调节自噬表达对A549细胞放疗敏感性改变的作用及机制研究
|
摘要
肺癌细胞的生物学发生是一个十分复杂的过程。寻找有效的抗癌方法与途径,在肺癌的发生阶段可以修复细胞成分的早期改变,肺癌形成之后能够清除突变或者损伤了的细胞器,甚至在肺癌形成后逆转肺癌细胞一直是近年来的研究热点。
近年来的研究显示:细胞器的代谢主要依靠自噬途径。在生命的进化过程中,自噬是一个古老的生物学现象,广泛存在于植物和其它较低级生命体中,是生物降解胞内蛋白,完成细胞器转化,保持内环境稳定的重要方式,也是哺乳动物清除癌细胞的手段之一。自噬是一个有大量小分子蛋白质参与的生物学过程,人类自噬过程中比较明确的和研究较多的是Beclin1基因和其编码蛋白。自噬体形成必需的微管相关蛋白轻链3 (MAPLC3)也常常用于自噬体的检测。
出于维持细胞内环境稳定的目的,正常情况下细胞自噬的基础水平保持在一个较低的状态,在某些“危机”状况下可以快速上调,如:生长激素缺乏、饥饿、细胞重建、细胞内出现过多的受损细胞器或代谢废物等。自噬受两种进化中形成的营养感受器调节:TOR激酶(target of rapamycin (TOR) kinase)在营养充分时关闭自噬信号,真核起始因子2激酶Gcn2(eukaryotic initiation factor 2 (eIF2 ) kinase Gcn2)及其下游靶位Gcn4-自噬基因的转录反式作用子,在饥饿时启动自噬信号。TOR激酶的下游是一些编码ATG蛋白的基因,参与自噬的发生,融合,成熟,再循环。雷帕霉素是TOR受体的拮抗剂,因此能够上调自噬。另外较常用3甲基腺嘌啉(3MA)抑制自噬。
自噬和癌细胞的形成、转化、治疗敏感性密切相关。癌细胞形成早期刺激自噬能够清除受损细胞器,分解细胞内过多的有害物质(如过氧化物等),阻止正常细胞向癌细胞转化,调控自噬的表达和化疗药物联合对肿瘤细胞的生长有协同或拮抗作用,表现为与单纯用化疗药物相比,调控自噬表达后肿瘤细胞的凋亡率发生了变化。在化疗或放疗中抑制自噬有可能导致癌细胞无法清除受损细胞器,加速细胞死亡,强化治疗效果。
为了研究自噬和非小细胞肺癌的关系,以及调节自噬的表达可能产生的放疗增敏效果,我们检测了经手术切除的非小细胞肺癌(NSCLC)临床标本中的自噬相关基因表达状态,探讨自噬的表达情况和NSCLC发生和发展的可能关系;观察了肺癌细胞系A549在γ射线照射下自噬和自噬上游信号传导系统的改变;研究了改变肺腺癌细胞系A549中自噬表达后,A549细胞对于放射治疗敏感性的变化以及自噬相关通道蛋白和一些耐药基因改变,检验肿瘤细胞周期和凋亡率,进而对其机制进行探讨,探究自噬与凋亡的内在联系和在程序性细胞死亡(PCD)的作用和意义。
本研究中,我们采集了2006年4月至2006年10月武汉协和医院胸外科手术切除的非小细胞肺癌新鲜标本72例,冰冻病理切片免疫荧光染色检测肺癌组织,癌旁组织,正常组织中自噬特异性基因Beclin1和MAPLC3表达;提取总蛋白和总RNA,采用Westernblot和RT-PCR检测Beclin1和MAPLC3蛋白表达和mRNA转录情况并且进行半定量分析。培养肺腺癌细胞系A549,对其采用γ射线照射,检测在射线作用下自噬相关蛋白和mRNA以及上游akt和mTOR的表达。用3甲基腺嘌呤(3-methyladenine 3MA)和雷帕霉素(Rapamycin)改变其自噬的表达(3MA抑制自噬表达,Rapamycin则上调自噬表达),收集细胞后检测不同自噬表达状态下其抑制率和细胞凋亡率,以及细胞周期改变情况;细胞集落试验检测不同自噬表达情况下射线对细胞增殖的抑制情况;Western blot检测MAPLC3、pakt、mTOR、COX2等蛋白表达,RTPCR检测MAPLC3、survivin、mTOR、Bcl-2、EIF-4E的mRNA转录情况水平。Beclin1和MAPLC3免疫荧光染色流式细胞检测A549细胞自噬相关蛋白表达率,分析凋亡和自噬在PCD中的关系。
对NSCLC临床切除标本免疫荧光染色和蛋白mRNA检测显示, Beclin1和MAPLC3在肺癌组织中表达低于在癌旁组织和非癌组织,而癌旁组织和正常组织中表达差异无统计学意义。γ射线能够上调A549细胞自噬的表达,且呈时间依赖性。3MA和rapamycin的自噬调节效果明显,3MA和放疗有拮抗作用,而rapamycin则有协同放疗作用,抑制自噬可能通过部分凋亡相关蛋白的改变和细胞周期的阻滞作用影响放疗效果。
本课题组对自噬和肺癌的关系做了一系列研究,有了一些新的发现。但是由于时间的关系,我们的实验尚局限于较浅的层面,在今后的工作中我们会作进一步深入研究。
第一部分自噬相关基因在NSCLC中的表达
目的:研究发现自噬相关基因表达的下调和肿瘤的发生关系密切。本研究检测人小细胞肺癌标本中自噬相关基因Beclin1和微管相关蛋白轻链3(Microtubule-associated protein 1 light chain 3 MAPLC3)的表达并探讨其意义。
方法:采用免疫荧光染色,逆转录-聚合酶链法(Reverse Transcription-Polymerase Chain Reaction RT-PCR)和Western blot法检测肺癌组织,癌旁组织,正常肺组织的Beclin1和MAPLC3蛋白、mRAN表达。
结果:免疫荧光染色显示Beclin1在肺癌组织中表达较低,阳性率为8.3%,在癌旁组织和正常组织阳性率均为100%(Χ2=199.40,p=0.00);MAPLC3在肺癌组织中表达阳性率为13.9%,在癌旁组织和正常组织阳性率均为100%(Χ2=182.75,P=0.00)。Beclin1 mRNA在肺癌组织中的相对表达量(1.30±0.07),与在癌旁组织(1.69±0.10)及正常组织中(1.67±0.08)的相对表达量比较差异有统计学意义(F=6.6, P= 0. 00);MAPLC3mRNA在肺癌组织中相对表达量(4.55±0.31),与在癌旁组织(6.73±0.37)及正常组织(6.90±0.37)中的相对表达量比较差异亦有统计学意义(F=14.1,P=0.00)。Western blot检测Beclin1蛋白在肺癌组织中的相对表达量(3.49±0.29),和癌旁组织(5.31±0.46),正常组织(6.33±0.58)中的相对表达量比较差异有统计学意义(F=9.73,P<0.01),MAPLC3蛋白在肺癌组织中的相对表达量(2.43±0.26),和癌旁组织(3.12±0.28),正常组织(3.41±0.30)中的相对表达量比较差异也有统计学意义(F=3.22,P=0.04)。
结论:自噬相关基因Beclin1和MAPLC3的mRNA及蛋白在正常肺组织和肺癌组织内均有表达,表达的相对量在肺癌组织中较癌旁组织和正常组织低,在癌旁和正常肺组织中表达接近。
第二部分放射治疗对A549细胞自噬及其相关基因和自噬上游信号传导途径的影响
目的:为了探讨γ电离辐射对于肺癌细胞A549的影响,本试验检测了γ射线照射不同时间后A549细胞自噬相关基因Beclin1和MAPLC3的表达和细胞周期改变,同时检测了自噬上游信号传导通道PI3K/AKT相关蛋白和mRNA的改变,并讨论其机制。
方法:设立对照组和照射后不同时间取材的试验组,采用细胞免疫荧光染色镜下计数检测照射不同时间后Beclin1、MAPLC3、pakt、mTOR的阳性细胞表达率,PI染色流式细胞仪检测照射不同时间后细胞周期的改变。RT-PCR和Western blot检测Beclin1、MAPLC3、pakt、mTOR的mRNA和蛋白表达。
结果:免疫荧光染色显示,未经照射的肺腺癌细胞系A549自噬相关蛋白免疫荧光阳性率较低(Beclin1:0.13±0.04,MAPLC3:0.25±0.05),自噬相关信号传导通道蛋白基础表达率也较低(mTOR:0.09±0.03,pakt:0.03±0.01),照射后上升,20h达到高峰后则较平稳(Beclin1:0.39±0.09;MAPLC3:0.59±0.12;pakt:0.09±0.02.较对照组均改变明显,P<0.05)。PI染色显示随照射后时间增加,细胞凋亡率逐渐增加,(照射前1.5%,照射后20h 3.3%),射线照射后A549细胞G2-M期细胞增多(照射前24.9%,照射后53.0%),S期细胞减少(照射前22.3%,照射后13.3%)。免疫荧光染色流式细胞仪检验自噬相关蛋白Beclin1和MAPLC3以及自噬相关信号传导途径蛋白akt和mTOR的表达:检验结果与免疫荧光染色共聚焦显微镜检验结果相似。射线照射后自噬相关基因Beclin1,MAPLC3,pakt蛋白和mRNA的表达水平都有所上升,随照射后时间推移上升明显。但是akt和mTOR蛋白和mTORmRNA表达水平没有明显改变。
结论:在γ射线的作用下,A549细胞内出现了自噬相关基因Beclin1和MAPLC3上调的情况,距照射时间越久,上升越明显,同时γ射线对A549细胞产生了G2-M期阻滞,增加了细胞的凋亡率,减少S期细胞。射线也造成了自噬上游信号pakt表达的上调。
第三部分调节A549细胞自噬的表达对其放疗敏感性的影响及机制探讨
目的:探讨3-MA和rapamycin对于A549细胞的不同作用,研究通过药物调节A549细胞自噬表达后对于放疗效果的影响,进而讨论其机制。
方法:分为对照组,单纯放疗组, 3MA治疗组,rapamycin治疗组,3MA联合放疗组,rapamycin联合放疗组;MTT法检测rapamycin和3MA细胞毒性;细胞集落试验检测各组细胞集落数和存活分数;采用免疫荧光染色细胞计数检测各组Beclin1, MAPLC3, pakt,mTOR蛋白表达;流式细胞仪检测每组细胞周期和Beclin1,MAPLC3,PAKT,mTOR蛋白表达率;RT-PCR检测MAPLC3,survivin, mTOR,Bcl-2,EIF-4EmRNA表达;Western blot检测MAPLC3、pakt、mTOR、COX2蛋白表达
结果:MTT法研究发现:3MA IC10:10Mm,rapamycin IC10:20nM。3MA单独使用时抑制了A549细胞自噬相关基因Beclin1,MAPLC3和pakt的表达,降低了细胞的凋亡率和COX2蛋白表达,对于Survivin,Bcl-2,eIF-2E的mRNA也有一定的抑制作用;rapamycin单独使用时提高了Beclin1,MAPLC3和pakt蛋白的表达率,降低了细胞的凋亡率mTOR蛋白表达,抑制eIF-2E的mRNA;3MA联合放疗组Beclin1,MAPLC3和pakt的表达下降,细胞的凋亡率和G0-G1期细胞明显上升,细胞集落试验显示存活分数明显下降, COX2蛋白表达下降, Bcl-2,eIF-2E mRNA转录减少;rapamycin联合放疗组Beclin1,MAPLC3和pakt的表达上升,细胞的凋亡率明显减少,G2-M期细胞明显上升,细胞集落试验显示存活分数明显增加,mTOR蛋白表达下降, mTOR和eIF-2E mRNA转录减少,Bcl-2增加。
结论:3MA抑制自噬,rapamycin上调自噬的表达;3MA对于放疗有协同作用,rapamycin对于放疗有拮抗作用;3MA放疗协同作用其机制可能与其抑制自噬体的产生,促进细胞凋亡,以及G0-G1期阻滞有关。
The tumorigenesis of lung cancer is a very complex procedure.Many researches were carried out to find effective anticancer methods, which could recover the impairment of organelles at early development of cancer,or eliminate mutated and damaged organelles while lung cancer cells formed ,or even reversed cancer cells to normal cells.
Datas from recent years indicated that the degradation and recycling of organelles were mainly depends on autophagy.Autophagy is a very primitive biological phenomena in the course of life evolution, which existed widely in plants and other lower creatures.Autophagy is also an important way in which protein degradated and organelles eliminated to keep homeostatic,it is a process to get rid of cancer cells in mammals.Many micromolecule protein participated in autophagy,what we understood well is Beclin1 gene and Beclin1 protein,microtubule-associated protein light chain3(MAPLC3) is another mark for autophagysome immunodetection. The basal autophagy level of cells keep lowly to maintain homeostatic situationin usually, autophagy level is up-regulated rapidly when crisis come. For example: growth factor deficiency、starvation、cell reconstruction、or too much damaged cell organelle or metabolic waste.Twe regulater of nutrition sensor regulates autophagy:target of rapamycin(TOR) kinase and eukaryotic initiation factor 2 (eIF2 ) kinase Gcn2. TOR shut off autophagy signal when nutrition adequacy;eIF2 start autophagy signal when starvation. There are many genes lies down-stream of TOR kinase, which participate autophagysome development、confluence、maturement、recycling. Rapamycin is antagon for TOR receptor and up-regulate autophagy,while 3-methyladenine 3MA inhibit autophagy formation.
Autophagy level is closely related cancer cells development, transformation, and sensitivity of therepy.At early stage of cancer cell formation, provoked autophagy help to eliminate damaged cell organelle、and degrade the noxious substance in endochylema(eg: peroxidate)as well, pretect normal cells from cancerization.Autophagy level modulation might be cooperation or rivalry with chemotherapy to cancer cells compares to single use of chemotherapy drugs, apoptosis rate of cancer cells fluctuates with autophagy level.Down-regulation of autophagy level would make organelles elimination breakdown and accelerate cell death to improve therapeutic efficacy.
In order to investigate the relationship between autophagy and NSCLC and the possibility sensitization effect of radiotherapy after autophagy expression alteration,we detected the autophagy-related gene expression level of clinical NSCLC samples to elucidate the relationship of autophagy level and NSCLC generation and development; tested autophagy and its up-stream signal conduction alteration of lung cancer cell line A549;investigated changes of radiotherapy sensitivity and autophagy correlated protein and some drug resistance gene when autophagy level of A549 altered. We also detect the A549 cell cycle and apoptosis rate to explain its mechanism to explain the association between autophagy and apoptosis.
In our research, Fresh 72 NSCLC tissue samples were obtained immediately from the resected specimens of NSCLC patients who underwent pulmonary lobectomy at the department of Thoracic Surgery, Union Hospital, between Apr 2006 and Oct 2006. The protein expression of Beclin1 and MAPLC3 in tumor tissues、adjucent noncancerous tissues、and normal tissues from 72 specimens of NSCLC cases were observed by immunofluorescence staining and Western blot, and mRNA expression were tested by reverse transcription polymerase chain reaction (RT-PCR). Then, we cultivated lung cancer cell line A549 and detect autophagy related protein and mRNA expression after radiation exposure,at the same time, the upper stream gene akt and mTOR were tested. We altered autophagy expression with 3MA or rapamycin, detected the inhibition ratio and apoptosis rate in each group together with the cell cycle changing; cell colony essay was used to detect different degree ofγ-ray suppression at distinct autophagy levels; MAPLC3、pakt、 mTOR、COX2 protein expression were detected by Western blot; MAPLC3、survivin、mTOR、Bcl-2、EIF-4E mRNA were detected by RTPCR. Immunofluorescence staining of Beclin1 and MAPLC3 was done for positive rate detection. All results were combined to analyze the relationship between autophagy and apoptosis.
Immunofluorescence staining and West blot and RT-CCR indicated that the expression of Beclin1 and MAPLC3 in NSCLC was significantly lower than that in adjucent non-cancerous tissues and normal tissues .γ-ray could up-regulate the autophagy related gene of A549 which is time-dependent. The regulation effect of 3MA and rapamycin were observed, 3MA was antagonism while rapamycin was in coordination with radiotherapy. The enhanced inhibition effect ofγray to A549 could be connected with the alteration of apoptosis related protein and cell cycle blocken by 3MA.
Our research group have done series of research about the relationship between autophagy and lung cancer. Also some original discovery were found which encourage us keep hardworkingon advanced work in the future.
PARTⅠExpression of Autophagy-Related Beclin1 and MAPLC3 Gene in Non-Small Cell Lung Cancer
OBJECTIVE:Some studies have indicated the down-regulation of autophagy-related gene might result in tumorigenisis. This study was to investigate the expression and significance of autophagy-related gene Beclin1 and Microtubule-associated protein 1 light chain 3 (MAPLC3) gene in human non-small cell lung cancer (NSCLC).
METHODS: The protein expression of Beclin1 and MAPLC3 in tumor tissues、adjucent noncancerous tissues、and normal tissues from 72 specimens of NSCLC cases were detected by immunofluorescence staining and Western blot, and mRNA expression were tested by reverse transcription polymerase chain reaction (RT-PCR).
RESULTS: Immunofluorescence staining showed that the expression rate of Beclin1 in NSCLC (8.3%) was significantly lower than that in adjucent non-cancerous tissues (100%) and normal tissues (100%) (Χ2=199.40, p=0.00); the expression rate of MAPLC3 in NSCLC(13.9%) was also significantly lower than that in adjucent non-cancerous tissues (100%) and normal tissues (100%) (Χ2=182.75,P=0.00).The mRNA of Beclin1 levels significantly lower in NSCLC (1.30±0.07)than that in adjucent noncancerous tissues(1.69±0.10) and normal tissues (1.67±0.08)(F=6.6, P= 0. 00); the mRNA level of MAPLC3 in NSCLC (4.55±0.31)also significantly lower than in adjucent noncancerous tissues(6.73±0.37 ) and normal tissues ( 6.90±0.37 )(F=14.1,P=0.00). Western blot indicated the similar results that there was significantly down-regulation of the protein level of Beclin1 and MAPLC3 in NSCLC than that of in adjucent noncancerous tissues and normal tissues (Beclin1:3.49+0.29, 5.31+0.46, 6.33+0.58, F=9.73, p<0.01; MAPLC3:2.43+0.26, 3.12+0.28, 3.41+0.30, F=3.22, p=0.04).。
CONCLUSIONS:The protein and mRNA expression of Beclin1 and MAPLC3 are significantly lower in NSCLC than in adjucent noncancerous tissues and normal tissues, there is no significantly difference in adjucent noncancerous and normal tissues.
PARTⅡ:The effect of radiotherapy to autophgy-related gene and signal transduction path up-stream autophagy of A549
OBJECTIVE:To investigate the effect ofγ-ionising radiation on lung cancer cell line A549, we detected the cell cycle and the expression of autophagy-related gene Beclin1 and MAPLC3 of A549 at different time spot after irradiation, and the PI3K/akt signal transduction path upstream of autophagy .
METHODS:Control group and experimental groups at different time spot after irradiation were setted up.In each group, Beclin1、MAPLC3、pakt、mTOR positive cells were counted after immunofluorescence staining by flow cytometry or under laser confocal microscope, cell cycle was detected after PI staining and by flow cytometry. Beclin1、MAPLC3、pakt、mTOR mRNA and protein were detected by RT-PCR and Western blot
RESULTS: Immunofluorescence staining showed that the expression rate of autophagy related gene in A549 was low(Beclin1:0.13±0.04,MAPLC3:0.25±0.05),the basal expression rate of signal transduction protein up stream of autophagy was low too(mTOR:0.09±0.03,pakt:0.03±0.01),positive rate ascensus after irradiation and was stable after 20h(Beclin1:0.39±0.09; MAPLC3:0.59±0.12; pakt:0.09±0.02,P<0.05).With time increasing after irradation, apoptosis rate raised(1.5% and 6.3% before and 20h after irradation),more G2-M stage cells(24.9% and 53.0% before and 20h after irradation)less S stage cells(22.3% and 3.3% before and 20h after irradation) were observed. Similar results were got when Beclin1、MAPLC3、pakt、mTOR were detected by flow cytometry after Immunofluorescence staining as under laser confocal microscope. Beclin1、MAPLC3、pakt protein and mRNA level of A549 raised after irradiation and more obviously with time-lapse,while akt and mTOR protein and mRNA level was stable.
CONCLUSIONS: Afterγray irradiation,autophagy related gene Beclin1 and MAPLC3 levels were upregulated and more obviously with time-lapse, there also be more G2-M stage cells and higher apoptosis rate but less S stage cells after irradiation, pakt upstreams autophagy level raised too.
PARTⅢ: The influence of autophagy expression modulation on A549 cell radiotherapy sensitivity and its mechanism
Objective:To investigate different effect on autophagy-related gene of 3MA and rapamycin, and the radiotherapy sensitivity to A549 at different autophagy levels with 3MA or rapamycin, to discuss its mechanism.
Methods:The subjects were divided into 6 groups: control group, radiotherapy group, 3MA therapy group, rapamycin therapy group, 3MA and radiotherapy group, rapamycin and radiotherapy group; cytotoxicity of 3MA and rapamycin were detected by MTT, cell colony number and survival score were count with cell colony essay; Beclin1, MAPLC3, pakt,mTOR protein expression rate were detected by flow cytometry after immunofluorescence staining; cell life cycle was too detected by flow cytometry; MAPLC3,survivin, mTOR,Bcl-2,EIF-4EmRNA expression was detected by RTPCR; MAPLC3、pakt、mTOR、COX2 protein expression was detected by Western blot
Results: 3MA IC10:10Mm,rapamycin IC10:20nM by MTT. Autophagy-related gene Beclin1,MAPLC3 and pakt were inhibited and apoptosis rate was low and so was Survivin、Bcl-2、EIF-4E mRNA,COX2 protein in 3MA therapy group; Beclin1、MAPLC3 and pakt protein level was raised,cell apaptosis rate and mTOR protein were inhibited in rapamycin therapy gruop; in 3MA and radiotherapy group,the expression of Beclin1,MAPLC3 and pakt gene was inhibited and threre were high apoptosis rate and more G0-G1 cells ,less COX2 protein and less Bcl-2 and EIF-4E mRNA ,survival score descend obviously; in rapamycin and radiotherapy group, Beclin1 , MAPLC3 and pakt were up-regulated,cell apoptosis rate descend; more G2-M cells, survival score increased,less mTOR and EIF-4E, more Bcl-2 expression.
Conclusions: 3MA down-regulated while rapamycin up-regulate autophagy expression; radiotherapy sensitivity was increased by 3MA while decreased by rapamycin; the mechanism may be related to autophagysome inhibition by 3MA, cell apoptosis enhancement, G0-G1 blockage.
近年来的研究显示:细胞器的代谢主要依靠自噬途径。在生命的进化过程中,自噬是一个古老的生物学现象,广泛存在于植物和其它较低级生命体中,是生物降解胞内蛋白,完成细胞器转化,保持内环境稳定的重要方式,也是哺乳动物清除癌细胞的手段之一。自噬是一个有大量小分子蛋白质参与的生物学过程,人类自噬过程中比较明确的和研究较多的是Beclin1基因和其编码蛋白。自噬体形成必需的微管相关蛋白轻链3 (MAPLC3)也常常用于自噬体的检测。
出于维持细胞内环境稳定的目的,正常情况下细胞自噬的基础水平保持在一个较低的状态,在某些“危机”状况下可以快速上调,如:生长激素缺乏、饥饿、细胞重建、细胞内出现过多的受损细胞器或代谢废物等。自噬受两种进化中形成的营养感受器调节:TOR激酶(target of rapamycin (TOR) kinase)在营养充分时关闭自噬信号,真核起始因子2激酶Gcn2(eukaryotic initiation factor 2 (eIF2 ) kinase Gcn2)及其下游靶位Gcn4-自噬基因的转录反式作用子,在饥饿时启动自噬信号。TOR激酶的下游是一些编码ATG蛋白的基因,参与自噬的发生,融合,成熟,再循环。雷帕霉素是TOR受体的拮抗剂,因此能够上调自噬。另外较常用3甲基腺嘌啉(3MA)抑制自噬。
自噬和癌细胞的形成、转化、治疗敏感性密切相关。癌细胞形成早期刺激自噬能够清除受损细胞器,分解细胞内过多的有害物质(如过氧化物等),阻止正常细胞向癌细胞转化,调控自噬的表达和化疗药物联合对肿瘤细胞的生长有协同或拮抗作用,表现为与单纯用化疗药物相比,调控自噬表达后肿瘤细胞的凋亡率发生了变化。在化疗或放疗中抑制自噬有可能导致癌细胞无法清除受损细胞器,加速细胞死亡,强化治疗效果。
为了研究自噬和非小细胞肺癌的关系,以及调节自噬的表达可能产生的放疗增敏效果,我们检测了经手术切除的非小细胞肺癌(NSCLC)临床标本中的自噬相关基因表达状态,探讨自噬的表达情况和NSCLC发生和发展的可能关系;观察了肺癌细胞系A549在γ射线照射下自噬和自噬上游信号传导系统的改变;研究了改变肺腺癌细胞系A549中自噬表达后,A549细胞对于放射治疗敏感性的变化以及自噬相关通道蛋白和一些耐药基因改变,检验肿瘤细胞周期和凋亡率,进而对其机制进行探讨,探究自噬与凋亡的内在联系和在程序性细胞死亡(PCD)的作用和意义。
本研究中,我们采集了2006年4月至2006年10月武汉协和医院胸外科手术切除的非小细胞肺癌新鲜标本72例,冰冻病理切片免疫荧光染色检测肺癌组织,癌旁组织,正常组织中自噬特异性基因Beclin1和MAPLC3表达;提取总蛋白和总RNA,采用Westernblot和RT-PCR检测Beclin1和MAPLC3蛋白表达和mRNA转录情况并且进行半定量分析。培养肺腺癌细胞系A549,对其采用γ射线照射,检测在射线作用下自噬相关蛋白和mRNA以及上游akt和mTOR的表达。用3甲基腺嘌呤(3-methyladenine 3MA)和雷帕霉素(Rapamycin)改变其自噬的表达(3MA抑制自噬表达,Rapamycin则上调自噬表达),收集细胞后检测不同自噬表达状态下其抑制率和细胞凋亡率,以及细胞周期改变情况;细胞集落试验检测不同自噬表达情况下射线对细胞增殖的抑制情况;Western blot检测MAPLC3、pakt、mTOR、COX2等蛋白表达,RTPCR检测MAPLC3、survivin、mTOR、Bcl-2、EIF-4E的mRNA转录情况水平。Beclin1和MAPLC3免疫荧光染色流式细胞检测A549细胞自噬相关蛋白表达率,分析凋亡和自噬在PCD中的关系。
对NSCLC临床切除标本免疫荧光染色和蛋白mRNA检测显示, Beclin1和MAPLC3在肺癌组织中表达低于在癌旁组织和非癌组织,而癌旁组织和正常组织中表达差异无统计学意义。γ射线能够上调A549细胞自噬的表达,且呈时间依赖性。3MA和rapamycin的自噬调节效果明显,3MA和放疗有拮抗作用,而rapamycin则有协同放疗作用,抑制自噬可能通过部分凋亡相关蛋白的改变和细胞周期的阻滞作用影响放疗效果。
本课题组对自噬和肺癌的关系做了一系列研究,有了一些新的发现。但是由于时间的关系,我们的实验尚局限于较浅的层面,在今后的工作中我们会作进一步深入研究。
第一部分自噬相关基因在NSCLC中的表达
目的:研究发现自噬相关基因表达的下调和肿瘤的发生关系密切。本研究检测人小细胞肺癌标本中自噬相关基因Beclin1和微管相关蛋白轻链3(Microtubule-associated protein 1 light chain 3 MAPLC3)的表达并探讨其意义。
方法:采用免疫荧光染色,逆转录-聚合酶链法(Reverse Transcription-Polymerase Chain Reaction RT-PCR)和Western blot法检测肺癌组织,癌旁组织,正常肺组织的Beclin1和MAPLC3蛋白、mRAN表达。
结果:免疫荧光染色显示Beclin1在肺癌组织中表达较低,阳性率为8.3%,在癌旁组织和正常组织阳性率均为100%(Χ2=199.40,p=0.00);MAPLC3在肺癌组织中表达阳性率为13.9%,在癌旁组织和正常组织阳性率均为100%(Χ2=182.75,P=0.00)。Beclin1 mRNA在肺癌组织中的相对表达量(1.30±0.07),与在癌旁组织(1.69±0.10)及正常组织中(1.67±0.08)的相对表达量比较差异有统计学意义(F=6.6, P= 0. 00);MAPLC3mRNA在肺癌组织中相对表达量(4.55±0.31),与在癌旁组织(6.73±0.37)及正常组织(6.90±0.37)中的相对表达量比较差异亦有统计学意义(F=14.1,P=0.00)。Western blot检测Beclin1蛋白在肺癌组织中的相对表达量(3.49±0.29),和癌旁组织(5.31±0.46),正常组织(6.33±0.58)中的相对表达量比较差异有统计学意义(F=9.73,P<0.01),MAPLC3蛋白在肺癌组织中的相对表达量(2.43±0.26),和癌旁组织(3.12±0.28),正常组织(3.41±0.30)中的相对表达量比较差异也有统计学意义(F=3.22,P=0.04)。
结论:自噬相关基因Beclin1和MAPLC3的mRNA及蛋白在正常肺组织和肺癌组织内均有表达,表达的相对量在肺癌组织中较癌旁组织和正常组织低,在癌旁和正常肺组织中表达接近。
第二部分放射治疗对A549细胞自噬及其相关基因和自噬上游信号传导途径的影响
目的:为了探讨γ电离辐射对于肺癌细胞A549的影响,本试验检测了γ射线照射不同时间后A549细胞自噬相关基因Beclin1和MAPLC3的表达和细胞周期改变,同时检测了自噬上游信号传导通道PI3K/AKT相关蛋白和mRNA的改变,并讨论其机制。
方法:设立对照组和照射后不同时间取材的试验组,采用细胞免疫荧光染色镜下计数检测照射不同时间后Beclin1、MAPLC3、pakt、mTOR的阳性细胞表达率,PI染色流式细胞仪检测照射不同时间后细胞周期的改变。RT-PCR和Western blot检测Beclin1、MAPLC3、pakt、mTOR的mRNA和蛋白表达。
结果:免疫荧光染色显示,未经照射的肺腺癌细胞系A549自噬相关蛋白免疫荧光阳性率较低(Beclin1:0.13±0.04,MAPLC3:0.25±0.05),自噬相关信号传导通道蛋白基础表达率也较低(mTOR:0.09±0.03,pakt:0.03±0.01),照射后上升,20h达到高峰后则较平稳(Beclin1:0.39±0.09;MAPLC3:0.59±0.12;pakt:0.09±0.02.较对照组均改变明显,P<0.05)。PI染色显示随照射后时间增加,细胞凋亡率逐渐增加,(照射前1.5%,照射后20h 3.3%),射线照射后A549细胞G2-M期细胞增多(照射前24.9%,照射后53.0%),S期细胞减少(照射前22.3%,照射后13.3%)。免疫荧光染色流式细胞仪检验自噬相关蛋白Beclin1和MAPLC3以及自噬相关信号传导途径蛋白akt和mTOR的表达:检验结果与免疫荧光染色共聚焦显微镜检验结果相似。射线照射后自噬相关基因Beclin1,MAPLC3,pakt蛋白和mRNA的表达水平都有所上升,随照射后时间推移上升明显。但是akt和mTOR蛋白和mTORmRNA表达水平没有明显改变。
结论:在γ射线的作用下,A549细胞内出现了自噬相关基因Beclin1和MAPLC3上调的情况,距照射时间越久,上升越明显,同时γ射线对A549细胞产生了G2-M期阻滞,增加了细胞的凋亡率,减少S期细胞。射线也造成了自噬上游信号pakt表达的上调。
第三部分调节A549细胞自噬的表达对其放疗敏感性的影响及机制探讨
目的:探讨3-MA和rapamycin对于A549细胞的不同作用,研究通过药物调节A549细胞自噬表达后对于放疗效果的影响,进而讨论其机制。
方法:分为对照组,单纯放疗组, 3MA治疗组,rapamycin治疗组,3MA联合放疗组,rapamycin联合放疗组;MTT法检测rapamycin和3MA细胞毒性;细胞集落试验检测各组细胞集落数和存活分数;采用免疫荧光染色细胞计数检测各组Beclin1, MAPLC3, pakt,mTOR蛋白表达;流式细胞仪检测每组细胞周期和Beclin1,MAPLC3,PAKT,mTOR蛋白表达率;RT-PCR检测MAPLC3,survivin, mTOR,Bcl-2,EIF-4EmRNA表达;Western blot检测MAPLC3、pakt、mTOR、COX2蛋白表达
结果:MTT法研究发现:3MA IC10:10Mm,rapamycin IC10:20nM。3MA单独使用时抑制了A549细胞自噬相关基因Beclin1,MAPLC3和pakt的表达,降低了细胞的凋亡率和COX2蛋白表达,对于Survivin,Bcl-2,eIF-2E的mRNA也有一定的抑制作用;rapamycin单独使用时提高了Beclin1,MAPLC3和pakt蛋白的表达率,降低了细胞的凋亡率mTOR蛋白表达,抑制eIF-2E的mRNA;3MA联合放疗组Beclin1,MAPLC3和pakt的表达下降,细胞的凋亡率和G0-G1期细胞明显上升,细胞集落试验显示存活分数明显下降, COX2蛋白表达下降, Bcl-2,eIF-2E mRNA转录减少;rapamycin联合放疗组Beclin1,MAPLC3和pakt的表达上升,细胞的凋亡率明显减少,G2-M期细胞明显上升,细胞集落试验显示存活分数明显增加,mTOR蛋白表达下降, mTOR和eIF-2E mRNA转录减少,Bcl-2增加。
结论:3MA抑制自噬,rapamycin上调自噬的表达;3MA对于放疗有协同作用,rapamycin对于放疗有拮抗作用;3MA放疗协同作用其机制可能与其抑制自噬体的产生,促进细胞凋亡,以及G0-G1期阻滞有关。
The tumorigenesis of lung cancer is a very complex procedure.Many researches were carried out to find effective anticancer methods, which could recover the impairment of organelles at early development of cancer,or eliminate mutated and damaged organelles while lung cancer cells formed ,or even reversed cancer cells to normal cells.
Datas from recent years indicated that the degradation and recycling of organelles were mainly depends on autophagy.Autophagy is a very primitive biological phenomena in the course of life evolution, which existed widely in plants and other lower creatures.Autophagy is also an important way in which protein degradated and organelles eliminated to keep homeostatic,it is a process to get rid of cancer cells in mammals.Many micromolecule protein participated in autophagy,what we understood well is Beclin1 gene and Beclin1 protein,microtubule-associated protein light chain3(MAPLC3) is another mark for autophagysome immunodetection. The basal autophagy level of cells keep lowly to maintain homeostatic situationin usually, autophagy level is up-regulated rapidly when crisis come. For example: growth factor deficiency、starvation、cell reconstruction、or too much damaged cell organelle or metabolic waste.Twe regulater of nutrition sensor regulates autophagy:target of rapamycin(TOR) kinase and eukaryotic initiation factor 2 (eIF2 ) kinase Gcn2. TOR shut off autophagy signal when nutrition adequacy;eIF2 start autophagy signal when starvation. There are many genes lies down-stream of TOR kinase, which participate autophagysome development、confluence、maturement、recycling. Rapamycin is antagon for TOR receptor and up-regulate autophagy,while 3-methyladenine 3MA inhibit autophagy formation.
Autophagy level is closely related cancer cells development, transformation, and sensitivity of therepy.At early stage of cancer cell formation, provoked autophagy help to eliminate damaged cell organelle、and degrade the noxious substance in endochylema(eg: peroxidate)as well, pretect normal cells from cancerization.Autophagy level modulation might be cooperation or rivalry with chemotherapy to cancer cells compares to single use of chemotherapy drugs, apoptosis rate of cancer cells fluctuates with autophagy level.Down-regulation of autophagy level would make organelles elimination breakdown and accelerate cell death to improve therapeutic efficacy.
In order to investigate the relationship between autophagy and NSCLC and the possibility sensitization effect of radiotherapy after autophagy expression alteration,we detected the autophagy-related gene expression level of clinical NSCLC samples to elucidate the relationship of autophagy level and NSCLC generation and development; tested autophagy and its up-stream signal conduction alteration of lung cancer cell line A549;investigated changes of radiotherapy sensitivity and autophagy correlated protein and some drug resistance gene when autophagy level of A549 altered. We also detect the A549 cell cycle and apoptosis rate to explain its mechanism to explain the association between autophagy and apoptosis.
In our research, Fresh 72 NSCLC tissue samples were obtained immediately from the resected specimens of NSCLC patients who underwent pulmonary lobectomy at the department of Thoracic Surgery, Union Hospital, between Apr 2006 and Oct 2006. The protein expression of Beclin1 and MAPLC3 in tumor tissues、adjucent noncancerous tissues、and normal tissues from 72 specimens of NSCLC cases were observed by immunofluorescence staining and Western blot, and mRNA expression were tested by reverse transcription polymerase chain reaction (RT-PCR). Then, we cultivated lung cancer cell line A549 and detect autophagy related protein and mRNA expression after radiation exposure,at the same time, the upper stream gene akt and mTOR were tested. We altered autophagy expression with 3MA or rapamycin, detected the inhibition ratio and apoptosis rate in each group together with the cell cycle changing; cell colony essay was used to detect different degree ofγ-ray suppression at distinct autophagy levels; MAPLC3、pakt、 mTOR、COX2 protein expression were detected by Western blot; MAPLC3、survivin、mTOR、Bcl-2、EIF-4E mRNA were detected by RTPCR. Immunofluorescence staining of Beclin1 and MAPLC3 was done for positive rate detection. All results were combined to analyze the relationship between autophagy and apoptosis.
Immunofluorescence staining and West blot and RT-CCR indicated that the expression of Beclin1 and MAPLC3 in NSCLC was significantly lower than that in adjucent non-cancerous tissues and normal tissues .γ-ray could up-regulate the autophagy related gene of A549 which is time-dependent. The regulation effect of 3MA and rapamycin were observed, 3MA was antagonism while rapamycin was in coordination with radiotherapy. The enhanced inhibition effect ofγray to A549 could be connected with the alteration of apoptosis related protein and cell cycle blocken by 3MA.
Our research group have done series of research about the relationship between autophagy and lung cancer. Also some original discovery were found which encourage us keep hardworkingon advanced work in the future.
PARTⅠExpression of Autophagy-Related Beclin1 and MAPLC3 Gene in Non-Small Cell Lung Cancer
OBJECTIVE:Some studies have indicated the down-regulation of autophagy-related gene might result in tumorigenisis. This study was to investigate the expression and significance of autophagy-related gene Beclin1 and Microtubule-associated protein 1 light chain 3 (MAPLC3) gene in human non-small cell lung cancer (NSCLC).
METHODS: The protein expression of Beclin1 and MAPLC3 in tumor tissues、adjucent noncancerous tissues、and normal tissues from 72 specimens of NSCLC cases were detected by immunofluorescence staining and Western blot, and mRNA expression were tested by reverse transcription polymerase chain reaction (RT-PCR).
RESULTS: Immunofluorescence staining showed that the expression rate of Beclin1 in NSCLC (8.3%) was significantly lower than that in adjucent non-cancerous tissues (100%) and normal tissues (100%) (Χ2=199.40, p=0.00); the expression rate of MAPLC3 in NSCLC(13.9%) was also significantly lower than that in adjucent non-cancerous tissues (100%) and normal tissues (100%) (Χ2=182.75,P=0.00).The mRNA of Beclin1 levels significantly lower in NSCLC (1.30±0.07)than that in adjucent noncancerous tissues(1.69±0.10) and normal tissues (1.67±0.08)(F=6.6, P= 0. 00); the mRNA level of MAPLC3 in NSCLC (4.55±0.31)also significantly lower than in adjucent noncancerous tissues(6.73±0.37 ) and normal tissues ( 6.90±0.37 )(F=14.1,P=0.00). Western blot indicated the similar results that there was significantly down-regulation of the protein level of Beclin1 and MAPLC3 in NSCLC than that of in adjucent noncancerous tissues and normal tissues (Beclin1:3.49+0.29, 5.31+0.46, 6.33+0.58, F=9.73, p<0.01; MAPLC3:2.43+0.26, 3.12+0.28, 3.41+0.30, F=3.22, p=0.04).。
CONCLUSIONS:The protein and mRNA expression of Beclin1 and MAPLC3 are significantly lower in NSCLC than in adjucent noncancerous tissues and normal tissues, there is no significantly difference in adjucent noncancerous and normal tissues.
PARTⅡ:The effect of radiotherapy to autophgy-related gene and signal transduction path up-stream autophagy of A549
OBJECTIVE:To investigate the effect ofγ-ionising radiation on lung cancer cell line A549, we detected the cell cycle and the expression of autophagy-related gene Beclin1 and MAPLC3 of A549 at different time spot after irradiation, and the PI3K/akt signal transduction path upstream of autophagy .
METHODS:Control group and experimental groups at different time spot after irradiation were setted up.In each group, Beclin1、MAPLC3、pakt、mTOR positive cells were counted after immunofluorescence staining by flow cytometry or under laser confocal microscope, cell cycle was detected after PI staining and by flow cytometry. Beclin1、MAPLC3、pakt、mTOR mRNA and protein were detected by RT-PCR and Western blot
RESULTS: Immunofluorescence staining showed that the expression rate of autophagy related gene in A549 was low(Beclin1:0.13±0.04,MAPLC3:0.25±0.05),the basal expression rate of signal transduction protein up stream of autophagy was low too(mTOR:0.09±0.03,pakt:0.03±0.01),positive rate ascensus after irradiation and was stable after 20h(Beclin1:0.39±0.09; MAPLC3:0.59±0.12; pakt:0.09±0.02,P<0.05).With time increasing after irradation, apoptosis rate raised(1.5% and 6.3% before and 20h after irradation),more G2-M stage cells(24.9% and 53.0% before and 20h after irradation)less S stage cells(22.3% and 3.3% before and 20h after irradation) were observed. Similar results were got when Beclin1、MAPLC3、pakt、mTOR were detected by flow cytometry after Immunofluorescence staining as under laser confocal microscope. Beclin1、MAPLC3、pakt protein and mRNA level of A549 raised after irradiation and more obviously with time-lapse,while akt and mTOR protein and mRNA level was stable.
CONCLUSIONS: Afterγray irradiation,autophagy related gene Beclin1 and MAPLC3 levels were upregulated and more obviously with time-lapse, there also be more G2-M stage cells and higher apoptosis rate but less S stage cells after irradiation, pakt upstreams autophagy level raised too.
PARTⅢ: The influence of autophagy expression modulation on A549 cell radiotherapy sensitivity and its mechanism
Objective:To investigate different effect on autophagy-related gene of 3MA and rapamycin, and the radiotherapy sensitivity to A549 at different autophagy levels with 3MA or rapamycin, to discuss its mechanism.
Methods:The subjects were divided into 6 groups: control group, radiotherapy group, 3MA therapy group, rapamycin therapy group, 3MA and radiotherapy group, rapamycin and radiotherapy group; cytotoxicity of 3MA and rapamycin were detected by MTT, cell colony number and survival score were count with cell colony essay; Beclin1, MAPLC3, pakt,mTOR protein expression rate were detected by flow cytometry after immunofluorescence staining; cell life cycle was too detected by flow cytometry; MAPLC3,survivin, mTOR,Bcl-2,EIF-4EmRNA expression was detected by RTPCR; MAPLC3、pakt、mTOR、COX2 protein expression was detected by Western blot
Results: 3MA IC10:10Mm,rapamycin IC10:20nM by MTT. Autophagy-related gene Beclin1,MAPLC3 and pakt were inhibited and apoptosis rate was low and so was Survivin、Bcl-2、EIF-4E mRNA,COX2 protein in 3MA therapy group; Beclin1、MAPLC3 and pakt protein level was raised,cell apaptosis rate and mTOR protein were inhibited in rapamycin therapy gruop; in 3MA and radiotherapy group,the expression of Beclin1,MAPLC3 and pakt gene was inhibited and threre were high apoptosis rate and more G0-G1 cells ,less COX2 protein and less Bcl-2 and EIF-4E mRNA ,survival score descend obviously; in rapamycin and radiotherapy group, Beclin1 , MAPLC3 and pakt were up-regulated,cell apoptosis rate descend; more G2-M cells, survival score increased,less mTOR and EIF-4E, more Bcl-2 expression.
Conclusions: 3MA down-regulated while rapamycin up-regulate autophagy expression; radiotherapy sensitivity was increased by 3MA while decreased by rapamycin; the mechanism may be related to autophagysome inhibition by 3MA, cell apoptosis enhancement, G0-G1 blockage.
引文
1. Cuervo A M, Dice J F. A receptor for the selective uptake and degradation of proteins by lysosomes. Science,1996,273(5274):501-503.
2. Takahiro Shintani, Daniel J Klionsky.Autophagy in Health and Disease:A Double Edged Sword.Science,2004,306(5698) :990-995.
3. Ogier-Denis E, Codogno P. Autophagy: a barrier or an adaptive response to cancer. Biochim Biophys Acta,2003, 1603(2):113-128.
4. Klionsky D J , Emr S D. Autophagy as a regulated pathway of cellular degradation. Science ,2000,290(5497):1717-1721.
5. Mortimore G E, Poso A R. Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr,1987,7:539-564.
6. Friedman L S.The search for BRACA1.Cancer Res.1994,54(24):6374-6382.
7. Edinger A L,Thompson C B.Defective autophagy leads to cancer.Cancer Cell,2003,4(6):422-424.
8. Qu X, Yu J,Bhagat G et al. Promotion of tumorgenesis by heterozygous disruption of the beclin1 autopahgy gene. J Clin Invest,2003, 112(12) : 1809 -1820.
9. Kabeya Y, Mizushima N, Ueno T,et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J,2003,19(21):5720-5728.
10. Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol.,2004, 36(12):2503-2518.
11. Mizushima N, Yamamoto A, Hatano M, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.J Cell Biol, 2001, 152(4): 657-668.
12. Toth S, Nagy K, Palfia Z,et al. Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up regulation in allpremalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation. Cell Tissue Res, 2002, 309(3) : 409 -416.
13. Cuervo A M. Autophagy: in sickness and in health. Trends Cell Biol,2004, 14(2): 70-77.
14.段振玲,彭芝兰,王赞宏.自噬基因Beclin1与上皮性卵巢癌发生和发展的相关性研究.癌症,2007,26(3):258-263.
15. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene,2004,23(16): 2891-2906.
16. Holm E,Hildebrandt W ,Kinscherf R. Low postabsorptive net protein degradation in male cancer patients: lack of sensitivity to regulatory amino acids.Oncol Rep,2007 ,17(3):695-700.
17. Botti J, Dajaheri-Merqny M, Pilatte Y. Autophagy signaling and the cogwheels of cancer.Autophagy. Autophagy. 2006 ,2(2): 67-73.
18. Klionsky D J . Autophagy,Curr Biol ,2005,15(8): 282-283.
19. Marino G, Lopez-Otin C. Autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol Life Sci,2004,61 (12): 1439-1454.
20. Eskelinen E L. Maturation of autophagic vacuoles in mammalian cells. Autophagy, 2005, 1(1): 001-010
21. D.J. Klionsky, S.D. Emr, Autophagy as a regulated pathway of cellular degradation, Science 290 (2000) 1717–1721.
22. Y. Ohsumi, Molecular dissection of autophagy: two ubiquitin-like systems, Nat. Rev. Mol. Cell. Biol. 2 (2001) 211–216.
23. N. Mizushima, Y. Ohsumi, T. Yoshimori, Autophagosome formation in mammalian cells, Cell. Struct. Funct. 27 (2002)421–429.
24. N. Mizushima, A. Yamamoto, M. Hatano, Y. Kobayashi, Y. Kabeya, K. Suzuki, T.Tokuhisa, Y. Ohsumi, T. Yoshimori,Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells, J. Cell Biol. 152 (2001) 657–667.
25. Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (2000)5720–5728
26. S. Yokota, M. Himeno, J. Roth, D. Brada, K. Kato, Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)phthalate treatment. II. Immunocytochemical analysis of early and late autophagosomes, Eur. J. Cell Biol. 62 (1993) 372–383.
27. R. Masaki, A. Yamamoto, Y. Tashiro, Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver, J. Cell Biol. 104 (1987) 1207–1215.
28. S.P. Elmore, T. Qian, S.F. Grissom, J.J. Lemasters, The mitochondrial permeability transition initiates autophagy in rat hepatocytes, FASEB J. 15 (2001) 2286–2287.
29. Bando K,Nagai H,Matsumoto S et al.Identification of 1-Mb common region at 16q24.1-24.2 deletied in hepatocellular carcinoma.Genes Chromosome Cancer.2000,28,38-44
30. Chen T,Sahi A,Aldaz CM,et al.Deletion map of chromosome 16q ductal incarcinoma in situ of breast :refining a putative tumor surpressor gene region.Cancer Res,1996, 56,5605-09
31. Elo JP,Harkonen P,Kellonen AP,et al.Loss of heterozygosity at 16q24.1-q24.2 is significantly associated with metastatic and aggressive behavior of prostate cancer..Caner Res.1997,57,3356-9
32. Suzuki S,Moore DH,Ginziger DG,et al.An approach to analysis of large scale correlations between genome changes and clinical endopoints in ovarian cancer.Cancer Res,2000,60,5382
1吴一龙,等.2007中国肺癌临床指南,人民卫生出版社,2007年,第一版
2 Cuervo AM. Autophagy: in sickness and in health. Trends Cell Biol,2004, 14: 70-77.
3 Clark AS, West K,Streicher S,et al.Constititive and inducible AKT activity promotes resistance to chemotherapy,trastuzumab,or tamoxifen in breast cancer cells.Mol Cancer Ther.2002 Jul;1(9):707-17
4 Canguilhem B, Pradines A, Baudouin C,et al.RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling. J Biol Chem,2005,280(52),43257-63
5殷蔚伯,谷铣之.肿瘤放射治疗学.北京中国协和医科大学出版社,2002,265
6 D.J. Klionsky, S.D. Emr, Autophagy as a regulated pathway of cellular degradation, Science 290 (2000) 1717–1721.
7 Y. Ohsumi, Molecular dissection of autophagy: two ubiquitin-like systems, Nat. Rev. Mol. Cell. Biol. 2 (2001) 211–216.
8 N. Mizushima, Y. Ohsumi, T. Yoshimori, Autophagosome formation in mammalian cells, Cell. Struct. Funct. 27 (2002)421–429.
9 N. Mizushima, A. Yamamoto, M. Hatano, Y. Kobayashi, Y.Kabeya, K. Suzuki, T. Tokuhisa, Y. Ohsumi, T. Yoshimori,Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells, J. Cell Biol. 152 (2001) 657–667.
10 Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako,T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (2000)5720–5728
11 S. Yokota, M. Himeno, J. Roth, D. Brada, K. Kato, Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)phthalate treatment. II. Immunocytochemical analysis of early and late autophagosomes, Eur. J. Cell
12 R. Masaki, A. Yamamoto, Y. Tashiro, Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver, J. Cell Biol. 104 (1987) 1207–1215.
13 S.P. Elmore, T. Qian, S.F. Grissom, J.J. Lemasters, The mitochondrial permeability transition initiates autophagy in rat hepatocytes, FASEB J. 15 (2001) 2286–2287.
14 Mortimore GE, Poso AR. (1987) Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr 7:539-564.
15 Klionsky DJ and Emr SD. (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717-1721.
16 Roberts P, Moshitch-Moshkovitz S, Kvam E, O’Toole E, Winey M, Goldfarb DS. (2003) Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol Biol Cell 14:129-141.
17 .Reggiori F and Klionsky DJ. (2005) Autophagosomes: biogenesis from scratch? Curr Opin Cell Biol 17(4):415-422.
18 Liang XH, Kleeman LK, Jiang HH et al. (1998) Protection against fetal Sindbis virus encephalitis by Beclin, a novel Bcl-2 interacting protein. J Virol 72:8586-8596.
19 Liang XH, Jackson S, Seaman M et al. (1999) Induction of autophagy and inhibition of tumorigenesis by Beclin 1. Nature 402: 672-676.
20 Thumm M,Kadowaki T.The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants.Mol Genet Genomics, 2001;266,651
21 Gupta VK, Park JO , Jaskowiak NT, et al . Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma . Ann Surg Oncol ,2002 ,9(5) :500～504.
22 Schimidt-Ullrich RK,Mikkelsen RB,Dent P,et al.Radiation-induced Proliferation of the human A431 squamouse carcinoma cells is dependent on EGFR tyrosine PhosPhorylation . Oncogene1997,15:1191-1197.
23 Brown EJ,AlbersMW, Shin TB, et a l. A mammalian p rotein targeted by G1-arresting rapamycin-receptor complex [ J ]. N a tu re ,1994, 369 (6483) : 756
24 Asnaghi L, Bruno P, Priulla M, et a l. mTOR: a p rotein kinase switching between life and death [ J ]. Pharm acol Res , 2004, 50(6) : 545
25 Martin KA, Rzucidlo EM, Merenick BL, et a l. The mTOR /p70 S6K1 pathway regulates vascular smooth muscle cell differentiation[ J ]. Am J Physiol Cell Physiol , 2004, 286 (3) : C507
26 Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt /PKB by the rictor2mTOR comp lex [ J ].S cience , 2005, 307 (5712) : 1098
27 Zhou X, Tna M, Stone Hawthorne V, et al1 Activation of the Akt/mammalian target of rapamycin /4E2BP1 pathway by ErbB2 overexp ression p redicts tumor progression in breast cancers1 Clin Cancer Res, 2004, 10 (20) : 6779267881
28 Klos KS, Wyszomierski SL,Sun M,et al.ErB2 increase vascular endothelial growth factor protein synthesis via activation of mamammalian taget of rapamycin /p70s6K leading to increased angiogenesis and spontaneous metastasis of human breast cancer cells{J}.Cancer Res,2006,66(4);2028
29 ItohN,Semba S,Ito M,et al.Phosphorylation of AKT/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colcrectal carcinoma. Cancer, 2002, 944,3127
30 Fruman DA,M eyer RE,Cantley LC.Phosphoinsitide kinases.Annu Rev Biochem,1998, 67,481
31 Blackhall FH,Pintilie M,Michael M,et al.Expression and prognostic significance of kit,protein kinase B,and mitrogen-activated protein kinase in patient with small cell lung cancer Clin Cancer Res,2003,9,2241
32 Wiederrecht G J, Sabers C J, Brunn G J, et al. Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in eukaryotic cells [ J] . Prog Cell Cycle Res, 1995, 1: 53- 71.
33 Edinger A L, Thompson C B. Akt maintains cell size and survival by ncreasing mTOR-dependent nutrient uptake [ J] . Mol Biol Cell, 2002, 13(7) : 2276- 2288.
34 Dann S G, Selvaraj A, Thomas G.mTOR Complex1-S6K1 signaling: at the crossroads of obesity, diabetes andcancer [ J] . Trends Mol Med, 2007,13( 6) : 252- 259.
35 Jacinto E et al. Nat Rev Mol Cell Biol, 2003, 4: 117
1. Vignot S, Faiver S, Aguirre D, et al1 mTOR2targeted therapy of cancerwith rapamycin drivatives1 Ann Oncol, 2005, 16(4) : 52525371
2. Rowinsky EK1 Targeting the molecular target of rapamycin(mTOR) 1 CurOp in Oncol, 2004, 16 (6) : 56425751
3. Shao, Y., Gao, Z., Marks, P. A. & Jiang, X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc. Natl Acad. Sci. USA, 101, 18030–18035 (2004).
4. Bursch, W. 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 17, 1595–1607 (1996
5.马湘一,王世宣,刘琰,等.雷帕霉素联合紫杉醇诱导卵巢癌细胞A2780和SKOV3凋亡及其分子机制[ J] .癌症, 2007, 26( 4) : 367- 370.
6. Busca R, Bertolotto C, Ortonne J P, et al. Inhibition of the phosphatidylinositol
3-kinase /p70 ( S6) -kinase pathway induces B16 melanoma cell differentiation [ J] . J Biol Chem, 1996, 271( 50) : 31824- 31830.
7. Eng C P, Sehgal S N, Vezina C. Activity of rapamycin (AY-22, 989) against transplanted tumors [ J] . J Antibiot ( Tokyo) , 1984, 37( 10) : 1231-1237.
8. Hosoi H, Dilling M B, Shikata T, et al. Rapamycin causes poorly reversible inhibition of mTOR and induces p53- independent apoptosis in human rhabdomyosarcoma cells [ J] . Cancer Res, 1999, 59( 4) : 886- 894.
9. Shi Y, Frankel A, Radvanyi L G, et al. Rapamycin enhances apoptosis and increases sensitivity to cisplatin in vitro [ J] . Cancer Res, 1995, 55 ( 9) : 1982-1988.
10. Wu Q, Kiguchi K, Kawamoto T, et al. Therapeutic effect of rapamycin on gallbladder cancer in a transgenic mouse model [ J] . Cancer Res, 2007, 67( 8) :3794- 3800.
11. Jimeno A, Kulesza P, Wheelhouse J, et al. Dual EGFR and mTOR targeting in squamous cell carcinoma models,and development of early markers of efficacy [ J] . Br J Cancer, 2007, 96( 6) :952- 959.
12. Semela D, Piguet A C, Kolev M, et al. Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma [ J] . J Hepatology, 2007, 46( 5) : 840- 848.
13. Kobayashi S, Kishimoto T, Kamata S, et al. Rapamycin, a specific inhibitor of the mammalian target of rapamycin, suppresses lymphangiogenesis and lymphatic metastasis [ J] . Cancer Sci, 2007, 98( 5) : 726- 733.
14. Granville C A, Warfel N, Tsurutani J, et al. Identification of a highly effective rapamycin schedule that markedly reduces the size, multiplicity, and phenotypic progression of tobacco carcinogen-induced murine lung tumors[ J] . Clin Cancer Res, 2007, 13( 7) :2281- 2289.
15. 15.Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H. & Balch, W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 130, 781–796 (1995).
16. 16.Inbal, B., Bialik, S., Sabanay, I., Shani, G. & Kimchi, A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J. Cell Biol. 157, 455–468 (2002).
17. Rhoads RE. Regulation of eukaryotic protein synthesis by initiation actors[J ] . J Biol Chem, 1993 ,268 (5) :301723021.
18. Lazatis KA , Montione KS , Sonenberg N. et al . Maglignant transformation by a eukaryotic initiation factor subunit that binds to mRNAs′cap [J ] . Nature ,1990 , 345(7) :5442547.
19. Rinker SCW, Graff JR , De Benedetti , et al . Decreasing the level of translation initiatio factor 4E with antisense RNA caused reversal of ras - mediatedtransformation and tumorigenesis of cloned rat embryo fibroblasts[J ] . Int J Cancer ,1993 ,55(5) :8412847.
20. Seki N , Takasu T , Mandai K, et al . Expression of eukaryotic initiation factor 4E in atypical adenomatous hyperplasia and adenocarcinoma of the human peripheral lung[J ] . Clin Cancer Res , 2002 ,8(10) :304623053.
21. Reed JC,Doctor K,Rojas A,et al.Comparative analysis of apoptosis and inflammation genes of mice and humans.Genome Res,2003,13,1376
22. Hanahan D,Wwinberg RA.The hallmarks of cancer.Cell,2000,100,57-70
23. Li LY,Luo X,Wang X,et al.Endonuclease G is an apaptotic Dnase when released from mitochondria.Nature,2001,412,95
24. Liston P,Fong WG,Korneluk RG.The inhibitors of apotosis:there is more to life than Bcl-2.Oncogene,2003,22,8568
25. Takaoka A,Adachi M,Okuda H,et al.Anti-cell death activity promotes pulmonary metastasis of melanoma cells.Oncogene,1997,14,2971
26. Del Bufalo D,Biroccio A,Leonetti C,et al.Bcl-2 overexpression enhances the metastastic of a human breast cancer line.FASEB J,1997,11,947
27. Martin SS,Leder P.Human MCF10A mammary epithelial cells undergo apoptisis following actin depolymerization that is independent of attachment and rescued by Bcl-2.Mol Cell Biol,2001,21,6529
28. Altieri DC.Survivin,versatile modulation of cell division and apoptosis in cancer. Oncogene,2003,22,8581
29. Kawasaki H,Altieri DC,Lu CD,et al.Inhibititor of apoptosis by surviving predicts shorter surviving rates in colorectal cancer. Cancer Res,1998;58(22):5071-5074
30. Ikehara M, Oshita F, Kameda Y, et al. Expression of surviving correlated with vessel invasion is a marker of poor prognosis in small adenocarcinoma of the lung. Oncol Rep,2002;9(4)835-838
31. Tamm I, Wang Y, Sausville E, et al. IAP-family protein suevivin inhibits caspaseactivity and apoptosis induced by Fas (CD95), Bax, caspase, and anticancer druds. Cancer Res, 1998, 58(23):5315-5320
32. Suzuki A,Ito T, Kawano H, et al. Suvivin initiates procaspase 3/p21 complex formation as a result of interaction with CDK4 to resist Fas-medizted cell death. Oncogene, 2000,19(10): 1346-1353
33. Johnstone RW,Ruefli AA.Lowe SW.Apoptosis:a link between cancer genetics and chemotherapy.Cell,2002,108,153
34. Hersey P,Zhang XD.Overcoming resistance of cancer cell to apoptosis.J Cell Physiol,2003,196:9
35. Hengartner MO.The biochemistry of apoptosis.Nature,2000,407,770
36. Degterev A,Boyce M,Yuan J.A decade of caspases.Oncogene,2003,22,8543
37. Arico S,Pattingre S,Bauvy C,et al.Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT29 cell line.J Biol Chem.2002,277,31,27613-21
38. Altieri DC. Survivin, versatile modulation of cell divisionand apoptosis in cancer [ J ]. Oncogene, 2003, 22 ( 53 ) :8 581-8 589
39.张燕,孙婷,曹祥荣. Survivin基因特异性siRNA腺病毒介导的肿瘤放射增敏作用[ J ].中国肿瘤生物治疗杂志, 2007, 14 (1) : 59-63
1. Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest 2005;115:2679–88.
2. Yokota, S., Himeno, M., Roth, J., Brada, D. & Kato, K. Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)phthalate treatment. II. Immunocytochemical analysis of early and late autophagosomes. Eur. J. Cell Biol. 62, 372–383 (1993).
3. Stromhaug, P. E., Berg, T. O., Fengsrud, M. & Seglen, P. O. Purification and characterization of autophagosomes from rat hepatocytes. Biochem. J. 335, 217–224 (1998).
4. Noda, T., Suzuki, K. & Ohsumi, Y. Yeast autophagosomes: de novo formation of a membrane structure. Trends Cell Biol. 12, 231–25 (2002).
5. Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nat Rev Cancer 2005;5:886–97.
6. Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 2004;16:663–9.
7. Kuma A, Hatano M, Matsui M, et al. The role of autophagy during the early neonatal starvation period. Nature 2004;432:1032–6.
8. Lum JJ, Bauer DE, Kong M, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 2005;120:237–48
9. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 2005;25:1025–40.
10. Degenhardt K, Mathew R, Beaudoin B, Bray K. Autophagy promotes tumor cell survivaland restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 2006; 51-64.
11. Liang XH, Kleeman LK, Jiang HH, et al. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol 1998;72:8586–96.
12. Liang XH, Jackson S, Seaman M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999;402:672–6.
13. 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;100:15077–82.
14. Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003;112:1809–20.
15. Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005;122:927–39.
16. Takacs-Vellai K, Vellai T, Puoti A, et al. Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Curr Biol 2005;15:1513–7.
17. Inoki K, Corradetti MN, Guan KL, Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 2005;37:19–24.
18. Kamada Y, Sekito T, Ohsumi Y. Autophagy in yeast: a TOR-mediated response to nutrient starvation. Curr Top Microbiol Immunol 2004;279:73–84.
19. Arico S, Petiot A, Bauvy C, et al. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem 2001;276:35243–6.
20. Feng Z, Zhang H, Levine AJ, Jin S. The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci USA 2005; 102:8204-9.
21. Tsuneoka M, Umata T, Kimura H, Koda Y, Nakajima M, Kosai K, Takahashi T, Takahashi Y, Yamamoto A. c-myc induces autophagy in rat 3Y1 fibroblast cells. Cell Struct Funct,2003; 28:195-204.
22. Pattingre S, Bauvy C, Codogno P. Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. J Biol Chem 2003; 278:16667-74.
23. Komata T, Kanzawa T, Takeuchi H, Germano IM, Schreiber M, Kondo Y, Kondo S.Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C),p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells. Br J Cancer,2003; 88:1277-80.
24. Browne GJ, Proud CG. A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin. Mol Cell Biol 2004;24:2986–97.
25. Meyuhas O. Synthesis of the translational apparatus is regulated at the translational level. Eur J Biochem 2000;267:6321–30.
26. Mamane Y, Petroulakis E, Rong L, Yoshida K, Ler LW, Sonenberg N. eIF4E-from translation to transformation. Oncogene 2004;23:3172–9.
27. Hardie DG, Carling D, Carlson M. The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 1998;67:821–55.
28. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003;115:577–90.
29. Ryazanov AG, Ward MD, Mendola CE, et al. Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc Natl Acad Sci U S A 1997;94:4884–9.
30. Chen Y, Matsushita M, Nairn AC, et al. Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels. Biochemistry 2001;40:11565–70.
31. Wang X, Li W, Williams M, Terada N, Alessi DR, Proud CG. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J 2001;20:4370–9.
32. Browne GJ, Finn SG, Proud CG. Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398. J Biol Chem,2004;279: 12220– 31.
33. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70.
34. Gunn, J. M., Clark, M. G., Knowles, S. E., Hopgood, M. F. & Ballard, F. J. Reduced rates of proteolysis in transformed cells. Nature 266, 58–60 (1977).
35. Kisen, G. O. et al. Reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells. Carcinogenesis 14, 2501–2505 (1993).
36. Kirkegaard, K., Taylor, M. P. & Jackson, W. T. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nature Rev. Microbiol. 2, 301–314 (2004).
37. Otsuka, H. & Moskowitz, M. Differences in the rates of protein degradation in untransformed and transformed cell lines. Exp. Cell Res. 112, 127–35 (1978).
38. Schwarze, P. E. & Seglen, P. O. Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats. Exp. Cell Res. 157, 15–28 (1985).
39. Russell SE, Hickey GI, Lowry WS, White P, Atkinson RJ. Allele loss from chromosome 17 in ovarian cancer. Oncogene 1990;5:1581–3
40. Futreal PA, Soderkvist P, Marks JR, et al. Detection of frequent allelic loss on proximal chromosome 17q in sporadic breast carcinoma using microsatellite length polymorphisms. Cancer Res 1992;52:2624–7.
41. Saito H, Inazawa J, Saito S, et al. Detailed deletion mapping of chromosome 17q in ovarian and breast cancers: 2-cM region on 17q21.3 often and commonly deleted in tumors. Cancer Res 1993;53:3382–5.
42. 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;17:1595–607.
43. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005;5:726–34.
44. Cuervo, A. M. Autophagy: in sickness and in health. Trends Cell Biol. 14, 70–77 (2004).
45. Edinger, A. L. & Thompson, C. B. Defective autophagy leads to cancer. Cancer Cell 4, 422–424 (2003).
46. Gozuacik, D. & Kimchi, A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23, 2891–2906 (2004
47. Ogier-Denis, E., Houri, J. J., Bauvy, C. & Codogno, P. Guanine nucleotide exchange on heterotrimeric GI3 protein controls autophagic sequestration in HT-29 cells. J. Biol. Chem. 271, 28593–28600 (1996).
48. Proikas-Cezanne, T. et al. WIPI-1 (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene 23, 9314–9325 (2004).
49. Susan, P. P. & Dunn, W. A. Jr. Starvation-induced lysosomal degradation of aldolase B requires glutamine 111 in a signal sequence for chaperone-mediated transport. J. Cell. Physiol. 187, 48–58 (2001).
50. Ito, H., Daido, S., Kanzawa, T., Kondo, S. & Kondo, Y. Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int. J. Oncol. 26, 1401–1410 (2005).
51. Takeuchi H, Kanzawa T, Kondo Y, Kondo S. Inhibition of platelet-derived growth factor signalling induces autophagy in malignant glioma cells. Br J Cancer 2004;90:1069–75.
52. 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;65:3336–46.
53. 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;11:448–57.
54. Wu H, Yang JM, Jin S, Zhang H, Hait WN. Elongation factor-2 kinase regulates autophagy in human glioblastoma cells. Cancer Res 2006;66:3015–23.
55. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004;36:2491–502.
56. SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004; 119:753-66.
57. Crotzer VL, Blum JS. Autophagy and intracellular surveillance: Modulating MHC class II antigen presentation with stress. Proc Natl Acad Sci USA 2005; 102:7779-80.
58. Komata T, Kanzawa T, Takeuchi H, et al. Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells. Br J Cancer 2003;88:1277–80.
59. Punnonen EL, Reunanen H. Effects of vinblastine, leucine, and histidine, and 3-methyladenine on autophagy in Ehrlich ascites cells. Exp Mol Pathol 1990;52:87–97.
60. Jia L, Dourmashkin RR, Allen PD, Gray AB, Newland AC, Kelsey SM. Inhibition of autophagy abrogates tumour necrosis factor induced apoptosis in human T-lymphoblastic leukaemic cells. Br J Haematol 1997;98:673–85.
61. Bauvy C, Gane P, Arico S, Codogno P, Ogier-Denis E. Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29. Exp Cell Res 2001;268:139–49.
62. Parmer TG, Ward MD, Yurkow EJ, Vyas VH, Kearney TJ, Hait WN. Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer. Br J Cancer 1999;79:59–64.
63. Arora S, Yang JM, Kinzy TG, et al. Identification and characterization of an inhibitor of eukaryotic elongation factor 2 kinase against human cancer cell lines. Cancer Res 2003;63:6894–9.
64. Bursch, W. 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 17, 1595–1607 (1996).
65. Scarlatti, F. et al. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of Beclin 1. J. Biol. Chem. 279, 18384–18391 (2004).
66. Shao, Y., Gao, Z., Marks, P. A. & Jiang, X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc. Natl Acad. Sci. USA, 101, 18030–18035 (2004).
67. Shinohara ET, Cao C, Niermann K, Mu Y, Zeng F, Hallahan DE, Lu B. Enhanced radiation damage of tumor vasculature by mTOR inhibitors. Oncogene 2005; 24:5414-22.
68. Hamada K, Sasaki T, Koni PA, Natsui M, Kishimoto H, Sasaki J, Yajima N, Horie Y,Hasegawa G, Naito M, Miyazaki J, Suda T, Itoh H, Nakao K, Mak TW, Nakano T, SuzukiA. The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis.Genes Dev 2005; 19:2054-65. Gutierrez MG, Master
69. Bursch, W., Ellinger, A., Gerner, C., Frohwein, U. & Schulte-Hermann, R. Programmed cell death (PCD). Apoptosis, autophagic PCD, or others? Ann. NY Acad. Sci. 926, 1–12 (2000).
70. Bursch, W. et al. Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments. J. Cell Sci. 113, 1189–1198 (2000).
71. Bursch, W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 8, 569–581 (2001).
72. Shimizu, S. et al. Role of BCL-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nature Cell Biol. 6, 1221–1228 (2004).
73. Lum, J. J. et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248 (2005).
74. Yu, L. et al. Regulation of an ATG7–Beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500–1502 (2004).
75. Boya, P. et al. Inhibition of macroautophagy triggers apoptosis. Mol. Cell. Biol., 25, 1025–1040 (2005).
76. Pelicano, H. et al. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J. Biol. Chem. 278, 37832–37839 (2003).
77. Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H. & Balch, W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 130, 781–796 (1995).
78. Inbal, B., Bialik, S., Sabanay, I., Shani, G. & Kimchi, A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J. Cell Biol. 157, 455–468 (2002).
79. Bursch, W. 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 17, 1595–1607 (1996).
80. Pelicano, H. et al. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J. Biol. Chem. 278, 37832–37839 (2003).
81. Piacentini, M., Evangelisti, C., Mastroberardino, P. G., Nardacci, R. & Kroemer, G. Does prothymosin- act as molecular switch between apoptosis and autophagy? Cell Death Differ. 10, 937–939 (2003).
82. K. Saeki, A. Yuo, E. Okuma, Y. Yazaki, S.A. Susin, G. Kroemer, F.Takaku, Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells[J]. Cell Death Differ. 7 (2000) 1263–1269.
83. N. Canu, R. Tufi, A.L. Serafino, G. Amadoro, M.T. Ciotti, P.Calissano, Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells J. Neurochem. 92 (2005) 1228–1242.
84. C. Cardenas-Aguayo Mdel, J. Santa-Olalla, J.M. Baizabal, L.M. Salgado, L. Covarrubias, Growth factor deprivation induces an alternative non-apoptotic death mechanism that is inhibited by Bcl2 in cells derived from neural precursor cellsJ. Hematother. Stem. Cell Res. 12 (2003) 774:735–748.
85. S. Pattingre, A. Tassa, X. Qu, R. Garuti, X.H. Liang, N. Mizushima, M. Packer, M.D. Schneider, B. Levine, Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagyCell 122 (2005): 927–939.
86. Cheney, I. W. et al. Suppression of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC1/PTEN gene transfer. Cancer Res. 58, 2331–2334 (1998).
87. Eshleman, J. S. et al. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res. 62, 7291–7297 (2002).
88. Su, B. & Karin, M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8, 402–411 (1996).
89. Chan, S. Targeting the mammalian target of rapamycin (mTOR): a new approach to treating cancer. Br. J. Cancer 91, 1420–1424 (2004).
90. Eshleman, J. S. et al. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res. 62, 7291–7297 (2002).
91. Mondesire, W. H. et al. Targeting mammalian target of rapamycin synergistically enhances chemotherapy-induced cytotoxicity in breast cancer cells. Clin. Cancer Res. 10, 7031–7042 (2004).
92. Stephan, S. et al. Effect of rapamycin alone and in combination with antiangiogenesis therapy in an orthotopic model of human pancreatic cancer. Clin. Cancer Res. 10, 6993–7000 (2004).
93. Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H. & Balch, W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 130, 781–796 (1995).
94. Inbal, B., Bialik, S., Sabanay, I., Shani, G. & Kimchi, A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J. Cell Biol. 157, 455–468 (2002).
95. Shimizu, S. et al. Role of BCL-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nature Cell Biol. 6, 1221–1228 (2004).
96. Lum, J. J. et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248 (2005).
97. Yu, L. et al. Regulation of an ATG7–Beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500–1502 (2004).
98. Folkman J. Angiogenesis and apoptosis. Seminars in Cancer Biology 2003; 13:159-67.
2. Takahiro Shintani, Daniel J Klionsky.Autophagy in Health and Disease:A Double Edged Sword.Science,2004,306(5698) :990-995.
3. Ogier-Denis E, Codogno P. Autophagy: a barrier or an adaptive response to cancer. Biochim Biophys Acta,2003, 1603(2):113-128.
4. Klionsky D J , Emr S D. Autophagy as a regulated pathway of cellular degradation. Science ,2000,290(5497):1717-1721.
5. Mortimore G E, Poso A R. Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr,1987,7:539-564.
6. Friedman L S.The search for BRACA1.Cancer Res.1994,54(24):6374-6382.
7. Edinger A L,Thompson C B.Defective autophagy leads to cancer.Cancer Cell,2003,4(6):422-424.
8. Qu X, Yu J,Bhagat G et al. Promotion of tumorgenesis by heterozygous disruption of the beclin1 autopahgy gene. J Clin Invest,2003, 112(12) : 1809 -1820.
9. Kabeya Y, Mizushima N, Ueno T,et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J,2003,19(21):5720-5728.
10. Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol.,2004, 36(12):2503-2518.
11. Mizushima N, Yamamoto A, Hatano M, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.J Cell Biol, 2001, 152(4): 657-668.
12. Toth S, Nagy K, Palfia Z,et al. Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up regulation in allpremalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation. Cell Tissue Res, 2002, 309(3) : 409 -416.
13. Cuervo A M. Autophagy: in sickness and in health. Trends Cell Biol,2004, 14(2): 70-77.
14.段振玲,彭芝兰,王赞宏.自噬基因Beclin1与上皮性卵巢癌发生和发展的相关性研究.癌症,2007,26(3):258-263.
15. Gozuacik D, Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene,2004,23(16): 2891-2906.
16. Holm E,Hildebrandt W ,Kinscherf R. Low postabsorptive net protein degradation in male cancer patients: lack of sensitivity to regulatory amino acids.Oncol Rep,2007 ,17(3):695-700.
17. Botti J, Dajaheri-Merqny M, Pilatte Y. Autophagy signaling and the cogwheels of cancer.Autophagy. Autophagy. 2006 ,2(2): 67-73.
18. Klionsky D J . Autophagy,Curr Biol ,2005,15(8): 282-283.
19. Marino G, Lopez-Otin C. Autophagy: molecular mechanisms, physiological functions and relevance in human pathology. Cell Mol Life Sci,2004,61 (12): 1439-1454.
20. Eskelinen E L. Maturation of autophagic vacuoles in mammalian cells. Autophagy, 2005, 1(1): 001-010
21. D.J. Klionsky, S.D. Emr, Autophagy as a regulated pathway of cellular degradation, Science 290 (2000) 1717–1721.
22. Y. Ohsumi, Molecular dissection of autophagy: two ubiquitin-like systems, Nat. Rev. Mol. Cell. Biol. 2 (2001) 211–216.
23. N. Mizushima, Y. Ohsumi, T. Yoshimori, Autophagosome formation in mammalian cells, Cell. Struct. Funct. 27 (2002)421–429.
24. N. Mizushima, A. Yamamoto, M. Hatano, Y. Kobayashi, Y. Kabeya, K. Suzuki, T.Tokuhisa, Y. Ohsumi, T. Yoshimori,Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells, J. Cell Biol. 152 (2001) 657–667.
25. Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (2000)5720–5728
26. S. Yokota, M. Himeno, J. Roth, D. Brada, K. Kato, Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)phthalate treatment. II. Immunocytochemical analysis of early and late autophagosomes, Eur. J. Cell Biol. 62 (1993) 372–383.
27. R. Masaki, A. Yamamoto, Y. Tashiro, Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver, J. Cell Biol. 104 (1987) 1207–1215.
28. S.P. Elmore, T. Qian, S.F. Grissom, J.J. Lemasters, The mitochondrial permeability transition initiates autophagy in rat hepatocytes, FASEB J. 15 (2001) 2286–2287.
29. Bando K,Nagai H,Matsumoto S et al.Identification of 1-Mb common region at 16q24.1-24.2 deletied in hepatocellular carcinoma.Genes Chromosome Cancer.2000,28,38-44
30. Chen T,Sahi A,Aldaz CM,et al.Deletion map of chromosome 16q ductal incarcinoma in situ of breast :refining a putative tumor surpressor gene region.Cancer Res,1996, 56,5605-09
31. Elo JP,Harkonen P,Kellonen AP,et al.Loss of heterozygosity at 16q24.1-q24.2 is significantly associated with metastatic and aggressive behavior of prostate cancer..Caner Res.1997,57,3356-9
32. Suzuki S,Moore DH,Ginziger DG,et al.An approach to analysis of large scale correlations between genome changes and clinical endopoints in ovarian cancer.Cancer Res,2000,60,5382
1吴一龙,等.2007中国肺癌临床指南,人民卫生出版社,2007年,第一版
2 Cuervo AM. Autophagy: in sickness and in health. Trends Cell Biol,2004, 14: 70-77.
3 Clark AS, West K,Streicher S,et al.Constititive and inducible AKT activity promotes resistance to chemotherapy,trastuzumab,or tamoxifen in breast cancer cells.Mol Cancer Ther.2002 Jul;1(9):707-17
4 Canguilhem B, Pradines A, Baudouin C,et al.RhoB protects human keratinocytes from UVB-induced apoptosis through epidermal growth factor receptor signaling. J Biol Chem,2005,280(52),43257-63
5殷蔚伯,谷铣之.肿瘤放射治疗学.北京中国协和医科大学出版社,2002,265
6 D.J. Klionsky, S.D. Emr, Autophagy as a regulated pathway of cellular degradation, Science 290 (2000) 1717–1721.
7 Y. Ohsumi, Molecular dissection of autophagy: two ubiquitin-like systems, Nat. Rev. Mol. Cell. Biol. 2 (2001) 211–216.
8 N. Mizushima, Y. Ohsumi, T. Yoshimori, Autophagosome formation in mammalian cells, Cell. Struct. Funct. 27 (2002)421–429.
9 N. Mizushima, A. Yamamoto, M. Hatano, Y. Kobayashi, Y.Kabeya, K. Suzuki, T. Tokuhisa, Y. Ohsumi, T. Yoshimori,Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells, J. Cell Biol. 152 (2001) 657–667.
10 Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako,T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (2000)5720–5728
11 S. Yokota, M. Himeno, J. Roth, D. Brada, K. Kato, Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)phthalate treatment. II. Immunocytochemical analysis of early and late autophagosomes, Eur. J. Cell
12 R. Masaki, A. Yamamoto, Y. Tashiro, Cytochrome P-450 and NADPH-cytochrome P-450 reductase are degraded in the autolysosomes in rat liver, J. Cell Biol. 104 (1987) 1207–1215.
13 S.P. Elmore, T. Qian, S.F. Grissom, J.J. Lemasters, The mitochondrial permeability transition initiates autophagy in rat hepatocytes, FASEB J. 15 (2001) 2286–2287.
14 Mortimore GE, Poso AR. (1987) Intracellular protein catabolism and its control during nutrient deprivation and supply. Annu Rev Nutr 7:539-564.
15 Klionsky DJ and Emr SD. (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717-1721.
16 Roberts P, Moshitch-Moshkovitz S, Kvam E, O’Toole E, Winey M, Goldfarb DS. (2003) Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol Biol Cell 14:129-141.
17 .Reggiori F and Klionsky DJ. (2005) Autophagosomes: biogenesis from scratch? Curr Opin Cell Biol 17(4):415-422.
18 Liang XH, Kleeman LK, Jiang HH et al. (1998) Protection against fetal Sindbis virus encephalitis by Beclin, a novel Bcl-2 interacting protein. J Virol 72:8586-8596.
19 Liang XH, Jackson S, Seaman M et al. (1999) Induction of autophagy and inhibition of tumorigenesis by Beclin 1. Nature 402: 672-676.
20 Thumm M,Kadowaki T.The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants.Mol Genet Genomics, 2001;266,651
21 Gupta VK, Park JO , Jaskowiak NT, et al . Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma . Ann Surg Oncol ,2002 ,9(5) :500～504.
22 Schimidt-Ullrich RK,Mikkelsen RB,Dent P,et al.Radiation-induced Proliferation of the human A431 squamouse carcinoma cells is dependent on EGFR tyrosine PhosPhorylation . Oncogene1997,15:1191-1197.
23 Brown EJ,AlbersMW, Shin TB, et a l. A mammalian p rotein targeted by G1-arresting rapamycin-receptor complex [ J ]. N a tu re ,1994, 369 (6483) : 756
24 Asnaghi L, Bruno P, Priulla M, et a l. mTOR: a p rotein kinase switching between life and death [ J ]. Pharm acol Res , 2004, 50(6) : 545
25 Martin KA, Rzucidlo EM, Merenick BL, et a l. The mTOR /p70 S6K1 pathway regulates vascular smooth muscle cell differentiation[ J ]. Am J Physiol Cell Physiol , 2004, 286 (3) : C507
26 Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt /PKB by the rictor2mTOR comp lex [ J ].S cience , 2005, 307 (5712) : 1098
27 Zhou X, Tna M, Stone Hawthorne V, et al1 Activation of the Akt/mammalian target of rapamycin /4E2BP1 pathway by ErbB2 overexp ression p redicts tumor progression in breast cancers1 Clin Cancer Res, 2004, 10 (20) : 6779267881
28 Klos KS, Wyszomierski SL,Sun M,et al.ErB2 increase vascular endothelial growth factor protein synthesis via activation of mamammalian taget of rapamycin /p70s6K leading to increased angiogenesis and spontaneous metastasis of human breast cancer cells{J}.Cancer Res,2006,66(4);2028
29 ItohN,Semba S,Ito M,et al.Phosphorylation of AKT/PKB is required for suppression of cancer cell apoptosis and tumor progression in human colcrectal carcinoma. Cancer, 2002, 944,3127
30 Fruman DA,M eyer RE,Cantley LC.Phosphoinsitide kinases.Annu Rev Biochem,1998, 67,481
31 Blackhall FH,Pintilie M,Michael M,et al.Expression and prognostic significance of kit,protein kinase B,and mitrogen-activated protein kinase in patient with small cell lung cancer Clin Cancer Res,2003,9,2241
32 Wiederrecht G J, Sabers C J, Brunn G J, et al. Mechanism of action of rapamycin: new insights into the regulation of G1-phase progression in eukaryotic cells [ J] . Prog Cell Cycle Res, 1995, 1: 53- 71.
33 Edinger A L, Thompson C B. Akt maintains cell size and survival by ncreasing mTOR-dependent nutrient uptake [ J] . Mol Biol Cell, 2002, 13(7) : 2276- 2288.
34 Dann S G, Selvaraj A, Thomas G.mTOR Complex1-S6K1 signaling: at the crossroads of obesity, diabetes andcancer [ J] . Trends Mol Med, 2007,13( 6) : 252- 259.
35 Jacinto E et al. Nat Rev Mol Cell Biol, 2003, 4: 117
1. Vignot S, Faiver S, Aguirre D, et al1 mTOR2targeted therapy of cancerwith rapamycin drivatives1 Ann Oncol, 2005, 16(4) : 52525371
2. Rowinsky EK1 Targeting the molecular target of rapamycin(mTOR) 1 CurOp in Oncol, 2004, 16 (6) : 56425751
3. Shao, Y., Gao, Z., Marks, P. A. & Jiang, X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc. Natl Acad. Sci. USA, 101, 18030–18035 (2004).
4. Bursch, W. 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 17, 1595–1607 (1996
5.马湘一,王世宣,刘琰,等.雷帕霉素联合紫杉醇诱导卵巢癌细胞A2780和SKOV3凋亡及其分子机制[ J] .癌症, 2007, 26( 4) : 367- 370.
6. Busca R, Bertolotto C, Ortonne J P, et al. Inhibition of the phosphatidylinositol
3-kinase /p70 ( S6) -kinase pathway induces B16 melanoma cell differentiation [ J] . J Biol Chem, 1996, 271( 50) : 31824- 31830.
7. Eng C P, Sehgal S N, Vezina C. Activity of rapamycin (AY-22, 989) against transplanted tumors [ J] . J Antibiot ( Tokyo) , 1984, 37( 10) : 1231-1237.
8. Hosoi H, Dilling M B, Shikata T, et al. Rapamycin causes poorly reversible inhibition of mTOR and induces p53- independent apoptosis in human rhabdomyosarcoma cells [ J] . Cancer Res, 1999, 59( 4) : 886- 894.
9. Shi Y, Frankel A, Radvanyi L G, et al. Rapamycin enhances apoptosis and increases sensitivity to cisplatin in vitro [ J] . Cancer Res, 1995, 55 ( 9) : 1982-1988.
10. Wu Q, Kiguchi K, Kawamoto T, et al. Therapeutic effect of rapamycin on gallbladder cancer in a transgenic mouse model [ J] . Cancer Res, 2007, 67( 8) :3794- 3800.
11. Jimeno A, Kulesza P, Wheelhouse J, et al. Dual EGFR and mTOR targeting in squamous cell carcinoma models,and development of early markers of efficacy [ J] . Br J Cancer, 2007, 96( 6) :952- 959.
12. Semela D, Piguet A C, Kolev M, et al. Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma [ J] . J Hepatology, 2007, 46( 5) : 840- 848.
13. Kobayashi S, Kishimoto T, Kamata S, et al. Rapamycin, a specific inhibitor of the mammalian target of rapamycin, suppresses lymphangiogenesis and lymphatic metastasis [ J] . Cancer Sci, 2007, 98( 5) : 726- 733.
14. Granville C A, Warfel N, Tsurutani J, et al. Identification of a highly effective rapamycin schedule that markedly reduces the size, multiplicity, and phenotypic progression of tobacco carcinogen-induced murine lung tumors[ J] . Clin Cancer Res, 2007, 13( 7) :2281- 2289.
15. 15.Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H. & Balch, W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 130, 781–796 (1995).
16. 16.Inbal, B., Bialik, S., Sabanay, I., Shani, G. & Kimchi, A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J. Cell Biol. 157, 455–468 (2002).
17. Rhoads RE. Regulation of eukaryotic protein synthesis by initiation actors[J ] . J Biol Chem, 1993 ,268 (5) :301723021.
18. Lazatis KA , Montione KS , Sonenberg N. et al . Maglignant transformation by a eukaryotic initiation factor subunit that binds to mRNAs′cap [J ] . Nature ,1990 , 345(7) :5442547.
19. Rinker SCW, Graff JR , De Benedetti , et al . Decreasing the level of translation initiatio factor 4E with antisense RNA caused reversal of ras - mediatedtransformation and tumorigenesis of cloned rat embryo fibroblasts[J ] . Int J Cancer ,1993 ,55(5) :8412847.
20. Seki N , Takasu T , Mandai K, et al . Expression of eukaryotic initiation factor 4E in atypical adenomatous hyperplasia and adenocarcinoma of the human peripheral lung[J ] . Clin Cancer Res , 2002 ,8(10) :304623053.
21. Reed JC,Doctor K,Rojas A,et al.Comparative analysis of apoptosis and inflammation genes of mice and humans.Genome Res,2003,13,1376
22. Hanahan D,Wwinberg RA.The hallmarks of cancer.Cell,2000,100,57-70
23. Li LY,Luo X,Wang X,et al.Endonuclease G is an apaptotic Dnase when released from mitochondria.Nature,2001,412,95
24. Liston P,Fong WG,Korneluk RG.The inhibitors of apotosis:there is more to life than Bcl-2.Oncogene,2003,22,8568
25. Takaoka A,Adachi M,Okuda H,et al.Anti-cell death activity promotes pulmonary metastasis of melanoma cells.Oncogene,1997,14,2971
26. Del Bufalo D,Biroccio A,Leonetti C,et al.Bcl-2 overexpression enhances the metastastic of a human breast cancer line.FASEB J,1997,11,947
27. Martin SS,Leder P.Human MCF10A mammary epithelial cells undergo apoptisis following actin depolymerization that is independent of attachment and rescued by Bcl-2.Mol Cell Biol,2001,21,6529
28. Altieri DC.Survivin,versatile modulation of cell division and apoptosis in cancer. Oncogene,2003,22,8581
29. Kawasaki H,Altieri DC,Lu CD,et al.Inhibititor of apoptosis by surviving predicts shorter surviving rates in colorectal cancer. Cancer Res,1998;58(22):5071-5074
30. Ikehara M, Oshita F, Kameda Y, et al. Expression of surviving correlated with vessel invasion is a marker of poor prognosis in small adenocarcinoma of the lung. Oncol Rep,2002;9(4)835-838
31. Tamm I, Wang Y, Sausville E, et al. IAP-family protein suevivin inhibits caspaseactivity and apoptosis induced by Fas (CD95), Bax, caspase, and anticancer druds. Cancer Res, 1998, 58(23):5315-5320
32. Suzuki A,Ito T, Kawano H, et al. Suvivin initiates procaspase 3/p21 complex formation as a result of interaction with CDK4 to resist Fas-medizted cell death. Oncogene, 2000,19(10): 1346-1353
33. Johnstone RW,Ruefli AA.Lowe SW.Apoptosis:a link between cancer genetics and chemotherapy.Cell,2002,108,153
34. Hersey P,Zhang XD.Overcoming resistance of cancer cell to apoptosis.J Cell Physiol,2003,196:9
35. Hengartner MO.The biochemistry of apoptosis.Nature,2000,407,770
36. Degterev A,Boyce M,Yuan J.A decade of caspases.Oncogene,2003,22,8543
37. Arico S,Pattingre S,Bauvy C,et al.Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT29 cell line.J Biol Chem.2002,277,31,27613-21
38. Altieri DC. Survivin, versatile modulation of cell divisionand apoptosis in cancer [ J ]. Oncogene, 2003, 22 ( 53 ) :8 581-8 589
39.张燕,孙婷,曹祥荣. Survivin基因特异性siRNA腺病毒介导的肿瘤放射增敏作用[ J ].中国肿瘤生物治疗杂志, 2007, 14 (1) : 59-63
1. Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest 2005;115:2679–88.
2. Yokota, S., Himeno, M., Roth, J., Brada, D. & Kato, K. Formation of autophagosomes during degradation of excess peroxisomes induced by di-(2-ethylhexyl)phthalate treatment. II. Immunocytochemical analysis of early and late autophagosomes. Eur. J. Cell Biol. 62, 372–383 (1993).
3. Stromhaug, P. E., Berg, T. O., Fengsrud, M. & Seglen, P. O. Purification and characterization of autophagosomes from rat hepatocytes. Biochem. J. 335, 217–224 (1998).
4. Noda, T., Suzuki, K. & Ohsumi, Y. Yeast autophagosomes: de novo formation of a membrane structure. Trends Cell Biol. 12, 231–25 (2002).
5. Kroemer G, Jaattela M. Lysosomes and autophagy in cell death control. Nat Rev Cancer 2005;5:886–97.
6. Edinger AL, Thompson CB. Death by design: apoptosis, necrosis and autophagy. Curr Opin Cell Biol 2004;16:663–9.
7. Kuma A, Hatano M, Matsui M, et al. The role of autophagy during the early neonatal starvation period. Nature 2004;432:1032–6.
8. Lum JJ, Bauer DE, Kong M, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 2005;120:237–48
9. Boya P, Gonzalez-Polo RA, Casares N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 2005;25:1025–40.
10. Degenhardt K, Mathew R, Beaudoin B, Bray K. Autophagy promotes tumor cell survivaland restricts necrosis, inflammation, and tumorigenesis. Cancer Cell 2006; 51-64.
11. Liang XH, Kleeman LK, Jiang HH, et al. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol 1998;72:8586–96.
12. Liang XH, Jackson S, Seaman M, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999;402:672–6.
13. 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;100:15077–82.
14. Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003;112:1809–20.
15. Pattingre S, Tassa A, Qu X, et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 2005;122:927–39.
16. Takacs-Vellai K, Vellai T, Puoti A, et al. Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans. Curr Biol 2005;15:1513–7.
17. Inoki K, Corradetti MN, Guan KL, Dysregulation of the TSC-mTOR pathway in human disease. Nat Genet 2005;37:19–24.
18. Kamada Y, Sekito T, Ohsumi Y. Autophagy in yeast: a TOR-mediated response to nutrient starvation. Curr Top Microbiol Immunol 2004;279:73–84.
19. Arico S, Petiot A, Bauvy C, et al. The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem 2001;276:35243–6.
20. Feng Z, Zhang H, Levine AJ, Jin S. The coordinate regulation of the p53 and mTOR pathways in cells. Proc Natl Acad Sci USA 2005; 102:8204-9.
21. Tsuneoka M, Umata T, Kimura H, Koda Y, Nakajima M, Kosai K, Takahashi T, Takahashi Y, Yamamoto A. c-myc induces autophagy in rat 3Y1 fibroblast cells. Cell Struct Funct,2003; 28:195-204.
22. Pattingre S, Bauvy C, Codogno P. Amino acids interfere with the ERK1/2-dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. J Biol Chem 2003; 278:16667-74.
23. Komata T, Kanzawa T, Takeuchi H, Germano IM, Schreiber M, Kondo Y, Kondo S.Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C),p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells. Br J Cancer,2003; 88:1277-80.
24. Browne GJ, Proud CG. A novel mTOR-regulated phosphorylation site in elongation factor 2 kinase modulates the activity of the kinase and its binding to calmodulin. Mol Cell Biol 2004;24:2986–97.
25. Meyuhas O. Synthesis of the translational apparatus is regulated at the translational level. Eur J Biochem 2000;267:6321–30.
26. Mamane Y, Petroulakis E, Rong L, Yoshida K, Ler LW, Sonenberg N. eIF4E-from translation to transformation. Oncogene 2004;23:3172–9.
27. Hardie DG, Carling D, Carlson M. The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu Rev Biochem 1998;67:821–55.
28. Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003;115:577–90.
29. Ryazanov AG, Ward MD, Mendola CE, et al. Identification of a new class of protein kinases represented by eukaryotic elongation factor-2 kinase. Proc Natl Acad Sci U S A 1997;94:4884–9.
30. Chen Y, Matsushita M, Nairn AC, et al. Mechanisms for increased levels of phosphorylation of elongation factor-2 during hibernation in ground squirrels. Biochemistry 2001;40:11565–70.
31. Wang X, Li W, Williams M, Terada N, Alessi DR, Proud CG. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J 2001;20:4370–9.
32. Browne GJ, Finn SG, Proud CG. Stimulation of the AMP-activated protein kinase leads to activation of eukaryotic elongation factor 2 kinase and to its phosphorylation at a novel site, serine 398. J Biol Chem,2004;279: 12220– 31.
33. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70.
34. Gunn, J. M., Clark, M. G., Knowles, S. E., Hopgood, M. F. & Ballard, F. J. Reduced rates of proteolysis in transformed cells. Nature 266, 58–60 (1977).
35. Kisen, G. O. et al. Reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells. Carcinogenesis 14, 2501–2505 (1993).
36. Kirkegaard, K., Taylor, M. P. & Jackson, W. T. Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nature Rev. Microbiol. 2, 301–314 (2004).
37. Otsuka, H. & Moskowitz, M. Differences in the rates of protein degradation in untransformed and transformed cell lines. Exp. Cell Res. 112, 127–35 (1978).
38. Schwarze, P. E. & Seglen, P. O. Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats. Exp. Cell Res. 157, 15–28 (1985).
39. Russell SE, Hickey GI, Lowry WS, White P, Atkinson RJ. Allele loss from chromosome 17 in ovarian cancer. Oncogene 1990;5:1581–3
40. Futreal PA, Soderkvist P, Marks JR, et al. Detection of frequent allelic loss on proximal chromosome 17q in sporadic breast carcinoma using microsatellite length polymorphisms. Cancer Res 1992;52:2624–7.
41. Saito H, Inazawa J, Saito S, et al. Detailed deletion mapping of chromosome 17q in ovarian and breast cancers: 2-cM region on 17q21.3 often and commonly deleted in tumors. Cancer Res 1993;53:3382–5.
42. 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;17:1595–607.
43. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer 2005;5:726–34.
44. Cuervo, A. M. Autophagy: in sickness and in health. Trends Cell Biol. 14, 70–77 (2004).
45. Edinger, A. L. & Thompson, C. B. Defective autophagy leads to cancer. Cancer Cell 4, 422–424 (2003).
46. Gozuacik, D. & Kimchi, A. Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23, 2891–2906 (2004
47. Ogier-Denis, E., Houri, J. J., Bauvy, C. & Codogno, P. Guanine nucleotide exchange on heterotrimeric GI3 protein controls autophagic sequestration in HT-29 cells. J. Biol. Chem. 271, 28593–28600 (1996).
48. Proikas-Cezanne, T. et al. WIPI-1 (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene 23, 9314–9325 (2004).
49. Susan, P. P. & Dunn, W. A. Jr. Starvation-induced lysosomal degradation of aldolase B requires glutamine 111 in a signal sequence for chaperone-mediated transport. J. Cell. Physiol. 187, 48–58 (2001).
50. Ito, H., Daido, S., Kanzawa, T., Kondo, S. & Kondo, Y. Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int. J. Oncol. 26, 1401–1410 (2005).
51. Takeuchi H, Kanzawa T, Kondo Y, Kondo S. Inhibition of platelet-derived growth factor signalling induces autophagy in malignant glioma cells. Br J Cancer 2004;90:1069–75.
52. 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;65:3336–46.
53. 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;11:448–57.
54. Wu H, Yang JM, Jin S, Zhang H, Hait WN. Elongation factor-2 kinase regulates autophagy in human glioblastoma cells. Cancer Res 2006;66:3015–23.
55. Mizushima N. Methods for monitoring autophagy. Int J Biochem Cell Biol 2004;36:2491–502.
56. SS, Singh SB, Taylor GA, Colombo MI, Deretic V. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 2004; 119:753-66.
57. Crotzer VL, Blum JS. Autophagy and intracellular surveillance: Modulating MHC class II antigen presentation with stress. Proc Natl Acad Sci USA 2005; 102:7779-80.
58. Komata T, Kanzawa T, Takeuchi H, et al. Antitumour effect of cyclin-dependent kinase inhibitors (p16(INK4A), p18(INK4C), p19(INK4D), p21(WAF1/CIP1) and p27(KIP1)) on malignant glioma cells. Br J Cancer 2003;88:1277–80.
59. Punnonen EL, Reunanen H. Effects of vinblastine, leucine, and histidine, and 3-methyladenine on autophagy in Ehrlich ascites cells. Exp Mol Pathol 1990;52:87–97.
60. Jia L, Dourmashkin RR, Allen PD, Gray AB, Newland AC, Kelsey SM. Inhibition of autophagy abrogates tumour necrosis factor induced apoptosis in human T-lymphoblastic leukaemic cells. Br J Haematol 1997;98:673–85.
61. Bauvy C, Gane P, Arico S, Codogno P, Ogier-Denis E. Autophagy delays sulindac sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29. Exp Cell Res 2001;268:139–49.
62. Parmer TG, Ward MD, Yurkow EJ, Vyas VH, Kearney TJ, Hait WN. Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer. Br J Cancer 1999;79:59–64.
63. Arora S, Yang JM, Kinzy TG, et al. Identification and characterization of an inhibitor of eukaryotic elongation factor 2 kinase against human cancer cell lines. Cancer Res 2003;63:6894–9.
64. Bursch, W. 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 17, 1595–1607 (1996).
65. Scarlatti, F. et al. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of Beclin 1. J. Biol. Chem. 279, 18384–18391 (2004).
66. Shao, Y., Gao, Z., Marks, P. A. & Jiang, X. Apoptotic and autophagic cell death induced by histone deacetylase inhibitors. Proc. Natl Acad. Sci. USA, 101, 18030–18035 (2004).
67. Shinohara ET, Cao C, Niermann K, Mu Y, Zeng F, Hallahan DE, Lu B. Enhanced radiation damage of tumor vasculature by mTOR inhibitors. Oncogene 2005; 24:5414-22.
68. Hamada K, Sasaki T, Koni PA, Natsui M, Kishimoto H, Sasaki J, Yajima N, Horie Y,Hasegawa G, Naito M, Miyazaki J, Suda T, Itoh H, Nakao K, Mak TW, Nakano T, SuzukiA. The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis.Genes Dev 2005; 19:2054-65. Gutierrez MG, Master
69. Bursch, W., Ellinger, A., Gerner, C., Frohwein, U. & Schulte-Hermann, R. Programmed cell death (PCD). Apoptosis, autophagic PCD, or others? Ann. NY Acad. Sci. 926, 1–12 (2000).
70. Bursch, W. et al. Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments. J. Cell Sci. 113, 1189–1198 (2000).
71. Bursch, W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ. 8, 569–581 (2001).
72. Shimizu, S. et al. Role of BCL-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nature Cell Biol. 6, 1221–1228 (2004).
73. Lum, J. J. et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248 (2005).
74. Yu, L. et al. Regulation of an ATG7–Beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500–1502 (2004).
75. Boya, P. et al. Inhibition of macroautophagy triggers apoptosis. Mol. Cell. Biol., 25, 1025–1040 (2005).
76. Pelicano, H. et al. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J. Biol. Chem. 278, 37832–37839 (2003).
77. Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H. & Balch, W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 130, 781–796 (1995).
78. Inbal, B., Bialik, S., Sabanay, I., Shani, G. & Kimchi, A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J. Cell Biol. 157, 455–468 (2002).
79. Bursch, W. 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 17, 1595–1607 (1996).
80. Pelicano, H. et al. Inhibition of mitochondrial respiration: a novel strategy to enhance drug-induced apoptosis in human leukemia cells by a reactive oxygen species-mediated mechanism. J. Biol. Chem. 278, 37832–37839 (2003).
81. Piacentini, M., Evangelisti, C., Mastroberardino, P. G., Nardacci, R. & Kroemer, G. Does prothymosin- act as molecular switch between apoptosis and autophagy? Cell Death Differ. 10, 937–939 (2003).
82. K. Saeki, A. Yuo, E. Okuma, Y. Yazaki, S.A. Susin, G. Kroemer, F.Takaku, Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells[J]. Cell Death Differ. 7 (2000) 1263–1269.
83. N. Canu, R. Tufi, A.L. Serafino, G. Amadoro, M.T. Ciotti, P.Calissano, Role of the autophagic-lysosomal system on low potassium-induced apoptosis in cultured cerebellar granule cells J. Neurochem. 92 (2005) 1228–1242.
84. C. Cardenas-Aguayo Mdel, J. Santa-Olalla, J.M. Baizabal, L.M. Salgado, L. Covarrubias, Growth factor deprivation induces an alternative non-apoptotic death mechanism that is inhibited by Bcl2 in cells derived from neural precursor cellsJ. Hematother. Stem. Cell Res. 12 (2003) 774:735–748.
85. S. Pattingre, A. Tassa, X. Qu, R. Garuti, X.H. Liang, N. Mizushima, M. Packer, M.D. Schneider, B. Levine, Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagyCell 122 (2005): 927–939.
86. Cheney, I. W. et al. Suppression of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC1/PTEN gene transfer. Cancer Res. 58, 2331–2334 (1998).
87. Eshleman, J. S. et al. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res. 62, 7291–7297 (2002).
88. Su, B. & Karin, M. Mitogen-activated protein kinase cascades and regulation of gene expression. Curr. Opin. Immunol. 8, 402–411 (1996).
89. Chan, S. Targeting the mammalian target of rapamycin (mTOR): a new approach to treating cancer. Br. J. Cancer 91, 1420–1424 (2004).
90. Eshleman, J. S. et al. Inhibition of the mammalian target of rapamycin sensitizes U87 xenografts to fractionated radiation therapy. Cancer Res. 62, 7291–7297 (2002).
91. Mondesire, W. H. et al. Targeting mammalian target of rapamycin synergistically enhances chemotherapy-induced cytotoxicity in breast cancer cells. Clin. Cancer Res. 10, 7031–7042 (2004).
92. Stephan, S. et al. Effect of rapamycin alone and in combination with antiangiogenesis therapy in an orthotopic model of human pancreatic cancer. Clin. Cancer Res. 10, 6993–7000 (2004).
93. Brown, W. J., DeWald, D. B., Emr, S. D., Plutner, H. & Balch, W. E. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J. Cell Biol. 130, 781–796 (1995).
94. Inbal, B., Bialik, S., Sabanay, I., Shani, G. & Kimchi, A. DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles during programmed cell death. J. Cell Biol. 157, 455–468 (2002).
95. Shimizu, S. et al. Role of BCL-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nature Cell Biol. 6, 1221–1228 (2004).
96. Lum, J. J. et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248 (2005).
97. Yu, L. et al. Regulation of an ATG7–Beclin 1 program of autophagic cell death by caspase-8. Science 304, 1500–1502 (2004).
98. Folkman J. Angiogenesis and apoptosis. Seminars in Cancer Biology 2003; 13:159-67.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |