喉癌中自噬基因Beclin1的表达及对顺铂药敏的实验研究
摘要
背景与研究目的
喉癌是头颈部常见的恶性肿瘤之一,严重威胁着患者的健康,寻找有效的治疗方法一直是临床的迫切需求,在喉癌的治疗中,基因治疗已成为手术、化疗放疗三大治疗外新的治疗手段。
自噬是细胞内成分被溶酶体降解过程的统称,在清除损伤或多余的细胞器,维持细胞内环境稳定方面起着重要作用。自噬可清除损伤的细胞器,避免如自由基等有毒性物质的产生,从而使突变率降低,抑制肿瘤的形成,而自噬的缺陷可导致肿瘤的发生。Beclin1基因是第一个被发现的哺乳动物的自噬基因,在多种肿瘤中表现为表达降低和缺失,Beclin1表达缺失明显增加肝癌、肺癌和淋巴瘤的发生率,但自噬基因Beclin1与喉癌的关系尚不清楚。
本研究通过检测喉癌组织中自噬基因Beclin1的表达,探讨自噬基因Beclin1与喉癌发生发展的关系；研究自噬基因Beclin1在喉癌Hep-2细胞中表达增强和缺失对细胞体外增殖、自噬、凋亡的作用及对喉癌Hep-2细胞顺铂敏感性的影响,探讨自噬诱导在喉癌治疗中的可行性,为喉癌的基因治疗提供新的思路。
方法：
1.采用免疫组化SP法对77例喉癌组织和22例正常喉组织进行Beclin1蛋白表达水平进行检测,分析Beclin1与喉癌临床分型、T分期、组织分化程度、淋巴结转移等临床病理因素的相关性；
2.通过RT-PCR法从人正常喉组织中获得目的基因Beclin1,将其插入真核表达载体pcDNA3.1中,构建真核表达载体pcDNA3.1/Beclin1;设计针对Beclin1的特异性RNA干扰序列,构建Beclin1基因的小发夹状RNA (shRNA)真核表达质粒pSUPER/Beclin1;
3.脂质体法将重组质粒pcDNA3.1/Beclinl和pSUPER/Beclin1转染人喉癌细胞株Hep-2,筛选稳定表达株,采用荧光定量RT-PCR及Western Blot分别在mRNA和蛋白质水平检测转染前后Beclin1、LC3和p62蛋白的表达变化；流式细胞仪检测自噬情况；Hoechs检测细胞凋亡；MTT法检测细胞的增殖变化；
4.顺铂处理Hep-2细胞后,MDC染色检测自噬情况；将重组质粒pcDNA3.1/Beclin1和pSUPER/Beclin1转染人喉癌细胞株Hep-2后,顺铂处理,MTT法检测细胞的增殖变化。
结果：
1.喉癌组织中Beclin1的蛋白表达阳性率低于正常喉组织(P<0.05),喉癌中常见Beclin1表达下调和缺失；Beclin1表达与喉癌的临床分型无关(P>0.05),与喉癌的T分期、分化程度及淋巴结转移相关(P<0.05)
2.利用PCR分析,酶切鉴定及DNA测序证实Beclin1基因的表达质粒pcDNA3.1/Beclin1(?)PRNA干扰质粒pSUPER/Beclin1构建成功；
3. pcDNA3.1/Beclin1转染Hep-2细胞后,Beclin1、LC3Ⅱ mRNA和蛋白的表达显著提高,p62mRNA和蛋白的表达显著降低(P<0.05),与未转染组相比,凋亡细胞和自噬细胞明显增加,细胞生长活性降低；
4. pSUPER/Beclin1转染Hep-2细胞后,Beclin1> LC3Ⅱ mRNA和蛋白的表达明显降低,p62mRNA和蛋白的表达显著增高(P<0.05),与未转染组相比,凋亡细胞和自噬细胞明显减少,细胞生长活性增强；
5.顺铂处理Hep-2细胞后,细胞内自噬增加,且呈浓度依赖关系,重组质粒pSUPER/Beclin1可以显著提高Hep-2细胞对顺铂的敏感性。
结论：
自噬基因Beclin1与喉癌的发生、发展有关,自噬基因Beclin1可有效抑制喉癌细胞的增殖,提高肿瘤细胞的自噬和凋亡能力,同时抑制自噬基因Beclin1表达能提高喉癌Hep-2细胞对顺铂的敏感性。
Background&Objective
Laryngeal cancer is one of the common malignant tumors in head and neck, anda serious threat to the health of patients. It is urgent to find effective treatment oflaryngeal cancer in clinical practice. Surgery, chemotherapy and radiation therapy areconventional treatment for laryngeal carcinoma, and gene therapy is regard as a newtreatment strategy for laryngeal carcinoma.
Autophagy plays a critical role in removing damaged or surplus organelles inorder to maintain cellular homeostasis. For example, by removing damagedorganelles, autophagy may limit the exposure of cellular DNA to genotoxic-stressessuch as free radicals. The removal of damaged organelles through autophagicdegradation would thus decrease the basal mutation rate and suppress oncogenesis.Beclin1, the first identified mammalian autophagy gene, is essential to formation ofautophagosome. Some studies reported that Beclin1expression was down-regulatedin several carcinomas, and loss of Beclin1would contribute to an increased incidenceof cancer, such as heptocellular carcinoma, lung adenocarcinoma and lymphoma. It isunclear that correlation of autophagy gene Beclin1to tumorigenesis anddevelopment of laryngeal cancer.
In the study, immunohistochemistry was employed to determine the expressionof Beclin1and explored its clinical significance in laryngeal cancer. We constructedthe eukaryotic expression vector pcDNA3.1/Beclin1and shRNA expression vectorpSUPER/Beclin1, transfected into Hep-2cells, to find the effect of Beclin1overexpression and downexpression on the growth, autophagy and apoptosis oflaryngeal cancer cell line Hep-2in vitro, and to investigate the role of the autophagyin the regulation of chemosensitivity to cisplatin in laryngeal cancer Hep-2cells, sowe explore the feasibility of induce of autophagy in treatment of laryngeal cancer.
Methods
1. The expression of Beclin1was detected with SP immunohistochemistry inspecimens of laryngeal carcinoma and normal laryngeal tissue. Correlations ofexpression of Beclin1gene to clinicopathologic factors of laryngeal carcinoma werestatistically analyzed;
2. The eukaryotic expression vector pcDNA3.1/Beclin1and shRNA expressionvector pSUPER/Beclin1were constructed;
3. The vectors were transfected into Hep-2cells via lipofectamine. Theexpression levels of Beclin1, LC3and p62mRNA and protein were detected byreal-time RT-PCR, Western Blot analysis in transfected cells. The autophagy,apoptosis and cell proliferations of Hep-2cells were measured with flow cytometry,Hoechs dye and MTT method after transfection;
4. Hep-2cells were treated with cisplatin and the autophagysonmes weredetected by MDC analysis. The vectors, pcDNA3.1/Beclin1and pSUPER/Beclin weretransfected into Hep-2cells, then, treated with cisplatin, and cell proliferations ofHep-2cells were measured with MTT method.
Results
1. Beclin1protein expression in tumor cells were down-regulation in laryngealcancer, significantly lower than that in normal laryngeal tissue (P <0.05); theexpression of Beclin1has no correlation with clinic types (P>0.05), and correlationwith T stage, lymph nodes metastases and tumor histological grade (P <0.05);
2. PCR analysis and DNA sequencing confirmed that the eukaryotic expressionvector pcDNA3.1(+)/Beclin1and shRNA expression vectors pSUPER/Beclin1wereconstructed successfully;
3. The eukaryotic expression vector pcDNA3.1/Beclin1significantly improvedthe expressin of mRNA and protein of Beclin1and LC3,and decreased theexpressin of mRNA and protein of p62in Hep-2cells (P <0.05), the cellproliferations of Hep-2cell were inhibited, while more apoptosis cells and moreautophagy cells were identified in these cells;
4. The shRNA expression vectors pSUPER Beclin1significantly inhibited theexpressin of mRNA and protein of Beclin1and LC3,and increased the expressin ofmRNA and protein of p62in Hep-2cells (P <0.05), and the cell proliferations ofHep-2cell were improved, while less apoptosis cells and less autophagy cells wereidentified;
5. Cisplatin significantly improved the expression of Beclin1and LC3protein((P <0.05)) and the pSUPER/Beclin1increased the sensitibity to cisplatin of Hep-2cell.
Conclusions
Beclin1expression was down-regulated in laryngeal cancer, which may becorrelated with the occurrence and development of laryngeal cancer. Beclin1overexpression can induce autophagy and apoptosis in Hep-2cell, inhibittumorigenesis of Hep-2cell in vitro, and defective autophagy may contribute to thesensitibity to cisplatin of Hep-2cell.
喉癌是头颈部常见的恶性肿瘤之一,严重威胁着患者的健康,寻找有效的治疗方法一直是临床的迫切需求,在喉癌的治疗中,基因治疗已成为手术、化疗放疗三大治疗外新的治疗手段。
自噬是细胞内成分被溶酶体降解过程的统称,在清除损伤或多余的细胞器,维持细胞内环境稳定方面起着重要作用。自噬可清除损伤的细胞器,避免如自由基等有毒性物质的产生,从而使突变率降低,抑制肿瘤的形成,而自噬的缺陷可导致肿瘤的发生。Beclin1基因是第一个被发现的哺乳动物的自噬基因,在多种肿瘤中表现为表达降低和缺失,Beclin1表达缺失明显增加肝癌、肺癌和淋巴瘤的发生率,但自噬基因Beclin1与喉癌的关系尚不清楚。
本研究通过检测喉癌组织中自噬基因Beclin1的表达,探讨自噬基因Beclin1与喉癌发生发展的关系；研究自噬基因Beclin1在喉癌Hep-2细胞中表达增强和缺失对细胞体外增殖、自噬、凋亡的作用及对喉癌Hep-2细胞顺铂敏感性的影响,探讨自噬诱导在喉癌治疗中的可行性,为喉癌的基因治疗提供新的思路。
方法：
1.采用免疫组化SP法对77例喉癌组织和22例正常喉组织进行Beclin1蛋白表达水平进行检测,分析Beclin1与喉癌临床分型、T分期、组织分化程度、淋巴结转移等临床病理因素的相关性；
2.通过RT-PCR法从人正常喉组织中获得目的基因Beclin1,将其插入真核表达载体pcDNA3.1中,构建真核表达载体pcDNA3.1/Beclin1;设计针对Beclin1的特异性RNA干扰序列,构建Beclin1基因的小发夹状RNA (shRNA)真核表达质粒pSUPER/Beclin1;
3.脂质体法将重组质粒pcDNA3.1/Beclinl和pSUPER/Beclin1转染人喉癌细胞株Hep-2,筛选稳定表达株,采用荧光定量RT-PCR及Western Blot分别在mRNA和蛋白质水平检测转染前后Beclin1、LC3和p62蛋白的表达变化；流式细胞仪检测自噬情况；Hoechs检测细胞凋亡；MTT法检测细胞的增殖变化；
4.顺铂处理Hep-2细胞后,MDC染色检测自噬情况；将重组质粒pcDNA3.1/Beclin1和pSUPER/Beclin1转染人喉癌细胞株Hep-2后,顺铂处理,MTT法检测细胞的增殖变化。
结果：
1.喉癌组织中Beclin1的蛋白表达阳性率低于正常喉组织(P<0.05),喉癌中常见Beclin1表达下调和缺失；Beclin1表达与喉癌的临床分型无关(P>0.05),与喉癌的T分期、分化程度及淋巴结转移相关(P<0.05)
2.利用PCR分析,酶切鉴定及DNA测序证实Beclin1基因的表达质粒pcDNA3.1/Beclin1(?)PRNA干扰质粒pSUPER/Beclin1构建成功；
3. pcDNA3.1/Beclin1转染Hep-2细胞后,Beclin1、LC3Ⅱ mRNA和蛋白的表达显著提高,p62mRNA和蛋白的表达显著降低(P<0.05),与未转染组相比,凋亡细胞和自噬细胞明显增加,细胞生长活性降低；
4. pSUPER/Beclin1转染Hep-2细胞后,Beclin1> LC3Ⅱ mRNA和蛋白的表达明显降低,p62mRNA和蛋白的表达显著增高(P<0.05),与未转染组相比,凋亡细胞和自噬细胞明显减少,细胞生长活性增强；
5.顺铂处理Hep-2细胞后,细胞内自噬增加,且呈浓度依赖关系,重组质粒pSUPER/Beclin1可以显著提高Hep-2细胞对顺铂的敏感性。
结论：
自噬基因Beclin1与喉癌的发生、发展有关,自噬基因Beclin1可有效抑制喉癌细胞的增殖,提高肿瘤细胞的自噬和凋亡能力,同时抑制自噬基因Beclin1表达能提高喉癌Hep-2细胞对顺铂的敏感性。
Background&Objective
Laryngeal cancer is one of the common malignant tumors in head and neck, anda serious threat to the health of patients. It is urgent to find effective treatment oflaryngeal cancer in clinical practice. Surgery, chemotherapy and radiation therapy areconventional treatment for laryngeal carcinoma, and gene therapy is regard as a newtreatment strategy for laryngeal carcinoma.
Autophagy plays a critical role in removing damaged or surplus organelles inorder to maintain cellular homeostasis. For example, by removing damagedorganelles, autophagy may limit the exposure of cellular DNA to genotoxic-stressessuch as free radicals. The removal of damaged organelles through autophagicdegradation would thus decrease the basal mutation rate and suppress oncogenesis.Beclin1, the first identified mammalian autophagy gene, is essential to formation ofautophagosome. Some studies reported that Beclin1expression was down-regulatedin several carcinomas, and loss of Beclin1would contribute to an increased incidenceof cancer, such as heptocellular carcinoma, lung adenocarcinoma and lymphoma. It isunclear that correlation of autophagy gene Beclin1to tumorigenesis anddevelopment of laryngeal cancer.
In the study, immunohistochemistry was employed to determine the expressionof Beclin1and explored its clinical significance in laryngeal cancer. We constructedthe eukaryotic expression vector pcDNA3.1/Beclin1and shRNA expression vectorpSUPER/Beclin1, transfected into Hep-2cells, to find the effect of Beclin1overexpression and downexpression on the growth, autophagy and apoptosis oflaryngeal cancer cell line Hep-2in vitro, and to investigate the role of the autophagyin the regulation of chemosensitivity to cisplatin in laryngeal cancer Hep-2cells, sowe explore the feasibility of induce of autophagy in treatment of laryngeal cancer.
Methods
1. The expression of Beclin1was detected with SP immunohistochemistry inspecimens of laryngeal carcinoma and normal laryngeal tissue. Correlations ofexpression of Beclin1gene to clinicopathologic factors of laryngeal carcinoma werestatistically analyzed;
2. The eukaryotic expression vector pcDNA3.1/Beclin1and shRNA expressionvector pSUPER/Beclin1were constructed;
3. The vectors were transfected into Hep-2cells via lipofectamine. Theexpression levels of Beclin1, LC3and p62mRNA and protein were detected byreal-time RT-PCR, Western Blot analysis in transfected cells. The autophagy,apoptosis and cell proliferations of Hep-2cells were measured with flow cytometry,Hoechs dye and MTT method after transfection;
4. Hep-2cells were treated with cisplatin and the autophagysonmes weredetected by MDC analysis. The vectors, pcDNA3.1/Beclin1and pSUPER/Beclin weretransfected into Hep-2cells, then, treated with cisplatin, and cell proliferations ofHep-2cells were measured with MTT method.
Results
1. Beclin1protein expression in tumor cells were down-regulation in laryngealcancer, significantly lower than that in normal laryngeal tissue (P <0.05); theexpression of Beclin1has no correlation with clinic types (P>0.05), and correlationwith T stage, lymph nodes metastases and tumor histological grade (P <0.05);
2. PCR analysis and DNA sequencing confirmed that the eukaryotic expressionvector pcDNA3.1(+)/Beclin1and shRNA expression vectors pSUPER/Beclin1wereconstructed successfully;
3. The eukaryotic expression vector pcDNA3.1/Beclin1significantly improvedthe expressin of mRNA and protein of Beclin1and LC3,and decreased theexpressin of mRNA and protein of p62in Hep-2cells (P <0.05), the cellproliferations of Hep-2cell were inhibited, while more apoptosis cells and moreautophagy cells were identified in these cells;
4. The shRNA expression vectors pSUPER Beclin1significantly inhibited theexpressin of mRNA and protein of Beclin1and LC3,and increased the expressin ofmRNA and protein of p62in Hep-2cells (P <0.05), and the cell proliferations ofHep-2cell were improved, while less apoptosis cells and less autophagy cells wereidentified;
5. Cisplatin significantly improved the expression of Beclin1and LC3protein((P <0.05)) and the pSUPER/Beclin1increased the sensitibity to cisplatin of Hep-2cell.
Conclusions
Beclin1expression was down-regulated in laryngeal cancer, which may becorrelated with the occurrence and development of laryngeal cancer. Beclin1overexpression can induce autophagy and apoptosis in Hep-2cell, inhibittumorigenesis of Hep-2cell in vitro, and defective autophagy may contribute to thesensitibity to cisplatin of Hep-2cell.
引文
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[1]Levine B, Klionsky DJ. Development by self-digestion; molecular mechanisms and biological functions of autophagy. Dev Cell2004;6:463-77.
[2]Mizushima N. The pleiotropic role of autophagy:From protein metabolism to bactericide. Cell Death Differ2005;12:1535-41.
[3]Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, Mukherjee C, Shi C, Gelinas C, Fan Y, Nelson Da, Jin S, White E. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell2006:51-64.
[4]Klionsky DJ. Autophagy. Current Biology,2005;15(8):282-283.
[5]Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol,2004;36(12):2405-2419.
[6]Yu L, Lenardo MJ, Baehreche EH. Autophagy and caspases:a new cell death program. Cell Cycle,2004;3(9):1124-1126.
[7]Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science2000;290:1717-21.
[8]Levine B, Klionsky DJ.Development by self-digestion:molecular mechanisma and biological functions of autophagy. Dev Cell2004;6:463-77.
[9]Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett1993;333:169-74.
[10]Thumm M, Egner R, Koch B, Schlumpberger M, Straub M, Veenhuis M, et al. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Lett1994;349:275-80.
[11]Harding TM, Morano KA, Scott SV, Klionsky DJ. Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. J Cell Bio1995;131:591-602
[12]Klionsky DJ, Cregg JM, Dunn WAJ, Emr SD, Sakai Y. Sandoval IV. et al. A unified nomenclature for yeast autophagy-related genes. Dev Cell2003;5:539-45.
[13] Ohsumi Y, Molecular dissection of autophagy: two ubiquitin-like systems.Nat Rev Moi Cell Biol2001;2:211-6.
[14] Reggiori E, Klionsky DJ. Autophagy in the eukaryotic cell. Eukaryot Cell2002;1:11-21.
[15] Baehrecke EH. How death shapes life during development. Nat Rev Moi CellBiol2002;3:779-87.
[16] Schweichel J-U, Merker H-J. The morphology of various types of cell deathin prenatal tissues. Teratology1973;7:253-66.
[17] Clarke PGH. Developmental cell death: morphological diversity and multiplemechanisms. Anat Embryol1990;181:195-213.
[18] Hengartner MO, The biochemistry of apoptosis. Nature2000;407:770-6.
[19] Jaattela M, Tschopp J. Caspase-independent cell death in T lymphocytes. NatImmunol2003;4:416-23.
[20] Chautan M, Chazal G, Cecconi F, Gruss P, Golstein P. Interdigial cell deathcan occur through a necrotic and caspase-independent pathway. Curr Biol1999;9:967-70.
[21] Oppenheim RW, Flavell RA, Vinsant S, Prevette D, Kuan CY, Rakic P.Programmed cell death of developing mammalian neurons after genetic deletion ofcaspases. J Neurosci2000;21:4752-60.
[22] Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S. et al. Regulation of anATG7-beclin1program of autophagic cell death by caspase-8. Science2004;304:1500-2.
[23] Onodera J, Ohsumi Y. Ald6p is a preferred target for autophagy in yeast,Saccharomyces cerevisiae. J Biol Chem2004;279:16071-6.
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[28] Lee C-Y, Clough EA, Yellon P, Teslovich TM, Stephan DA, Baehrecke EH.Genome-wide analysis of steroid-and radiation-triggered programmed cell death inDrosophila. Curr boil2003;13:350-7.
[29] Martin DN, Baehrecke EH. Caspase function in autophagic cell death inDrosophila. Development2004;131:275-84.
[30]Chun HJ, Zheng L, Ahmad M, Wang J, Speirs CK, Siegel RM. et al.Pleiotropic defects in lymphocyte activations caused by caspase-8mutations lead tohuman immunodeficiency. Nature2002;419:395-9.
[31] Arama E, Agapite J, Steller H. Caspase activity and a specific cytochrome care require for sperm differentination in Drosophila. Dev Cell2003;4:687-97.
[32]Huh JR, Vernooy SY, Yu H, Yan N, Shi Y, Guo M. et al. Multiple apoptoticcaspase cascades are required in nonapoptotic roles for Drosophila spermatidindividualization. Plos Biol2004;2:E15.
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[35] Danial NN, Korsmeyer SJ. Cell death: Critical control points. Cell2004;116:205–19.
[36] Degenhardt K, Chen G, Lindsten T, White E. Bax and Bak mediatep53-independent suppression of tumorigenesis. Cancer Cell2002;2:193–203.
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[38] Folkman J. Angiogenesis and apoptosis. Seminars in Cancer Biology2003;13:159–67.
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[42] Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin1, an autophagy geneessential for early embryonic development, is a haploinsufficient tumor suppressor.Proc Natl Acad Sci USA2003;100:15077–82.
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[1]Levine B, Klionsky DJ. Development by self-digestion; molecular mechanisms and biological functions of autophagy. Dev Cell2004;6:463-77.
[2]Mizushima N. The pleiotropic role of autophagy:From protein metabolism to bactericide. Cell Death Differ2005;12:1535-41.
[3]Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, Mukherjee C, Shi C, Gelinas C, Fan Y, Nelson Da, Jin S, White E. Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell2006:51-64.
[4]Klionsky DJ. Autophagy. Current Biology,2005;15(8):282-283.
[5]Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol,2004;36(12):2405-2419.
[6]Yu L, Lenardo MJ, Baehreche EH. Autophagy and caspases:a new cell death program. Cell Cycle,2004;3(9):1124-1126.
[7]Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science2000;290:1717-21.
[8]Levine B, Klionsky DJ.Development by self-digestion:molecular mechanisma and biological functions of autophagy. Dev Cell2004;6:463-77.
[9]Tsukada M, Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett1993;333:169-74.
[10]Thumm M, Egner R, Koch B, Schlumpberger M, Straub M, Veenhuis M, et al. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Lett1994;349:275-80.
[11]Harding TM, Morano KA, Scott SV, Klionsky DJ. Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. J Cell Bio1995;131:591-602
[12]Klionsky DJ, Cregg JM, Dunn WAJ, Emr SD, Sakai Y. Sandoval IV. et al. A unified nomenclature for yeast autophagy-related genes. Dev Cell2003;5:539-45.
[13] Ohsumi Y, Molecular dissection of autophagy: two ubiquitin-like systems.Nat Rev Moi Cell Biol2001;2:211-6.
[14] Reggiori E, Klionsky DJ. Autophagy in the eukaryotic cell. Eukaryot Cell2002;1:11-21.
[15] Baehrecke EH. How death shapes life during development. Nat Rev Moi CellBiol2002;3:779-87.
[16] Schweichel J-U, Merker H-J. The morphology of various types of cell deathin prenatal tissues. Teratology1973;7:253-66.
[17] Clarke PGH. Developmental cell death: morphological diversity and multiplemechanisms. Anat Embryol1990;181:195-213.
[18] Hengartner MO, The biochemistry of apoptosis. Nature2000;407:770-6.
[19] Jaattela M, Tschopp J. Caspase-independent cell death in T lymphocytes. NatImmunol2003;4:416-23.
[20] Chautan M, Chazal G, Cecconi F, Gruss P, Golstein P. Interdigial cell deathcan occur through a necrotic and caspase-independent pathway. Curr Biol1999;9:967-70.
[21] Oppenheim RW, Flavell RA, Vinsant S, Prevette D, Kuan CY, Rakic P.Programmed cell death of developing mammalian neurons after genetic deletion ofcaspases. J Neurosci2000;21:4752-60.
[22] Yu L, Alva A, Su H, Dutt P, Freundt E, Welsh S. et al. Regulation of anATG7-beclin1program of autophagic cell death by caspase-8. Science2004;304:1500-2.
[23] Onodera J, Ohsumi Y. Ald6p is a preferred target for autophagy in yeast,Saccharomyces cerevisiae. J Biol Chem2004;279:16071-6.
[24] Jesenberger V, Jentsch S. Deadly encounter: ubiquitin meets apoptosis. NatRev Moi Cell Biol2002;3:112-21.
[25] Debnath J, Mills KR, Collins NL, Reginato MJ, Muthuswamy SK, Brugge JS.The role of apoptosis in creating and maintaining luminal space within normal andoncogene-expressing mammary acini. Cell2002;111:29-40.
[26] Mills KR, Reginato M, Debnath J, Queenan B, Brugge JS. Tumor necrosisfactor-relateed apoptosis-inducing ligand(TRAIL) is required for induction ofautophagy during lumen formation in vitro. Proc Natl Acad Sci USA2004;101:3438-43.
[27] Lee C-Y, Baehrecke EH. Steroid regulation of autophagic programmed celldeath during development. Development2001;128:1443-55.
[28] Lee C-Y, Clough EA, Yellon P, Teslovich TM, Stephan DA, Baehrecke EH.Genome-wide analysis of steroid-and radiation-triggered programmed cell death inDrosophila. Curr boil2003;13:350-7.
[29] Martin DN, Baehrecke EH. Caspase function in autophagic cell death inDrosophila. Development2004;131:275-84.
[30]Chun HJ, Zheng L, Ahmad M, Wang J, Speirs CK, Siegel RM. et al.Pleiotropic defects in lymphocyte activations caused by caspase-8mutations lead tohuman immunodeficiency. Nature2002;419:395-9.
[31] Arama E, Agapite J, Steller H. Caspase activity and a specific cytochrome care require for sperm differentination in Drosophila. Dev Cell2003;4:687-97.
[32]Huh JR, Vernooy SY, Yu H, Yan N, Shi Y, Guo M. et al. Multiple apoptoticcaspase cascades are required in nonapoptotic roles for Drosophila spermatidindividualization. Plos Biol2004;2:E15.
[33] Toth S, Nagy K, Palfia Z, Rez G. Cellular autophagic capacity changes duringazaserine-induced tumour progression in the rat pancreas. Cell Tissue Res è2002×309:409–416.
[34] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell2000;100:57–70.
[35] Cory S, Huang DC, Adams JM. The Bcl-2family: Roles in cell survival andoncogenesis. Oncogene2003;22:8590–607.
[35] Danial NN, Korsmeyer SJ. Cell death: Critical control points. Cell2004;116:205–19.
[36] Degenhardt K, Chen G, Lindsten T, White E. Bax and Bak mediatep53-independent suppression of tumorigenesis. Cancer Cell2002;2:193–203.
[37] Nelson D, Tan TT, Rabson AB, Anderson D, Degenhardt K, White E.Hypoxia and defective apoptosis drive genomic instability and tumorigenesis. GenesDev2004;18:2095–107.
[38] Folkman J. Angiogenesis and apoptosis. Seminars in Cancer Biology2003;13:159–67.
[39] Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T, Thompson CB.Growth factor regulation of autophagy and cell survival in the absence of apoptosis.Cell2004;120:237–48.
[40] Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T,Ohsumi Y, Tokuhisa T, Mizushima N. The role of autophagy during the earlyneonatal starvation period. Nature2004;432:1032–6.
[41] Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, EskelinenEL, Mizushima N, Ohsumi Y, Cattoretti G, Levine B. Promotion of tumorigenesis byheterozygous disruption of the beclin1autophagy gene. J Clin Invest2003;112:1809–20.
[42] Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin1, an autophagy geneessential for early embryonic development, is a haploinsufficient tumor suppressor.Proc Natl Acad Sci USA2003;100:15077–82.
[43] Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H,Levine B. Induction of autophagy and inhibition of tumorigenesis by beclin1. Nature1999;402:672–6.
[44] Komatsu M, Waguri S, Ueno T, Iwata J, Murato S, Tanida I, Ezaki J,Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J Cell Biol2005;169:425–34.
[45] Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, KoikeM, Uchiyama Y, Kominami E, Tanaka K. Loss of autophagy in the central nervoussystem causes neurodegeneration in mice. Nature2006:880–4.
[46] Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y,Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N.Suppression of basal autophagy in neural cells causes neurodegenerative disease inmice. Nature2006;441:885–9.
[47] Jin S. Autophagy, mitochondrial quality control and oncogenesis. Autophagy2006;2:80–4.
[48] Zhang Y, Qi H, Taylor R, Xu W, Liu LF, Jin S. Autophagy is required formitochondria degradation and optimal mitochondrial function in S. cerevisiae.2006Submitted
[49] Warburg O. On respiratory impairment in cancer cells. Science1956;124:269–70.
[50] Tan TT, Degenhardt K, Nelson DA, Beaudoin B, Nieves-Neira W, Bouillet P,Villunger A, Adams JM, White E. Key roles of BIM-driven apoptosis in epithelialtumors and rational chemotherapy. Cancer Cell2005;7:227–38.
[51] Iwaya K, Mukai K. Accumulation of ubiqutin-conjugated cytokeratinfragments in tumor cells. Semin Cancer Biol2005;15:309–18.
[52] Balkwill F, Charles KA, Mantovani A. Smoldering and polarizedinflammation in the initiation and promotion of malignant disease. Cancer Cell2005;7:211–7.
[53] Balkwill F, Coussens LM. Cancer: An inflammatory link. Nature2004;431:405–6.
[54] Zeh HJI, Lotze MT. Addicted to death. J Immunother2005;28:1–9.