mTOR靶向抑制剂雷帕霉素对人急性T淋巴细胞白血病细胞的作用及机理研究
|
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。
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 Cells
Firstly,the effects of rapamycin on the cell proliferation in T-ALL cell lines(Jurkat andMolt-4)and primary cells were assessed by
3-(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 Telomerase
Activity in T-ALL Cells
Firstly,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 Cells
For 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.
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。
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 Cells
Firstly,the effects of rapamycin on the cell proliferation in T-ALL cell lines(Jurkat andMolt-4)and primary cells were assessed by
3-(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 Telomerase
Activity in T-ALL Cells
Firstly,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 Cells
For 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.
引文
1.Gokbuget N,Hoelzer D.Recent approaches in acute lymphoblastic leukemia in adults.Rev Clin Exp Hematol 2002;6 (2):114.
2.Schrappe M,Camitta B,Pui CH,et al.Long-term results of large prospective trials in childhood acute lymphoblastic leukemia.Leukemia 2000;14 (12):2193.
3.Dennis PB,Jaeschke A,Saitoh M,Fowler B,Kozma SC,Thomas G Mammalian TOR:a homeostatic ATP sensor.Science 2001;294 (5544):1102.
4.Schmelzle T,Hall MN.TOR,a central controller of cell growth.Cell 2000;103 (2):253.
5.West KA,Castillo SS,Dennis PA.Activation of the PI3K/Akt pathway and chemotherapeutic resistance.Drug Resist Updat 2002;5 (6):234.
6.Knuefermann C,Lu Y,Liu B,et al.HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells.Oncogene 2003;22 (21):3205.
7.Brognard J,Clark AS,Ni Y,Dennis PA.Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation.Cancer Res 2001;61 (10):3986.
8.Cullion K,Draheim KM,Hermance N,et al.Targeting the Notch 1 and mTOR pathways in a mouse T-ALL model.Blood 2009.
9.Xu Q,Simpson SE,Scialla TJ,Bagg A,Carroll M.Survival of acute myeloid leukemia cells requires PI3 kinase activation.Blood 2003;102 (3):972.
10.Ly C,Arechiga AF,Melo JV,Walsh CM,Ong ST.Bcr-Abl kinase modulates the translation regulators ribosomal protein S6 and 4E-BP1 in chronic myelogenous leukemia cells via the mammalian target of rapamycin.Cancer Res 2003;63(18):5716.
11.Rowinsky EK.Targeting the molecular target of rapamycin (mTOR).Curr Opin Oncol 2004;16 (6):564.
12.Burchert A,Wang Y,Cai D,et al.Compensatory PI3-kinase/Akt/mTor activation regulates imatinib resistance development.Leukemia 2005;19(10):1774.
13.Sillaber C,Mayerhofer M,Bohm A,et al.Evaluation of antileukaemic effects of rapamycin in patients with imatinib-resistant chronic myeloid leukaemia.Eur J Clin Invest 2008;38(1):43.
14.Mohi MG,Boulton C,Gu TL,et al.Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs.Proc Natl Acad Sci U S A2004;101 (9):3130.
15.Recher C,Beyne-Rauzy O,Demur C,et al.Antileukemic activity of rapamycin in acute myeloid leukemia.Blood 2005;105 (6):2527.
16.Yilmaz OH,Valdez R,Theisen BK,et al.Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells.Nature 2006;441 (7092):475.
17.Palomero T,Dominguez M,Ferrando AA.The role of the PTEN/AKT Pathway in NOTCH 1-induced leukemia.Cell Cycle 2008;7 (8):965.
18.Gutierrez A,Look AT.NOTCH and PI3K-AKT pathways intertwined.Cancer Cell 2007;12 (5):411.
19.Laurent G,Jaffrezou JP.Signaling pathways activated by daunorubicin.Blood 2001;98 (4):913.
20.Takeuchi H,Kondo Y,Fujiwara K,et al.Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors.Cancer Res 2005;65 (8):3336.
21.Laport GF,Larson RA.Treatment of adult acute lymphoblastic leukemia.Semin Oncol 1997;24(1):70.
22.Mungamuri SK,Yang X,Thor AD,Somasundaram K.Survival signaling by Notchl:mammalian target of rapamycin (mTOR)-dependent inhibition of p53.Cancer Res 2006;66 (9):4715.
23.Minowada J,Onuma T,Moore GE.Rosette-forming human lymphoid cell lines.I.Establishment and evidence for origin of thymus-derived lymphocytes.J Natl Cancer Inst 1972;49 (3):891.
24.Hosoi H,Dilling MB,Shikata T,et al.Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells.Cancer Res 1999;59 (4):886.
25.Pene F,Claessens YE,Muller O,et al.Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.Oncogene 2002;21 (43):6587.
26.Avellino R,Romano S,Parasole R,et al.Rapamycin stimulates apoptosis of childhood acute lymphoblastic leukemia cells.Blood 2005;106 (4):1400.
27.Hirase C,Maeda Y,Takai S,Kanamaru A.Hypersensitivity of Ph-positive lymphoid cell lines to rapamycin:Possible clinical application of mTOR inhibitor.Leuk Res 2009;33 (3):450.
28.Crazzolara R,Cisterne A,Thien M,et al.Potentiating effects of RAD001 (Everolimus) on vincristine therapy in childhood acute lymphoblastic leukemia.Blood2009;113(14):3297.
29.Yoshimori T.Autophagy:a regulated bulk degradation process inside cells.Biochem Biophys Res Commun 2004;313 (2):453.
30.Klionsky DJ,Emr SD.Autophagy as a regulated pathway of cellular degradation.Science 2000;290 (5497):1717.
31.Ogier-Denis E,Codogno P.Autophagy:a barrier or an adaptive response to cancer.Biochim Biophys Acta 2003;1603 (2):113.
32.Schwarze PE,Seglen PO.Reduced autophagic activity,improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats.Exp Cell Res 1985;157 (1):15.
33.Ertmer A,Huber V,Gilch S,et al.The anticancer drug imatinib induces cellular autophagy.Leukemia 2007;21 (5):936.
34.Bursch W,Ellinger A,Kienzl H,et al.Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture:the role of autophagy.Carcinogenesis 1996;17 (8):1595.
35.Kanzawa T,Germano IM,Komata T,Ito H,Kondo Y,Kondo S.Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells.Cell Death Differ 2004;11 (4):448.
36.Kanzawa T,Kondo Y,Ito H,Kondo S,Germano I.Induction of autophagic cell death in malignant glioma cells by arsenic trioxide.Cancer Res 2003;63 (9):2103.
37.Qu X,Yu J,Bhagat G,et al.Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.J Clin Invest 2003;112(12):1809.
38.Yue Z,Jin S,Yang C,Levine AJ,Heintz N.Beclin 1,an autophagy gene essential for early embryonic development,is a haploinsufficient tumor suppressor.Proc Natl Acad Sci U S A 2003;100 (25):15077.
39.Xue L,Fletcher GC,Tolkovsky AM.Autophagy is activated by apoptotic signalling in sympathetic neurons:an alternative mechanism of death execution.Mol Cell Neurosci 1999;14 (3):180.
40.Blommaart EF,Luiken JJ,Blommaart PJ,van Woerkom GM,Meijer AJ.Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes.J Biol Chem 1995;270 (5):2320.
41.Klionsky DJ,Meijer AJ,Codogno P.Autophagy and p70S6 kinase.Autophagy 2005;1 (1):59.
42.Bae-Jump VL,Zhou C,Gehrig PA,Whang YE,Boggess JF.Rapamycin inhibits hTERT telomerase mRNA expression,independent of cell cycle arrest.Gynecol Oncol 2006;100 (3):487.
43.Kawauchi K,Ihjima K,Yamada O.IL-2 increases human telomerase reverse transcriptase activity transcriptionally and posttranslationally through phosphatidylinositol 3'-kinase/Akt,heat shock protein 90,and mammalian target of rapamycin in transformed NK cells.J Immunol 2005;174 (9):5261.
44.Loewith R,Jacinto E,Wullschleger S,et al.Two TOR complexes,only one of which is rapamycin sensitive,have distinct roles in cell growth control.Mol Cell 2002;10 (3):457.
45.Oshiro N,Yoshino K,Hidayat S,et al.Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function.Genes Cells 2004;9 (4):359.
46.Sarbassov DD,Ali SM,Sengupta S,et al.Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Mol Cell 2006;22 (2):159.
47.Fingar DC,Richardson CJ,Tee AR,Cheatham L,Tsou C,Blenis J.mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BPl/eukaryotic translation initiation factor 4E.Mol Cell Biol 2004;24 (1):200.
48.Decker T,Hipp S,Ringshausen I,et al.Rapamycin-induced Gl arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3,cyclin E,cyclin A,and survivin.Blood 2003;101 (1):278.
49.C.J.Sherr.Cancer cell cycle.Science 1996;274:1672
50.B.D.Dynlacht.Regulation of transcription by proteins that control the cell cycle,.Nature 1997;389:149.
51.C.J.Sherr,J.M.Roberts,.Inhibitors of mammalian Gl cyclin-dependent kinases,.Genes Dev 1995;9:1149
52.M.Peter,I.Herskowitz.Joining the complexxyclin-dependent kinase inhibitory proteins and the cell cycle,.Cell 1994;79:181
53.Lovec H,Sewing A,Lucibello FC,Muller R,Moroy T.Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras:link between cell cycle control and malignant transformation.Oncogene 1994;9 (1):323.
54.Bodrug SE,Warner BJ,Bath ML,Lindeman GJ,Harris AW,Adams JM.Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.Embo J 1994;13 (9):2124.
55.Wang TC,Cardiff RD,Zukerberg L,Lees E,Arnold A,Schmidt EV.Mammary hyperplasia and carcinoma in MMTV-cyclin Dl transgenic mice.Nature 1994;369 (6482):669.
56.Nakayama K,Ishida N,Shirane M,et al.Mice lacking p27(Kip1) display increased body size,multiple organ hyperplasia,retinal dysplasia,and pituitary tumors.Cell 1996;85 (5):707.
57.Fero ML,Rivkin M,Tasch M,et al.A syndrome of multiorgan hyperplasia with features of gigantism,tumorigenesis,and female sterility in p27(Kip1)-deficient mice.Cell 1996;85 (5):733.
58.Yang RM,Naitoh J,Murphy M,et al.Low p27 expression predicts poor disease-free survival in patients with prostate cancer.J Urol 1998;159 (3):941.
59.Loda M,Cukor B,Tarn SW,et al.Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas.Nat Med 1997;3 (2):231.
60.Mori M,Mimori K,Shiraishi T,et al.p27 expression and gastric carcinoma.Nat Med 1997;3 (6):593.
61.Yang W,Shen J,Wu M,et al.Repression of transcription of the p27(Kip1)cyclin-dependent kinase inhibitor gene by c-Myc.Oncogene 2001;20 (14):1688.
62.Vita M,Henriksson M.The Myc oncoprotein as a therapeutic target for human cancer.Semin Cancer Biol 2006;16 (4):318.
63.Kleideiter E,Bangerter U,Schwab M,et al.Telomeres and telomerase in paediatric patients with T-cell acute lymphoblastic leukaemia (T-ALL).Leukemia 2005;19 (2):296.
64.Ohyashiki JH,Ohyashiki K,Iwama H,Hayashi S,Toyama K,Shay JW.Clinical implications of telomerase activity levels in acute leukemia.Clin Cancer Res 1997;3 (4):619.
65.Kang SS,Kwon T,Kwon DY,Do SI.Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit.J Biol Chem 1999;274 (19):13085.
66.Zhu X,Kumar R,Mandal M,et al.Cell cycle-dependent modulation of telomerase activity in tumor cells.Proc Natl Acad Sci U S A 1996;93 (12):6091.
67.Zhao YM,Li JY,Lan JP,et al.Cell cycle dependent telomere regulation by telomerase in human bone marrow mesenchymal stem cells.Biochem Biophys Res Commun 2008;369 (4):1114.
68.Lee SH,Kim JW,Oh SH,et al.IFN-gamma/IRF-1-induced p27kip1 down-regulates telomerase activity and human telomerase reverse transcriptase expression in human cervical cancer.FEBS Lett 2005;579 (5):1027.
69.Zhao L,Wientjes MG,Au JL.Evaluation of combination chemotherapy:integration of nonlinear regression,curve shift,isobologram,and combination index analyses.Clin Cancer Res 2004;10 (23):7994.
70.Yan H,Frost P,Shi Y,et al.Mechanism by which mammalian target of rapamycin inhibitors sensitize multiple myeloma cells to dexamethasone-induced apoptosis.Cancer Res 2006;66 (4):2305.
71.Stromberg T,Dimberg A,Hammarberg A,et al.Rapamycin sensitizes multiple myeloma cells to apoptosis induced by dexamethasone.Blood 2004;103 (8):3138.
72.Gu L,Gao J,Li Q,et al.Rapamycin reverses NPM-ALK-induced glucocorticoid resistance in lymphoid rumor cells by inhibiting mTOR signaling pathway,enhancing Gl cell cycle arrest and apoptosis.Leukemia 2008;22 (11):2091.
73.Wanner K,Hipp S,Oelsner M,et al.Mammalian target of rapamycin inhibition induces cell cycle arrest in diffuse large B cell lymphoma (DLBCL) cells and sensitises DLBCL cells to rituximab.Br J Haematol 2006;134 (5):475.
74.Romano MF,Avellino R,Petrella A,Bisogni R,Romano S,Venuta S. Rapamycin inhibits doxorabicin-induced NF-kappaB/Rel nuclear activity and enhances the apoptosis of melanoma cells.Eur J Cancer 2004;40 (18):2829.
75.McCubrey JA,Abrams SL,Ligresti G,et al.Involvement of p53 and Raf/MEK/ERK pathways in hematopoietic drug resistance.Leukemia 2008;22 (11):2080.
76.Yu HG,Ai YW,Yu LL,et al.Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death.Int J Cancer 2008;122 (2):433.
77.Garcia MG,Alaniz LD,Cordo Russo RI,Alvarez E,Hajos SE.PI3K/Akt inhibition modulates multidrug resistance and activates NF-kappaB in murine lymphoma cell lines.Leuk Res 2009;33 (2):288.
78.Green DR,Reed JC.Mitochondria and apoptosis.Science 1998;281 (5381):1309.
79.He J,Whitacre CM,Xue LY,Berger NA,Oleinick NL.Protease activation and cleavage of poly(ADP-ribose) polymerase:an integral part of apoptosis in response to photodynamic treatment.Cancer Res 1998;58 (5):940.
80.Wolter KG,Hsu YT,Smith CL,Nechushtan A,Xi XQ Youle RJ.Movement of Bax from the cytosol to mitochondria during apoptosis.J Cell Biol 1997;139 (5):1281.
81.Nakayama K,Kamihira S.Survivin an important determinant for prognosis in adult T-cell leukemia:a novel biomarker in practical hemato-oncology.Leuk Lymphoma 2002;43 (12):2249.
1．Hanahan D， Weinberg RA．The hallmarks of cancer．Cell 2000； 100 (1)： 57．
2．Collins K， Mitchell JR．Telomerase in the human organism．Oncogene 2002； 21(4)： 564．
3．Feng J， Funk WD， Wang SS， et al．The RNA component of human telomerase．Science 1995； 269 (5228)： 1236．
4．Niida H， Matsumoto T， Satoh H， et al．Severe growth defect in mouse cells lacking the telomerase RNA component．Nat Genet 1998； 19 (2)： 203．
5．Morin GB．The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats．Cell 1989； 59 (3)： 521．
6．Ly H， Blackburn EH， Parslow TG．Comprehensive structure-function analysis of the core domain of human telomerase RNA．Mol Cell Biol 2003； 23(19)： 6849．
7．Marrone A， Mason PJ．Dyskeratosis congenita．Cell Mol Life Sci 2003； 60 (3)： 507．
8．Vulliamy TJ， Marrone A， Knight SW， Walne A， Mason PJ， Dokal I．Mutations in dyskeratosis congenita： their impact on telomere length and the diversity of clinical presentation．Blood 2006； 107 (7)： 2680．
9．Walne AJ， Dokal I．Telomerase dysfunction and dyskeratosis congenita．Cytotechnology 2004； 45 (1-2)： 13．
10．Dokal I．Dyskeratosis congenita．A disease of premature ageing．Lancet 2001； 358 Suppl:S27.
11.Fernandez PC,Frank SR,Wang L,et al.Genomic targets of the human c-Myc protein.Genes Dev 2003;17 (9):1115.
12.Xu D,Popov N,Hou M,et al.Switch from Myc/Max to Madl/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells.Proc Natl Acad Sci U S A 2001;98 (7):3826.
13.Hu X,Yu N,Fu L,et al.Downregulation of human telomerase reverse transcriptase through anti-C-myc siRNA in human colon cancer Colo 320 cells.Nucleosides Nucleotides Nucleic Acids 2009;28 (1):1.
14.Yatabe N,Kyo S,Maida Y,et al.HIF-1-mediated activation of telomerase in cervical cancer cells.Oncogene 2004;23 (20):3708.
15.Dessain SK,Yu H,Reddel RR,Beijersbergen RL,Weinberg RA.Methylation of the human telomerase gene CpG island.Cancer Res 2000;60 (3):537.
16.Zaffaroni N,Villa R,Pastorino U,et al.Lack of telomerase activity in lung carcinoids is dependent on human telomerase reverse transcriptase transcription and alternative splicing and is associated with long telomeres.Clin Cancer Res 2005;11 (8):2832.
17.Liu L,Lai S,Andrews LG,Tollefsbol TO.Genetic and epigenetic modulation of telomerase activity in development and disease.Gene 2004;340 (1):1.
18.Li H,Zhao LL,Funder JW,Liu JP.Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells.J Biol Chem 1997;272 (27):16729.
19.Kang SS,Kwon T,Kwon DY,Do SI.Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit.J Biol Chem 1999;274 (19):13085.
20.Holt SE,Aisner DL,Baur J,et al.Functional requirement of p23 and Hsp90 in telomerase complexes.Genes Dev 1999;13 (7):817.
21.Saito T,Matsuda Y,Suzuki T,et al.Comparative gene mapping of the human and mouse TEP1 genes,which encode one protein component of telomerases.Genomics 1997;46 (1):46.
22.Kim NW,Wu F.Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP).Nucleic Acids Res 1997;25 (13):2595.
23.Murakami J,Nagai N,Ohama K,Tahara H,Ide T.Telomerase activity in ovarian tumors.Cancer 1997;80 (6):1085.
24.Nakatani K,Yoshimi N,Mori H,et al.The significant role of telomerase activity in human brain tumors.Cancer 1997;80 (3):471.
25.Stewart SA,Hahn WC,O'Connor BF,et al.Telomerase contributes to tumorigenesis by a telomere length-independent mechanism.Proc Natl Acad Sci USA2002;99(20):12606.
26.Lansdorp PM,Poon S,Chavez E,et al.Telomeres in the haemopoietic system.Ciba Found Symp 1997;211:209.
27.Murnane JP,Sabatier L,Marder BA,Morgan WF.Telomere dynamics in an immortal human cell line.Embo J 1994;13 (20):4953.
28.Henson JD,Neumann AA,Yeager TR,Reddel RR.Alternative lengthening of telomeres in mammalian cells.Oncogene 2002;21 (4):598.
29.Yeager TR,Neumann AA,Englezou A,Huschtscha LI,Noble JR,Reddel RR.Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res 1999;59 (17):4175.
30.Perrem K,Bryan TM,Englezou A,Hackl T,Moy EL,Reddel RR.Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.Oncogene 1999;18 (22):3383.
31.Grobelny JV,Godwin AK,Broccoli D.ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle.J Cell Sci 2000;113 Pt 24:4577.
32.Wu G,Lee WH,Chen PL.NBS1 and TRF1 colocalize at promyelocytoic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells.Implication of NBS1 in alternative lengthening of telomeres.J Biol Chem 2000;275 (39):30618.
33.Jiang WQ,Zhong ZH,Henson JD,Neumann AA,Chang AC,Reddel RR.Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex.Mol Cell Biol 2005;25 (7):2708.
34.Zhou XZ,Perrem K,Lu KP.Role of Pin2/TRF1 in telomere maintenance and cell cycle control.J Cell Biochem 2003;89 (1):19.
35.Stansel RM,de Lange T,Griffith JD.T-loop assembly in vitro involves binding of TRF2 near the 3' telomeric overhang.Embo J 2001;20 (19):5532.
36.Le S,Moore JK,Haber JE,Greider CW.RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase.Genetics 1999;152(1):143.
37.Londono-Vallejo JA,Der-Sarkissian H,Cazes L,Bacchetti S,Reddel RR.Alternative lengthening of telomeres is characterized by high rates of telomeric exchange.Cancer Res 2004;64 (7):2324.
38.Bailey SM,Brenneman MA,Goodwin EH.Frequent recombination in telomeric DNA may extend the proliferative life of telomerase-negative cells.Nucleic Acids Res 2004;32 (12):3743.
39.Dunham MA,Neumann AA,Fasching CL,Reddel RR.Telomere maintenance by recombination in human cells.Nat Genet 2000;26 (4):447.
40.Griffith JD,Comeau L,Rosenfield S,et al.Mammalian telomeres end in a large duplex loop.Cell 1999;97 (4):503.
41.Baumann P,Cech TR.Potl,the putative telomere end-binding protein in fission yeast and humans.Science 2001;292 (5519):1171.
42.Wang RC,Smogorzewska A,de Lange T.Homologous recombination generates T-loop-sized deletions at human telomeres.Cell 2004;119 (3):355.
43.Cesare AJ,Griffith JD.Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops.Mol Cell Biol 2004;24 (22):9948.
44.Henson JD,Hannay JA,McCarthy SW,et al.A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas.Clin Cancer Res 2005;11(1):217.
45.Ulaner GA,Huang HY,Otero J,et al.Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma.Cancer Res 2003;63 (8):1759.
2.Schrappe M,Camitta B,Pui CH,et al.Long-term results of large prospective trials in childhood acute lymphoblastic leukemia.Leukemia 2000;14 (12):2193.
3.Dennis PB,Jaeschke A,Saitoh M,Fowler B,Kozma SC,Thomas G Mammalian TOR:a homeostatic ATP sensor.Science 2001;294 (5544):1102.
4.Schmelzle T,Hall MN.TOR,a central controller of cell growth.Cell 2000;103 (2):253.
5.West KA,Castillo SS,Dennis PA.Activation of the PI3K/Akt pathway and chemotherapeutic resistance.Drug Resist Updat 2002;5 (6):234.
6.Knuefermann C,Lu Y,Liu B,et al.HER2/PI-3K/Akt activation leads to a multidrug resistance in human breast adenocarcinoma cells.Oncogene 2003;22 (21):3205.
7.Brognard J,Clark AS,Ni Y,Dennis PA.Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation.Cancer Res 2001;61 (10):3986.
8.Cullion K,Draheim KM,Hermance N,et al.Targeting the Notch 1 and mTOR pathways in a mouse T-ALL model.Blood 2009.
9.Xu Q,Simpson SE,Scialla TJ,Bagg A,Carroll M.Survival of acute myeloid leukemia cells requires PI3 kinase activation.Blood 2003;102 (3):972.
10.Ly C,Arechiga AF,Melo JV,Walsh CM,Ong ST.Bcr-Abl kinase modulates the translation regulators ribosomal protein S6 and 4E-BP1 in chronic myelogenous leukemia cells via the mammalian target of rapamycin.Cancer Res 2003;63(18):5716.
11.Rowinsky EK.Targeting the molecular target of rapamycin (mTOR).Curr Opin Oncol 2004;16 (6):564.
12.Burchert A,Wang Y,Cai D,et al.Compensatory PI3-kinase/Akt/mTor activation regulates imatinib resistance development.Leukemia 2005;19(10):1774.
13.Sillaber C,Mayerhofer M,Bohm A,et al.Evaluation of antileukaemic effects of rapamycin in patients with imatinib-resistant chronic myeloid leukaemia.Eur J Clin Invest 2008;38(1):43.
14.Mohi MG,Boulton C,Gu TL,et al.Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs.Proc Natl Acad Sci U S A2004;101 (9):3130.
15.Recher C,Beyne-Rauzy O,Demur C,et al.Antileukemic activity of rapamycin in acute myeloid leukemia.Blood 2005;105 (6):2527.
16.Yilmaz OH,Valdez R,Theisen BK,et al.Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells.Nature 2006;441 (7092):475.
17.Palomero T,Dominguez M,Ferrando AA.The role of the PTEN/AKT Pathway in NOTCH 1-induced leukemia.Cell Cycle 2008;7 (8):965.
18.Gutierrez A,Look AT.NOTCH and PI3K-AKT pathways intertwined.Cancer Cell 2007;12 (5):411.
19.Laurent G,Jaffrezou JP.Signaling pathways activated by daunorubicin.Blood 2001;98 (4):913.
20.Takeuchi H,Kondo Y,Fujiwara K,et al.Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors.Cancer Res 2005;65 (8):3336.
21.Laport GF,Larson RA.Treatment of adult acute lymphoblastic leukemia.Semin Oncol 1997;24(1):70.
22.Mungamuri SK,Yang X,Thor AD,Somasundaram K.Survival signaling by Notchl:mammalian target of rapamycin (mTOR)-dependent inhibition of p53.Cancer Res 2006;66 (9):4715.
23.Minowada J,Onuma T,Moore GE.Rosette-forming human lymphoid cell lines.I.Establishment and evidence for origin of thymus-derived lymphocytes.J Natl Cancer Inst 1972;49 (3):891.
24.Hosoi H,Dilling MB,Shikata T,et al.Rapamycin causes poorly reversible inhibition of mTOR and induces p53-independent apoptosis in human rhabdomyosarcoma cells.Cancer Res 1999;59 (4):886.
25.Pene F,Claessens YE,Muller O,et al.Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.Oncogene 2002;21 (43):6587.
26.Avellino R,Romano S,Parasole R,et al.Rapamycin stimulates apoptosis of childhood acute lymphoblastic leukemia cells.Blood 2005;106 (4):1400.
27.Hirase C,Maeda Y,Takai S,Kanamaru A.Hypersensitivity of Ph-positive lymphoid cell lines to rapamycin:Possible clinical application of mTOR inhibitor.Leuk Res 2009;33 (3):450.
28.Crazzolara R,Cisterne A,Thien M,et al.Potentiating effects of RAD001 (Everolimus) on vincristine therapy in childhood acute lymphoblastic leukemia.Blood2009;113(14):3297.
29.Yoshimori T.Autophagy:a regulated bulk degradation process inside cells.Biochem Biophys Res Commun 2004;313 (2):453.
30.Klionsky DJ,Emr SD.Autophagy as a regulated pathway of cellular degradation.Science 2000;290 (5497):1717.
31.Ogier-Denis E,Codogno P.Autophagy:a barrier or an adaptive response to cancer.Biochim Biophys Acta 2003;1603 (2):113.
32.Schwarze PE,Seglen PO.Reduced autophagic activity,improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats.Exp Cell Res 1985;157 (1):15.
33.Ertmer A,Huber V,Gilch S,et al.The anticancer drug imatinib induces cellular autophagy.Leukemia 2007;21 (5):936.
34.Bursch W,Ellinger A,Kienzl H,et al.Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture:the role of autophagy.Carcinogenesis 1996;17 (8):1595.
35.Kanzawa T,Germano IM,Komata T,Ito H,Kondo Y,Kondo S.Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells.Cell Death Differ 2004;11 (4):448.
36.Kanzawa T,Kondo Y,Ito H,Kondo S,Germano I.Induction of autophagic cell death in malignant glioma cells by arsenic trioxide.Cancer Res 2003;63 (9):2103.
37.Qu X,Yu J,Bhagat G,et al.Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.J Clin Invest 2003;112(12):1809.
38.Yue Z,Jin S,Yang C,Levine AJ,Heintz N.Beclin 1,an autophagy gene essential for early embryonic development,is a haploinsufficient tumor suppressor.Proc Natl Acad Sci U S A 2003;100 (25):15077.
39.Xue L,Fletcher GC,Tolkovsky AM.Autophagy is activated by apoptotic signalling in sympathetic neurons:an alternative mechanism of death execution.Mol Cell Neurosci 1999;14 (3):180.
40.Blommaart EF,Luiken JJ,Blommaart PJ,van Woerkom GM,Meijer AJ.Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes.J Biol Chem 1995;270 (5):2320.
41.Klionsky DJ,Meijer AJ,Codogno P.Autophagy and p70S6 kinase.Autophagy 2005;1 (1):59.
42.Bae-Jump VL,Zhou C,Gehrig PA,Whang YE,Boggess JF.Rapamycin inhibits hTERT telomerase mRNA expression,independent of cell cycle arrest.Gynecol Oncol 2006;100 (3):487.
43.Kawauchi K,Ihjima K,Yamada O.IL-2 increases human telomerase reverse transcriptase activity transcriptionally and posttranslationally through phosphatidylinositol 3'-kinase/Akt,heat shock protein 90,and mammalian target of rapamycin in transformed NK cells.J Immunol 2005;174 (9):5261.
44.Loewith R,Jacinto E,Wullschleger S,et al.Two TOR complexes,only one of which is rapamycin sensitive,have distinct roles in cell growth control.Mol Cell 2002;10 (3):457.
45.Oshiro N,Yoshino K,Hidayat S,et al.Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function.Genes Cells 2004;9 (4):359.
46.Sarbassov DD,Ali SM,Sengupta S,et al.Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Mol Cell 2006;22 (2):159.
47.Fingar DC,Richardson CJ,Tee AR,Cheatham L,Tsou C,Blenis J.mTOR controls cell cycle progression through its cell growth effectors S6K1 and 4E-BPl/eukaryotic translation initiation factor 4E.Mol Cell Biol 2004;24 (1):200.
48.Decker T,Hipp S,Ringshausen I,et al.Rapamycin-induced Gl arrest in cycling B-CLL cells is associated with reduced expression of cyclin D3,cyclin E,cyclin A,and survivin.Blood 2003;101 (1):278.
49.C.J.Sherr.Cancer cell cycle.Science 1996;274:1672
50.B.D.Dynlacht.Regulation of transcription by proteins that control the cell cycle,.Nature 1997;389:149.
51.C.J.Sherr,J.M.Roberts,.Inhibitors of mammalian Gl cyclin-dependent kinases,.Genes Dev 1995;9:1149
52.M.Peter,I.Herskowitz.Joining the complexxyclin-dependent kinase inhibitory proteins and the cell cycle,.Cell 1994;79:181
53.Lovec H,Sewing A,Lucibello FC,Muller R,Moroy T.Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras:link between cell cycle control and malignant transformation.Oncogene 1994;9 (1):323.
54.Bodrug SE,Warner BJ,Bath ML,Lindeman GJ,Harris AW,Adams JM.Cyclin D1 transgene impedes lymphocyte maturation and collaborates in lymphomagenesis with the myc gene.Embo J 1994;13 (9):2124.
55.Wang TC,Cardiff RD,Zukerberg L,Lees E,Arnold A,Schmidt EV.Mammary hyperplasia and carcinoma in MMTV-cyclin Dl transgenic mice.Nature 1994;369 (6482):669.
56.Nakayama K,Ishida N,Shirane M,et al.Mice lacking p27(Kip1) display increased body size,multiple organ hyperplasia,retinal dysplasia,and pituitary tumors.Cell 1996;85 (5):707.
57.Fero ML,Rivkin M,Tasch M,et al.A syndrome of multiorgan hyperplasia with features of gigantism,tumorigenesis,and female sterility in p27(Kip1)-deficient mice.Cell 1996;85 (5):733.
58.Yang RM,Naitoh J,Murphy M,et al.Low p27 expression predicts poor disease-free survival in patients with prostate cancer.J Urol 1998;159 (3):941.
59.Loda M,Cukor B,Tarn SW,et al.Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas.Nat Med 1997;3 (2):231.
60.Mori M,Mimori K,Shiraishi T,et al.p27 expression and gastric carcinoma.Nat Med 1997;3 (6):593.
61.Yang W,Shen J,Wu M,et al.Repression of transcription of the p27(Kip1)cyclin-dependent kinase inhibitor gene by c-Myc.Oncogene 2001;20 (14):1688.
62.Vita M,Henriksson M.The Myc oncoprotein as a therapeutic target for human cancer.Semin Cancer Biol 2006;16 (4):318.
63.Kleideiter E,Bangerter U,Schwab M,et al.Telomeres and telomerase in paediatric patients with T-cell acute lymphoblastic leukaemia (T-ALL).Leukemia 2005;19 (2):296.
64.Ohyashiki JH,Ohyashiki K,Iwama H,Hayashi S,Toyama K,Shay JW.Clinical implications of telomerase activity levels in acute leukemia.Clin Cancer Res 1997;3 (4):619.
65.Kang SS,Kwon T,Kwon DY,Do SI.Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit.J Biol Chem 1999;274 (19):13085.
66.Zhu X,Kumar R,Mandal M,et al.Cell cycle-dependent modulation of telomerase activity in tumor cells.Proc Natl Acad Sci U S A 1996;93 (12):6091.
67.Zhao YM,Li JY,Lan JP,et al.Cell cycle dependent telomere regulation by telomerase in human bone marrow mesenchymal stem cells.Biochem Biophys Res Commun 2008;369 (4):1114.
68.Lee SH,Kim JW,Oh SH,et al.IFN-gamma/IRF-1-induced p27kip1 down-regulates telomerase activity and human telomerase reverse transcriptase expression in human cervical cancer.FEBS Lett 2005;579 (5):1027.
69.Zhao L,Wientjes MG,Au JL.Evaluation of combination chemotherapy:integration of nonlinear regression,curve shift,isobologram,and combination index analyses.Clin Cancer Res 2004;10 (23):7994.
70.Yan H,Frost P,Shi Y,et al.Mechanism by which mammalian target of rapamycin inhibitors sensitize multiple myeloma cells to dexamethasone-induced apoptosis.Cancer Res 2006;66 (4):2305.
71.Stromberg T,Dimberg A,Hammarberg A,et al.Rapamycin sensitizes multiple myeloma cells to apoptosis induced by dexamethasone.Blood 2004;103 (8):3138.
72.Gu L,Gao J,Li Q,et al.Rapamycin reverses NPM-ALK-induced glucocorticoid resistance in lymphoid rumor cells by inhibiting mTOR signaling pathway,enhancing Gl cell cycle arrest and apoptosis.Leukemia 2008;22 (11):2091.
73.Wanner K,Hipp S,Oelsner M,et al.Mammalian target of rapamycin inhibition induces cell cycle arrest in diffuse large B cell lymphoma (DLBCL) cells and sensitises DLBCL cells to rituximab.Br J Haematol 2006;134 (5):475.
74.Romano MF,Avellino R,Petrella A,Bisogni R,Romano S,Venuta S. Rapamycin inhibits doxorabicin-induced NF-kappaB/Rel nuclear activity and enhances the apoptosis of melanoma cells.Eur J Cancer 2004;40 (18):2829.
75.McCubrey JA,Abrams SL,Ligresti G,et al.Involvement of p53 and Raf/MEK/ERK pathways in hematopoietic drug resistance.Leukemia 2008;22 (11):2080.
76.Yu HG,Ai YW,Yu LL,et al.Phosphoinositide 3-kinase/Akt pathway plays an important role in chemoresistance of gastric cancer cells against etoposide and doxorubicin induced cell death.Int J Cancer 2008;122 (2):433.
77.Garcia MG,Alaniz LD,Cordo Russo RI,Alvarez E,Hajos SE.PI3K/Akt inhibition modulates multidrug resistance and activates NF-kappaB in murine lymphoma cell lines.Leuk Res 2009;33 (2):288.
78.Green DR,Reed JC.Mitochondria and apoptosis.Science 1998;281 (5381):1309.
79.He J,Whitacre CM,Xue LY,Berger NA,Oleinick NL.Protease activation and cleavage of poly(ADP-ribose) polymerase:an integral part of apoptosis in response to photodynamic treatment.Cancer Res 1998;58 (5):940.
80.Wolter KG,Hsu YT,Smith CL,Nechushtan A,Xi XQ Youle RJ.Movement of Bax from the cytosol to mitochondria during apoptosis.J Cell Biol 1997;139 (5):1281.
81.Nakayama K,Kamihira S.Survivin an important determinant for prognosis in adult T-cell leukemia:a novel biomarker in practical hemato-oncology.Leuk Lymphoma 2002;43 (12):2249.
1．Hanahan D， Weinberg RA．The hallmarks of cancer．Cell 2000； 100 (1)： 57．
2．Collins K， Mitchell JR．Telomerase in the human organism．Oncogene 2002； 21(4)： 564．
3．Feng J， Funk WD， Wang SS， et al．The RNA component of human telomerase．Science 1995； 269 (5228)： 1236．
4．Niida H， Matsumoto T， Satoh H， et al．Severe growth defect in mouse cells lacking the telomerase RNA component．Nat Genet 1998； 19 (2)： 203．
5．Morin GB．The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats．Cell 1989； 59 (3)： 521．
6．Ly H， Blackburn EH， Parslow TG．Comprehensive structure-function analysis of the core domain of human telomerase RNA．Mol Cell Biol 2003； 23(19)： 6849．
7．Marrone A， Mason PJ．Dyskeratosis congenita．Cell Mol Life Sci 2003； 60 (3)： 507．
8．Vulliamy TJ， Marrone A， Knight SW， Walne A， Mason PJ， Dokal I．Mutations in dyskeratosis congenita： their impact on telomere length and the diversity of clinical presentation．Blood 2006； 107 (7)： 2680．
9．Walne AJ， Dokal I．Telomerase dysfunction and dyskeratosis congenita．Cytotechnology 2004； 45 (1-2)： 13．
10．Dokal I．Dyskeratosis congenita．A disease of premature ageing．Lancet 2001； 358 Suppl:S27.
11.Fernandez PC,Frank SR,Wang L,et al.Genomic targets of the human c-Myc protein.Genes Dev 2003;17 (9):1115.
12.Xu D,Popov N,Hou M,et al.Switch from Myc/Max to Madl/Max binding and decrease in histone acetylation at the telomerase reverse transcriptase promoter during differentiation of HL60 cells.Proc Natl Acad Sci U S A 2001;98 (7):3826.
13.Hu X,Yu N,Fu L,et al.Downregulation of human telomerase reverse transcriptase through anti-C-myc siRNA in human colon cancer Colo 320 cells.Nucleosides Nucleotides Nucleic Acids 2009;28 (1):1.
14.Yatabe N,Kyo S,Maida Y,et al.HIF-1-mediated activation of telomerase in cervical cancer cells.Oncogene 2004;23 (20):3708.
15.Dessain SK,Yu H,Reddel RR,Beijersbergen RL,Weinberg RA.Methylation of the human telomerase gene CpG island.Cancer Res 2000;60 (3):537.
16.Zaffaroni N,Villa R,Pastorino U,et al.Lack of telomerase activity in lung carcinoids is dependent on human telomerase reverse transcriptase transcription and alternative splicing and is associated with long telomeres.Clin Cancer Res 2005;11 (8):2832.
17.Liu L,Lai S,Andrews LG,Tollefsbol TO.Genetic and epigenetic modulation of telomerase activity in development and disease.Gene 2004;340 (1):1.
18.Li H,Zhao LL,Funder JW,Liu JP.Protein phosphatase 2A inhibits nuclear telomerase activity in human breast cancer cells.J Biol Chem 1997;272 (27):16729.
19.Kang SS,Kwon T,Kwon DY,Do SI.Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit.J Biol Chem 1999;274 (19):13085.
20.Holt SE,Aisner DL,Baur J,et al.Functional requirement of p23 and Hsp90 in telomerase complexes.Genes Dev 1999;13 (7):817.
21.Saito T,Matsuda Y,Suzuki T,et al.Comparative gene mapping of the human and mouse TEP1 genes,which encode one protein component of telomerases.Genomics 1997;46 (1):46.
22.Kim NW,Wu F.Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP).Nucleic Acids Res 1997;25 (13):2595.
23.Murakami J,Nagai N,Ohama K,Tahara H,Ide T.Telomerase activity in ovarian tumors.Cancer 1997;80 (6):1085.
24.Nakatani K,Yoshimi N,Mori H,et al.The significant role of telomerase activity in human brain tumors.Cancer 1997;80 (3):471.
25.Stewart SA,Hahn WC,O'Connor BF,et al.Telomerase contributes to tumorigenesis by a telomere length-independent mechanism.Proc Natl Acad Sci USA2002;99(20):12606.
26.Lansdorp PM,Poon S,Chavez E,et al.Telomeres in the haemopoietic system.Ciba Found Symp 1997;211:209.
27.Murnane JP,Sabatier L,Marder BA,Morgan WF.Telomere dynamics in an immortal human cell line.Embo J 1994;13 (20):4953.
28.Henson JD,Neumann AA,Yeager TR,Reddel RR.Alternative lengthening of telomeres in mammalian cells.Oncogene 2002;21 (4):598.
29.Yeager TR,Neumann AA,Englezou A,Huschtscha LI,Noble JR,Reddel RR.Telomerase-negative immortalized human cells contain a novel type of promyelocytic leukemia (PML) body.Cancer Res 1999;59 (17):4175.
30.Perrem K,Bryan TM,Englezou A,Hackl T,Moy EL,Reddel RR.Repression of an alternative mechanism for lengthening of telomeres in somatic cell hybrids.Oncogene 1999;18 (22):3383.
31.Grobelny JV,Godwin AK,Broccoli D.ALT-associated PML bodies are present in viable cells and are enriched in cells in the G(2)/M phase of the cell cycle.J Cell Sci 2000;113 Pt 24:4577.
32.Wu G,Lee WH,Chen PL.NBS1 and TRF1 colocalize at promyelocytoic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells.Implication of NBS1 in alternative lengthening of telomeres.J Biol Chem 2000;275 (39):30618.
33.Jiang WQ,Zhong ZH,Henson JD,Neumann AA,Chang AC,Reddel RR.Suppression of alternative lengthening of telomeres by Sp100-mediated sequestration of the MRE11/RAD50/NBS1 complex.Mol Cell Biol 2005;25 (7):2708.
34.Zhou XZ,Perrem K,Lu KP.Role of Pin2/TRF1 in telomere maintenance and cell cycle control.J Cell Biochem 2003;89 (1):19.
35.Stansel RM,de Lange T,Griffith JD.T-loop assembly in vitro involves binding of TRF2 near the 3' telomeric overhang.Embo J 2001;20 (19):5532.
36.Le S,Moore JK,Haber JE,Greider CW.RAD50 and RAD51 define two pathways that collaborate to maintain telomeres in the absence of telomerase.Genetics 1999;152(1):143.
37.Londono-Vallejo JA,Der-Sarkissian H,Cazes L,Bacchetti S,Reddel RR.Alternative lengthening of telomeres is characterized by high rates of telomeric exchange.Cancer Res 2004;64 (7):2324.
38.Bailey SM,Brenneman MA,Goodwin EH.Frequent recombination in telomeric DNA may extend the proliferative life of telomerase-negative cells.Nucleic Acids Res 2004;32 (12):3743.
39.Dunham MA,Neumann AA,Fasching CL,Reddel RR.Telomere maintenance by recombination in human cells.Nat Genet 2000;26 (4):447.
40.Griffith JD,Comeau L,Rosenfield S,et al.Mammalian telomeres end in a large duplex loop.Cell 1999;97 (4):503.
41.Baumann P,Cech TR.Potl,the putative telomere end-binding protein in fission yeast and humans.Science 2001;292 (5519):1171.
42.Wang RC,Smogorzewska A,de Lange T.Homologous recombination generates T-loop-sized deletions at human telomeres.Cell 2004;119 (3):355.
43.Cesare AJ,Griffith JD.Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops.Mol Cell Biol 2004;24 (22):9948.
44.Henson JD,Hannay JA,McCarthy SW,et al.A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas.Clin Cancer Res 2005;11(1):217.
45.Ulaner GA,Huang HY,Otero J,et al.Absence of a telomere maintenance mechanism as a favorable prognostic factor in patients with osteosarcoma.Cancer Res 2003;63 (8):1759.