重组人TRAIL及突变体的原核表达和抗肿瘤生物活性研究
摘要
肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL,也称为Apo-2L)是Wiley 1995年首次克隆成功的一种新的抗肿瘤细胞因子,属于TNF家族的一个成员,在人体的多种组织中如脾、肺、前列腺、胸腺和外周血淋巴细胞等正常组织均可发现其表达。人TRAIL蛋白编码281个氨基酸,它的基因位于染色体3q26。TRAIL与目前发现的TNF家族的其它主要成员,如TNF-α,TNF-β(也称为淋巴毒素或LT-α),淋巴毒素-β(LT-β),41BBL,OX40L,CD27L,CD30L,CD40L和FasL(CD95L,也称为Apo-1L)等配体均为Ⅱ型膜蛋白,可水解形成可溶性同源三聚体,参与调节机体内的许多重要生物功能。TRAIL除了在免疫调节和炎性反应方面起重要的作用外,还与TNF家族中的TNF-α、TNF-β、FasL配体可导致易感细胞凋亡。TRAIL与这些配体的区别在于,除可特异性导致肿瘤细胞发生凋亡外,不损伤正常细胞。这些细胞因子的跨膜受体含有死亡结构阈,与配体结合导致引起凋亡的一系列蛋白酶反应。本研究首先对合成的TRAIL寡核苷酸全长进行了诱导表达和纯化,获得了有活性的TRAIL蛋白,并验证其抗肿瘤效应。同时采用分子模建方法,对TRATL结构进行了缺失突变,得到了两个突变体,并对它们进行了热诱导表达,以便深入的研究结构与功能的关系,为TRAIL开发成新的抗肿瘤药物打下基础。
1.人TRAIL寡核苷酸全长合成及在大肠杆菌中表达和纯化
人TRAIL寡核甘酸全长843个碱基,我们对其分段合成,然后进行全长拼接,并改变其中的30个密码子,以纠正氨基酸偏性,更利于在原核载体中进行表达。核苷酸自动测序结果,与预想的序列一致。合成的产物亚克隆至pGEX-2T质粒构成TRAIL-pGEX-2T,然后转染受体菌DH-5α大肠杆菌。我们在低温30℃,用IPTG 0.4mM/L诱导TRAIL融合蛋白表达,得到了TRAIL融合蛋白。然后采用亲和层析方法纯化TRAIL蛋白。
2.TRAIL的抗肿瘤生物活性
用A172人胶质细胞瘤,B104神经母细胞瘤,SKNSH人神经母细胞瘤,
TRAIL/Apo2 (TNF-related apoptosis inducing ligand), a novel cytokine, is a member of the tumor necrosis factor(TNF) family and was first cloned and identified by Willy in 1995, which can be detected in a varity of human tissue, most predominantly in spleen, lung, prostate,thymus and peripheral blood lymphocytes. TRAIL consists of 281 amino acids, and its gene is located on chromosome 3 at position 3q25. TRAIL as well as other members of TNF family, including TNF α , lymphotoxin β (LT β ),and ligands for CD40, CD30, CD27,OX40L is type II membrane protein, which can be hydrolyzed to soluble homotrimers. TRAIL play an important role in regulating many biological functions, such as prominent mediators of immune regulation and inflammatory responses, especially in inducing apoptosis in a wide variety of transformed cell lines and tumor cells. The key point between TRAIL and other apoptosis inducing ligands of the TNF family is that TRAIL effectively kills many tumor cells by apoptosis while leaving normal cells unharmed. These cytotoxic lignads induce the apoptosis (programmed cell death) by binding the receptors, which contain death domains.The full-length of TRAIL oligonucleotide was synthesized, and prokaryotic expression vector was constructed. After gene transfecting, the stable expression E coli DH-5α were obtained. Fusion TRAIL protein was expressed in E coli DH-5 α and purified by affinity chromatography. Subsequently, the purified protein was
tested to assess its ability to induce apoptosis in various tumor cell lines such as BGC832 lung cancer and A172 glioma cells. We also got two TRAIL mutants through homology modeling. These two TRAIL mutants were expressed in E coli DH-5 α ,after their oligonucleotide, fragments were inserted into pBV220, a thermo-sensitive expression vector.The synthesis of full-length of TRAIL oligonucleotides, expression of TRAIL in E coli DH-5α and purification of TRAIL: The full-length of human TRAIL oligonucleotide codes for 841 bases, which was synthesized and thirty of which were replaced to make TRAIL expressed in E coli DH-5α efficiently. The DNA sequence analysis proved the full-length of human TRAIL oligonucleotide synthesized was the same as which we wanted. The mutant TRAIL oligonucleotide was inserted into pGEX-2T vector, which contains a GST protein code to be purified easily by affinity chromatography, then transfecting E coli DH-5 a . The fusion TRAIL protein was expressed in E coli DH-5 a induced by 0.4mM/L IPTG for 4 hours at 28℃, and purified by affinity chromatography through GSTrap column.Antitumor activity of TRAIL: The cytotoxic activities of TRAIL were measured on A172 huanm glioma cells, B104 murine neuroblastoma, SKNSH human neuroblastoma, BGC823 stomach cancer cells, A549 lung cancer cells, H69 lung cancer cells, KB nasopharyngeal cancer cells, etc. TRAIL to a final concentration of 1μg/ml is capable to induce apoptosis of these tumor cell lines efficiently.TRAIL mutant design based on homology modeling: TRAIL belongs to a member of TNF family, which gene codes for a polypeptide of 281 amino acids. The soluble active extracellular segment of TRAIL ranged from amino acids 95-281. The two TRAIL mutants were obtained
by homology modeling, one is residues 130-145 and 210-281, and the other is residues 145-281. The primers were designed according to oligonuclotide sequence of TRAIL mutants, 5' primer with EcoR I restriction enzyme site and 3' primer with BamH I restriction site. The mutants oligonucleotide segments were obtained by PCR, and then inserted into pUC18-T vector. The result of sequence analysis of mutants proved properly.Expression and purification of TRAIL mutants: The mutants oligonucleotide segments were inserted into pBV220.The recombinant plasmid pBV-TRAIL mutant identified by digestion with EcoR I AND BamH I was transformed to the competent cells of E coli DH-5α , and then the transformed cells were induced to be expressed by shifting culture from 37℃ to 42℃. The TRAIL mutants were expressed in the form of inclusion bodies and soluble protein. The inclusion bodies were washed with 8M urea, and then purified preparatively.
1.人TRAIL寡核苷酸全长合成及在大肠杆菌中表达和纯化
人TRAIL寡核甘酸全长843个碱基,我们对其分段合成,然后进行全长拼接,并改变其中的30个密码子,以纠正氨基酸偏性,更利于在原核载体中进行表达。核苷酸自动测序结果,与预想的序列一致。合成的产物亚克隆至pGEX-2T质粒构成TRAIL-pGEX-2T,然后转染受体菌DH-5α大肠杆菌。我们在低温30℃,用IPTG 0.4mM/L诱导TRAIL融合蛋白表达,得到了TRAIL融合蛋白。然后采用亲和层析方法纯化TRAIL蛋白。
2.TRAIL的抗肿瘤生物活性
用A172人胶质细胞瘤,B104神经母细胞瘤,SKNSH人神经母细胞瘤,
TRAIL/Apo2 (TNF-related apoptosis inducing ligand), a novel cytokine, is a member of the tumor necrosis factor(TNF) family and was first cloned and identified by Willy in 1995, which can be detected in a varity of human tissue, most predominantly in spleen, lung, prostate,thymus and peripheral blood lymphocytes. TRAIL consists of 281 amino acids, and its gene is located on chromosome 3 at position 3q25. TRAIL as well as other members of TNF family, including TNF α , lymphotoxin β (LT β ),and ligands for CD40, CD30, CD27,OX40L is type II membrane protein, which can be hydrolyzed to soluble homotrimers. TRAIL play an important role in regulating many biological functions, such as prominent mediators of immune regulation and inflammatory responses, especially in inducing apoptosis in a wide variety of transformed cell lines and tumor cells. The key point between TRAIL and other apoptosis inducing ligands of the TNF family is that TRAIL effectively kills many tumor cells by apoptosis while leaving normal cells unharmed. These cytotoxic lignads induce the apoptosis (programmed cell death) by binding the receptors, which contain death domains.The full-length of TRAIL oligonucleotide was synthesized, and prokaryotic expression vector was constructed. After gene transfecting, the stable expression E coli DH-5α were obtained. Fusion TRAIL protein was expressed in E coli DH-5 α and purified by affinity chromatography. Subsequently, the purified protein was
tested to assess its ability to induce apoptosis in various tumor cell lines such as BGC832 lung cancer and A172 glioma cells. We also got two TRAIL mutants through homology modeling. These two TRAIL mutants were expressed in E coli DH-5 α ,after their oligonucleotide, fragments were inserted into pBV220, a thermo-sensitive expression vector.The synthesis of full-length of TRAIL oligonucleotides, expression of TRAIL in E coli DH-5α and purification of TRAIL: The full-length of human TRAIL oligonucleotide codes for 841 bases, which was synthesized and thirty of which were replaced to make TRAIL expressed in E coli DH-5α efficiently. The DNA sequence analysis proved the full-length of human TRAIL oligonucleotide synthesized was the same as which we wanted. The mutant TRAIL oligonucleotide was inserted into pGEX-2T vector, which contains a GST protein code to be purified easily by affinity chromatography, then transfecting E coli DH-5 a . The fusion TRAIL protein was expressed in E coli DH-5 a induced by 0.4mM/L IPTG for 4 hours at 28℃, and purified by affinity chromatography through GSTrap column.Antitumor activity of TRAIL: The cytotoxic activities of TRAIL were measured on A172 huanm glioma cells, B104 murine neuroblastoma, SKNSH human neuroblastoma, BGC823 stomach cancer cells, A549 lung cancer cells, H69 lung cancer cells, KB nasopharyngeal cancer cells, etc. TRAIL to a final concentration of 1μg/ml is capable to induce apoptosis of these tumor cell lines efficiently.TRAIL mutant design based on homology modeling: TRAIL belongs to a member of TNF family, which gene codes for a polypeptide of 281 amino acids. The soluble active extracellular segment of TRAIL ranged from amino acids 95-281. The two TRAIL mutants were obtained
by homology modeling, one is residues 130-145 and 210-281, and the other is residues 145-281. The primers were designed according to oligonuclotide sequence of TRAIL mutants, 5' primer with EcoR I restriction enzyme site and 3' primer with BamH I restriction site. The mutants oligonucleotide segments were obtained by PCR, and then inserted into pUC18-T vector. The result of sequence analysis of mutants proved properly.Expression and purification of TRAIL mutants: The mutants oligonucleotide segments were inserted into pBV220.The recombinant plasmid pBV-TRAIL mutant identified by digestion with EcoR I AND BamH I was transformed to the competent cells of E coli DH-5α , and then the transformed cells were induced to be expressed by shifting culture from 37℃ to 42℃. The TRAIL mutants were expressed in the form of inclusion bodies and soluble protein. The inclusion bodies were washed with 8M urea, and then purified preparatively.
引文
1. Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of new member of the TNF family that induces apoptosis . Immunity, 1995, 3(6): 673-682
2. Jeremias I, Herr I, Boehler T, et al. TRAIL/Apo-2-ligand-induced apoptosis in human T cells. Eur J Immunol, 1998, 28: 143-152
3. Phillip TA, Ni J, Pan G, et al. TRAIL (Apo-2L) and TRAIL receptors in human placentas: implications for immune privilege. J Immunol, 1999,162: 6053-6059
4. Musgrave BL, Phu T, Butler LL, et al. Murine TRAIL (TNF-related apoptosis inducing ligand) expressed by T cell activation is blocked by rapamycin, cyclosporinA, and inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and protein tyrosine kinases: evidence for TRAIL induction via the T cell receptor signaling pathway. Exp Cell Res, 1999, 252: 96-103
5. Schulze-Osthoff K, Ferrari D, Los M, et al. Apoptosis signaling by death receptors. Eur J Biochem, 1998, 254: 439-459
6. Cha SS, Kim MS, Choi YH, et al. 2.8 A Resolution crystal structure of human TRAIL, a cytokine with selective antitumor activity. Immunity, 1999, 11: 253-261
7. Kagan BL, Baldwin RL, Yuan WL, et al. Formation of ion-permeable channels by tumor necrosis factor-alpha. Science 1992; 255: 1427-1430
8. Eck MJ, Sprang SR. The structure of tumor necrosis factor-alpha at 2.6A resolution. Implications for receptor binding J. Biol. Chem 1989; 264: 17596-17605
9. Eck MJ, Ultsch M, Rinderknecht E, et al. The structure of human lymphotoxin (tumor necrosis factor-beta) at 1.9-A resolution J. Biol Chem 1992; 267: 2119-2122
10. Walczak H: Tumoricidal activitiy of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157-163
11. Corti A, Fassina G, Marcucci F, et al. Oligomeric tumor necrosis factor α slowly converts into inactive forms at biological levels. Biochem J, 1992, 284: 905-910
12. Pan G, O-Rourke K, Chinaiyan AM, et al. The receptor for cytotoxic ligand TRAIL. Science, 1997, 276: 111-113
13. Pan G, Ni Jian, Wei Ying-fei, et al. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science, 1997, 277: 815-818
14. Sheridan JP, Marsters SA, Pitti RM, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors, science, 1997, 277: 818-821
15. Pan G, Ni J, Yu G, et al. TRUNDD: a new member of the TRAIL receptor family that antagonizes TRAIL signaling. FEBS Letters, 1998, 424: 41-45
16. Degli-Esposti MA, Smolak PJ, Walczak H, et al. Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J Exp Med, 1997, 186: 1165-1170
17. Marsters SA, Sheridan JP, Pitti RM, et al. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr Biol, 1997, 7: 1003-1006
18. Emery JG, McDonnell P, Burke MB, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem, 1998, 273: 14363-14367
19. Walczak H, Degli-Esposti MA, Johnson RS, et al. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J, 1997,16:5386-5397
20. Schneider P, Thome M, Burns K, et al. TRAIL receptors 1(DR4) and 2(DR5) signal FADD-dependent apoptosis and activate NF-kappaB. Immunity,1997,7:831-836
21. Smith CA: The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 1994;76:959-962
22. Van Ostade:Structure-activity studies of human tumour necrosis factors. Protein Eng.1994:7:5-22
23. Banner DW: Crystal structure of the soluble human 55kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 1993;73:431-445
24. Creasey,AA:Biological effects of recombinant human tumor necrosis factors and its novel muteins on tumor and normal cell lines. Cancer Res, 1987;47:145-149
25. Thomas WD, Hersey P. TNF-related apoptosis-inducing ligand(TRAIL) induces apoptosis in Fas ligand-resistent melanoma cells and mediates CD4 T cell killing at target cells. J Immunol, 1998, 16:2195-2200
26. Gazitt Y. TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma patients and is not cytotoxic to hematopoietic stem cells. Leukemia, 1999,13:1817-1124
27. Gliniak B, Le T. Tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11 Cancer Res, 1999,59:6153-6158
28. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.
Nat Med. 1999,5:157-163
29. Nakamura M, Rieger J, Weller M, et al. Apo2L/TRAIL expression in human brain tumors. Acta Neuropathol (Berl), 2000, 99:1-6
30. Rieger J, Ohgaki H, Kleihues P, et al. Human astrocytic brain tumors express AP02L/TRAIL. Acta Neuropathol(Berl), 2000,97:1-4
31. Roth W, Isenmann S, Naumann U, et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem Biophys Res Comun, 1999,265:479-483
32. Keane MM, Ettenberg SA, Nau MM, et al. Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer Res, 1999,59:734-741
33. Cuello M, Ettenberg SA, Nau MM, et al. synergistic induction of apoptosis by the combination of trail and chemotherapy in chemoresistant ovarian cancer cells. Gynecol Oncol, 2001,81:380-390
34. Johnson HM, Torres BA, Green MM, et al. Hypothesis: ligand/receptor-assisted nuclear translocation of STATS. Proc Soc Exp Biol Med, 1998,218:149-155
35. Kischkel FC, Lawrence DA, Chuntharapai A, et al. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity, 2000,12:611-620
36. Sprick MR, Weigand MA, Rieser E, et al. FADD/MORTl and caspase-8 are recruited to TRAIL receptors 1 and 2 are essential for apoptosis mediated by TRAIL receptor 2. Immunity, 2000,12:599-609
37. Kuang AA, Diehl GE, Zhang J, et al. FADD is required for DR4- and DR5- mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts. J Biol Chem,
2000,275:25065-25068
38. Bodmer JL, Holler N, Reynard S, et al. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol, 2000,2:241-243
39. Chinnaiyan AM, O'Rourke K, Tewari M, et al. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell, 1995,81:505-512
40. Thome M, Schneider P, Hofmann K, et al. Viral FLICE-inhibitory proteins(FLIPs) prevent apoptosis induced by death receptors. Nature, 1997,386:517-521
41. Wang CY, Mayo MW, Korneluk RG, et al. NF-k B anti-apoptosis: induction of TRAF1 and TRAF2 and C-IAP2 to suppress caspase-8 activation. Science, 1998,281:1680-1683
2. Jeremias I, Herr I, Boehler T, et al. TRAIL/Apo-2-ligand-induced apoptosis in human T cells. Eur J Immunol, 1998, 28: 143-152
3. Phillip TA, Ni J, Pan G, et al. TRAIL (Apo-2L) and TRAIL receptors in human placentas: implications for immune privilege. J Immunol, 1999,162: 6053-6059
4. Musgrave BL, Phu T, Butler LL, et al. Murine TRAIL (TNF-related apoptosis inducing ligand) expressed by T cell activation is blocked by rapamycin, cyclosporinA, and inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and protein tyrosine kinases: evidence for TRAIL induction via the T cell receptor signaling pathway. Exp Cell Res, 1999, 252: 96-103
5. Schulze-Osthoff K, Ferrari D, Los M, et al. Apoptosis signaling by death receptors. Eur J Biochem, 1998, 254: 439-459
6. Cha SS, Kim MS, Choi YH, et al. 2.8 A Resolution crystal structure of human TRAIL, a cytokine with selective antitumor activity. Immunity, 1999, 11: 253-261
7. Kagan BL, Baldwin RL, Yuan WL, et al. Formation of ion-permeable channels by tumor necrosis factor-alpha. Science 1992; 255: 1427-1430
8. Eck MJ, Sprang SR. The structure of tumor necrosis factor-alpha at 2.6A resolution. Implications for receptor binding J. Biol. Chem 1989; 264: 17596-17605
9. Eck MJ, Ultsch M, Rinderknecht E, et al. The structure of human lymphotoxin (tumor necrosis factor-beta) at 1.9-A resolution J. Biol Chem 1992; 267: 2119-2122
10. Walczak H: Tumoricidal activitiy of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157-163
11. Corti A, Fassina G, Marcucci F, et al. Oligomeric tumor necrosis factor α slowly converts into inactive forms at biological levels. Biochem J, 1992, 284: 905-910
12. Pan G, O-Rourke K, Chinaiyan AM, et al. The receptor for cytotoxic ligand TRAIL. Science, 1997, 276: 111-113
13. Pan G, Ni Jian, Wei Ying-fei, et al. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science, 1997, 277: 815-818
14. Sheridan JP, Marsters SA, Pitti RM, et al. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors, science, 1997, 277: 818-821
15. Pan G, Ni J, Yu G, et al. TRUNDD: a new member of the TRAIL receptor family that antagonizes TRAIL signaling. FEBS Letters, 1998, 424: 41-45
16. Degli-Esposti MA, Smolak PJ, Walczak H, et al. Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family. J Exp Med, 1997, 186: 1165-1170
17. Marsters SA, Sheridan JP, Pitti RM, et al. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr Biol, 1997, 7: 1003-1006
18. Emery JG, McDonnell P, Burke MB, et al. Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. J Biol Chem, 1998, 273: 14363-14367
19. Walczak H, Degli-Esposti MA, Johnson RS, et al. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J, 1997,16:5386-5397
20. Schneider P, Thome M, Burns K, et al. TRAIL receptors 1(DR4) and 2(DR5) signal FADD-dependent apoptosis and activate NF-kappaB. Immunity,1997,7:831-836
21. Smith CA: The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell 1994;76:959-962
22. Van Ostade:Structure-activity studies of human tumour necrosis factors. Protein Eng.1994:7:5-22
23. Banner DW: Crystal structure of the soluble human 55kd TNF receptor-human TNF beta complex: implications for TNF receptor activation. Cell 1993;73:431-445
24. Creasey,AA:Biological effects of recombinant human tumor necrosis factors and its novel muteins on tumor and normal cell lines. Cancer Res, 1987;47:145-149
25. Thomas WD, Hersey P. TNF-related apoptosis-inducing ligand(TRAIL) induces apoptosis in Fas ligand-resistent melanoma cells and mediates CD4 T cell killing at target cells. J Immunol, 1998, 16:2195-2200
26. Gazitt Y. TRAIL is a potent inducer of apoptosis in myeloma cells derived from multiple myeloma patients and is not cytotoxic to hematopoietic stem cells. Leukemia, 1999,13:1817-1124
27. Gliniak B, Le T. Tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo is enhanced by the chemotherapeutic agent CPT-11 Cancer Res, 1999,59:6153-6158
28. Walczak H, Miller RE, Ariail K, et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.
Nat Med. 1999,5:157-163
29. Nakamura M, Rieger J, Weller M, et al. Apo2L/TRAIL expression in human brain tumors. Acta Neuropathol (Berl), 2000, 99:1-6
30. Rieger J, Ohgaki H, Kleihues P, et al. Human astrocytic brain tumors express AP02L/TRAIL. Acta Neuropathol(Berl), 2000,97:1-4
31. Roth W, Isenmann S, Naumann U, et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem Biophys Res Comun, 1999,265:479-483
32. Keane MM, Ettenberg SA, Nau MM, et al. Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer Res, 1999,59:734-741
33. Cuello M, Ettenberg SA, Nau MM, et al. synergistic induction of apoptosis by the combination of trail and chemotherapy in chemoresistant ovarian cancer cells. Gynecol Oncol, 2001,81:380-390
34. Johnson HM, Torres BA, Green MM, et al. Hypothesis: ligand/receptor-assisted nuclear translocation of STATS. Proc Soc Exp Biol Med, 1998,218:149-155
35. Kischkel FC, Lawrence DA, Chuntharapai A, et al. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity, 2000,12:611-620
36. Sprick MR, Weigand MA, Rieser E, et al. FADD/MORTl and caspase-8 are recruited to TRAIL receptors 1 and 2 are essential for apoptosis mediated by TRAIL receptor 2. Immunity, 2000,12:599-609
37. Kuang AA, Diehl GE, Zhang J, et al. FADD is required for DR4- and DR5- mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts. J Biol Chem,
2000,275:25065-25068
38. Bodmer JL, Holler N, Reynard S, et al. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol, 2000,2:241-243
39. Chinnaiyan AM, O'Rourke K, Tewari M, et al. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell, 1995,81:505-512
40. Thome M, Schneider P, Hofmann K, et al. Viral FLICE-inhibitory proteins(FLIPs) prevent apoptosis induced by death receptors. Nature, 1997,386:517-521
41. Wang CY, Mayo MW, Korneluk RG, et al. NF-k B anti-apoptosis: induction of TRAF1 and TRAF2 and C-IAP2 to suppress caspase-8 activation. Science, 1998,281:1680-1683