促凋亡蛋白PUMA对小鼠肝细胞凋亡的影响及其分子机制研究
摘要
PUMA是2001年发现的一种促凋亡蛋白,最早发现于直结肠癌细胞中。该基因可以被p53快速诱导,具有强大的促凋亡的作用,故称其为puma。它属于凋亡相关蛋白Bcl-2家族,仅拥有一个BH3结构域,是促凋亡蛋白BH3-only亚族的重要成员。BH3-only亚族通过变构效应调节Bcl-2家族促凋亡和抗凋亡成员的平衡,在凋亡信号通路中处于中心位置。
既往研究表明PUMA基础表达量较低,在各种应激刺激(如DNA损伤,缺氧,糖皮质激素刺激及血清撤除等)影响下,可以通过p53依赖和非依赖两种途径被诱导表达,发挥着重要的促凋亡作用。由上述应激因素所诱导的细胞凋亡过程中,PUMA的作用是必需的,也就是说没有PUMA参与,就不会引发细胞凋亡。现有研究表明puma是p53的直接靶基因,也是介导p53凋亡通路的主要媒介分子。
在研究人类结直肠癌细胞中PUMA介导的细胞凋亡的分子机制时发现,PUMA通过其BH3结构域分别和Bcl-2家族多种抗凋亡成员(如Bcl-XL、Bcl-2等)产生广泛的相互作用,使原本结合于抗凋亡蛋白上的Bax解离并多聚化,进而转位激活线粒体依赖的内源性凋亡通路,诱导结肠癌细胞凋亡。此外Bax的缺失也会引起细胞凋亡障碍。因此PUMA对Bax的调控可能是一条重要的细胞凋亡通路。
我们的前期工作已经发现促凋亡基因puma在肝脏再生早期下调了5倍,其下调的意义是什么?是否提示PUMA低表达对肝细胞增殖的发动有显著作用?puma基因是否是调控肝脏再生的重要或关键靶点?其调控的机制是什么?我们对这些问题非常有兴趣,希望通过在体、离体实验揭示其规律。本课题通过离体实验研究PUMA对小鼠肝细胞凋亡的影响及其分子机制,对进一步揭示puma在肝脏再生过程中的作用有重要意义。同时,PUMA腺病毒有作为基因治疗药物的广阔应用前景(Biomed Pharmacother,2007)。运用腺病毒载体进行肝细胞离体实验研究,有助于初步了解PUMA腺病毒作为在体进行基因治疗的载体及其对正常肝细胞毒副作用。
课题研究的内容包括以下三个主要部分:
1.用HEK293 low passage(LP)细胞包装制备重组PUMA腺病毒(Ad-puma)和GFP(Ad-GFP)腺病毒及其鉴定。
我们实验用239LP细胞反复包装多次制备的PUMA腺病毒存储液。经Millipore 10 kDa超滤离心管浓缩富集后得到浓缩病毒液。通过普通PCR、Westernblot检测证明Ad-PUMA在目的细胞中有效高表达。且Ad-GFP在BNL CL.2细胞中有效表达绿色荧光蛋白。通过壳蛋白免疫法、GFP荧光法计算Ad-PUMA和Ad-GFP的感染性滴度,所制备的病毒浓缩液Ad-PUMA的滴度为2.0×108 ifu/ml;Ad-GFP滴度为2.1×109 ifu/ml。为进一步的生物学实验打下了良好的基础。
2.过表达PUMA后,对小鼠肝细胞BNL CL.2生物学功能的影响(包括周期和凋亡等生物学功能指标)。
在该部分生物学功能实验中,以MOI=8的Ad-PUMA和Ad-GFP感染细胞。经流式细胞仪检测分析ANNEXINⅤ标记的早期凋亡细胞,Ad-PUMA感染组比Ad-GFP对照组早期凋亡细胞高出约10倍;Hoechst 33342核染色发现,BNL CL.2细胞有明显的细胞凋亡形态特征,且随时间点推移凋亡细胞数明显增加;同时实验中我们利用Westernblot还检测发现,Puma介导的细胞凋亡效应也与线粒体凋亡通路Caspase级联效应有关。G0期同步化后的细胞周期发现,36小时S期细胞明显增多。36小时S/G2期细胞比Ad-GFP感染组和阴性对照组高出近4倍。PUMA过表达后可能使细胞从S期发展为G2期时发生了停留或阻滞。联系先前实验中puma在过程肝再生中下调,puma基因通过对肝细胞凋亡的调节和对细胞周期的影响参与到肝再生过程中。
3. PUMA对肝细胞生物学功能影响的分子机制研究。
我们运用免疫共沉淀实验进一步分析了BCL-2家族成员间在PUMA介导的凋亡中的相互作用。实验中分别研究PUMA、Bax与Bcl-XL的关系时发现:在腺病毒介导的PUMA过表达后,运用PUMA、Bax共沉淀抗体均可沉淀出Bcl-XL,即PUMA和Bax均可强烈结合Bcl-XL。而且Bax与Bcl-XL的结合,随时间点推移减低。在存在争议的PUMA与Bax的相互作用中,我们发现无论是HA共沉淀抗体pulldown检测Bax,还是用Bax共沉淀抗体pulldown检测PUMA,PUMA都能与Bax结合。因此我们推断,在BNL CL.2细胞中,PUMA促凋亡的分子机制既可能是通过与Bax竞争性结合Bcl-XL从而释放Bax,改变线粒体膜通透性而促凋亡,也可通过PUMA直接激活Bax的方式起作用。
PUMA (p53 up - regulated modulator of apoptosis) was discovered in colon carcinoma cells in 2001. PUMA expression causes rapid p53-dependent apoptosis and growth inhibition. It belongs to Bcl-2 family and consists of a BH3 domain. It is a member of BH3-only subgroups. BH3-only proteins sharing only the short BH3 interaction domain, by which they can bind to the pro-survival relatives to induce apoptosis. BH3-only proteins play a critical role in regulating apoptosis initiation through the mitochondria. BH3-only proteins function through multidomain Bcl-2 family members to induce apoptosis, either by antagonizing antiapoptotic proteins or by directly activating proapoptotic proteins.
The basal protein expression level of PUMA is relatively low according to past researches. However, in response to a range of stress signals,(Such as the DNA-damage, hypoxia and serum deprivation). PUMA could be induced in a p53 dependent or independent manner, and shows its potent pro-apoptotic activity. PUMA, an essential initiator of programmed cell death, can transduct signals of distinct apoptotic stimuli such as above factors. PUMA is a downstream target of the p53 tumor suppressor gene.
Previous studies in colon cancer cells showed that PUMA interacts with several antiapoptotic members of Bcl-2 family through its BH3 domain to induce apoptosis。PUMA initiates apoptosis partly by promoting Bax multimerization and mitochondrial translocation via dissociating Bax and Bcl-XL, thereby promoting Bax multimerization and mitochondrial translocation. Disrupting Bax Multimerization or Mitochondrial Localization could block PUMA-induced apoptosis. Therefore, it may be an important pathway of apoptosis.
In our previous works, we found that the PUMA expression in the early phase of liver regeneration was down-regulated transcriptionally. What does this finding suggest? Whether or not there is a close relationship between puma gene and liver regeneration? What is its the regulatory mechanism?We are very interasted in these problems and hope to solve these problems by experiment in vitro and in vivo. This paper mainly investigates its function in PUMA-mediated apoptosis and its mechanisms in mouse liver cells in vitro. It may help to explain functions and molecular mechanisms of PUMA in hepatocyte and provide envidence for exploring its role in liver regeneration. The next, respecting PUMA induce apoptosis in most mammalian cells, many studies indicated that PUMA adenovirus have broad applied prospect in gene therapy. Therefore, an experimental study investigat- ing the biological effects,toxicity and adverse reactions about adenovirus and adenovirus vector would be necessary.
This study consists of the following three parts:
1. The packaging and preparation of adenoviruses expressing PUMA (Ad-PUMA) and Adenoviruses expressing GFP (Ad-GFP).
2. The cellular biological function of BNL CL.2 cells after infected by Ad-PUMA.
3. Molecular mechanisms of the effection of PUMA on mouse liver cells.
The results indicated that the packaging and preparation of Adenoviruses expressing PUMA (Ad-PUMA) was available and effective expression in mouse liver cell BNL CL.2. PUMA could induce apoptosis in these cells and the number of cells at S stage displayed increasing tendency with the infection time. The caspase activation also seems to be dependent on these changes. Taking into account above factors, we then analyzed that PUMA induction affects the interactions between Bax and Bcl-XL, between PUMA and Bcl-XL, as well as the interactions between Bax and PUMA by immunoprecipitation.
既往研究表明PUMA基础表达量较低,在各种应激刺激(如DNA损伤,缺氧,糖皮质激素刺激及血清撤除等)影响下,可以通过p53依赖和非依赖两种途径被诱导表达,发挥着重要的促凋亡作用。由上述应激因素所诱导的细胞凋亡过程中,PUMA的作用是必需的,也就是说没有PUMA参与,就不会引发细胞凋亡。现有研究表明puma是p53的直接靶基因,也是介导p53凋亡通路的主要媒介分子。
在研究人类结直肠癌细胞中PUMA介导的细胞凋亡的分子机制时发现,PUMA通过其BH3结构域分别和Bcl-2家族多种抗凋亡成员(如Bcl-XL、Bcl-2等)产生广泛的相互作用,使原本结合于抗凋亡蛋白上的Bax解离并多聚化,进而转位激活线粒体依赖的内源性凋亡通路,诱导结肠癌细胞凋亡。此外Bax的缺失也会引起细胞凋亡障碍。因此PUMA对Bax的调控可能是一条重要的细胞凋亡通路。
我们的前期工作已经发现促凋亡基因puma在肝脏再生早期下调了5倍,其下调的意义是什么?是否提示PUMA低表达对肝细胞增殖的发动有显著作用?puma基因是否是调控肝脏再生的重要或关键靶点?其调控的机制是什么?我们对这些问题非常有兴趣,希望通过在体、离体实验揭示其规律。本课题通过离体实验研究PUMA对小鼠肝细胞凋亡的影响及其分子机制,对进一步揭示puma在肝脏再生过程中的作用有重要意义。同时,PUMA腺病毒有作为基因治疗药物的广阔应用前景(Biomed Pharmacother,2007)。运用腺病毒载体进行肝细胞离体实验研究,有助于初步了解PUMA腺病毒作为在体进行基因治疗的载体及其对正常肝细胞毒副作用。
课题研究的内容包括以下三个主要部分:
1.用HEK293 low passage(LP)细胞包装制备重组PUMA腺病毒(Ad-puma)和GFP(Ad-GFP)腺病毒及其鉴定。
我们实验用239LP细胞反复包装多次制备的PUMA腺病毒存储液。经Millipore 10 kDa超滤离心管浓缩富集后得到浓缩病毒液。通过普通PCR、Westernblot检测证明Ad-PUMA在目的细胞中有效高表达。且Ad-GFP在BNL CL.2细胞中有效表达绿色荧光蛋白。通过壳蛋白免疫法、GFP荧光法计算Ad-PUMA和Ad-GFP的感染性滴度,所制备的病毒浓缩液Ad-PUMA的滴度为2.0×108 ifu/ml;Ad-GFP滴度为2.1×109 ifu/ml。为进一步的生物学实验打下了良好的基础。
2.过表达PUMA后,对小鼠肝细胞BNL CL.2生物学功能的影响(包括周期和凋亡等生物学功能指标)。
在该部分生物学功能实验中,以MOI=8的Ad-PUMA和Ad-GFP感染细胞。经流式细胞仪检测分析ANNEXINⅤ标记的早期凋亡细胞,Ad-PUMA感染组比Ad-GFP对照组早期凋亡细胞高出约10倍;Hoechst 33342核染色发现,BNL CL.2细胞有明显的细胞凋亡形态特征,且随时间点推移凋亡细胞数明显增加;同时实验中我们利用Westernblot还检测发现,Puma介导的细胞凋亡效应也与线粒体凋亡通路Caspase级联效应有关。G0期同步化后的细胞周期发现,36小时S期细胞明显增多。36小时S/G2期细胞比Ad-GFP感染组和阴性对照组高出近4倍。PUMA过表达后可能使细胞从S期发展为G2期时发生了停留或阻滞。联系先前实验中puma在过程肝再生中下调,puma基因通过对肝细胞凋亡的调节和对细胞周期的影响参与到肝再生过程中。
3. PUMA对肝细胞生物学功能影响的分子机制研究。
我们运用免疫共沉淀实验进一步分析了BCL-2家族成员间在PUMA介导的凋亡中的相互作用。实验中分别研究PUMA、Bax与Bcl-XL的关系时发现:在腺病毒介导的PUMA过表达后,运用PUMA、Bax共沉淀抗体均可沉淀出Bcl-XL,即PUMA和Bax均可强烈结合Bcl-XL。而且Bax与Bcl-XL的结合,随时间点推移减低。在存在争议的PUMA与Bax的相互作用中,我们发现无论是HA共沉淀抗体pulldown检测Bax,还是用Bax共沉淀抗体pulldown检测PUMA,PUMA都能与Bax结合。因此我们推断,在BNL CL.2细胞中,PUMA促凋亡的分子机制既可能是通过与Bax竞争性结合Bcl-XL从而释放Bax,改变线粒体膜通透性而促凋亡,也可通过PUMA直接激活Bax的方式起作用。
PUMA (p53 up - regulated modulator of apoptosis) was discovered in colon carcinoma cells in 2001. PUMA expression causes rapid p53-dependent apoptosis and growth inhibition. It belongs to Bcl-2 family and consists of a BH3 domain. It is a member of BH3-only subgroups. BH3-only proteins sharing only the short BH3 interaction domain, by which they can bind to the pro-survival relatives to induce apoptosis. BH3-only proteins play a critical role in regulating apoptosis initiation through the mitochondria. BH3-only proteins function through multidomain Bcl-2 family members to induce apoptosis, either by antagonizing antiapoptotic proteins or by directly activating proapoptotic proteins.
The basal protein expression level of PUMA is relatively low according to past researches. However, in response to a range of stress signals,(Such as the DNA-damage, hypoxia and serum deprivation). PUMA could be induced in a p53 dependent or independent manner, and shows its potent pro-apoptotic activity. PUMA, an essential initiator of programmed cell death, can transduct signals of distinct apoptotic stimuli such as above factors. PUMA is a downstream target of the p53 tumor suppressor gene.
Previous studies in colon cancer cells showed that PUMA interacts with several antiapoptotic members of Bcl-2 family through its BH3 domain to induce apoptosis。PUMA initiates apoptosis partly by promoting Bax multimerization and mitochondrial translocation via dissociating Bax and Bcl-XL, thereby promoting Bax multimerization and mitochondrial translocation. Disrupting Bax Multimerization or Mitochondrial Localization could block PUMA-induced apoptosis. Therefore, it may be an important pathway of apoptosis.
In our previous works, we found that the PUMA expression in the early phase of liver regeneration was down-regulated transcriptionally. What does this finding suggest? Whether or not there is a close relationship between puma gene and liver regeneration? What is its the regulatory mechanism?We are very interasted in these problems and hope to solve these problems by experiment in vitro and in vivo. This paper mainly investigates its function in PUMA-mediated apoptosis and its mechanisms in mouse liver cells in vitro. It may help to explain functions and molecular mechanisms of PUMA in hepatocyte and provide envidence for exploring its role in liver regeneration. The next, respecting PUMA induce apoptosis in most mammalian cells, many studies indicated that PUMA adenovirus have broad applied prospect in gene therapy. Therefore, an experimental study investigat- ing the biological effects,toxicity and adverse reactions about adenovirus and adenovirus vector would be necessary.
This study consists of the following three parts:
1. The packaging and preparation of adenoviruses expressing PUMA (Ad-PUMA) and Adenoviruses expressing GFP (Ad-GFP).
2. The cellular biological function of BNL CL.2 cells after infected by Ad-PUMA.
3. Molecular mechanisms of the effection of PUMA on mouse liver cells.
The results indicated that the packaging and preparation of Adenoviruses expressing PUMA (Ad-PUMA) was available and effective expression in mouse liver cell BNL CL.2. PUMA could induce apoptosis in these cells and the number of cells at S stage displayed increasing tendency with the infection time. The caspase activation also seems to be dependent on these changes. Taking into account above factors, we then analyzed that PUMA induction affects the interactions between Bax and Bcl-XL, between PUMA and Bcl-XL, as well as the interactions between Bax and PUMA by immunoprecipitation.
引文
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[2] Yu J, Wang Z , Kinzler KW, et al . PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci U S A, 2003 , 100 (4) : 1 93126.
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[1] Yu J, Zhang L, Hwang PM. PUMA induces the rapid apoptosis of colorectal cancer cells [J]. Mol Cell, 2001, 7 (3) : 673-682.
[2] Nakano K,Vousden KH. PUMA, a novel p roapop totic gene, is induced by p53 [J]. Mol Cell, 2001, 7 (3): 683-694. [3 ] Han JW, Flemington C, Houghton AB, et al. Exp ression of bbc, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals [J]. PNAS, 2001, 98 (20): 11318-11323.
[4] Yu J, Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic response to p53 in colorectal cancer cells[ J ]. PNAS, 2003, 100 (4) :1931-1936.
[5] Shi Y. Mechanisms of caspase activation and inhibition during apoptosis.[J]. Mol Cell, 2002; 9(3):459-470.
[6] Vousden KH , Lu X. Live or let die : the cell’s response to p53. Nat Rev Cancer , 2002 , 2 (8) : 594-604.
[7] Shi Y. Mechanisms of caspase activation and inhibition during apoptosis [J]. Mol Cell, 2002; 9(3) :459-470.
[8] Ran RV, Hermel E, Castro-Obregon S, et a1. Coupling endoplasmic reticulum stress to the cell death program:mechanism of caspase activation [J]. J Biol Chem, 200l; 276 (36):33869-33874.
[9] Lan L, Zhao F, W ang Y, Zeng H. The mechanism of apoptosis induced by a nove1 thioredoxin reductase inhibitor in A549 cells: possible involvement of nuclear factor-kappaB-dependent pathway [J]. Eur J Pharmaco1, 2007; 555(2-3):83-92.
[10] Zhao F, Yan J, Deng S, Lan L,et al. A thioredoxin reductase inhibitor induces growth inhibition and apoptosis in five cultured human carcinoma cel1 1ines [J]. Cancer Lett, 2006; 236(1):46—53.
[11] Liu JW,Chandra D ,Tang SH , et al . Identification and characterization of Bimγ,a novel proapoptotic BH3-only splice variant of Bim[J]. Cancer Res ,2002 ,62 (10) :2976-2981.
[12] JiangM, Wei Q, Wang J , et al. Regulation of PUMA -alpha by p53 in cisplatin - induced renal cell apop tosis. Oncogene. 2006, 25 (29)∶4 056
[13] Yoo NJ, Lee JW, Jeong EG, Lee SH. Immunohistochemical analysis of pro-apoptotic PUMA protein and mutational analysis of PUMA gene in gastric carcinomas. Dig Liver Dis. 2007; 39: 222-227.
[14] Kim MR, Jeong EG, Chae B, Lee JW, Soung YH, HNam SW, Lee JY, Yoo NJ, Lee SH. Pro-apoptotic PUMA and anti-apoptotic phospho-BAD are highly expressed in colorectal carcinomas. Dig Dis Sci. 2007; 52: 2751-2756.
[15] Ahn CH, Jeong EG, Kim SS, Lee JW, Lee SH, Kim SH, Kim MS, Yoo NJ, Lee SH. Expressional and mutational analysis of pro-apoptotic Bcl-2 member PUMA in hepatocellular carcinomas. Dig Dis Sci. 2008; 53: 1395-1399.
[16] Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer [J]. Nature, 2001; 411(6835):342-348.
[1] Yu J, Zhang L, Hwang PM. PUMA induces the rapid apoptosis of colorectal cancer cells [J]. Mol Cell, 2001, 7 (3): 673-682.
[2] Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53 [J]. Mol Cell, 2001, 7 (3) : 683-694.
[3] Han JW, Flemington C, Houghton AB, et al. Expression of bbc, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals[ J ]. PNAS, 2001, 98 (20):11318-11323.
[4] Yu J,Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic response to p53 in colorectal cancer cells[ J ]. PNAS, 2003, 100 (4) :1931-1936.
[5] HemannMT, Zilfou JT, Zhao Z, et al. Suppression of tumorigenesis by the p53 target PUMA [J]. PNAS, 2004, 101 (25):9333-9338.
[6] Ming L, Wang P, Bank A, et al. PUMA Dissociates Bax and Bcl - X(L) to induce apoptosis in colon cancer cells. J Biol Chem, 2006, 281 (23)∶16034
[7] Zong WX. BH3-only proteins t hat bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax andBak[J ]. Genes Dev ,2001 , 15 :1481-1486
[8] Green DR , Kroemer G. The pathophysiology of mitochondrialcell death[J ] . Science ,2004 , 305 :626-629.
[9] Adams JM, Cory S. Bcl-2 regulated apoptosis mechanism and therapeutic potential [ J ]. Curr Op inImmunol, 2007, 19: 488 - 496
[10] Chipuk JE, Bouchier-Hayes L, Kuwana T, et al. PUMA couples the nuclear and cytoplasmic proapoptotic function of p53 [J]. Science, 2005, 309 (5741) : 1732-1735.
[11] Letai A, Bassik MC, Walensky LD, et al. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics [J]. Cancer Cell, 2002, 2 (3): 183-192.
[1] Cory S , Huang DCS , Adams JM. The Bcl-2 family : roles in cell survival and oncogenesis [J] . Oncogene , 2003 , 22 :8590-8607.
[2] Suzuki M , Youle RJ , Tjandra N. Structure of Bax : coregulation of dimer formation and intracellular localization. Cell , 2000 , 103 (4) : 645254.
[3] Yamaguchi H , Wang H.Bcl-XL protects BimEL-induced Bax conformational change and cytochrome c release independent of interacting with Bax or BimEL. J Biol Chem ,2002 , 277 : 41 604212.
[4] Cheng EH , Wei MC , Weiler S , et al . BCL-2 ,Bcl-XL sequester BH3 domain-only molecules preventing BAX-and BAK-mediated mitochondrial apoptosis. Mol Cell ,2001 , 8 (3) : 705211.
[5] Letai A , Bassik MC , Walensky LD , et al . Distinct BH3 domains either sensitize or activate mitochondrial apoptosis , serving as prototype cancer therapeutics. Cancer Cell ,2002 , 2 (3):183-92.
[6]药学学报Acta Pharmaceutica Sinica 2008, 43 (7) : 669 - 677·669·
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[8] Evan G, Little Wood T. A matter of life and cell death [ J ]. Science, 1998, 281 (5381) : 1317 - 1322.
[9] Eva B, Andrea H, Jin P, et al. Nuclear topography and expression of the BCR /ABL fusion gene and its protein level influenced by cell differentiation and RNA interference [ J ]. Leukemia Research, 2005, 29: 901 - 913.
[10] Tak H C, Tony K H , Keith WK, et al. Apotosis– Related Proteins in Cervical Intraep ithelial Neop lasia and Squamous Cell Carcinoma of the Cervix [J]. Gynecologic Oncology, 2002, 86: 14 - 18.
[11] Zong WX. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak [J] . Genes Dev, 2001, 15 :148121486
[12] Green DR , Kroemer G. The pathophysiology of mitochondrialcell death [J] . Science ,2004 , 305 :6262629.
[13] Adams JM, Cory S. Bcl-2 regulated apoptosis:mechanism and therapeutic potential [ J ]. Curr Op inImmunol, 2007, 19: 488 - 496.
[14] KarstAM, DaiDL, MartinkaM, et al. PUMA expression is significantly reduced in human cutaneous melanomas.Oncogene, 2005, 24 (6)∶1 111
[15] Karst AM, Dai DL, Martinka M , et al . PUMA expression is significantly reduced in human cutaneous melanomas [ J ] .Oncogene , 2005 ,24 :1111-1116.
[16] Hoque MO, Begum S, SommerM. PUMA in head and neck cancer. Cancer Lett. 2003, 199 (1)∶75
[17] Wang H, Qian H, Yu J , et al. Administration of PUMA adenovirus increases the sensitivity of esophageal cancer cells to anticancer drugs. Cancer Biol Ther. 2006, 5 (4)∶380
[18] Ming L, Wang P, Bank A, et al. PUMA Dissociates Bax and Bcl - X(L) to induce apoptosis in colon cancer cells. J Biol Chem, 2006, 281 (23)∶16034
[19] Jeffers JR, Parganas E, Lee Y. Puma is an essentialmediator of p53 dependent and independent apop- totic pathways. Cancer Cell, 2003, 4 (4)∶321.
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[21] Yu J , Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci USA. 2003, 100 (4)∶1931
[22] JiangM, Wei Q, Wang J , et al. Regulation of PUMA -alpha by p53 in cisplatin - induced renal cell apop tosis. Oncogene. 2006, 25 (29)∶4 056
[23] Wang X, Li M, Wang J, et al. The BH3 - only protein, PUMA, is involved in oxaliplatin - induced apoptosis in colon cancer cells. Biochem Pharmacol, 2006, 71 (11)∶1 540
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[26] Sunters A , Fernandez de Mattos S , Stahl M , et al . Foxo3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines [J]. J Biol Chem, 2003, 278(50) : 49795-49805.
[27] Liu JW,Chandra D ,Tang SH , et al . Identification and characterization of Bimγ,a novel proapoptotic BH3-only splice variant of Bim[J]. Cancer Res ,2002 ,62 (10) :2976-2981.
[28] Egle A , Harris AW, Bouillet P, et al. Bim is a suppressor of Myc induced mouse B cell leukaemia[J] . Proc N at l Acad Sci US A ,2004 , 101 :616426169.
[29] Tagawa H, Karnan S, Suzuki R, et al. Genome-wide arraybased CGH for mantle cell lymphoma: identification of homozygous deletions of the proapoptotic gene BIM [J]. Oncogene, 2005, 24:1348- 1358.
[30] Bouillet P , Metcalf D , Huang DCS , et al . Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses leukocyte homeostasis, and to preclude autoimmunity [J] Science, 1999, 286 : 1735-1738.
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[41] Itoh M,Noutomi T, Chiba H, et al. Bcl-XL antisense treatment sensitizes Bcl-XL overexpressing squamous cell carcinoma cells to carboplatin. Oral Oncol,2002,38/(8):725-756.
[42] Hopkins Donaldson S, et al. Induction of apoptosis and chemosensitization of mesothelioma cells by Bcl-2 and Bcl-XL antisense treatment Int J Cancer,2003,106(2):160-166
[43]Takehara T, Takahashi H. Suppression of Bcl-xL deamidation in human hepatocellular carcinomas[J] . Cancer Res, 2003, 63 (12):305423057.
[44] Deverman BE ,Cook BL ,Manson SR , et al .Bcl-XL deamidation is acritical switch in the regulation of the response to DNA damage [J ] .Cell ,2002 ,111 (1) :51-62.
[45]医学临床研究2006年5月第23卷第5期J Clin Res , May 2006 , Vol 23 ,№5·801·
[46] Cory S, Huang DCS, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis [J] . Oncogene, 2003, 22 :8590-8607.
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[48]刘雪梅,黄剑飞. Bcl-2、bad在乳腺癌中表达的临床意义[J].河南肿瘤学杂志, 2005; 18 (1)∶14
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[1] Yu J, Zhang L, Hwang PM. PUMA induces the rapid apoptosis of colorectal cancer cells [J]. Mol Cell, 2001, 7 (3) : 673-682.
[2] Nakano K,Vousden KH. PUMA, a novel p roapop totic gene, is induced by p53 [J]. Mol Cell, 2001, 7 (3): 683-694. [3 ] Han JW, Flemington C, Houghton AB, et al. Exp ression of bbc, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals [J]. PNAS, 2001, 98 (20): 11318-11323.
[4] Yu J, Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic response to p53 in colorectal cancer cells[ J ]. PNAS, 2003, 100 (4) :1931-1936.
[5] Shi Y. Mechanisms of caspase activation and inhibition during apoptosis.[J]. Mol Cell, 2002; 9(3):459-470.
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[1] Yu J, Zhang L, Hwang PM. PUMA induces the rapid apoptosis of colorectal cancer cells [J]. Mol Cell, 2001, 7 (3): 673-682.
[2] Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, is induced by p53 [J]. Mol Cell, 2001, 7 (3) : 683-694.
[3] Han JW, Flemington C, Houghton AB, et al. Expression of bbc, a pro-apoptotic BH3-only gene, is regulated by diverse cell death and survival signals[ J ]. PNAS, 2001, 98 (20):11318-11323.
[4] Yu J,Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic response to p53 in colorectal cancer cells[ J ]. PNAS, 2003, 100 (4) :1931-1936.
[5] HemannMT, Zilfou JT, Zhao Z, et al. Suppression of tumorigenesis by the p53 target PUMA [J]. PNAS, 2004, 101 (25):9333-9338.
[6] Ming L, Wang P, Bank A, et al. PUMA Dissociates Bax and Bcl - X(L) to induce apoptosis in colon cancer cells. J Biol Chem, 2006, 281 (23)∶16034
[7] Zong WX. BH3-only proteins t hat bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax andBak[J ]. Genes Dev ,2001 , 15 :1481-1486
[8] Green DR , Kroemer G. The pathophysiology of mitochondrialcell death[J ] . Science ,2004 , 305 :626-629.
[9] Adams JM, Cory S. Bcl-2 regulated apoptosis mechanism and therapeutic potential [ J ]. Curr Op inImmunol, 2007, 19: 488 - 496
[10] Chipuk JE, Bouchier-Hayes L, Kuwana T, et al. PUMA couples the nuclear and cytoplasmic proapoptotic function of p53 [J]. Science, 2005, 309 (5741) : 1732-1735.
[11] Letai A, Bassik MC, Walensky LD, et al. Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics [J]. Cancer Cell, 2002, 2 (3): 183-192.
[1] Cory S , Huang DCS , Adams JM. The Bcl-2 family : roles in cell survival and oncogenesis [J] . Oncogene , 2003 , 22 :8590-8607.
[2] Suzuki M , Youle RJ , Tjandra N. Structure of Bax : coregulation of dimer formation and intracellular localization. Cell , 2000 , 103 (4) : 645254.
[3] Yamaguchi H , Wang H.Bcl-XL protects BimEL-induced Bax conformational change and cytochrome c release independent of interacting with Bax or BimEL. J Biol Chem ,2002 , 277 : 41 604212.
[4] Cheng EH , Wei MC , Weiler S , et al . BCL-2 ,Bcl-XL sequester BH3 domain-only molecules preventing BAX-and BAK-mediated mitochondrial apoptosis. Mol Cell ,2001 , 8 (3) : 705211.
[5] Letai A , Bassik MC , Walensky LD , et al . Distinct BH3 domains either sensitize or activate mitochondrial apoptosis , serving as prototype cancer therapeutics. Cancer Cell ,2002 , 2 (3):183-92.
[6]药学学报Acta Pharmaceutica Sinica 2008, 43 (7) : 669 - 677·669·
[7] Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics [ J ]. Br J Cancer, 1972, 26 (4) : 239 - 2571
[8] Evan G, Little Wood T. A matter of life and cell death [ J ]. Science, 1998, 281 (5381) : 1317 - 1322.
[9] Eva B, Andrea H, Jin P, et al. Nuclear topography and expression of the BCR /ABL fusion gene and its protein level influenced by cell differentiation and RNA interference [ J ]. Leukemia Research, 2005, 29: 901 - 913.
[10] Tak H C, Tony K H , Keith WK, et al. Apotosis– Related Proteins in Cervical Intraep ithelial Neop lasia and Squamous Cell Carcinoma of the Cervix [J]. Gynecologic Oncology, 2002, 86: 14 - 18.
[11] Zong WX. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak [J] . Genes Dev, 2001, 15 :148121486
[12] Green DR , Kroemer G. The pathophysiology of mitochondrialcell death [J] . Science ,2004 , 305 :6262629.
[13] Adams JM, Cory S. Bcl-2 regulated apoptosis:mechanism and therapeutic potential [ J ]. Curr Op inImmunol, 2007, 19: 488 - 496.
[14] KarstAM, DaiDL, MartinkaM, et al. PUMA expression is significantly reduced in human cutaneous melanomas.Oncogene, 2005, 24 (6)∶1 111
[15] Karst AM, Dai DL, Martinka M , et al . PUMA expression is significantly reduced in human cutaneous melanomas [ J ] .Oncogene , 2005 ,24 :1111-1116.
[16] Hoque MO, Begum S, SommerM. PUMA in head and neck cancer. Cancer Lett. 2003, 199 (1)∶75
[17] Wang H, Qian H, Yu J , et al. Administration of PUMA adenovirus increases the sensitivity of esophageal cancer cells to anticancer drugs. Cancer Biol Ther. 2006, 5 (4)∶380
[18] Ming L, Wang P, Bank A, et al. PUMA Dissociates Bax and Bcl - X(L) to induce apoptosis in colon cancer cells. J Biol Chem, 2006, 281 (23)∶16034
[19] Jeffers JR, Parganas E, Lee Y. Puma is an essentialmediator of p53 dependent and independent apop- totic pathways. Cancer Cell, 2003, 4 (4)∶321.
[20] KelekarA, Th omp son CB. Bcl-2 family p roteins: The role of the BH3 domain in apoptosis. Trends Cell Biol, 1998, 8 (8)∶324
[21] Yu J , Wang Z, Kinzler KW, et al. PUMA mediates the apoptotic response to p53 in colorectal cancer cells. Proc Natl Acad Sci USA. 2003, 100 (4)∶1931
[22] JiangM, Wei Q, Wang J , et al. Regulation of PUMA -alpha by p53 in cisplatin - induced renal cell apop tosis. Oncogene. 2006, 25 (29)∶4 056
[23] Wang X, Li M, Wang J, et al. The BH3 - only protein, PUMA, is involved in oxaliplatin - induced apoptosis in colon cancer cells. Biochem Pharmacol, 2006, 71 (11)∶1 540
[24] Bouillet P ,Zhang LC ,Huang DC , et al . Gene structure, alternative splicing, and chromosomallocalization of pro-apoptotic Bcl-2 relative Bim[J]. Mamm Genome, 2001, 12 (2) :163-168.
[25] Zhou XM, Liu Y, Payne G, et al. Growth factors inactivate the cell death promoter BAD by phosphorylation of its BH3 domain on Ser155 [J] . J Biol Chem,2000 ,275 (32) :25046-25051
[26] Sunters A , Fernandez de Mattos S , Stahl M , et al . Foxo3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines [J]. J Biol Chem, 2003, 278(50) : 49795-49805.
[27] Liu JW,Chandra D ,Tang SH , et al . Identification and characterization of Bimγ,a novel proapoptotic BH3-only splice variant of Bim[J]. Cancer Res ,2002 ,62 (10) :2976-2981.
[28] Egle A , Harris AW, Bouillet P, et al. Bim is a suppressor of Myc induced mouse B cell leukaemia[J] . Proc N at l Acad Sci US A ,2004 , 101 :616426169.
[29] Tagawa H, Karnan S, Suzuki R, et al. Genome-wide arraybased CGH for mantle cell lymphoma: identification of homozygous deletions of the proapoptotic gene BIM [J]. Oncogene, 2005, 24:1348- 1358.
[30] Bouillet P , Metcalf D , Huang DCS , et al . Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses leukocyte homeostasis, and to preclude autoimmunity [J] Science, 1999, 286 : 1735-1738.
[31] Tsuj Imoyo Y , Finger L R , Yun IS J , et al . Cloning of the chromosome break point of neoplastic cellswith the t ( 14 -18 ) chromosome trattslocation [ J ]. Science, 1984, 226: 1097- 1099 .
[32] L iu XJ , Zhang LQ, L iu XL, et al. Role for the Bcl-2 family proteins and BH3 domain in apoptosi [J]. Prog Biochem Biophys (生物化学与生物物理进展), 2006,33:221–222
[33] Liu X , Dai S , Zhu Y, et al . The st ructure of a Bcl-XL/ Bim fragment complex: Implications for Bim function [J] .Immunity , 2003, 19:341-352.
[34] De Cesare M, Perego P, Righetti SC, et al. Enhanced antitumour efficacy of gimatecan in combination tith Bcl-2 antisense oligonucleotide in human melanoma xenografts. Eur J Cancer, 2005, 41(8):1213-1222.
[35] Kim R, Emi M, Tanabe K, et al. Therapeutic potential of antisense Bcl-2 as a chemosensitizer for cancer therapy. Cancer, 2004, 101(11);2492-2502
[36] Kim R,Tanabe K, Emi M, et al. Modulation of tamoxifen sensitivity by antisense Bcl-2 and trastuzumab in breast carcinoma cells. Cancer, 2005, 103(10):2199-2207
[37] Sinicrope FA, Ruan SB, Cleary KR, Stephens LC, Lee JJ, Levin B. Bcl-2 and p53 oncoprotein expression during colorectal tumorigenesis. Cancer Res 1995; 55: 237-241
[38] Dursun A, Poyraz A, Suer O, Sezer C, Akyol G. Expression of Bcl-2 and c-ErbB-2 in colorectal neoplasia. Pathol Oncol Res 2001; 7: 24-27
[39] Ferlini C, Raspaglio G, Mozzetti S et al. Bcl-2 down-regulation is a novel mechanism of paclitaxelresistance[J]. Mol Pharmacol, 2003,64 (1): 51-58
[40] Kim R, Emi M, Tanabe K, et al. Therapeutic potential of antisense Bcl-2 as a chemosensitizer for cancer therapy[J] . Tutorial Cancer,2004, 101(11):2491-2502
[41] Itoh M,Noutomi T, Chiba H, et al. Bcl-XL antisense treatment sensitizes Bcl-XL overexpressing squamous cell carcinoma cells to carboplatin. Oral Oncol,2002,38/(8):725-756.
[42] Hopkins Donaldson S, et al. Induction of apoptosis and chemosensitization of mesothelioma cells by Bcl-2 and Bcl-XL antisense treatment Int J Cancer,2003,106(2):160-166
[43]Takehara T, Takahashi H. Suppression of Bcl-xL deamidation in human hepatocellular carcinomas[J] . Cancer Res, 2003, 63 (12):305423057.
[44] Deverman BE ,Cook BL ,Manson SR , et al .Bcl-XL deamidation is acritical switch in the regulation of the response to DNA damage [J ] .Cell ,2002 ,111 (1) :51-62.
[45]医学临床研究2006年5月第23卷第5期J Clin Res , May 2006 , Vol 23 ,№5·801·
[46] Cory S, Huang DCS, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis [J] . Oncogene, 2003, 22 :8590-8607.
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