抗人DR5单抗AD5-10诱导肺癌细胞凋亡敏感性的作用及其分子机制的研究
摘要
AD5-10是一株新的鼠抗人死亡受体5(death receptor 5，DR5)单克隆抗体，为本实验室在前期研究中获得，能够强烈诱导多种肿瘤细胞系发生凋亡，抑制小鼠体内移植瘤的生长，对人正常肝细胞和人外周血淋巴细胞没有明显的诱导毒性，已成为研发生物类药物的候选者，具有抗肿瘤作用的应用前景。但是研究发现，某些肿瘤细胞对AD5-10的诱导却表现为明显的抗性，其抗性是否具有一定的表现规律，其内在的调控机制又是什么，仍然是目前一个有待阐明的科学问题。本论文拟选用肺癌细胞系A549、SCLC、H460、H1299和Calu-3，分析细胞表面DR5表达水平与细胞对AD5-10诱导凋亡敏感性之间的关系；并进一步寻找凋亡相关调节分子，研究其调节细胞对AD5-10诱导凋亡敏感性的内在分子机制。本研究所选的五株肺癌细胞中，A549和SCLC细胞对AD5-10中度敏感，其他三株细胞对AD5-10不太敏感。研究表明，这五株肺癌细胞对AD5-10的敏感性与细胞中DR5的表达水平无关。但发现，使用AD5-10处理，敏感或中度敏感细胞中，c-FLIP_L发生明显的切割，c-FLIP_L的表达量显著下降；而不太敏感或抗性的细胞中的c-FLIP_L的表达水平没有明显变化。表明c-FLIP_L是参与调节AD5-10诱导细胞凋亡的关键分子。
进一步研究证明，c-FLIP_L过量表达可以部分抑制人T淋巴细胞白血病细胞SVT35、人肺癌细胞A549对AD5-10的敏感性；而亚毒性浓度的化疗药物环磷酰胺(CHX)、放线菌素D(ActD)能通过下调细胞中c-FLIP_L蛋白表达水平，增强AD5-10对肺癌细胞的杀伤活性。此外，通过c-FLIP_L siRNA特异抑制细胞中的c-FLIP_L的表达，可以显著增强多种肺癌细胞对AD5-10诱导凋亡的敏感性。其调节机制是c-FLIP_L siRNA和AD5-10能激活细胞中caspase依赖的信号通路，下调Bcl-2家族蛋白Bid和Bcl-X_L的表达，进而促进线粒体膜电位的降低以及细胞色素C的释放。并且，研究证明c-FLIP_L siRNA和AD5-10联合作用对正常人细胞没有毒性。
最后，我们检测了人肺癌组织中c-FLIP_L的表达，结果显示c-FLIP_L特异高水平表达于人肺癌纽织中的肿瘤细胞中，而在正常组织和肿瘤细胞周围间质细胞中表达量很低，两者在统计学上具有显著差异。以上研究提示特异下调c-FLIP_L可能是一种非常有潜力的治疗肺癌的策略，特别是针对那些对功能性单抗单独作用产生抗性的肿瘤病人。
第二部分研究中，本文还利用AD5-10可变区基因，构建了表达鼠单链抗体(ScFv)的原核表达载体，并获成功表达。初步证明了它们的肿瘤杀伤活性。同时，我们将AD5-10的可变区基因和人抗体恒定区基因，分别克隆入表达载体pKN100VL和pG1D105VH，通过筛选获得了稳定表达和分泌人-鼠嵌合抗体(anti-DR5 mV-hH)的重组细胞；分泌表达的嵌合抗体与人DR5蛋白具有特异性结合活性，并且能明显杀伤体外培养的人T淋巴细胞白血病Jurkat细胞，为发展人源化抗体药物的研究奠定了基础。
Lung cancer is the leading cause of cancer-related mortality in the world.Non-small cell lung cancers (NSCLCs) account for about 75-80% of all types of lungcancers. Surgery offers substantial cure rates for those NSCLC patients with earlystage disease (stagesⅠandⅡ). However, 70% of the lung cancer patients present atstageⅢ/Ⅳ, and most of these patients continue to die of disease progression becauseof resistance to chemotherapeutic drugs or radiation therapy. Clearly, new treatmentstrategies are necessary to improve lung cancer therapy.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is amember of the structurally related TNF family. Up to date, five TRAIL receptors havebeen identified, such as TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1,TRID), TRAIL-R4 (DcR2, TRUNDD) and OPG (osteoprotegerin). Agonisticantibodies against death receptor 4 and 5 are cytotoxic to various cancer cells. In thepresent study, the sensitivity and molecular mechanism of five human lung cancer celllines to previously reported agonistic antibody against DR5, AD5-10, wereinvestigated. Of these cell lines, A549 and SCLC showed a moderate sensitivity toAD5-10 and other three cell lines were resistant. We showed that the varioussensitivities of lung cancer cell lines to AD5-10 were not related to the expressionlevel of DR5, but the expression and cleavage of c-FLIP_L in these cells. Inhibition ofendogenous c-FLIP_L expression by siRNA significantly enhanced AD5-10 inducedcell death in these lung cancer cells. And this sensitizing effect was associated withdecreased expression of Bid and Bcl-X_L, change of mitochondrial membrane potential,release of cytochrome c from mitochondria, and caspase activation. Therefore, thesedata provided evidences that c-FLIP_L expression and cleavage are involved in thesensitivity to DR5-mediated lung cancer cell apoptosis. Moreover, we demonstratedthat c-FLIP was expressed in 87.9% (29 of 33) of the tissues from lung carcinoma patients, but little in the normal controls, suggesting that inhibition of c-FLIPexpression might be a potential strategy for lung cancer therapy.
In the present study, we have successfully constructed the single chain fragmentvariable (scFv) and human/mouse chimeric antibody of AD5-10, a functionalanti-DR5 MAb. The scFv gene of anti-DR5 MAb was amplified and cloned into thepET-15b, then expressed in E. coli. Then detect the tumoricidal activity of the purifiedscFv protein by MTS-PMS assay. The results showed that purified anti-DR5-ScFvinduces tumor cell cell death in vitro. And the VL and VH genes of anti-DR5 MAbwere amplified and cloned into the light and heavy chain expression vectorsrespectively, then the recombinant plasmids were co-transfected into CHO-dhfr~- cellsfor expression. Screen for the positive clone by the two selective genes (neo and dhfr).Then identify the humanization and specificity of chimeric antibody by ELISA andweatern blot. And detect the tumoricidal activity of the expressed chimeric antibodyby MTS-PMS assay. The expression vectors can stably express chimeric antibody inCHO-dhfr~- cells. In the cell supernatant of the F4'clone, we identified the human IgGheavy constant region Cγl and light constant region Cκ. Moreover, the secretedchimeric antibody retained the binding capacity to the antigen (DR5) and decreasedthe cell viability of Jurkat cells by 26.85% in vitro. The human/mouse anti-DR5chimeric antibody has been expressed successfully in eukaryotic cells, which has thehuman IgG constant region, antigen binding specificity and tumoricidal activity. Thisstudy provides the evidences that gene engineering antibody against TRAIL receptorDR5 has potential application in the future of tumor antibody therapy.
进一步研究证明，c-FLIP_L过量表达可以部分抑制人T淋巴细胞白血病细胞SVT35、人肺癌细胞A549对AD5-10的敏感性；而亚毒性浓度的化疗药物环磷酰胺(CHX)、放线菌素D(ActD)能通过下调细胞中c-FLIP_L蛋白表达水平，增强AD5-10对肺癌细胞的杀伤活性。此外，通过c-FLIP_L siRNA特异抑制细胞中的c-FLIP_L的表达，可以显著增强多种肺癌细胞对AD5-10诱导凋亡的敏感性。其调节机制是c-FLIP_L siRNA和AD5-10能激活细胞中caspase依赖的信号通路，下调Bcl-2家族蛋白Bid和Bcl-X_L的表达，进而促进线粒体膜电位的降低以及细胞色素C的释放。并且，研究证明c-FLIP_L siRNA和AD5-10联合作用对正常人细胞没有毒性。
最后，我们检测了人肺癌组织中c-FLIP_L的表达，结果显示c-FLIP_L特异高水平表达于人肺癌纽织中的肿瘤细胞中，而在正常组织和肿瘤细胞周围间质细胞中表达量很低，两者在统计学上具有显著差异。以上研究提示特异下调c-FLIP_L可能是一种非常有潜力的治疗肺癌的策略，特别是针对那些对功能性单抗单独作用产生抗性的肿瘤病人。
第二部分研究中，本文还利用AD5-10可变区基因，构建了表达鼠单链抗体(ScFv)的原核表达载体，并获成功表达。初步证明了它们的肿瘤杀伤活性。同时，我们将AD5-10的可变区基因和人抗体恒定区基因，分别克隆入表达载体pKN100VL和pG1D105VH，通过筛选获得了稳定表达和分泌人-鼠嵌合抗体(anti-DR5 mV-hH)的重组细胞；分泌表达的嵌合抗体与人DR5蛋白具有特异性结合活性，并且能明显杀伤体外培养的人T淋巴细胞白血病Jurkat细胞，为发展人源化抗体药物的研究奠定了基础。
Lung cancer is the leading cause of cancer-related mortality in the world.Non-small cell lung cancers (NSCLCs) account for about 75-80% of all types of lungcancers. Surgery offers substantial cure rates for those NSCLC patients with earlystage disease (stagesⅠandⅡ). However, 70% of the lung cancer patients present atstageⅢ/Ⅳ, and most of these patients continue to die of disease progression becauseof resistance to chemotherapeutic drugs or radiation therapy. Clearly, new treatmentstrategies are necessary to improve lung cancer therapy.
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is amember of the structurally related TNF family. Up to date, five TRAIL receptors havebeen identified, such as TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1,TRID), TRAIL-R4 (DcR2, TRUNDD) and OPG (osteoprotegerin). Agonisticantibodies against death receptor 4 and 5 are cytotoxic to various cancer cells. In thepresent study, the sensitivity and molecular mechanism of five human lung cancer celllines to previously reported agonistic antibody against DR5, AD5-10, wereinvestigated. Of these cell lines, A549 and SCLC showed a moderate sensitivity toAD5-10 and other three cell lines were resistant. We showed that the varioussensitivities of lung cancer cell lines to AD5-10 were not related to the expressionlevel of DR5, but the expression and cleavage of c-FLIP_L in these cells. Inhibition ofendogenous c-FLIP_L expression by siRNA significantly enhanced AD5-10 inducedcell death in these lung cancer cells. And this sensitizing effect was associated withdecreased expression of Bid and Bcl-X_L, change of mitochondrial membrane potential,release of cytochrome c from mitochondria, and caspase activation. Therefore, thesedata provided evidences that c-FLIP_L expression and cleavage are involved in thesensitivity to DR5-mediated lung cancer cell apoptosis. Moreover, we demonstratedthat c-FLIP was expressed in 87.9% (29 of 33) of the tissues from lung carcinoma patients, but little in the normal controls, suggesting that inhibition of c-FLIPexpression might be a potential strategy for lung cancer therapy.
In the present study, we have successfully constructed the single chain fragmentvariable (scFv) and human/mouse chimeric antibody of AD5-10, a functionalanti-DR5 MAb. The scFv gene of anti-DR5 MAb was amplified and cloned into thepET-15b, then expressed in E. coli. Then detect the tumoricidal activity of the purifiedscFv protein by MTS-PMS assay. The results showed that purified anti-DR5-ScFvinduces tumor cell cell death in vitro. And the VL and VH genes of anti-DR5 MAbwere amplified and cloned into the light and heavy chain expression vectorsrespectively, then the recombinant plasmids were co-transfected into CHO-dhfr~- cellsfor expression. Screen for the positive clone by the two selective genes (neo and dhfr).Then identify the humanization and specificity of chimeric antibody by ELISA andweatern blot. And detect the tumoricidal activity of the expressed chimeric antibodyby MTS-PMS assay. The expression vectors can stably express chimeric antibody inCHO-dhfr~- cells. In the cell supernatant of the F4'clone, we identified the human IgGheavy constant region Cγl and light constant region Cκ. Moreover, the secretedchimeric antibody retained the binding capacity to the antigen (DR5) and decreasedthe cell viability of Jurkat cells by 26.85% in vitro. The human/mouse anti-DR5chimeric antibody has been expressed successfully in eukaryotic cells, which has thehuman IgG constant region, antigen binding specificity and tumoricidal activity. Thisstudy provides the evidences that gene engineering antibody against TRAIL receptorDR5 has potential application in the future of tumor antibody therapy.
引文
Buchsbaum DJ, Zhou T, Grizzle WE, Oliver PG, Hammond CJ, Zhang S et al. (2003). Antitumor efficacy of TRA-8 anti-DR5 monoclonal antibody alone or in combination with chemotherapy and/or radiation therapy in a human breast cancer model. Clin Cancer Res 9:3731-3741.
Boatright KM, Deis C, Denault DP, Salvesen GS. (2004). Activation of caspases-8 and-10 by FLIPL. Biochem J 382:651-657.
Cario-Stella C, Lavazza C, Locatelli A, Vigano L, Gianni AM, Gianni L. (2007). Targeting TRAIL agonistic receptors for cancer therapy. Clin Cancer Res 13: 2313-2317.
Chang DW, Xing Z, Pan Y et al. (2002). c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J 21:3704-3714.
Chaudhary PM, Eby MT, Jasmin A, Kumar A, Liu L, Hood L. (2000). Activation of the NF-κB pathway by caspase 8 and its homologs. Oncogene 19:4451-4460.
Chuntharapai A., Dodge K, Grimmer K, Schroeder K, Marsters SA, Koeppen H et al. (2001). Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4. J Immunol 166: 4891-4898.
Djerbi M, Darreh-Shori T, Zhivotovsky B, Grandien A. (2001). Characterization of the human FLICE-inhibitory protein locus and comparison of the anti-apoptotic activity of four different flip isoforms. Scand J Immunol 54:180-189.
Fang JM, Qian JJ, Yi SL, et al. (2005). Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 23:584-590.
Ferreira CG, Huisman C, Giaccone G. (2002). Novel approaches to the treatment of non-small cell lung cancer. Crit Rev Oncol Hematol 41: 57-77.
Ferrigno D, Buccheri G. (2000). Second-line chemotherapy for recurrent non-small cell lung cancer: do new agents make a difference. Lung Cancer 29: 91-104.
Fisher MJ,Virmani AK,Wu L,Aplenc R,Harper JC,Powell SM,Rebbeck TR,Sidransky D,Gazdar AF,El-Deiry WS.(2001).Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer.Clin Cancer Res 7(6):1688-1697.
Georgakis GV,Li Y,Humphreys R et al.(2005).Activity of selective fully human agonistic antibodies to the TRAIL death receptors TRAIL-R1 and TRAIL-R2 in primary and cultured lymphoma cells:induction of apoptosis and enhancement of doxorubicin-and bortezomib-induced cell death.British Journal of Haematology 130:501-510.
Golks A,Brenner D,Fritsch C,Krammer PH,Lavrik IN.(2005).c-FLIPR,a New Regulator of Death Receptor-induced Apoptosis.J Biol Chem 280:14507-14513.
Gross A,Yin XM,Wang K,Wei MC,Jockel J,Milliman C,Erdjument-Bromage H,Tempst P,Korsmeyer,SJ.(1999).Caspase cleaved BID targets mitochondria and is required for cytochrome c release,while BCL-XL prevents this release but not tumor necrosis factor-Rl/Fas death.J Biol Chem 274(2):1156-1163.
Guo Y,Chen C,Zheng Y,Zhang J,Tao X,Liu S et al.(2005).A novel anti-human DR5 monoclonal antibody with tumoricidal activity induces caspase-dependent and caspase-independent cell death.J Biol Chem 280:41940-41952.
Han DK,Chaudhary PM,Wright ME,Friedman C,Trask BJ,Riedel RT et al.(1997).MRIT,a novel death-effector domain-containing protein,interacts with caspases and BclXL and initiates cell death.Proc Natl Acad Sci U S A 94:11333-11338.
Hoffman PC,Mauer AM,Vokes EE.(2000).Lung cancer.Lancet 355:479-485.
Horak P,Pils D,Kaider A,Pinter A,Elandt K,Sax C et al.(2005).Perturbation of theTumor Necrosis Factor Related Apoptosis-Inducing Ligand Cascade in Ovarian Cancer:overexpression of FLIPL and Deregulation of the Functional Receptors DR4 and DR5.Clin Cancer Res 11:8585-8591.
Hu S,Vincenz C,Ni J,Gentz R,Dixit VM.(1997).I-FLICE,a novel inhibitor of tumor necrosis factor receptor 1 and CD95 induced apoptosis.J Biol Chem 272:17255-17257.
Hu WH,Johnson H,Shu HB.(2000).Activation of NF-kB by FADD,Casper,and caspase-8.J Biol Chem 275:10838-10844.
Ibrahim SM,Ringel J,Schmidt C,Ringel B,M(?)ller P,Koczan D et al.(2001).Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death.Pancreas 23:72-79.
Ichikawa K,Liu W,Fleck M et al.(2003).TRAIL-R2(DR5)mediates apoptosis of synovial fibroblasts in rheumatoid arthritis.The Journal of Immunology 171:1061-1069.
Ichikawa K,Liu W,Zhao L et al.(2001).Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.Nat Med 7(8):954-60.
Irmler M,Thome M,Hahne M,Schneider P,Hofmann K,Steiner V et al.(1997).Inhibition of death receptor signals by cellular FLIP.Nature 388:190-195.
Itoh N,Nagata S.(1993).A novel protein domain required for apoptosis.Mutational analysis of human Fas antigen.J Biol Chem 268:10932-10937.
Jo M,Kim TH,Seol DW,Esplen JE,Dorko K,Billiar TR et al.(2000).Apoptosis induced in normal human hepatocytes by tumor necrosis factorrelated apoptosis-inducing ligand.Nat Med 6:564-567.
Johnson BE,Grayson J,Makuch RW,Linnoila RI,Anderson MJ,Cohen MH et al.(1990).Ten-year survival of patients with small-cell lung cancer treated with combination chemotherapy with or without irradiation.J Clin Oncol 8:396-401.
Juo P,Kou CJ,Yuan J,Blenis J.(1998).Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade.Curr Biol 8:1001-1008.
Kaliberov S,Stackhouse MA,Kaliberova L et al.(2004).Enhanced apoptosis following treatment with TRA-8 anti-human DR5 monoclonal antibody and overexpression of exogenous Bax in human glioma cells.Gene Therapy 11:658-667.
Kataoka T,Budd RC,Holler N,Thome M,Martinon F,Irmler M et al.(2000).The caspase-8 inhibitor FLIP promotes activation of NF-kB and Erk signaling pathways.Curr Biology 10:640-648.
Kawahara A,Ohsawa Y,Matsumura H,Uchiyama Y,Nagata S.(1998).Caspase-independent cell killing by Fas-associated protein with death domain.J Cell Biol 143:1353-1360.
Kischkel FC,Hellbardt S,Behrmann I,Germer M,Pawlita M,Krammer PH,Peter ME.(1995).Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC)with the receptor.EMBO J 14:5579-5588.
Kischkel FC,Lawrence DA,Chuntharapai A,Schow P,Kim KJ,Ashkenazi A.(2000).Apo2L/TRAILdependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.Immunity 12:611-620.
Koornstra JJ,Kleibeuker JH,van Geelen CM,Rijcken FE,Hollema H,de Vries EG et al.(2003).Expression of TRAIL (TNF-related apoptosis inducing ligand)and its receptors in normal colonic mucosa,adenomas,and carcinomas.J Pathol 200:327-335.
Kuang AA,Diehl GE,Zhang J,Winoto A.(2000).FADD is required for DR4-and DR5-mediated apoptosis:lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts.J Biol Chem 275(33):25065-25068.
Lacour S,Hammann A,Wotawa A,Corcos L,Solary E,Dimanche-Boitrel MT.(2001).Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand mediated caspase-8 activation and apoptosis.Cancer Res 6:1645-1651.
Lawrence D,Shahrokh Z,Marsters S,Achilles K,Shih D,Mounho B et al.(2001).Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions.Nat Med 7:383-385.
LeBlanc HN,Ashkenazi A.(2003).Apo2L/TRAIL and its death and decoy receptors.Cell Death Differ 10:66-75.
Lee SH,Shin MS,Kim HS,Lee HK,Park WS,Kim SY et al.(1999).Alterations of the DR5/TRAIL receptor 2 gene in non-small cell lung cancers.Cancer Res 59:5683-5686.
Levy R,Miller RA.(1983).Biological and clinical implication of lymphocytes hybridomas:tumor therapy with monoclonal antibodies.Annu Rev Med 34:107-116.
Li H,Zhu H,Xu CJ,Yuan J.(1998).Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.Cell 94(4):491-501.
Longley DB,Wilson TR,McEwan M,Allen WL,McDermott U,Galligan L et al.(2006).c-FLIP inhibits chemotherapy-induced colorectal cancer cell death.Oncogene 25:838-848.
Medema JP,Scaffidi C,Kischkel FC,Shevchenko A,Mann M,Krammer PH,Peter ME.(1997).FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).EMBO J 16:2794-2804.
Menoret E,Gomez-Bougie P,Geffroy-Luseau A et al.(2006).Mcl-1L cleavage is involved in TRAIL-R1 and TRAIL-R2 mediated apoptosis induced by HGS-ETR1 and HGS-ETR2 human mAb in myeloma cells.Blood 108(4):1346-1352.
Mitsiades N,Poulaki V,Mitsiades C,Tsokos M.(2001).Ewing's sarcoma family tumors are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)and express DR4 and DR5 receptors.Cancer Res 61(6):2704-2712.
Mitsiades N,Poulaki V,Tseleni-Balafouta S,Koutras DA,Stamenkovic I.(2000).Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand.Cancer Res 60:4122-4129.
Mongkolsapaya J,Cowper AE,Xu XN,Morris G,McMichael AJ,Bell JI et al.(1998).Lymphocyte inhibitor of TRAIL (TNF related apoptosis-inducing ligand):A new receptor protecting lymphocytes from the death ligand TRAIL.J Immunol 160:3-6.
Pai SI,Wu GS,Ozoren N,Wu L,Jen J,Sidransky D et al.(1998).Rare loss-of-function mutation of a death receptor gene in head and neck cancer.Cancer Res 58:3513-3518.
Rasper DM,Vaillancourt JP,Hadano S,Houtzager VM,Seiden I,Keen SL et al.(1998).Cell death attenuation by 'Usurpin',a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas,APO-1)receptor complex.Cell Death Differ 5:271-288.
Ren YG,Wagner KW,Knee DA,Aza-Blanc P,Nasoff M,Deveraux QL.(2004).Differential Regulation of the TRAIL Death Receptors DR4 and DR5 by the Signal Recognition Particle.Mol Biol Cell 15:5064-5074.
Rippo MR,Moretti S,Vescovi S,Tomasetti M,Orecchia S,Amici G et al.(2004).FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells.Oncogene 23:7753-7760.
Ryu BK,Lee MG,Chi SG,Kim YW,Park JH.(2001).Increased expression of cFLIP(L)in colonic adenocarcinoma.J Pathol 194:15-19.
Shankar S,Chen X,Srivastava RK.(2005).Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo.Prostate 62:165-186.
Shi J,Liu Y,Zheng Y,Guo Y,Zhang J,Cheung PT et al.(2006).Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice.Cancer Res 66:11946-11953.
Shi J,Zhang J,Chang X,Liu S,Liu Y,Zheng,D.(2003).Expression and Biological Activity of the Soluble Recombinant Tumor Necrosis Factor Related Apoptosis Inducing Ligand.Chin JBioengineering 23:46-49.
Shin MS,Kim HS,Lee SH,Park WS,Kim SY,Park JY et al.(2001).Mutations of tumor necrosis factor-related Apoptosis inducing ligand receptor 1 (TRAIL-R1)and receptor 2 (TRAIL-R2)genes in metastatic breast cancers.Cancer Res 61:4942-4946.
Shu HB,Halpin DR,Goeddel DV.(1997).Casper is a FADD-and caspase-related inducer of apoptosis.Immunity 6:751-763.
Spierings DC,de Vries EG,Timens W,Groen HJ,Boezen HM,de Jong S.(2003).Expression of TRAIL and TRAIL Death Receptors in Stage Ⅲ Non-Small Cell Lung Cancer Tumors.Clin Cancer Res 9:3397-3405.
Sprick MR,Weigand MA,Rieser E,et al.(2000).FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2.Immunity 12:599-609.
Straughn JM Jr,Oliver PG,Zhou T,Wang W,Alvarez RD,Grizzle WE et al.(2006).Anti-tumor activity of TRA-8 anti-death receptor 5 (DR5)monoclonal antibody in combination with chemotherapy and radiation therapy in a cervical cancer model.Gynecol Oncol 101:46-54.
Takeda K,Yamaguchi N,Akiba H,Kojima Y,Hayakawa Y,Tanner JE et al.(2004).Induction of Tumor-specific T Cell Immunity by Anti-DR5 Antibody.Therapy J Exp Med 199:437-448.
Thompson CB.(1995).Apoptosis in the pathogenesis and treatment of disease.Science 267:1456-1462.
Walczak H,Miller RE,Ariail K,Gliniak B,Griffith TS,Kubin M et al.(1999).Tumoricidal activity of tumor necrosis factor-related apoptos-inducing ligand in vivo.Nat Med 5:157-163.
Wang S,El-Deiry WS.(2003).Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy.Proc Natl Acad Sci U S A 100:15095-15100.
Wiley SR,Schooley K,Smolak PJ,Din WS,Huang CP,Nicholl JK et al.(1995).Identification and characterization of a new member of the TNF family that inducesapoptosis.Immunity 3:673-682.
Wyllie AH,Kerr JF,Currie AR.(1980).Cell death:the significance of apoptosis.Int Rev Cytol 68:251-306.
Yamaguchi Y,Shiraki K,Fuke H,Inoue T,Miyashita K,Yamanaka Y et al.(2006).Adenovirus-mediated transfection of caspase-8 sensitizes hepatocellular carcinoma to TRAIL-and chemotherapeutic agent-induced cell death.Biochim Biophys Acta 1763:844-853.
Yeh WC,Itie A,Elia AJ,Ng M,Shu HB,Wakeham A et al.(2000).Requirement for Casper (c-FLIP)in regulation of death receptor-induced apoptosis and embryonic development.Immunity 12:633-642.
Zeng Y, Wu XX, Fiscella Met al. (2006). Monoclonai antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) induces apoptosis in primary renal cell carcinoma cells in vitro and inhibits tumor growth in vivo. Int J Oncol 28(2): 421-30.
Zhang L, Zhang X, Barrisford GW, Olumi AF. (2007). Lexatumumab (TRAIL-receptor 2 mAb) induces expression of DR5 and promotes apoptosis in primary and metastatic renal cell carcinoma in a mouse orthotopic model. Cancer Lett 251: 146-157.
Zhou XD, Yu JP, Liu J, Luo HS, Chen HX, Yu HG. (2004). Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma. Clin Sci (London) 106: 397-405.
邵晓枫，徐晨，许元富，等。抗CD_3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定。生物工程学报2003 19(5)：527-531．
向军俭，李洪庆，邓 宁，等。抗rh-bFGF嵌合抗体真核表达载体的构建与表达。细胞与分子免疫学杂志2004 20(2)：163-167．
1. Igney FH, Karmmer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2002; 2:277-288.
2. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-1316.
3. Huang DS, Strasser A. BH3-only proteins- essential initiators of apoptotic cell death. Cell 2000; 103:839-842.
4. Zou H, Li Y, Liu X, Wang X. An APAF-1 : Cytochrome c multimeric complex is a functional apoptosome that activates Procaspase-9. J Biol Chem 1999; 274:11549-11556.
5. Cain K, Brown DG, LanglaisA C, Cohen GM. Caspase activation involves the formation of the aposome, a large(70 kDa) caspase-activating complex. J Biol Chem 1999; 274:22686-22692.
6. Iton N, Yonehara S, Ishii A et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991; 66:233-243.
7. Nagata S, Goistein P. The Fas death factor. Science 1995; 267:1449-1456.
8.Chinnaiyan AM,Tepper CG,Seldin MF et al.FADD/MORT1 is a common mediator of CD95 (Fas/APO-1)and tumour necrosis factor receptor induced apoptosis.J Biol Chem 1996;271:4961-4965.
9.Kischkel FC,Hellbardt S,Behrmann I et al.Cytotoxicity dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC)with the receptor.EMBO J 1995;14:5579-5588.
10.Kischkel FC,Lawrence DA,Tinel A et al.Death receptor recruitment of endogenous Caspase-10 and apoptosis initiation in the absence of Caspase-8.J Biol Chem 2001 276:46639-46646.
11.Smyth MJ,Takede K,Hayakawa Y et al.Nature's TRAIL-on a path to cancer immunotherapy.Immunity 2003;18:1-6.
12.Sprick MR,Wigand MA,Reiser E et al.FADD/MORT1 and Caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2.Immunity 2000;12:599-609.
13.Kischkel FC,Lawrence DA,Chuntharapai A,Schow P,Kim KJ,Ashkenazi A.Apo2L/TRAIL dependent recruitment of endogenous FADD and Caspase 8 to death receptors 4 and 5.Immunity 2000;12:611-620.
14.Kelley SK,Ashkenazi A.Targeting death receptors in cancer with Apo2L/TRAIL.Curr Opin Pharmacol 2004;4:333-339.
15.Bouralexis S,Findlay DM,Evdokiou A.Death to the bad guys:targeting cancer via Apo2L/TRAIL.Apoptosis 2005;10:35-51.
16.Liu X,Yue P,Khuri FR,Sun SY:Decoy receptor 2 (DcR2)is a p53 target gene and regulates chemosensitivity.Cancer Res 2005;65:9169-9175.
17.Ashkenazi A.Targeting death and decoy receptors of the tumour-necrosis factor superfamily.Nat Rev Cancer 2002;2:420-430.
18.Thome M,Schneider P,Hofmann K et al.Viral FLICE inhibitory protein (FLIPs)prevents apoptosis induced by death receptors.Nature 1997;386:517-521.
19.Irmler M,Thome M,Hahne M et al.Inhibition of death receptor signals by cellular FLIP.Nature 1997;388:190-195.
20.Shu HB,Halpin DR,Goeddel DV.Casper is a FADD-and caspase-related inducer of apoptosis.Immunity 1997;6:751-763.
21.Goltsev YV,Kovalenko AV,Arnold E et al.CASH,a novel caspase homolgue with death effector domains.J Biol Chem 1997;272:19641-19644.
22.Han DK,Chaudhary PM,Wright ME et al.MRIT,a novel death-effector domain containing protein,interacts with caspases and BclXL and initiates cell death.Proc Natl Acad Sci USA 1997;94:11333-11338.
23.Rasper DM,Vaillancourt JP,Hadano S et al.Cell death attenuation by‘Usurpin’,a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by CD-95 (Fas,APO-1)receptor complex.Cell Death Differ 1998;5:271-288.
24.Scaffidi C,Schmitz I,Krammer PH,Peter ME.The role of c-FLIP in modulation of CD95-induced apoptosis.J Biol Chem 1999;274:1541-1548.
25.Krueger A,Schmitz I,Baumann S,Krammer PH,Kirchhoff S.Cellular FLICE inhibitory protein splice variants inhibit different steps of Caspase 8 activation at the CD95 death induced signalling complex.J Biol Chem 2001 276:20633-20640.
26.Micheau O,Thome M,Schneider P et al.The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signalling complex.J Biol Chem 2002 277:45162-45171.
27.Boatright KM,Deis C,Denault DP,Salvesen GS.Activation of caspases-8 and-10 by FLIPL.Biochem J 2004;382:651-657.
28.Chang DW,Xing Z,Pan Y et al.c-FLIP(L)is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis.EMBO J 2002;21:3704-3714.
29.Medema JP,Dejong J,Van Hall T,Malief CJ,Offringa R.Immune escape of tumours in vivo by expression of cellular FLICE-inhibitory protein.J Exp Med 1999 190:1033-1038.
30.Djerbi M,Screpanti V,Catrina AL,Bogen B,Biberfeld P,Grandien A:FLICE inhibitory protein defines a new class of tumour progression factors.J Exp Med 1999;190:1025-1032.
31.Dutton A,O'Neil JD,Milner AE et al.Expression of the Cellular FLICE inhibitory protein (c-FLIP)protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.Proc Natl Acad Sci USA 2004;101:6611-6616.
32.Mathas S,Lietz A,Anagnostopoulos et al.c-FLIP mediates resistance of Hodgkin/ Reed-Sternberg cells to death receptorinduced apoptosis.J Exp Med 2004;199:1041-1052.
33.Ganter TM,Haas TL,Sykora J et al.Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs.Cell Death Diff 2004;11:S86-S96.
34.Okano H,Shiraki K,Inoue H et al.Cellular FLICE/Caspase-8-inhibitory protein as a principle regulator of cell death and survival in human hepatocellular carcinoma.Lab Invest 2003 83:1033-1043.
35.Shivapurkar N,Reddy J,Matta H et al.Loss of expression of death-inducing signalling complex (DISC)components in lung cancer cell lines and the influence of MYC amplification.Oncogene 2002;21:8510-8514.
36.EInemr A,Ohta T,Yachie A et al.Human pancreatic cancer cells disable function of Fas receptors at several levels in Fas signal transduction pathway.Int J Oncol 2001;18:311-316.
37.Bullani RR,Huard B,Viard-Leweugle I et al.Selective expression of FLIP in malignant melanocytic skin lesions.J Invest Dermatol 2001;117:360-364.
38.Rye BK,Lee MG,Chi SG et al.Increased expression of cFLIP (L)in colonic adenocarcinoma.J Pathol 2001;194:15-19.
39.Baumler C,Duan F,Onel K et al.Differential recruitment of Caspase 8 to cFLIP confers sensitivity or resistance to Fas mediated apoptosis in a subset of familial lymphoma patients.Leuk Res 2003;27:841-851.
40.Thomas RK,Kallenborn A,Wickenhauser C.Constitutive expression of c-FLIP in Hodgkin and Reed-Sternberg cells.Am J Pathol 2002;160:1521-1528.
41.Siegmund D,Hadwiger P,Pfizenmaier K,Vornlocher HP,Wajant H.Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitise tumour cells for TRAIL-induced apoptosis.Mol Med 2002;8:725-732.
42.Ashkenazi A,Pai RC,Fong S et al.Safety and antitumor activity of recombinant soluble Apo2 ligand.Clin Invest 1999;104:155-162.
43.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.
44.Leverkus M,Meumann M,Mengling T et al.Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes.Cancer Res 2000 60:553-559.
45.Hu W,Johnson H,Shu HB:Activation of NF-kB by FADD,Casper,and Caspase-8.J Biol Chem 2000;275:10838-10844.
46.Kataoka T,Budd RC,Holler N et al.The caspase-8 inhibitor FLIP promotes activation of NF-kB and Erk signaling pathways.Curr Biol 2000;10:640-648.
47.Chaudhary PM,Eby MT,Jasmin A,Kumar A,Liu L,Hood L:Activation of the NF-kB pathway by caspase 8 and its homologs.Oncogene 2000;19:4451-4460.
48.Kataoka T,Tschopp J.N-terminal fragment of c-FLIP(L)processed by caspase 8 specifically interacts with TRAF2 and induces activation of the NF-kB signaling pathway.Mol Cell Biol 2004;24:2627-2636.
49.Kreuz S,Siegmund D,Rumpf JJ et al.NFkappaB activation by Fas is mediated through FADD,Caspase-8,and RIP and is inhibited by FLIP.J Cell Biol 2004;166:369-380.
50.Watcher T,Sricks M,Hausmann D,Kerstan A et al.cFLIPL inhibits tumour necrosis factor related apoptosis inducing ligand-mediated NF-kB activation at the death signaling complex in human keratinocytes.J Biol Chem 2004;51:52824-52834.
51.Zhang N,He YW.An essential role for c-FLIP in the efficient development of T lymphocytes.J Exp Med 2005;202:395-404.
52.Sioud M.siRNA delivery in vivo.Methods Mol Biol 2005;309:237-249.
53.Takei Y,Kadomatsu K,Yuzawa Y et al.Small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics.Cancer Res 2004;64:3365-3370.
54.Morrissey DV,Lockridge JA,Shaw L et al.Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.Nat Biotechnol 2005;23:1002-1007.
55.Schiffelers RM,Ansari A,Xu J et al.Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle.Nucleic Acids Res 2004;32:e 149.
56.Song E,Zhu P,Lee SK et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat Biotech 2005;23:709-717.
57.Karagiannis TC,El-osta A.RNA interference and potential therapeutic applications of short interfering RNAs.Cancer Gene Ther 2005;12:787-795.
Boatright KM, Deis C, Denault DP, Salvesen GS. (2004). Activation of caspases-8 and-10 by FLIPL. Biochem J 382:651-657.
Cario-Stella C, Lavazza C, Locatelli A, Vigano L, Gianni AM, Gianni L. (2007). Targeting TRAIL agonistic receptors for cancer therapy. Clin Cancer Res 13: 2313-2317.
Chang DW, Xing Z, Pan Y et al. (2002). c-FLIP(L) is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis. EMBO J 21:3704-3714.
Chaudhary PM, Eby MT, Jasmin A, Kumar A, Liu L, Hood L. (2000). Activation of the NF-κB pathway by caspase 8 and its homologs. Oncogene 19:4451-4460.
Chuntharapai A., Dodge K, Grimmer K, Schroeder K, Marsters SA, Koeppen H et al. (2001). Isotype-dependent inhibition of tumor growth in vivo by monoclonal antibodies to death receptor 4. J Immunol 166: 4891-4898.
Djerbi M, Darreh-Shori T, Zhivotovsky B, Grandien A. (2001). Characterization of the human FLICE-inhibitory protein locus and comparison of the anti-apoptotic activity of four different flip isoforms. Scand J Immunol 54:180-189.
Fang JM, Qian JJ, Yi SL, et al. (2005). Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 23:584-590.
Ferreira CG, Huisman C, Giaccone G. (2002). Novel approaches to the treatment of non-small cell lung cancer. Crit Rev Oncol Hematol 41: 57-77.
Ferrigno D, Buccheri G. (2000). Second-line chemotherapy for recurrent non-small cell lung cancer: do new agents make a difference. Lung Cancer 29: 91-104.
Fisher MJ,Virmani AK,Wu L,Aplenc R,Harper JC,Powell SM,Rebbeck TR,Sidransky D,Gazdar AF,El-Deiry WS.(2001).Nucleotide substitution in the ectodomain of trail receptor DR4 is associated with lung cancer and head and neck cancer.Clin Cancer Res 7(6):1688-1697.
Georgakis GV,Li Y,Humphreys R et al.(2005).Activity of selective fully human agonistic antibodies to the TRAIL death receptors TRAIL-R1 and TRAIL-R2 in primary and cultured lymphoma cells:induction of apoptosis and enhancement of doxorubicin-and bortezomib-induced cell death.British Journal of Haematology 130:501-510.
Golks A,Brenner D,Fritsch C,Krammer PH,Lavrik IN.(2005).c-FLIPR,a New Regulator of Death Receptor-induced Apoptosis.J Biol Chem 280:14507-14513.
Gross A,Yin XM,Wang K,Wei MC,Jockel J,Milliman C,Erdjument-Bromage H,Tempst P,Korsmeyer,SJ.(1999).Caspase cleaved BID targets mitochondria and is required for cytochrome c release,while BCL-XL prevents this release but not tumor necrosis factor-Rl/Fas death.J Biol Chem 274(2):1156-1163.
Guo Y,Chen C,Zheng Y,Zhang J,Tao X,Liu S et al.(2005).A novel anti-human DR5 monoclonal antibody with tumoricidal activity induces caspase-dependent and caspase-independent cell death.J Biol Chem 280:41940-41952.
Han DK,Chaudhary PM,Wright ME,Friedman C,Trask BJ,Riedel RT et al.(1997).MRIT,a novel death-effector domain-containing protein,interacts with caspases and BclXL and initiates cell death.Proc Natl Acad Sci U S A 94:11333-11338.
Hoffman PC,Mauer AM,Vokes EE.(2000).Lung cancer.Lancet 355:479-485.
Horak P,Pils D,Kaider A,Pinter A,Elandt K,Sax C et al.(2005).Perturbation of theTumor Necrosis Factor Related Apoptosis-Inducing Ligand Cascade in Ovarian Cancer:overexpression of FLIPL and Deregulation of the Functional Receptors DR4 and DR5.Clin Cancer Res 11:8585-8591.
Hu S,Vincenz C,Ni J,Gentz R,Dixit VM.(1997).I-FLICE,a novel inhibitor of tumor necrosis factor receptor 1 and CD95 induced apoptosis.J Biol Chem 272:17255-17257.
Hu WH,Johnson H,Shu HB.(2000).Activation of NF-kB by FADD,Casper,and caspase-8.J Biol Chem 275:10838-10844.
Ibrahim SM,Ringel J,Schmidt C,Ringel B,M(?)ller P,Koczan D et al.(2001).Pancreatic adenocarcinoma cell lines show variable susceptibility to TRAIL-mediated cell death.Pancreas 23:72-79.
Ichikawa K,Liu W,Fleck M et al.(2003).TRAIL-R2(DR5)mediates apoptosis of synovial fibroblasts in rheumatoid arthritis.The Journal of Immunology 171:1061-1069.
Ichikawa K,Liu W,Zhao L et al.(2001).Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.Nat Med 7(8):954-60.
Irmler M,Thome M,Hahne M,Schneider P,Hofmann K,Steiner V et al.(1997).Inhibition of death receptor signals by cellular FLIP.Nature 388:190-195.
Itoh N,Nagata S.(1993).A novel protein domain required for apoptosis.Mutational analysis of human Fas antigen.J Biol Chem 268:10932-10937.
Jo M,Kim TH,Seol DW,Esplen JE,Dorko K,Billiar TR et al.(2000).Apoptosis induced in normal human hepatocytes by tumor necrosis factorrelated apoptosis-inducing ligand.Nat Med 6:564-567.
Johnson BE,Grayson J,Makuch RW,Linnoila RI,Anderson MJ,Cohen MH et al.(1990).Ten-year survival of patients with small-cell lung cancer treated with combination chemotherapy with or without irradiation.J Clin Oncol 8:396-401.
Juo P,Kou CJ,Yuan J,Blenis J.(1998).Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade.Curr Biol 8:1001-1008.
Kaliberov S,Stackhouse MA,Kaliberova L et al.(2004).Enhanced apoptosis following treatment with TRA-8 anti-human DR5 monoclonal antibody and overexpression of exogenous Bax in human glioma cells.Gene Therapy 11:658-667.
Kataoka T,Budd RC,Holler N,Thome M,Martinon F,Irmler M et al.(2000).The caspase-8 inhibitor FLIP promotes activation of NF-kB and Erk signaling pathways.Curr Biology 10:640-648.
Kawahara A,Ohsawa Y,Matsumura H,Uchiyama Y,Nagata S.(1998).Caspase-independent cell killing by Fas-associated protein with death domain.J Cell Biol 143:1353-1360.
Kischkel FC,Hellbardt S,Behrmann I,Germer M,Pawlita M,Krammer PH,Peter ME.(1995).Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC)with the receptor.EMBO J 14:5579-5588.
Kischkel FC,Lawrence DA,Chuntharapai A,Schow P,Kim KJ,Ashkenazi A.(2000).Apo2L/TRAILdependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.Immunity 12:611-620.
Koornstra JJ,Kleibeuker JH,van Geelen CM,Rijcken FE,Hollema H,de Vries EG et al.(2003).Expression of TRAIL (TNF-related apoptosis inducing ligand)and its receptors in normal colonic mucosa,adenomas,and carcinomas.J Pathol 200:327-335.
Kuang AA,Diehl GE,Zhang J,Winoto A.(2000).FADD is required for DR4-and DR5-mediated apoptosis:lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts.J Biol Chem 275(33):25065-25068.
Lacour S,Hammann A,Wotawa A,Corcos L,Solary E,Dimanche-Boitrel MT.(2001).Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand mediated caspase-8 activation and apoptosis.Cancer Res 6:1645-1651.
Lawrence D,Shahrokh Z,Marsters S,Achilles K,Shih D,Mounho B et al.(2001).Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions.Nat Med 7:383-385.
LeBlanc HN,Ashkenazi A.(2003).Apo2L/TRAIL and its death and decoy receptors.Cell Death Differ 10:66-75.
Lee SH,Shin MS,Kim HS,Lee HK,Park WS,Kim SY et al.(1999).Alterations of the DR5/TRAIL receptor 2 gene in non-small cell lung cancers.Cancer Res 59:5683-5686.
Levy R,Miller RA.(1983).Biological and clinical implication of lymphocytes hybridomas:tumor therapy with monoclonal antibodies.Annu Rev Med 34:107-116.
Li H,Zhu H,Xu CJ,Yuan J.(1998).Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.Cell 94(4):491-501.
Longley DB,Wilson TR,McEwan M,Allen WL,McDermott U,Galligan L et al.(2006).c-FLIP inhibits chemotherapy-induced colorectal cancer cell death.Oncogene 25:838-848.
Medema JP,Scaffidi C,Kischkel FC,Shevchenko A,Mann M,Krammer PH,Peter ME.(1997).FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).EMBO J 16:2794-2804.
Menoret E,Gomez-Bougie P,Geffroy-Luseau A et al.(2006).Mcl-1L cleavage is involved in TRAIL-R1 and TRAIL-R2 mediated apoptosis induced by HGS-ETR1 and HGS-ETR2 human mAb in myeloma cells.Blood 108(4):1346-1352.
Mitsiades N,Poulaki V,Mitsiades C,Tsokos M.(2001).Ewing's sarcoma family tumors are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)and express DR4 and DR5 receptors.Cancer Res 61(6):2704-2712.
Mitsiades N,Poulaki V,Tseleni-Balafouta S,Koutras DA,Stamenkovic I.(2000).Thyroid carcinoma cells are resistant to FAS-mediated apoptosis but sensitive to tumor necrosis factor-related apoptosis-inducing ligand.Cancer Res 60:4122-4129.
Mongkolsapaya J,Cowper AE,Xu XN,Morris G,McMichael AJ,Bell JI et al.(1998).Lymphocyte inhibitor of TRAIL (TNF related apoptosis-inducing ligand):A new receptor protecting lymphocytes from the death ligand TRAIL.J Immunol 160:3-6.
Pai SI,Wu GS,Ozoren N,Wu L,Jen J,Sidransky D et al.(1998).Rare loss-of-function mutation of a death receptor gene in head and neck cancer.Cancer Res 58:3513-3518.
Rasper DM,Vaillancourt JP,Hadano S,Houtzager VM,Seiden I,Keen SL et al.(1998).Cell death attenuation by 'Usurpin',a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas,APO-1)receptor complex.Cell Death Differ 5:271-288.
Ren YG,Wagner KW,Knee DA,Aza-Blanc P,Nasoff M,Deveraux QL.(2004).Differential Regulation of the TRAIL Death Receptors DR4 and DR5 by the Signal Recognition Particle.Mol Biol Cell 15:5064-5074.
Rippo MR,Moretti S,Vescovi S,Tomasetti M,Orecchia S,Amici G et al.(2004).FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells.Oncogene 23:7753-7760.
Ryu BK,Lee MG,Chi SG,Kim YW,Park JH.(2001).Increased expression of cFLIP(L)in colonic adenocarcinoma.J Pathol 194:15-19.
Shankar S,Chen X,Srivastava RK.(2005).Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo.Prostate 62:165-186.
Shi J,Liu Y,Zheng Y,Guo Y,Zhang J,Cheung PT et al.(2006).Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice.Cancer Res 66:11946-11953.
Shi J,Zhang J,Chang X,Liu S,Liu Y,Zheng,D.(2003).Expression and Biological Activity of the Soluble Recombinant Tumor Necrosis Factor Related Apoptosis Inducing Ligand.Chin JBioengineering 23:46-49.
Shin MS,Kim HS,Lee SH,Park WS,Kim SY,Park JY et al.(2001).Mutations of tumor necrosis factor-related Apoptosis inducing ligand receptor 1 (TRAIL-R1)and receptor 2 (TRAIL-R2)genes in metastatic breast cancers.Cancer Res 61:4942-4946.
Shu HB,Halpin DR,Goeddel DV.(1997).Casper is a FADD-and caspase-related inducer of apoptosis.Immunity 6:751-763.
Spierings DC,de Vries EG,Timens W,Groen HJ,Boezen HM,de Jong S.(2003).Expression of TRAIL and TRAIL Death Receptors in Stage Ⅲ Non-Small Cell Lung Cancer Tumors.Clin Cancer Res 9:3397-3405.
Sprick MR,Weigand MA,Rieser E,et al.(2000).FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2.Immunity 12:599-609.
Straughn JM Jr,Oliver PG,Zhou T,Wang W,Alvarez RD,Grizzle WE et al.(2006).Anti-tumor activity of TRA-8 anti-death receptor 5 (DR5)monoclonal antibody in combination with chemotherapy and radiation therapy in a cervical cancer model.Gynecol Oncol 101:46-54.
Takeda K,Yamaguchi N,Akiba H,Kojima Y,Hayakawa Y,Tanner JE et al.(2004).Induction of Tumor-specific T Cell Immunity by Anti-DR5 Antibody.Therapy J Exp Med 199:437-448.
Thompson CB.(1995).Apoptosis in the pathogenesis and treatment of disease.Science 267:1456-1462.
Walczak H,Miller RE,Ariail K,Gliniak B,Griffith TS,Kubin M et al.(1999).Tumoricidal activity of tumor necrosis factor-related apoptos-inducing ligand in vivo.Nat Med 5:157-163.
Wang S,El-Deiry WS.(2003).Requirement of p53 targets in chemosensitization of colonic carcinoma to death ligand therapy.Proc Natl Acad Sci U S A 100:15095-15100.
Wiley SR,Schooley K,Smolak PJ,Din WS,Huang CP,Nicholl JK et al.(1995).Identification and characterization of a new member of the TNF family that inducesapoptosis.Immunity 3:673-682.
Wyllie AH,Kerr JF,Currie AR.(1980).Cell death:the significance of apoptosis.Int Rev Cytol 68:251-306.
Yamaguchi Y,Shiraki K,Fuke H,Inoue T,Miyashita K,Yamanaka Y et al.(2006).Adenovirus-mediated transfection of caspase-8 sensitizes hepatocellular carcinoma to TRAIL-and chemotherapeutic agent-induced cell death.Biochim Biophys Acta 1763:844-853.
Yeh WC,Itie A,Elia AJ,Ng M,Shu HB,Wakeham A et al.(2000).Requirement for Casper (c-FLIP)in regulation of death receptor-induced apoptosis and embryonic development.Immunity 12:633-642.
Zeng Y, Wu XX, Fiscella Met al. (2006). Monoclonai antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) induces apoptosis in primary renal cell carcinoma cells in vitro and inhibits tumor growth in vivo. Int J Oncol 28(2): 421-30.
Zhang L, Zhang X, Barrisford GW, Olumi AF. (2007). Lexatumumab (TRAIL-receptor 2 mAb) induces expression of DR5 and promotes apoptosis in primary and metastatic renal cell carcinoma in a mouse orthotopic model. Cancer Lett 251: 146-157.
Zhou XD, Yu JP, Liu J, Luo HS, Chen HX, Yu HG. (2004). Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma. Clin Sci (London) 106: 397-405.
邵晓枫，徐晨，许元富，等。抗CD_3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定。生物工程学报2003 19(5)：527-531．
向军俭，李洪庆，邓 宁，等。抗rh-bFGF嵌合抗体真核表达载体的构建与表达。细胞与分子免疫学杂志2004 20(2)：163-167．
1. Igney FH, Karmmer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2002; 2:277-288.
2. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998; 281: 1312-1316.
3. Huang DS, Strasser A. BH3-only proteins- essential initiators of apoptotic cell death. Cell 2000; 103:839-842.
4. Zou H, Li Y, Liu X, Wang X. An APAF-1 : Cytochrome c multimeric complex is a functional apoptosome that activates Procaspase-9. J Biol Chem 1999; 274:11549-11556.
5. Cain K, Brown DG, LanglaisA C, Cohen GM. Caspase activation involves the formation of the aposome, a large(70 kDa) caspase-activating complex. J Biol Chem 1999; 274:22686-22692.
6. Iton N, Yonehara S, Ishii A et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991; 66:233-243.
7. Nagata S, Goistein P. The Fas death factor. Science 1995; 267:1449-1456.
8.Chinnaiyan AM,Tepper CG,Seldin MF et al.FADD/MORT1 is a common mediator of CD95 (Fas/APO-1)and tumour necrosis factor receptor induced apoptosis.J Biol Chem 1996;271:4961-4965.
9.Kischkel FC,Hellbardt S,Behrmann I et al.Cytotoxicity dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC)with the receptor.EMBO J 1995;14:5579-5588.
10.Kischkel FC,Lawrence DA,Tinel A et al.Death receptor recruitment of endogenous Caspase-10 and apoptosis initiation in the absence of Caspase-8.J Biol Chem 2001 276:46639-46646.
11.Smyth MJ,Takede K,Hayakawa Y et al.Nature's TRAIL-on a path to cancer immunotherapy.Immunity 2003;18:1-6.
12.Sprick MR,Wigand MA,Reiser E et al.FADD/MORT1 and Caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2.Immunity 2000;12:599-609.
13.Kischkel FC,Lawrence DA,Chuntharapai A,Schow P,Kim KJ,Ashkenazi A.Apo2L/TRAIL dependent recruitment of endogenous FADD and Caspase 8 to death receptors 4 and 5.Immunity 2000;12:611-620.
14.Kelley SK,Ashkenazi A.Targeting death receptors in cancer with Apo2L/TRAIL.Curr Opin Pharmacol 2004;4:333-339.
15.Bouralexis S,Findlay DM,Evdokiou A.Death to the bad guys:targeting cancer via Apo2L/TRAIL.Apoptosis 2005;10:35-51.
16.Liu X,Yue P,Khuri FR,Sun SY:Decoy receptor 2 (DcR2)is a p53 target gene and regulates chemosensitivity.Cancer Res 2005;65:9169-9175.
17.Ashkenazi A.Targeting death and decoy receptors of the tumour-necrosis factor superfamily.Nat Rev Cancer 2002;2:420-430.
18.Thome M,Schneider P,Hofmann K et al.Viral FLICE inhibitory protein (FLIPs)prevents apoptosis induced by death receptors.Nature 1997;386:517-521.
19.Irmler M,Thome M,Hahne M et al.Inhibition of death receptor signals by cellular FLIP.Nature 1997;388:190-195.
20.Shu HB,Halpin DR,Goeddel DV.Casper is a FADD-and caspase-related inducer of apoptosis.Immunity 1997;6:751-763.
21.Goltsev YV,Kovalenko AV,Arnold E et al.CASH,a novel caspase homolgue with death effector domains.J Biol Chem 1997;272:19641-19644.
22.Han DK,Chaudhary PM,Wright ME et al.MRIT,a novel death-effector domain containing protein,interacts with caspases and BclXL and initiates cell death.Proc Natl Acad Sci USA 1997;94:11333-11338.
23.Rasper DM,Vaillancourt JP,Hadano S et al.Cell death attenuation by‘Usurpin’,a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by CD-95 (Fas,APO-1)receptor complex.Cell Death Differ 1998;5:271-288.
24.Scaffidi C,Schmitz I,Krammer PH,Peter ME.The role of c-FLIP in modulation of CD95-induced apoptosis.J Biol Chem 1999;274:1541-1548.
25.Krueger A,Schmitz I,Baumann S,Krammer PH,Kirchhoff S.Cellular FLICE inhibitory protein splice variants inhibit different steps of Caspase 8 activation at the CD95 death induced signalling complex.J Biol Chem 2001 276:20633-20640.
26.Micheau O,Thome M,Schneider P et al.The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signalling complex.J Biol Chem 2002 277:45162-45171.
27.Boatright KM,Deis C,Denault DP,Salvesen GS.Activation of caspases-8 and-10 by FLIPL.Biochem J 2004;382:651-657.
28.Chang DW,Xing Z,Pan Y et al.c-FLIP(L)is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis.EMBO J 2002;21:3704-3714.
29.Medema JP,Dejong J,Van Hall T,Malief CJ,Offringa R.Immune escape of tumours in vivo by expression of cellular FLICE-inhibitory protein.J Exp Med 1999 190:1033-1038.
30.Djerbi M,Screpanti V,Catrina AL,Bogen B,Biberfeld P,Grandien A:FLICE inhibitory protein defines a new class of tumour progression factors.J Exp Med 1999;190:1025-1032.
31.Dutton A,O'Neil JD,Milner AE et al.Expression of the Cellular FLICE inhibitory protein (c-FLIP)protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.Proc Natl Acad Sci USA 2004;101:6611-6616.
32.Mathas S,Lietz A,Anagnostopoulos et al.c-FLIP mediates resistance of Hodgkin/ Reed-Sternberg cells to death receptorinduced apoptosis.J Exp Med 2004;199:1041-1052.
33.Ganter TM,Haas TL,Sykora J et al.Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs.Cell Death Diff 2004;11:S86-S96.
34.Okano H,Shiraki K,Inoue H et al.Cellular FLICE/Caspase-8-inhibitory protein as a principle regulator of cell death and survival in human hepatocellular carcinoma.Lab Invest 2003 83:1033-1043.
35.Shivapurkar N,Reddy J,Matta H et al.Loss of expression of death-inducing signalling complex (DISC)components in lung cancer cell lines and the influence of MYC amplification.Oncogene 2002;21:8510-8514.
36.EInemr A,Ohta T,Yachie A et al.Human pancreatic cancer cells disable function of Fas receptors at several levels in Fas signal transduction pathway.Int J Oncol 2001;18:311-316.
37.Bullani RR,Huard B,Viard-Leweugle I et al.Selective expression of FLIP in malignant melanocytic skin lesions.J Invest Dermatol 2001;117:360-364.
38.Rye BK,Lee MG,Chi SG et al.Increased expression of cFLIP (L)in colonic adenocarcinoma.J Pathol 2001;194:15-19.
39.Baumler C,Duan F,Onel K et al.Differential recruitment of Caspase 8 to cFLIP confers sensitivity or resistance to Fas mediated apoptosis in a subset of familial lymphoma patients.Leuk Res 2003;27:841-851.
40.Thomas RK,Kallenborn A,Wickenhauser C.Constitutive expression of c-FLIP in Hodgkin and Reed-Sternberg cells.Am J Pathol 2002;160:1521-1528.
41.Siegmund D,Hadwiger P,Pfizenmaier K,Vornlocher HP,Wajant H.Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitise tumour cells for TRAIL-induced apoptosis.Mol Med 2002;8:725-732.
42.Ashkenazi A,Pai RC,Fong S et al.Safety and antitumor activity of recombinant soluble Apo2 ligand.Clin Invest 1999;104:155-162.
43.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.
44.Leverkus M,Meumann M,Mengling T et al.Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes.Cancer Res 2000 60:553-559.
45.Hu W,Johnson H,Shu HB:Activation of NF-kB by FADD,Casper,and Caspase-8.J Biol Chem 2000;275:10838-10844.
46.Kataoka T,Budd RC,Holler N et al.The caspase-8 inhibitor FLIP promotes activation of NF-kB and Erk signaling pathways.Curr Biol 2000;10:640-648.
47.Chaudhary PM,Eby MT,Jasmin A,Kumar A,Liu L,Hood L:Activation of the NF-kB pathway by caspase 8 and its homologs.Oncogene 2000;19:4451-4460.
48.Kataoka T,Tschopp J.N-terminal fragment of c-FLIP(L)processed by caspase 8 specifically interacts with TRAF2 and induces activation of the NF-kB signaling pathway.Mol Cell Biol 2004;24:2627-2636.
49.Kreuz S,Siegmund D,Rumpf JJ et al.NFkappaB activation by Fas is mediated through FADD,Caspase-8,and RIP and is inhibited by FLIP.J Cell Biol 2004;166:369-380.
50.Watcher T,Sricks M,Hausmann D,Kerstan A et al.cFLIPL inhibits tumour necrosis factor related apoptosis inducing ligand-mediated NF-kB activation at the death signaling complex in human keratinocytes.J Biol Chem 2004;51:52824-52834.
51.Zhang N,He YW.An essential role for c-FLIP in the efficient development of T lymphocytes.J Exp Med 2005;202:395-404.
52.Sioud M.siRNA delivery in vivo.Methods Mol Biol 2005;309:237-249.
53.Takei Y,Kadomatsu K,Yuzawa Y et al.Small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics.Cancer Res 2004;64:3365-3370.
54.Morrissey DV,Lockridge JA,Shaw L et al.Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.Nat Biotechnol 2005;23:1002-1007.
55.Schiffelers RM,Ansari A,Xu J et al.Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle.Nucleic Acids Res 2004;32:e 149.
56.Song E,Zhu P,Lee SK et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat Biotech 2005;23:709-717.
57.Karagiannis TC,El-osta A.RNA interference and potential therapeutic applications of short interfering RNAs.Cancer Gene Ther 2005;12:787-795.