【作者】 赵妍敏；

【导师】 黄河；

【作者基本信息】 浙江大学 ， 临床医学， 2009， 博士

【摘要】 急性T淋巴细胞白血病(T cell acute lymphoblastic leukemia，T-ALL)是一种恶性程度高、预后差的淋巴系统恶性克隆性疾病。近年来随着大剂量联合化疗方案的应用，T-ALL的预后有了较大改善，但仍有部分T-ALL患者对标准治疗耐药，出现难治或复发，5年无病生存率仅为28-39％。在急性白血病中，mTOR(mammalian target ofrapamycin，哺乳动物雷帕霉素靶蛋白)信号通路的异常激活不仅参与白血病细胞的增殖、存活、细胞周期进程和葡萄糖转运等多种细胞功能的调节，而且可能与白血病对化疗药物的耐药性相关。现已提出以TOR信号通路的关键分子为靶点的肿瘤治疗策略，雷帕霉素(Rapamycin)作为mTOR的靶向抑制剂对恶性血液系统肿瘤有着广泛的治疗前景。近年来国内外研究发现雷帕霉素对于多种实体肿瘤如前列腺癌、小细胞肺癌、肾细胞癌、乳癌等，均有显著的抗癌作用。而对血液系统肿瘤尤其是T-ALL的作用则鲜有报道。因此本研究以人T-ALL细胞株(Jurkat、Molt-4)和原代细胞为实验对象，旨在探索mTOR抑制剂雷帕霉素对T-ALL细胞的作用及机理，为雷帕霉素在T-ALL中的临床应用提供实验依据。本研究共分为三个部分：第一部分雷帕霉素对急性T淋巴细胞白血病细胞的生长抑制作用本部分研究首先采用MTT比色法分析雷帕霉素对T-ALL细胞株Jurkat、Molt-4和原代细胞增殖的影响。结果显示：浓度为0．1nmol／L-100nmol／L的雷帕霉素对Jurkat、Molt-4细胞和原代T-ALL细胞具有生长抑制作用，且其抑制作用呈现时间浓度依赖效应。雷帕霉素作用Jurkat细胞、Molt-4细胞和原代T-ALL细胞72h的半数抑制浓度(50％inhibition concentration，IC50)分别为16nmol／L、23nmol／L、16nmol／L；而作用于人正常骨髓单个核细胞72h的IC50为461nmol／L，已经远大于雷帕霉素的临床允许血药浓度。为探讨雷帕霉素抑制T-ALL细胞生长的作用机制，我们从细胞凋亡、自噬两个方面做了进一步的研究。以Jurkat细胞为研究对象，采用DNA片段化分析和PARP活性检测，并结合AnnexinV染色流式细胞仪分析技术，对细胞凋亡现象进行定性或定量检测。结果显示：100nmol／L的雷帕霉素作用Jurkat细胞72小时，未出现DNA梯形条带，且无PARP激活带。1nmol／L、10nmol／L、100nmol／L的雷帕霉素作用于Jurkat细胞72小时，AnnexinV阳性的凋亡细胞百分数分别为5．33±1．33％，5．78±2．26％，5．69±2．20，与空白对照组5．37±2．19％相仿(p＞0．05)，表明雷帕霉素不能诱导Jurkat细胞凋亡。除凋亡外，自噬被认为是另一种不同于凋亡的程序性细胞死亡方式。我们采用透射电镜观察，可见经100nmol／L雷帕霉素处理72h的Jurkat细胞胞质内有大量自噬体和自噬溶酶体。结合吖啶橙染色流式细胞仪定量检测，发现Jurkat细胞经100nmol／L雷帕霉素作用72h后，自噬细胞比例为44．56±13．79％，较空白对照组(2．55±1．50％)明显升高(p＜0．05)。自噬抑制剂3-methyladenine(3-MA)可以显著抑制100nmol／L雷帕霉素诱导的自噬，而不增加凋亡细胞的比例。经RT-PCR和Westemblot检测不同浓度雷帕霉素处理72h后Beclin1的mRNA和蛋白表达，发现此经典自噬相关蛋白的表达显著上升，呈剂量依赖性。第二部分雷帕霉素对急性T淋巴细胞白血病细胞的mTOR信号通路、细胞周期及端粒酶活性的影响本部分首先明确了原代T-ALL细胞和细胞株均存在mTOR信号通路的激活。10nmol／L雷帕霉素作用Jurkat细胞12h后即可见mTOR下游蛋白P70S6K和S6的磷酸化受抑制。随着时间的延长，磷酸化蛋白p-S6、p-P70S6K的表达逐渐减少而总S6、总P70S6K基本不变。对于mTOR的上游蛋白Akt，雷帕霉素同样能降低其磷酸化水平。已知mTOR信号通路参与细胞周期的调控，因而我们研究了mTOR抑制剂雷帕霉素对T-ALL细胞周期的影响。采用PI染色流式细胞仪检测发现，100nmol／L雷帕霉素作用Jurkat和Molt-4细胞，16h后可见G1期细胞比例明显上升，而S期比例下降。采用血清饥饿法同步化处理Jurkat细胞，再通过血清释放刺激细胞进入S期，S期和Gl期细胞比例分别为40．3％、39．1％，而经lnmol／L、10nmol／L、100nmol／L的雷帕霉素处理24h后，S期比例分别为38．6％、19．9％、17．5％，G1期比例则分别为41．2％、64．3％、66．4％，说明雷帕霉素能显著阻断血清诱导的细胞周期从G1期到S期的转化。为进一步探讨雷帕霉素诱导的G1期阻滞的机制，我们用Westem blot检测了细胞周期相关蛋白CDK4、CDK6、Cyclin D2、Cyclin D3、P21waf1、P27kip1、c-Myc的表达变化。发现随着雷帕霉素作用时间的延长，Jurkat细胞中P21waf1和P27kip1有上调的趋势，而Cyclin D3表达逐渐减少。雷帕霉素对Cyclin D2、CDK4、CDK6均无明显影响，但能呈浓度依赖性地抑制c-Myc蛋白表达。另一方面，我们研究了雷帕霉素对Jurkat细胞的端粒酶活性的影响。TRAP法检测提示雷帕霉素作用24h后，端粒酶活性开始下降，且其抑制作用呈现浓度和时间依赖性。其次，用半定量RT-PCR检测了不同浓度的雷帕霉素作用48h对hTERT的mRNA表达的影响，显示随着药物浓度的增加，hTERT的转录逐渐减少。同时，Westernblot法检测hTERT的蛋白含量，发现雷帕霉素能下调其蛋白含量，变化趋势与hTERTmRNA表达相一致。第三部分雷帕霉素联合去甲氧柔红霉素的抗白血病作用蒽环类抗生素参与构成T-ALL的联合化疗方案，本部分主要探讨蒽环类代表药物去甲氧柔红霉素(idarubicin，IDA)联合雷帕霉素的抗白血病效应及机理。首先采用MTT法检测发现IDA作用Jurkat细胞和Molt-4细胞48h的IC50分别为21．8nmol／L和2．21nmol／L。当IDA联合10nmol／L雷帕霉素作用Jurkat细胞和Molt-4时，IDA的IC50分别降为2．95nmol／L和1．28nmol／L。联合指数小于1，说明两药的联用具有协同作用。利用电镜形态学观察和Annexin染色流式细胞仪检测发现，雷帕霉素能明显增强IDA的诱导凋亡作用。低浓度IDA单药处理Jurkat细胞和Molt-4细胞48h，凋亡细胞比例分别为17．51±3．20％和22．78±3．54％，两药联用使凋亡细胞比例分别增加为50．17±5．19％和47．33±9．03％(p＜0．05)。同样，两药联用组较IDA单用组更显著地激活了Caspase3和底物PARP，而caspase8和caspase9在两药联用组均呈现出最明显的激活，说明两药联用后是通过细胞膜和线粒体双途径影响细胞凋亡的。为进一步研究凋亡的线粒体途径，我们采用Westem分析了启动线粒体凋亡途径的重要蛋白Bax、Bcl-2、Survivin，发现雷帕霉素能逆转IDA引起的Bcl-2的上调，且两药联用组的Bax／Bcl-2比值较两药单用组明显显著升高。对于Survivin，两药联用较两药单用对Survivin下调无明显增强作用。此外，IDA能使mTOR信号通路上的Akt、P85S6K和P70S6K发生磷酸化激活，联用雷帕霉素后能使P85S6K、P70S6K和Akt磷酸化水平明显降低。单用IDA或单用雷帕霉素对ERK的磷酸化均无明显影响，但两药一旦联用，则能协同下调ERK的磷酸化水平。说明两药联用能通过抑制Akt／mTOR、ERK信号通路而发挥抗T-ALL作用。综上所述，本研究得出以下结论：(1)雷帕霉素可直接抑制T-ALL细胞的生长，具有剂量时间依赖性。(2)单用雷帕霉素并不能引起细胞凋亡，但可诱导自噬，并伴有自噬相关蛋白Beclinl的表达增加。3-MA阻断自噬不能促使向凋亡转化。(3)雷帕霉素对mTOR上游Akt和下游P70S6K、S6的磷酸化均有抑制作用。(4)雷帕霉素可使T-ALL细胞周期受阻于G1期，并下调Cyclin D3和c-Myc，上调P21waf1、P27kip1的表达。(5)雷帕霉素能够抑制细胞端粒酶活性，并下调hTERT的mRNA及蛋白表达水平。(6)雷帕霉素和IDA对T-ALL细胞的生长抑制具有协同作用。两药联用时能通过线粒体和细胞膜双途径增强凋亡效应，且通过调控Bax、Bcl-2、Survivin等影响线粒体途径的调亡。(7)雷帕霉素能逆转IDA引起的Bcl-2的上调和Akt／mTOR信号通路的激活，抑制ERK信号的活化，为临床上两药的联用提供实验证据。更多还原

【Abstract】 T-cell acute lymphoblastic leukemia(T-cell acute lymphoblastic leukemia,T-ALL)is aclonal lymphoid malignancy with poor prognosis.Over the last two decades,theoutcome for patients with T-ALL has improved dramatically with current therapy,however,some relapsed or refractory patients often develop resistance to standardchemotherapy.The 5-year disease-free survival rate is only 28-39%.For acute leukemia,the mammalian target of rapamycin(mTOR)pathway plays animportant role in energy metabolism,the regulation of cell proliferation,and promotingleukemia cell survival.The over-activation of the mTOR pathway is also implicated inhuman leukemia resistance to conventional chemotherapy.Thus,the development ofnovel therapeutic agents override the resistance,and directly targeting this signalingnetwork is crucial.One potential class of novel therapeutics is rapamycin.Preclinicalstudies have shown that rapamycin may be effective for the treatment of prostate,smallcell lung,glioblastoma,renal cell,and breast cancer.However,the use of rapamycin islimitedly documented in hematological malignancies,especially for T-ALL.Thus in thepresent study,we used two T-ALL cell lines(Jurkat and Molt-4)and T-ALL primarycells to explore the antileukemic mechanism of rapamycin in T-ALL and evaluate the potential clinical implications in the treatment of T-ALL.There are three sections in ourresearch.Section 1:Effect of Rapamycin on Growth in T-ALL CellsFirstly,the effects of rapamycin on the cell proliferation in T-ALL cell lines(Jurkat andMolt-4)and primary cells were assessed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay.Exposureto rapamycin(0.1-100 nmol/L)resulted in a significant inhibitory effect on theproliferative activity ofT-ALL cells:an effect that was dose and time dependent.The50% inhibitory concentration(IC50)values for Jurkat,Molt-4 and primary cells at 72 hwere 16,23 and 16 nmol/L,respectively.However,for normal human bone marrowmononucleated cells(MNCs),the IC50 concentration was over 461 nmol/L,which is farbeyond the clinical usage.To determine the mechanism of growth inhibition byrapamycin in more detail,we used Jurkat cells to analyze the cell apoptosis andautophagy profile after treatment with rapamycin.The classic method,such as genomic DNA fragmentation assay and PARP cleavageimmunoblotting assay,were used to detect apoptosis qualitatively.It showed nogenomic DNA fragmentation and PARP cleavage in Jurkat cells treated with 10nmol/Lrapamycin for 72h.The cell apoptosis index was investigated quantitatively by AnnexinV/propidium iodide(AV/PI)staining followed by flow cytometry analysis.Exposure tolnmol/L,10nmol/L and 100nmol/L rapamycin for 72h caused 5.33±1.33%,5.78±2.26%,5.69±2.20 apoptotic cells,which was comparable to that of the untreated group(5.37±2.19%)(p＞0.05),indicating rapamycin could not induce apoptosis in Jurkatcells.Autophagy,another form of programmed cell death is thought to be as prevalent asapoptosis.Using electron microscope,some large autolysosome and vacuoles wereobserved in Jurkat cells treated with 100nmol/L rapamycin for 72h.Furthermore,using acridine orange staining followed by flow cytometry analysis,cell autophagy index wasinvestigated quantitatively.Exposure to 100 nmol/L rapamycin for 72h,the autophagyindex was apparently increased to 44.56±13.79%,while in the untreated control group,the autophagy index was 2.55±1.50%(p＜0.05).3-methyladenine(3-MA),whichinhibits autophagosome sequestration,could decrease the autophagy index to 6.02±3.91%,but it could not promote the apoptotic cell death.Finally,Jurkat cells treatedwith 1-100 nmol/L rapamycin showed a dose-dependently up-regulation of Beclin-1mRNA and protein expression,the classic marker for autophagy.Section 2:Effect of Rapamycin on mTOR pathway Cell Cycle and TelomeraseActivity in T-ALL CellsFirstly,we confirmed that the PI3K/Akt/mTOR signaling pathway is over-activated inJurkat and primary T-ALL cells.Rapamycin at 10nmol/L markedly reduced thephosphorylation ofp70S6K and S6 in Jurkat cells.This effect was seen as early as 12 hafter exposure to rapamycin,and the total proteins of the pan-S6 and p70S6Kmaintained stable levels.Interestingly,rapamycin could also slightly downregulate thephosphorylation of Akt,which functioned upstream ofmTOR.Since mTOR signaling relates to cell cycle progression,we therefore determine theregulation of cell cycle by rapamycin.The cell cycle profile was analyzed by flowcytometry with PI staining.As expected,G1 phase arrest was induced by rapamycin(100nmol/L)after 16 h in Jurkat and Molt-4 cells,and the ratio of G1 continued to growas treatment was prolonged to 32 h.Furthermore,we evaluated the effect of rapamycinat different concentrations on the cell cycles arrest.Jurkat cells were synchronized cellsby serum starvation.Serum stimulation resulted in the transition of cells from the G1phase to the S phase(G1:40.3%,S:39.1%).Rapamycin significantly blockedserum-induced entry to the S phase in a dose-dependent manner in Jurkat cells.The G1phase index was 41.2%,64.3% and 66.4% for lnmol/L,10nmol/L and 100nmol/L, respectively.To further determine the mechanism of G1 phase arrest by rapamycin,we investigatedthe effects of rapamycin on cell cycle related proteins,specifically CDK4,CDK6,cyclin D2,cyclin D3,p27Kipl,p21 wafl and c-Myc.Rapamycin treatment maintainedelevated levels of p27Kipl and p21 wafl in Jurkat cells,and downregulated cyclin D3without affecting the cyclin D2 protein levels.It decreased the c-Myc expression but didnot affect the protein levels of either CDK4 or CDK6.On the other hand,we investigate the regulation of telomerase by rapamycin in Jurkatcells.First,telomerase activity was measured by the TRAP assay.When the cells weretreated with rapamycin,telomerase activity was downregulated within 24 h and had areduced tendency that was dose and time dependent.Because telomerase activitycorrelates with the expression of hTERT mRNA,RT-PCR was used to examine theexpression of hTERT mRNA in Jurkat cells treated with rapamycin for 48 h.We foundthat treatment with varying concentrations of rapamycin significantly reduced thehTERT mRNA level in a dose-dependent manner.Furthermore,by Western blotting,thehTERT protein level decreased and correlated with these changes in the expression ofhTERT mRNA induced by rapamycin.Section 3:Synergistic Cytotoxic Effect of Rapamycin and Idarubicin by inhibitionof mTOR pathway in T-ALL CellsFor patients with T-ALL,the induction chemotherapy regimens usually includeanthracyclines.Thus in this section,we tried to determine the antileukemic effect ofidarubicin combined with the the anthracycline idarubicin(IDA).The IC50 of IDA forJurkat and Molt-4 was 21.8nmol/L and 2.21 nmol/L,respectively.When combined with10nmol/L rapamycin,the IC50 of IDA for those two cell lines was decreased to2.95nmol/L and 1.28nmol/L.The combine index was＜1,indicating the synergisticcytotoxic effect of IDA and rapamycin. Both electron microscope and Annexin staining flow cytometry analysis revealed thatrapamycin significantly increased apoptotic sensitivity to IDA.Treatment of Jurkat andMolt-4 cells with IDA at a low concentration for 48h resulted in 17.51±3.20% and22.78±3.54% apoptosis,respectively.However,when combined with 10nmol/Lrapamycin,the apoptosis index increased to 50.17±5.19% and 47.33±9.03%,respectively(p＜0.05).The combination of IDA with rapamycin was prone to activateCaspase3,PARP,Caspase8 and Caspase9,indicating both extrinsic and intrinsicapoptotic pathways were involved.To further investigate the mechanism of the intrinsic apoptosis,we analyzed Bcl-2family members,which regulate cytochrome c release from mitochondria.The resultsshowed rapamycin could reverse the up-regulation of Bcl-2 protein triggered by IDA.The Bax/Bcl-2 ratio is significantly higher in two-agent combination group than that inIDA or rapamycin single agent group.However,the two-agent combination did notaugment the inhibition of Survivin expression.Besides,the mTOR signaling upstream Akt and downstream S6K were activated byphosphorylation in response to IDA treatment.Rapamycin significantly inhibit mTORsignaling activation triggered by IDA.Furthermore,rapamycin alone or IDA alone didnot affect the ERK phosphorylation,but the combination of the two agents led tosynergistic inhibition of ERK phosphorylation.Thus,combination of IDA withrapamycin exerts antileukemic effect via inhibition ofmTOR and ERK signaling inT-ALL.In summary:(1)Rapamycin directly inhibits the proliferative activity of T-ALL celllines(Jurkat and Molt-4)and primary T-ALL cells in dose and time dependent manner(2)As a single agent,rapamycin could induce cell autophagy but not apoptosis in Jurkatcells,accompanied with up-regulation of Beclin-1 expression,the marker for autophagy.Inhibition of autophagy by 3-MA could not enhance the apoptotic cell death.(3) Rapamycin reduces the phosphorylation of p70S6K and S6 which belong to mTORdownstream proteins,as well as the phosphorylation of Akt,which functions upstreamof mTOR.(4)Rapamycin induces Gl phase arrest,up-regulates the protein level ofP21 wafl as well as P27kipl,and downregulates cyclinD3 and c-Myc.(5)Exposure torapamycin reduced telomerase activity,and reduced hTERT mRNA and proteinexpression.(6)Combination of IDA with rapamycin exerted a synergisticanti-poliferative effect and promoted apoptosis by both extrinsic and intrinsic apoptoticpathways.The intrinsic apoptotic pathway was triggered via regulation of Bax,Bcl-2and Survivin.(7)Rapamycin could reverse up-regulation of Bcl-2 protein andover-activation ofmTOR signaling induced by IDA.The findings provide a strongrationale for developing therapy with rapamycin for T-ALL.更多还原