNAtemplatesolutionasanegativecontroltoassurethequalityoftheprimersweresynthesized:senseprimer:5′-TTGGCAGGTGCCTGTTGAAT-3′，antisenseprimer:5′-AGCCAGTCCCCCACAGCAT-3′.Theamplifiedproductshouldbe465bp［3］.cyclinE2primerwasdesignedwitholigoaccordingtogenegi3885975:senseprimer5′-TGGCTTTTAGAGGTATGTGAA-3′，antisenseprimer5′-TAATGAATCAATGGCTAGAAT-3′product426bp，β-actinsenseprimer5′-TCATCACCATTGGCAATGAG-3′，antisenseprimer5-CACTGTGTTGGCGTACAGGT-3′，product155bp。

PCRanalysisofproducts

Theelectrophoresisof10μlPCRproductswasperformedat85Vfor90minin2%agarosegelcontainedmg/LethidiumthebandswereviewedandphotographedunderUVillumination.ThetranscriptswereestimatedtheopticaldensityratioofcyclinE2orsurvivintoβ-ratioofpositivestandardwaslargerthan

Statisticalanalysis

Thedatawasregisteredasmean±betweendifferentgroupswereevaluatedbythechi-squaretest，correctionforcontinuitychi-squaretest，andexactprobabilitiesin2×2test:analysisofPearsoncorrelation，PvaluesoflessthanwasconsiderdstatisticallyanalysiswasperformedusingSPSScomputersoftware.

Results

ExpressionofcyclinE2andsurvivinmRNAinK562cells

TheexpressionofbothcyclinE2andsurvivinintheK562celllinewaspositive，andcouldbeusedasapositivecontrol，showedinFigure1，2.

ExpressionofcyclinE2inALandthecontrol

ExpressionofcyclinE2mRNAwasdeterminedin84casesofALwere20negative(Figure1).TherateofexpressionofcyclinE2inALwas%andmuchhigherthan0%incontrols(χ2＝，）.

ExpressionofsurvivininALandthecontrol

ExpressionofsurvivinmRNAwasdeterminedin84casesofAL，outofwhich61caseswerepositiveand23caseswerenegative，that6in20controlswerepositiveand14werenegative(Figure2).TherateofexpressionofsurvivininALwas%andmuchhigherthanthatincontrol30%，.

ThecorrelationbetweenexpressionsofcyclinE2andsurvivininAL

ExpressionofcyclinE2wassignificantlypositivelycorrelatedwithexpressionofsurvivinin84casesofAL(Figure3)(r=，).

%(24/43)ofremissionof43cyclinE2-positivepatientswithdenovoALwerelowerthan%(15/17)of17cyclinE2-negativepatientswithdenovoAL;thedifferencewassignificant(χ2＝，）.Allof14patientswithdenovoM3obtainedcompelteremissionafterreceivinginductionrateofremissionof8cyclinE2-positivepatientswithdenovoM3was100%(8/8)，andthatof6cyclinE2-negativepatientswithdenovoM3wasalso100%(6/6)，andtherewerenodifferencebetweenthem.Theremissionrateof35cyclinE2-positivepatientswithdenovoALexceptM3was%(16/35)lowerthan%(9/11)of11cyclinE2-negativepatientswithdenovoALexceptM3;andthedifferencewassignificant(χ2＝，）.39ofthe60patientswhoobtainedremissionhadbeenclinicallyobservedfor24months，anditprovedthat24remissionpatientswithdenovoALincyclinE2-positivegrouphasarelapserateof%(10/24)，whichishigherthan20%(3/15)ofthe15remissionpatientswithdenovoALincyclinE2-negativegroup，butthedifferencewasnotstatisticallysignificant(exactprobabilitiesin2×2table，).16remissionpatientswithdenovoALexceptM3incyclinE2-positivegrouphasarelapserateof%(10/16)，whichishigherthan%(3/9)ofthe9remissionpatientswithdenovoALexceptM3incyclinE2-negativegroup，ExpressionofcyclinE2inrelapsegroup，denovoALgroupandCCRgroupwasshowedinrateofcyclinE2expressioninrelapsegroupwasthehighestinthethreegroups，(100%vs%，χ2＝，;100%vs0%，exactprobabilitiesin2×2table，);therateofcyclinE2expressioninCCRgroupwasthelowestinthethreegroups，(0%vs%，χ2＝，).ofcyclinE2mRNAinrelapsegroup，denovoALgroupandCCRgroup

Discussion

CyclinE2，asanovelG1cyclin，consistsof404aminoacidresidues，whichshares47%similaritywithcyclinE2shares70％identityinthedomainofcyclinboxwithcyclin，cyclinE2playsaphysiologicalrolesimilartocyclinE1，andcyclinE2bindstoCdk2toformthecyclinE2-Cdk2kinasecomplexthatpromotesthetransitionofcellfromG1toSactivityofcyclinE2-Cdk2kinasecomplexisinhibitedbyp27Kip1andthedifferencebetweencyclinE2andcyclinE1isthepointthatalowlevelofcyclinE1mRNA，butnodetectablecyclinE2mRNAwaspresentinnormalproliferatingcells;theirmRNAvariablelevelswerepresentinhumancancercellssuchaslungtumorcelllines，breastcancercells，maybecyclinE2isamarkerforcelltransformation［2］.However，theexpressionofcyclinE2inleukemiacellsisunknown，andnoreportaboutitwasthisstudy，theexpressionofcyclinE2andsurvivininleukemiacelllineK562wasexaminedbyoftheirmRNAwerepresentinK562cells，andthenwetookK562cellsasthepositive，expressionofcyclinE2mRNAinbonemarrowMNCfrom84adultpatientswithALand20controlswasexaminedbysemi-quantitative%ofcyclinE2-positiveexpressionrateinALwassignificantlyhigherthanincontrols0%.TheresultindicatedthattheexpressionofcyclinE2geneinAlwasamplified，andtheabnormalexpressionofcyclinE2probablycorrelatedwiththemalignantbehaviorofleukemiaresultwassimilartothatinlung，breastanduteruscancerreportedbyGudasetal［2］.Survivinasainhibitorofapoptosisplaysacentralroleincancerprogressionandresistancetoapoptosisindiversetumortypesexceptforleukemia［3，4］.%expressionrateofsurvivininALwashigherthan30%inresultwassimilartothatreportedinliterature［3］.

SubsequentstudiesshowedthatexpressionofcyclinE2positivelycorrelatedwiththatofsurvivinin84Al(r=)，andtheirsynergeticexpressionwasfoundin68casesoutof84ALpatientsenrolledintheshowedthatretinoblastomaprotein(pRb)caninteractwiththesurvivinpromoterandrepresssurvivintranscription，andE2Factivatorscanbindtothesurvivinpromoterandinducesurvivintranscription［4］.CyclinE2bindstoCdk2toformcyclinE2-Cdk2complexthatphosphorylatespRbandinactivatespRb，thenpRbreleasesE2Factivator［5］.ItisimportantthattheactivityofcyclinE2-Cdk2complexpositivelycorrelateswiththecontentofcyclinE2［2］.So，thepositivecorrelationbetweenexpressionofcyclinE2andsurvivinsuggeststhattheoverexpressionofcyclinE2boundtoCdk2formsmorecyclinE2-Cdk2complexwhichphosphorylatedandinactivatedpRb，andthenpRbreleasedmoreE2Factivators;asaresult，survivintranscriptionwasincreasinglytheotherhand，survivinalsointeractswiththecellcycleregulatorCdk4leadingtocyclinE2-Cdk2activation，andplaystheroleofapoptosisinhibitors［6］.Overall，intensivestudiesoftheinteractionbetweencyclinE2andsurvivinarevaluabletoillustratethemechanismofcyclinE2action.

FurtherclinicalinvestigationshowedthatlowremissionrateandhighrelapseratewerepresentedincyclinE2-positiveALpatientswithdenovoAL;sodidinpatientswithALexceptofcyclinE2waspresentedinrelapsegroup，whilenoexpressionofcyclinE2waspresentedinCCRgroupandnumberofstudieshavedemonstratedastrongcorrelationbetweentheelevatedlevelsofthemRNAandproteinofcyclinE1andacuteleukemiaprogressionandmortality，whileadetectablelowlevelofcyclinE1mRNAandproteinwasexpressedinproliferatingnormalcellssuchasbronchialepithelialcells，bonemarrowMNC［2，7］.CyclinE2mRNAwasdetectablyexpressedindiverselungtumorcelllinesandbreastcancerwasdemonstratedthatnodetectablelevelsofcyclinE2mRNAwereintheproliferatingnormalcellslikebronchialepithelialcells.Uptonow，therehavebeennostudiesfocusingontheexpressionofcyclinE2anditsinfluenceonclinicalprogressionofleukemiainvestigationresultsuggestedthatcyclinE2wasmoreoftenexpressedinrelapseanddenovoAL，whilenoexpressionofcyclinE2inCCRgroupandcontrols;theaberrantexpressionofcyclinE2implicatedthatALwasinprogressionandcyclinE2mightbeamarkerforMRD，andthecorrelationbetweencyclinE2expressionandprogressionofALshouldbeintensivelycarriedsignificantdifferenceofrelapseratebetweencyclinE2-positivepatientsandcyclinE2-negativepatientswasfoundasthecasesenrolledinanalysesandthetimeofinvestigationwasthenumberofpatientsenrolledinanalysisandprolongingtheinvestigationtimecontributetointensivelyevaluatetheinfluenceofcyclinE2onrelapserate.

ThestudyofcyclinE2andclinicalfeatureinALpatientsshowedthat%ofcyclinE2expressionrateinAMLwaslowerthan96%ofthatinwasmoreapttorelapse，worseclinicaloutcomeandlessdisease-freesurvivalthanAML;theaberrantoverexpressioncyclinE2inALLsuggestedthatcyclinE2correlatedwiththepoorprognosisofE2asapositiveregulatorincellcyclewasaberrantlyexpressedinALofcyclinE2shouldhavebeencorrelatedwiththeWBCcountsofpatientswithAL，iftheaberrantexpressionofcyclinE2acceleratedtheproliferatingofcells，anddecreasedtheapoptosisofinterestingly，theWBCcountsofpatientswithALatdiagnosiswerenotcorrelatedwiththeexpressionofcyclinE2，interpretationonthisresultwasthatthefluctuationintheWBCcountswasinfluencedbytheoccasionofdiagnosis，andsubsequentlywouldinfluencetheresultaccuracy;ontheotherhand，theresultperhapssuggestedafunctionofcyclinE2outsideofcellcyclereportedbyZariwala［8］hasshownthatcyclinE2isexpressedinbrain，anorgancontainspredominantlypostmitoticcells，whilethecyclinE1transcriptisund