c-FLIP(L)在乳腺癌中的表达及对TRAIL通路调节作用的初步探讨
摘要
研究目的
1．观察c-FLIP(L)蛋白及TRAIL凋亡通路受体DR4／DR5在乳腺癌临床标本及体外培养细胞系中的表达，结合临床病理资料加以分析，初步探讨c-FLIP(L)蛋白、DR4／DR5受体在乳腺癌中的存在意义。
2．在乳腺癌组织和细胞系中，观察c-FLIP(L)蛋白与细胞凋亡敏感性的相关性，明确c-FLIP(L)蛋白对乳腺癌TRAIL凋亡通路的调节作用。
3．以凋亡信号转导中caspases蛋白酶级联活化程序为切入点，通过研究c-FLIP(L)蛋白和caspase-8分子的相互作用，初步探讨c-FLIP(L)促进乳腺癌细胞TRAIL凋亡通路转导的作用机理。
研究方法
1．选取乳腺浸润性导管癌和配对的癌旁乳腺组织各150例，应用免疫组织化学方法检测c-FLIP(L)蛋白的表达，搜集患者的临床病理参数，以DR4、DR5的染色评价TRAIL通路受体表达水平，所得结果进行统计学分析。
2．体外培养乳腺癌细胞系，应用半定量RT-PCR和Western blot技术从mRNA和蛋白两个水平检测c-FLIP(L)表达水平，通过间接免疫荧光流式技术检测DR4、DR5受体的阳性率，比较不同细胞间上述指标的差异。
3．通过TUNEL技术和对Ki-67的免疫组化检测，评价乳腺癌细胞的凋亡指数和增殖指数，分析c-FLIP(L)蛋白表达水平与二者的相关性。
4．在乳腺癌细胞系MDA-MB-231细胞中，转染特异性表达质粒，应用RT-PCR和Western blot技术检测c-FLIP(L)mRNA和蛋白的表达水平。在c-FLIP(L)高表达的前提下，外源给予TRAIL诱导细胞凋亡，MTT法绘制TRAIL作用的剂量曲线和时间曲线，选择最佳的诱导条件。在TRAIL处理下，应用Annexin V流式细胞分析术、DNA Ladder和相差显微镜技术观察MDA-MB-231细胞在高表达c-FLIP(L)后凋亡敏感性的变化。
5．以转染c-FLIP(L)表达质粒的乳腺癌细胞系MDA-MB-231为研究对象，通过caspases蛋白酶活性分析，观察c-FLIP(L)蛋白对酶活性的影响。应用Westem blot技术，检测c-FLIP(L)蛋白与caspase-8分子的酶切水解产物，推测它们的活化过程，并进一步应用免疫共沉淀技术分析二者的相互作用。
研究结果
1．c-FLIP(L)蛋白在乳腺浸润性导管癌组织中呈低～中等程度表达(阳性率为36．36％)，在癌旁乳腺组织中呈高表达(阳性率为75％)；其中强阳性染色病例在乳腺癌组织中占14．29％，在癌旁乳腺组织中占57．58％；两组比较有显著性差别(P＜0．01)。统计分析结果显示，c-FLIP(L)蛋白高表达与患者年龄(＜35岁)、绝经前、未发生远处转移、较早的pTNM分期(Ⅰ-Ⅱ期)密切相关以及ER、PR蛋白的表达水平相关(P＜0．05)。
2．在体外培养的不同乳腺癌细胞系中，均可检测到c-FLIP(L)mRNA和蛋白的表达，但c-FLIP(L)在不同的细胞间存在表达差异。TRAIL通路受体DR4、DR5在不同的乳腺癌细胞中表达水平不同，相对于每一株细胞来说，二者的阳性率呈现一致性。
3．免疫组化染色发现，c-FLIP(L)蛋白的表达与DR4受体的表达呈正相关，二者之间关系有统计学意义(r_s=0．452，P=0．035)；对于DR5受体，虽然呈现出c-FLIP(L)蛋白表达越高，DR5受体表达越高的趋势，但二者之间无明显相关性(r_s=0．419，P=0．066)。此外，c-FLIP(L)蛋白的表达与凋亡指数正相关(r_s=0．554，P=0．005)；该蛋白表达升高增殖指数有所下降，但二者之间无显著相关性。
4．转染特异性表达质粒发现，转染后24h，c-FLIP(L)mRNA和蛋白含量出现升高；转染后48h和72h mRNA含量仍能维持较高水平，蛋白的表达量有所下降。在此基础上，TRAIL诱导细胞4h，抑制率出现变化，随时间延长呈现逐渐升高的趋势，其中处理24h可达到较高水平；每个观察时间点下TRAIL浓度为10ng/ml时的抑制率均较高，随浓度加大仍能维持。
5．TRAIL诱导后，在高表达c-FLIP(L)的MDA-MB-231细胞中，凋亡细胞比例可升至51．65％；经电泳分析，其DNA断裂呈规律的阶梯状；此时大部分细胞失去原有形态，胞体圆缩，间隙增大，折光性减弱，分裂像少见。
6．应用TRAIL诱导凋亡后，在高表达c-FLIP(L)的MDA-MB-231细胞中，caspase-8活性提升5倍，caspase-3活性提升3倍，caspase-9、caspase-2活性变化不明显。
7．与转染空载体细胞相比，TRAIL可诱导c-FLIP(L)高表达细胞内的caspase-8蛋白发生酶切降解，全长的p55 caspase-8含量减少，胞浆中出现p43、p18和p10亚片段；同时c-FLIP(L)p55片段含量亦出现降低，p43和p12片段含量增多。通过免疫共沉淀实验发现，c-FLIP(L)蛋白与caspase-8蛋白能够发生相互作用，此外作用复合物上尚可检测到二者的p43片段。
研究结论
1．通过检测c-FLIP(L)在癌和癌旁组织中的表达、明确该蛋白调节细胞凋亡的作用机制，从体内和体外不同角度证实，c-FLIP(L)在乳腺癌中具有促进TRAIL凋亡通路的作用，其阳性表达提示较为良好的临床预后。
2．c-FLIP(L)作为天然存在的蛋白酶类似物，在不同条件下可能存在不同的表达水平、具有截然不同的凋亡调节功能，具体机制仍需要进一步的研究。
3．鉴于c-FLIP(L)在不同类型的肿瘤组织和不同个体间存在表达差异，在将其开发为有意义的预测因子和治疗靶点之前，要充分考虑这一特点，注意诊治的个体化。
Objective
1.To study the expression of c-FLIP(L) and TRAIL-DR4/DR5 in human breastcancer tissue and cell lines and analysis the correlation between c-FLIP(L) expressionand clinicopathologic parameters.
2.To observe the association of c-FLIP(L) expression and apoptotic sensitivity inbreast cancer tissue and cell lines and primarily confirm the exact regulation ofc-FLIP(L) in TRAIL pathway in breast cancer.
3.To study the co-operation between c-FLIP(L) and caspase-8 and make sure themechanism of c-FLIP(L) in TRAIL pathway in breast cancer primarily.
Methods
1.Use the technique of IHC to detect the expression of c-FLIP(L) and DR4/DR5 inbreast cancer tissue and its counterparts (150 cases each).Analysis the association ofthese factors and the correlation between c-FLIP(L) and clinicopathologic parameters.
2.Detect c-FLIP(L) in mRNA and protein level by RT-PCR and Western blot andevaluate the difference of DR4/DR5 by indirect immunofluorescence FCM in breastcell lines.
3.Calculate the apoptosis index and proliferate index by TUNEL and Ki-67immunohistochemistry and evaluate their correlation.
4.Establish c-FLIP(L) high-expressed MDA-MB-231 breast cancer cell line bytransfecting c-FLIP(L) plasmids.Under TRAIL inducing,map out dose-dependentand time-dependent curve and select the perfect condition by MTT analysis.Detectapoptosis by Annexin V-FITC,DNA Ladder and phase contrast microscope inc-FLIP(L) high-expressed MDA-MB-231 cells.
5.Use caspases assay plates,Western blot and Co-immunoprecipitation (Co-IP) toanalysis the co-operation between c-FLIP(L) and caspase-8.
Results
1.c-FLIP(L) is low or moderate in breast invasive ductal cancer tissue (36.36%),while it is high-expressed in normal counterparts (75%) (P＜0.01).The positive rate ofexpression level of+++ is 14.29% in malignant and 57.58% in normal breast tissue (P＜0.01).c-FLIP(L) expression is positively associated with age,menopausal status,distant metastasis and pathologic stage (P＜0.05).Still c-FLIP(L) is positive in the ERand/or PR positive cases and it has no correlation with Her-2 expression.
2.c-FLIP(L) and DR4/DR5 are present in four breast cancer cell lines.Although thereare different levels of these factors,DR4 and DR5 are consistent in single cell line.
3.DR4 is high-expressed in the cases which has high level of c-FLIP(L) and thecorrelation of them is 0.452 (P=0.035).As to DR5 and c-FLIP(L),the association hasno statistic significance (r_s=0.419,P=0.066).There is positive correlation betweenc-FLIP(L) expression and apoptotic index (r_s=0.554,P=0.005).When c-FLIP(L) ishigh-expressed,proliferate index changed into low or moderate and they are notstatistic correlated.
4.The mRNA and protein level of c-FLIP(L) are increased after transfecting plasmids24h,and the mRNA level is still after 48h and 72h,while protein level is decreasedslightly.The growth inhibition rate begins to change after TRAIL induction 4h and ishigh after 24h.Under each time point,the rate increases obviously when TRAIL is10ng/ml compared with other doses.
5.After TRAIL inducing,apoptotic rate increases to 51.56% in c-FLIP(L)high-expressed MDA-MB-231 cells.DNA Ladder can be detected and apoptoticmorphologic changes can be observed by phase contrast microscope.
6.Under TRAIL inducing,the caspase-8 activity increases to 5-folds and caspase-3to 3-folds in c-FLIP(L) high-expressed MDA-MB-231 cell line,while the activitiesofcaspase-2 and caspase-9 do not change obviously.
7.Compared with cells transfected with pEGFP-N1,there are some changes in thecells with c-FLIP(L) plasmids:the p55 segment of caspase-8 decreases and the p43,p 18,and p 10 increase;the p55 of c-FLIP(L) also decreases and p43 and p 12 increase.c-FLIP(L) can co-operate with caspase-8 by Co-IP analysis,and the p43 segment ofthese two factors can be detected in their complex.
Conclusion
1.We confirm that c-FLIP(L) protein can promote TRAIL pathway in breast cancerby ditecting the expression of c-FLIP(L) in cancer and its counterparts and observingthe exact regulation of c-FLIP(L) in apoptosis.We also report that the low-expressed c-FLIP(L) indicats good prognosis in breast cancer.
2.c-FLIP(L) may have different expression level and apoptotic modulation underdifferent conditions,which needs further research to confirm.
3.We should consider the different of c-FLIP(L) in diverse cancer types and distinctindividuals before use it as a significative target for prognosis and therapy.
1．观察c-FLIP(L)蛋白及TRAIL凋亡通路受体DR4／DR5在乳腺癌临床标本及体外培养细胞系中的表达，结合临床病理资料加以分析，初步探讨c-FLIP(L)蛋白、DR4／DR5受体在乳腺癌中的存在意义。
2．在乳腺癌组织和细胞系中，观察c-FLIP(L)蛋白与细胞凋亡敏感性的相关性，明确c-FLIP(L)蛋白对乳腺癌TRAIL凋亡通路的调节作用。
3．以凋亡信号转导中caspases蛋白酶级联活化程序为切入点，通过研究c-FLIP(L)蛋白和caspase-8分子的相互作用，初步探讨c-FLIP(L)促进乳腺癌细胞TRAIL凋亡通路转导的作用机理。
研究方法
1．选取乳腺浸润性导管癌和配对的癌旁乳腺组织各150例，应用免疫组织化学方法检测c-FLIP(L)蛋白的表达，搜集患者的临床病理参数，以DR4、DR5的染色评价TRAIL通路受体表达水平，所得结果进行统计学分析。
2．体外培养乳腺癌细胞系，应用半定量RT-PCR和Western blot技术从mRNA和蛋白两个水平检测c-FLIP(L)表达水平，通过间接免疫荧光流式技术检测DR4、DR5受体的阳性率，比较不同细胞间上述指标的差异。
3．通过TUNEL技术和对Ki-67的免疫组化检测，评价乳腺癌细胞的凋亡指数和增殖指数，分析c-FLIP(L)蛋白表达水平与二者的相关性。
4．在乳腺癌细胞系MDA-MB-231细胞中，转染特异性表达质粒，应用RT-PCR和Western blot技术检测c-FLIP(L)mRNA和蛋白的表达水平。在c-FLIP(L)高表达的前提下，外源给予TRAIL诱导细胞凋亡，MTT法绘制TRAIL作用的剂量曲线和时间曲线，选择最佳的诱导条件。在TRAIL处理下，应用Annexin V流式细胞分析术、DNA Ladder和相差显微镜技术观察MDA-MB-231细胞在高表达c-FLIP(L)后凋亡敏感性的变化。
5．以转染c-FLIP(L)表达质粒的乳腺癌细胞系MDA-MB-231为研究对象，通过caspases蛋白酶活性分析，观察c-FLIP(L)蛋白对酶活性的影响。应用Westem blot技术，检测c-FLIP(L)蛋白与caspase-8分子的酶切水解产物，推测它们的活化过程，并进一步应用免疫共沉淀技术分析二者的相互作用。
研究结果
1．c-FLIP(L)蛋白在乳腺浸润性导管癌组织中呈低～中等程度表达(阳性率为36．36％)，在癌旁乳腺组织中呈高表达(阳性率为75％)；其中强阳性染色病例在乳腺癌组织中占14．29％，在癌旁乳腺组织中占57．58％；两组比较有显著性差别(P＜0．01)。统计分析结果显示，c-FLIP(L)蛋白高表达与患者年龄(＜35岁)、绝经前、未发生远处转移、较早的pTNM分期(Ⅰ-Ⅱ期)密切相关以及ER、PR蛋白的表达水平相关(P＜0．05)。
2．在体外培养的不同乳腺癌细胞系中，均可检测到c-FLIP(L)mRNA和蛋白的表达，但c-FLIP(L)在不同的细胞间存在表达差异。TRAIL通路受体DR4、DR5在不同的乳腺癌细胞中表达水平不同，相对于每一株细胞来说，二者的阳性率呈现一致性。
3．免疫组化染色发现，c-FLIP(L)蛋白的表达与DR4受体的表达呈正相关，二者之间关系有统计学意义(r_s=0．452，P=0．035)；对于DR5受体，虽然呈现出c-FLIP(L)蛋白表达越高，DR5受体表达越高的趋势，但二者之间无明显相关性(r_s=0．419，P=0．066)。此外，c-FLIP(L)蛋白的表达与凋亡指数正相关(r_s=0．554，P=0．005)；该蛋白表达升高增殖指数有所下降，但二者之间无显著相关性。
4．转染特异性表达质粒发现，转染后24h，c-FLIP(L)mRNA和蛋白含量出现升高；转染后48h和72h mRNA含量仍能维持较高水平，蛋白的表达量有所下降。在此基础上，TRAIL诱导细胞4h，抑制率出现变化，随时间延长呈现逐渐升高的趋势，其中处理24h可达到较高水平；每个观察时间点下TRAIL浓度为10ng/ml时的抑制率均较高，随浓度加大仍能维持。
5．TRAIL诱导后，在高表达c-FLIP(L)的MDA-MB-231细胞中，凋亡细胞比例可升至51．65％；经电泳分析，其DNA断裂呈规律的阶梯状；此时大部分细胞失去原有形态，胞体圆缩，间隙增大，折光性减弱，分裂像少见。
6．应用TRAIL诱导凋亡后，在高表达c-FLIP(L)的MDA-MB-231细胞中，caspase-8活性提升5倍，caspase-3活性提升3倍，caspase-9、caspase-2活性变化不明显。
7．与转染空载体细胞相比，TRAIL可诱导c-FLIP(L)高表达细胞内的caspase-8蛋白发生酶切降解，全长的p55 caspase-8含量减少，胞浆中出现p43、p18和p10亚片段；同时c-FLIP(L)p55片段含量亦出现降低，p43和p12片段含量增多。通过免疫共沉淀实验发现，c-FLIP(L)蛋白与caspase-8蛋白能够发生相互作用，此外作用复合物上尚可检测到二者的p43片段。
研究结论
1．通过检测c-FLIP(L)在癌和癌旁组织中的表达、明确该蛋白调节细胞凋亡的作用机制，从体内和体外不同角度证实，c-FLIP(L)在乳腺癌中具有促进TRAIL凋亡通路的作用，其阳性表达提示较为良好的临床预后。
2．c-FLIP(L)作为天然存在的蛋白酶类似物，在不同条件下可能存在不同的表达水平、具有截然不同的凋亡调节功能，具体机制仍需要进一步的研究。
3．鉴于c-FLIP(L)在不同类型的肿瘤组织和不同个体间存在表达差异，在将其开发为有意义的预测因子和治疗靶点之前，要充分考虑这一特点，注意诊治的个体化。
Objective
1.To study the expression of c-FLIP(L) and TRAIL-DR4/DR5 in human breastcancer tissue and cell lines and analysis the correlation between c-FLIP(L) expressionand clinicopathologic parameters.
2.To observe the association of c-FLIP(L) expression and apoptotic sensitivity inbreast cancer tissue and cell lines and primarily confirm the exact regulation ofc-FLIP(L) in TRAIL pathway in breast cancer.
3.To study the co-operation between c-FLIP(L) and caspase-8 and make sure themechanism of c-FLIP(L) in TRAIL pathway in breast cancer primarily.
Methods
1.Use the technique of IHC to detect the expression of c-FLIP(L) and DR4/DR5 inbreast cancer tissue and its counterparts (150 cases each).Analysis the association ofthese factors and the correlation between c-FLIP(L) and clinicopathologic parameters.
2.Detect c-FLIP(L) in mRNA and protein level by RT-PCR and Western blot andevaluate the difference of DR4/DR5 by indirect immunofluorescence FCM in breastcell lines.
3.Calculate the apoptosis index and proliferate index by TUNEL and Ki-67immunohistochemistry and evaluate their correlation.
4.Establish c-FLIP(L) high-expressed MDA-MB-231 breast cancer cell line bytransfecting c-FLIP(L) plasmids.Under TRAIL inducing,map out dose-dependentand time-dependent curve and select the perfect condition by MTT analysis.Detectapoptosis by Annexin V-FITC,DNA Ladder and phase contrast microscope inc-FLIP(L) high-expressed MDA-MB-231 cells.
5.Use caspases assay plates,Western blot and Co-immunoprecipitation (Co-IP) toanalysis the co-operation between c-FLIP(L) and caspase-8.
Results
1.c-FLIP(L) is low or moderate in breast invasive ductal cancer tissue (36.36%),while it is high-expressed in normal counterparts (75%) (P＜0.01).The positive rate ofexpression level of+++ is 14.29% in malignant and 57.58% in normal breast tissue (P＜0.01).c-FLIP(L) expression is positively associated with age,menopausal status,distant metastasis and pathologic stage (P＜0.05).Still c-FLIP(L) is positive in the ERand/or PR positive cases and it has no correlation with Her-2 expression.
2.c-FLIP(L) and DR4/DR5 are present in four breast cancer cell lines.Although thereare different levels of these factors,DR4 and DR5 are consistent in single cell line.
3.DR4 is high-expressed in the cases which has high level of c-FLIP(L) and thecorrelation of them is 0.452 (P=0.035).As to DR5 and c-FLIP(L),the association hasno statistic significance (r_s=0.419,P=0.066).There is positive correlation betweenc-FLIP(L) expression and apoptotic index (r_s=0.554,P=0.005).When c-FLIP(L) ishigh-expressed,proliferate index changed into low or moderate and they are notstatistic correlated.
4.The mRNA and protein level of c-FLIP(L) are increased after transfecting plasmids24h,and the mRNA level is still after 48h and 72h,while protein level is decreasedslightly.The growth inhibition rate begins to change after TRAIL induction 4h and ishigh after 24h.Under each time point,the rate increases obviously when TRAIL is10ng/ml compared with other doses.
5.After TRAIL inducing,apoptotic rate increases to 51.56% in c-FLIP(L)high-expressed MDA-MB-231 cells.DNA Ladder can be detected and apoptoticmorphologic changes can be observed by phase contrast microscope.
6.Under TRAIL inducing,the caspase-8 activity increases to 5-folds and caspase-3to 3-folds in c-FLIP(L) high-expressed MDA-MB-231 cell line,while the activitiesofcaspase-2 and caspase-9 do not change obviously.
7.Compared with cells transfected with pEGFP-N1,there are some changes in thecells with c-FLIP(L) plasmids:the p55 segment of caspase-8 decreases and the p43,p 18,and p 10 increase;the p55 of c-FLIP(L) also decreases and p43 and p 12 increase.c-FLIP(L) can co-operate with caspase-8 by Co-IP analysis,and the p43 segment ofthese two factors can be detected in their complex.
Conclusion
1.We confirm that c-FLIP(L) protein can promote TRAIL pathway in breast cancerby ditecting the expression of c-FLIP(L) in cancer and its counterparts and observingthe exact regulation of c-FLIP(L) in apoptosis.We also report that the low-expressed c-FLIP(L) indicats good prognosis in breast cancer.
2.c-FLIP(L) may have different expression level and apoptotic modulation underdifferent conditions,which needs further research to confirm.
3.We should consider the different of c-FLIP(L) in diverse cancer types and distinctindividuals before use it as a significative target for prognosis and therapy.
引文
[1] Ramaswam M, Efimova EV, Martinez 0, et al. IG20 (MADD splice variant-5), a proapoptotic protein, interacts with DR4/DR5 and enhances TRAIL-induced apoptosis by increasing recruitment of FADD and caspase-8 to the DISC[J].Oncogene, 2004,23(36):6083-6094.
[2] Thomas LR, Henson A, Reed JC, et al. Direct binding of Fas-associated death domain (FADD) to the tumor necrosis factor-related apoptosis-inducing ligand receptor DR5 is regulated by the death effector domain of FADD[J]. J Biol Chem,2004,279(31):32780-32785.
[3] Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by celluar FLIP[J]. Nature, 1997,388:190-195.
[4] Goltsev YV, Kovalenko AV, Arnold E, et al. CASH, a novel caspase homologue with death effector domains[J]. J Biol Chem, 1997,272(32): 19641 -19645.
[5] Heijink DM, Kleibeuker JH, Jalving M, et al. Independent induction of caspase-8 and cFLIP expression during colorectal carcinogenesis in sporadic and HNPCC adenomas and carcinomas [J]. Cell Oncol, 2007,29(5):409-419.
[6] Korkolopoulou P, Saetta AA, Levidou G, et al. c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications[J].Histopathology, 2007,51:150-156.
[7] Valnet-Rabier MB, Challier B, Thiebault S, et al. c-Flip protein expression in Burkitt's lymphomas is associated with a poor clinical outcome[J]. Brit J Haemtol,2005,128:767-773.
[8] Uherova P, Olson S, Thompson MA, et al. Expression of c-FLIP in classic and nodular lymphocyte-predominant Hodgkin lymphoma[J]. Appl Immunohistochem Mol Morphol, 2004,12:105-110.
[9] Yeh WC, Itie A, Elia AJ, et al. Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development[ J]. Immunity, 2000,12 (6):633-642.
[10] Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma[J]. Clin Sci (Lond), 2004,106(4):397-405.
[11]Koomstra JJ,Kleibeuker JH,van Geelen CM,et al.Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa,adenomas,and carcinomas[J].J Pathol,2003,200(3):327-335.
[12]Thomas RK,Kallenborn A,Wickenhauser C,et al.Constitutive expression of c-FLIP in Hodgkin and Reed-Sternberg cells[J].Am J Pathol,2002,160(4):1521-1528.
[13]Chen F,Guo J,Zhang Y,et al.Knockdown of c-FLIP enhanced AD5-10 anti-death receptor 5 monoclonal antibody-induced apoptosis in human lung cancer cells[J].Cancer Sci,2009,100(5):940-947.
[14]杨奎忠，杜庆聪，项继舜，黄凤昌．食管癌组织中c-FLIP蛋白的表达及意义[J]．山东医药，2007，47(3)：77-78．
[15]van Houdt IS,Muris JJ,Hesselink AT,et al.Expression of c-FLIP is primarily detected in diffuse large B-cell lymphoma and Hodgkin's lymphoma and correlates with lack of caspase 8 activation[J].Histopathology,2007,51(6):778-784.
[16]Valnet-Rabier MB,Challier B,Thiebault S,et al.c-Flip protein expression in Burkitt's lymphomas is associated with a poor clinical outcome[J].Br J Haematol,2005,128(6):767-773.
[17]陈红霞，刘元姣，黎辉，等．cFLIP基因在子宫内膜腺癌组织中的表达[J]．癌症，2004，23(3)：254-258．
[18]Kim SY,Song SY,Kim MS,et al.Immunohistochemical analysis of Fas and FLIP in prostate cancers[J].APMIS,2009,117(1):28-33.
[19]Hammond ME,Fitzgibbons PL,Compton CC,et al.College of American Pathologists Conference ⅩⅩⅩⅤ:solid tumor prognostic factors-which,how and so what? Summary Document and recommendations for implementation.Cancer Committeee and Conference Participants[J].Arch Pathol Lab Med,2000,124:958-965.
[20]Crowe JP,Gordon NH,Hubay CA,et al.Estrogen receptor determination and long term survival of patients with carcinoma of the breast[J].Surg Gynecol Obstet,1991,173:273-278.
[21]MacFarlane M.TRAIL-induced signaling and apoptosis[J].Toxicol Lett,2003,139(2-3):89-97.
[22]Romaswamy M,Efimova EV,Martinez O,et al.IG20 (MADD splice variant-5), a proapoptotic protein, interacts with DR4/DR5 and enhanced TRAIL-induced apoptosis by increasing recruitment of FADD and caspase-8 to the DISC[J].Oncogene, 2004,23(36):6083-6094.
[23] Sanlioglu AD, Korcum AF, Pestereli E, et al. TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma[J]. Int J Radiation Oncology Biol Phys, 2007,69(3):716-723.
[24] Day TW, Huang S, Safa AR. c-FLIP knockdown induces ligand-independent DR5-, FADD-, caspase-8-, and caspase-9-dependent apoptosis in breast cancer cells.Biochem Pharmacol, 2008,76(12): 1694-704.
[25] Kazhdan I, Marciniak RA. Death receptor 4 (DR4) efficiently kills breast cancer cells irrespective of their sensitivity to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) [J]. Cancer Gene Ther, 2004,11(10):691-698.
[26] Guseva NV, Rokhlin OW, Taghiyev AF, et al. Unique resistance of breast carcinoma cell line T47D to TRAIL but not anti-Fas is linked to p43cFLIP(L) [J].Breast Cancer Res Treat, 2008,107(3):349-357.
[27] Hyer ML, Croxton R, Krajewska M, et al. Synthetic triterpenoids cooperate with tumor necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer cells[J]. Cancer Res, 2005,65(11):4799-4808.
[28] Kim IK, Jung YK, Noh DY, et al. Functional screening of genes suppressing TRAIL-induced apoptosis: distinct inhibitory activities of Bcl-XL and Bcl-2[J]. Br J Cancer, 2003,88(6):910-917.
[29] Kim S, Lee TJ, Leem J,et al. Sanguinarine-induced apoptosis: generation of ROS, down-regulation of Bcl-2, c-FLIP, and synergy with TRAIL[J]. J Cell Biochem, 2008,104(3):895-907.
[30] Park C, Moon DO, Ryu CH, et al. Beta-sitosterol sensitizes MDA-MB-231 cells to TRAIL-induced apoptosis[J]. Acta Pharmacol Sin, 2008,29(3):341-348.
[31] Zhang Y, Yoshida T, Zhang B. TRAIL induces endocytosis of its death receptors in MDA-MB-231 breast cancer cells[J]. Cancer Biol Ther, 2008,8(10).[Epub ahead of print]
[32] Yu JW, Shi Y. FLIP and the death effector domain family[J]. Oncogene, 2008,27:6216-6227.
[33] Zang FL, Sun BC, Zhao XL, et al. Critical role for c-FLIP_L on Fas resistance in colon carcinoma cell line HT-29[J]. Cell Biol Inter, 2008,32:329-336.
[34] Zhang N, Hopkins K, He YW. The long isoform of cellular FLIP is essencial for T lymphocyte proliferation through an NF-κB-independent pathway[J]. J Immuno,2008,180:5506-5511.
[35] Chang DW, Xing Z, Pan Y, et al. c-FLIP_L is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis[J]. EMBO, 2002,21(14):3704-3714.
[36] Krueger A, Baumann S, Krammer PH, et al. FLICE-inhibitory proteins: regulations of death receptor-mediated apoptosis[J]. Mol Cell Bio, 2001, 21(24):8247-8254.
[37] Kurbanov BM, Fecker LF, Geilen CC, et al. Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-kappaB but is related to downregulation of initiator caspases and DR4[J]. Oncogene, 2007,26(23):3364-3377.
[38] Boatright KM, Deis C, Denault JB, et al. Activation of caspases-8 and -10 by FLIP(L)[J]. Biochem J, 2004;382(Pt 2):651-657.
[39] Micheau O, Thome M, Schneider P, et al. The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex[J]. J Biol Chem, 2002;277 (47): 45162-45171.
[40] Dohrman A, Kataoka T, Cuenin S, et al. Cellular FLIP (long form) regulates CD8~+ T cell activation through caspase-8-dependent NF-kappa B activation[J]. J Immunol, 2005,174(9):5270-5278.
[41] Mitsiades CS, Poulaki V, Fanourakis G, et al. Fas signaling in thyroid carcinomas is diverted from apoptosis to proliferation. Clin Cancer Res[J]. 2006, 12(12):3705-3712.
[42] Lee TJ, Lee JT, Park JW, et al. Acquired TRAIL resistance in human breast cancer cells are caused by the sustained cFLIP(L) and XIAP protein levels and ERK activation[J]. Biochem Biophys Res Commun, 2006,351(4):1024-1030.
[43] Thai le M, Labrinidis A, Hay S, et al. Apo21/Tumor necrosis factor-related apoptosis-inducing ligand prevents breast cancer-induced bone destruction in a mouse model. Cancer Res, 2006,66(10):5363-70.
[1]Krammer PH.CD95's deadly mission in the immune system[J].Nature,2000,407(6805):789-795.Review.
[2]Thome M,Schneider P,Hofmann K,et al.Viral FLICE inhibitory proteins (FLIP) prevent apoptosis induced by death receptors[J].Nature,1997,386(6624):517-521.
[3]Irmler M,Thome M,Hahne M,et al.Inhibition of death receptor signals by celluar FLIP[J].Nature,1997,388(6624):190-195.
[4]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 the CD95 (Fas,APO-1) receptor complex[J].Cell Death Differ,1998,5(4):271-288.
[5]Schmitz IS,Kirchhoff PH,Krammer PH.Regulation of death receptor-mediated apoptosis pathways[J].Int J Biochem Cell Biol,2000,32:1123-1136.
[6] Oehme I, Neumann F, Bosser S, et al. Transgenic overexpression of the caspase-8 inhibitor FLIP(short) leads to impaired T cell proliferation and an increased memory T cell pool after staphylococcal enterotoxin B injection[J]. Eur J Immunol, 2005,35(4):1240-1249.
[7] Boatright KM, Deis C, Denault JB, et al. Activation of caspases-8 and -10 by FLIP(L) [J]. Biochem J, 2004,382(Pt 2):651-657.
[8] Dohrman A, Russell JQ, Cuenin S, et al. Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase-8 [J]. J Immunol, 2005,175(l):311-318.
[9] Goltsev YV, Kovalenko E, Arnold, et al. CASH, a novel caspase homologue with death effector domain[J]. J Biol Chem, 1997,272:19641-19644.
[10] Shu HB, Halpin DR, Goeddel DV. Casper is a FADD- and caspase-related inducer of apoptosis [J]. Immunity, 1997,6:751 -763.
[11] Srinivasula SM, Ahmad M, Ottilie S, et al. FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis[J]. J Biol Chem,1997,272:18542-18545.
[12] Han DK, Chaudhary PM, Wright ME, et al. MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclX_L and initiates cell death[J]. Proc Natl Acad Sci USA, 1997,94:11333-11338.
[13] Hu S, Vincenz C, Ni J, et al. I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis[J]. J Biol Chem, 1997,272:17255-17257.
[14] Kataoka T, Budd RC, Holler N, et al. The caspase 8 inhibitor FLIP promotes activation of NF-kB and Erk signaling pathways[J]. Curr Biol, 2000,10:640-648.
[15] Scaffidi C, Schmitz I, Krammer PH, et al. The role of c-FLIP in modulation of CD95-induced apoptosis[J]. J Biol Chem, 1999,274:1514-1548.
[16] Krueger A, Schmitz I, Baumann S, et al. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase 8 activation at the CD95 death- inducing signaling complex[J]. J Biol Chem, 2001,276:20633-20640.
[17] Earnshaw WC, Martins LM. Mammalian caspases: structure, activation, substrates, and functuions during apoptosis[J]. Annu Rev Biochem, 1999,68:383-424.
[18] Muzio M, Stockwell BR, Stennicke HR, et al. An induced proximity model for caspase 8 activation[J]. J Biol Chem, 1998,273:2926-2930.
[19] Zeiss CJ. The apoptosis-necrosis continuum: insights from genetically altered mice[J]. Vet Pathol, 2003,40(5):481-495. Review.
[20] 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-kappaB signaling pathway[J]. Mol Cell Biol, 2004,24(7):2627-2636.
[21] Kim YY, Park BJ, Seo GJ, et al. Long form of cellular FLICE-inhibitory protein interacts with Daxx and prevents Fas-induced JNK activation[J]. Biochem Biophys Res Commun, 2003,312(2):426-433.
[22] Wang X, Wang Y, Zhang J, et al. FLIP protects against hypoxia/reoxygenation- induced endothelial cell apoptosis by inhibiting Bax activation [J]. Mol Cell Biol,2005, 25(11):4742-4751.
[23] Susanne M, Lens A. The caspase 8 inhibitor c-FLIPL modulates T-cell receptor-induced proliferation but not activation-induced cell death oflymphocytes[J]. Mol Cell Biol, 2002,22(15):5419-5433.
[24] Grambihler A, Higuchi H, Bronk SF, et al. cFLIP-L inhibits p38 MAPK activation: an additional anti-apoptotic mechanism in bile acid-mediated apoptosis[J].J Biol Chem, 2003,278(29):26831-26837.
[25] Nakagiri S, Murakami A, Takada S, et al. Viral FLIP enhances Wnt signaling downstream of stabilized beta-catenin, leading to control of cell growth[J]. Mol Cell Biol, 2005,25(21):9249-9258.
[26] Cudrici C, Niculescu F, Jensen T, et al. C5b-9 terminal complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and upregulating FLIP[J]. J Immunol, 2006,176(5):3173-3180.
[27] Yeh JH, Hsu SC, Han SH, et al. Mitogen-activated protein kinase kinase antagonized fas-associated death domain protein-mediated apoptosis by induced FLICE-inhibitory protein expression[J]. J Exp Med, 1998,188:1795-1802.
[28] Schlapbach R, Spanaus KS, Malipiero U, et al. TGF-beta induces the expression of the FLICE-inhibitory protein and inhibits Fas-mediated apoptosis of microglia[J].Eur J Immunol, 2000,30:3680-3688.
[29] Holmstr(?)m TH, Schmitz I, S(?)derstr(?)m TS, et al. MAPK/ERK signaling in activated T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly[J], EMBO J, 2000,19:5418-5428.
[30] Panka DJ, Mano T, Suhara T, et al. Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells[J]. J Biol Chem, 2001,276:6893-6896.
[31] Kreuz SD, Siegmund, et al. NF-kB inducers upregulate c-FLIP, a cycloheximide-sensitive inhibitor of death receptor signaling[J]. Mol Cell Biol, 2001,21:3964-3973.
[32] Micheau O, Lens S, Gaide O, et al. NF-kB signals induce the expression of c-FLIP[J]. Mol Cell Biol, 2001,21:5299-5305.
[33] Ricci MS, Jin Z, Dews M, et al. Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity [J]. Mol Cell Biol,2004,24(19):8541-8555.
[34] Zhou XD, Yu JP, Chen HX, et al. Expression of cellular FLICE-inhibitory protein and its association with p53 mutation in colon cancer[J]. World J Gastroenterol, 2005,11 (16):2482-2485.
[35] Wang Q, Wang X, Zhou Y, et al. PKCdelta-mediated regulation of FLIP expression in human colon cancer cells[J]. Int J Cancer, 2006,118(2):326-334.
[36] Toh Y, Pencil SD, Nicolson GL. A novel candidate metastasis asscoiate gene,MTA1, diferentially expressed in highly metastatic mammary adenocarcinoma cell lines[J]. J Biol Chem, 1994,269(37):22958-22963.
[37] Okamoto K, Fujisawa J, Reth M, et al. Human T-cell leukemia virus T type-Ⅰ oncoprotein tax inhibits Fas-mediated apoptosis by inducing cellular FLIP through activation of NF kappaB[J]. Genes Cells, 2006,11(2):177-191.
[38] Horie R, Watanabe M, Okamura T, et al. DHMEQ, a new NF-kappaB inhibitor, induces apoptosis and enhances fludarabine effects on chronic lymphocytic leukemia cells[J]. Leukemia, 2006,20(5):800-806.
[39] Pencil SD, Toh Y, Nicolson GL. Candidate metastasis-associated gene of the rat 13762 NF mammary adenoearcinoma[J]. Breast Cancer Res Treat, 1993,25(2): 165-174.
[40] Toh Y, Kuninaka S, Endo K, et al. Molecular analysis of a candidate metastasis associated gene, MTA1: possible interaction with histone descetylase[J]. J Exp Clin Cancer Res, 2000,19(1): 105-111.
[41] Moil T, Doi R, Toyoda E, et al. Regulation of the resistance to TRAIL-induced apoptosis as a new strategy for pancreatic cancer[J]. Surgery, 2005,138(1):71-77.
[42] Bullani RR, Huard B, Viard-Leveugle I, et al. Selective expression of FLIP in malignant melanocytic skin lesions[J]. J Invest Dermatol, 2001,117(2):360-364.
[43] Penelope K, Athirm G, Cerassimos V, et al. c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations [J]. Urology, 2004,63(1):1198-1204.
[44] Xiao C, Yang BF, Asadi N, et al. Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cell[J]. J Biol Chem,2002,277(28):25020-25025.
[45] Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma[J]. Clin Sci (Lond), 2004,106(4): 3 97-405.
[46] Takeda K, Hayakawa Y, Smyth MJ, et al. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in NK cell mediated and IFN-gamma-dependent suppression of subcutaneous tumor growth[J]. Cell Immunol, 2001,214(2):194-200.
[47] French LE, Tschopp J. Defective death receptor signaling as a cause of tumor immune escapes [J]. Semin Cancer Biol, 2002,12(1):51-55.
[48] Thomas PK, Kallenbom A, Wickenhauser C, et al. Constitutive expression of c-FLIP in Hodgkin and Reed-Stemberg cells[J]. Am J Pathol, 2002,160(4): 1521-1528.
[49] Song JH, Song DK, Herlyn M, et al. Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells[J]. Clin Cancer Res, 2003,9(11):4255-4266.
[50] Abedi, Mohammad R, Qiu Q, et al. Possible role of FLICE-like inhibitory protein(FLIP) in chemoresistant ovarian cancer cells in vitron[J]. Oncogene,2004,23(42): 6997-7004.
[51]. Iwase M, Watanabe H, Kondo G, et al.Enhanced susceptibiligy of oral squamous cell carcinoma cell lines to FAS-mediated apoptosis by cisplatin and 5fluorouracil[J].Int J Cancer,2003,106(4):619-625.
[52] Eggert A, Grotzer MA, Zuzak TJ, et al. Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression[J]. Cancer Res,2001,61(4):1314-1319
[53] Fulda S, Kufer MU, Meyer E, et al. Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer[J]. Oncogene, 2001,20(41):5865-5877.
[54] Harada K, Toyooka S, Shivapurkar N, et al. Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines[J]. Cancer Res, 2002,62(20):5897-5901.
[55] Takita J, Yaog HW, Chen YY, et al. Allelic imbalance on chomosome 2q and alterations of the caspase-8 gene in neuroblastoma[J]. Oncogene, 2001,20(32):4424-4432.
[56] Poulaki V, Mitsiades CS, McMullan C, et al. Human retinoblastoma cells are resistant to apoptosis induced by death receptors: role of caspase-8 gene silencing[J].Invest Ophthalmol Vis Sci, 2005,46(1):358-366.
[57] Yoshikawa H, Nakajima Y, Tasakal K. Enhanced expression of fas-associated death domain-like IL-1-convening enzyme(FLICE)-inhibitory protein induces resistance to fas-mediated apoptosis in activated mast cells[J]. J Immunol, 2000,165(11): 6262-6269.
[58] Wu W, Rinaldi L, Former KA, et al. Cellular FLIP long form-transgenic mice manifest a Th2 cytokine bias and enhanced allergic airway inflammation[J]. J Immunol, 2004,172(8):4724-4732.
[59] Wei Y, Chen K, Sharp GC, et al. FLIP and FasI expression by inflammatory cells vs thyrocytes can be predictive of chronic inflammation or resolution of autoimmune ethyroiditis[J]. Clin Immunol, 2003,108(3):221-233.
[60] Tepper CG, Seldin MF. Modulation of caspase-8 and FLICE-inhibitory protein expression as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt's lymphoma[J]. Blood, 1999,94(5): 1772-1737.
[61] Catrina AI, Ulfgren AK, Lindblad S, et al. Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium[J]. Ann Rheum Dis,2002,61(10): 934-936.
[62] Perlman H, Liu H, Georganas C, et al. Differential expression pattern of the antiapoptotic proteins, Bcl-2 and FLIP, in experimental arthritis[J]. Arthritis Rheum,2001,44(12):2899-2908.
[63] Kobayashi T, Okamoto K, Kobata T, et al. Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor alpha and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules[J]. J Immunol, 2000,43(5):1106-1114.
[64] Engidawork E, Gulesserian T, Yoo BC, et al. Alteration of caspases and apoptosis related proteins in brains of patients with Alzheimer s disease[J]. Biochem Biophys Res Commun, 2001,281(1):84-93.
[65] Zhao M, Cribbs DH, Anderson AJ, et al. The induction of TNFalpha death domain signaling pathway in Alzheimer s disease brain[J]. Neurochem Res,2003,28(2): 307-318.
[66] Kishimoto H, Sprent J. A defect in central tolerance in NOD mice[J]. Nat immunol, 2001,2(11): 1025-1031.
[67] Arreaza G, Salojin K, Yang W, et al. Deficient activation and resistance to activation-induced apoptosis of CD8 T cells is associated with defective peripheral tolerance in nonobese diabetic mice[J]. Clin Immunol, 2003,107(2): 103-115.
[68] Maedler K, Fontana A, Ris F, et al. FLIP switches Fas-mediated glucose signaling in human pancreatic beta cells from apoptosis to cell replication[J]. Proc Natl Acad Sci USA, 2002,99(12):8236-8241.
[69] Steenbergen C, Afshari CA, Petranka JG, et al .Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure[J]. Am J Physiol Heart Circ Physiol, 2003,284(1): 268-276.
[70] The Swiss HIV Cohort Study. Persistent apoptosis in HIV-1-infected individuals receiving potent antiretroviral therapy is associated with poor recovery of CD4 T lymphocytes[J]. J Acquir Immune Defic Syndr, 2004,36(2):671-685.
[71] Imanishi T, McBride J, Ho Q, et al. Expression of cellular FLICE-inhibitory protein in human coronary arteries and in a rat vascular injury model[J]. Am J Pathol, 2000,156(1):125-137.
[72] Yeh WC, Itie A, Elia AJ, et al. Requirement for caspase (c-FLIP) in regulation of death receptor induced apoptosis and embryonic development[J]. Immunity, 2000,12(6): 633-642.
[73] Davidson SM, Stephanou A, Latehman DS. FLIP protects eardiomyocytes from apoptosis induced by simulated ischemia/reoxygenation, as demonstrated by short hair pin-induced(shRNA)silencing of FLIP Mrna[J]. J Mol Cell Cardiol, 2003,35(11):1359-1364.
[2] Thomas LR, Henson A, Reed JC, et al. Direct binding of Fas-associated death domain (FADD) to the tumor necrosis factor-related apoptosis-inducing ligand receptor DR5 is regulated by the death effector domain of FADD[J]. J Biol Chem,2004,279(31):32780-32785.
[3] Irmler M, Thome M, Hahne M, et al. Inhibition of death receptor signals by celluar FLIP[J]. Nature, 1997,388:190-195.
[4] Goltsev YV, Kovalenko AV, Arnold E, et al. CASH, a novel caspase homologue with death effector domains[J]. J Biol Chem, 1997,272(32): 19641 -19645.
[5] Heijink DM, Kleibeuker JH, Jalving M, et al. Independent induction of caspase-8 and cFLIP expression during colorectal carcinogenesis in sporadic and HNPCC adenomas and carcinomas [J]. Cell Oncol, 2007,29(5):409-419.
[6] Korkolopoulou P, Saetta AA, Levidou G, et al. c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications[J].Histopathology, 2007,51:150-156.
[7] Valnet-Rabier MB, Challier B, Thiebault S, et al. c-Flip protein expression in Burkitt's lymphomas is associated with a poor clinical outcome[J]. Brit J Haemtol,2005,128:767-773.
[8] Uherova P, Olson S, Thompson MA, et al. Expression of c-FLIP in classic and nodular lymphocyte-predominant Hodgkin lymphoma[J]. Appl Immunohistochem Mol Morphol, 2004,12:105-110.
[9] Yeh WC, Itie A, Elia AJ, et al. Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development[ J]. Immunity, 2000,12 (6):633-642.
[10] Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma[J]. Clin Sci (Lond), 2004,106(4):397-405.
[11]Koomstra JJ,Kleibeuker JH,van Geelen CM,et al.Expression of TRAIL (TNF-related apoptosis-inducing ligand) and its receptors in normal colonic mucosa,adenomas,and carcinomas[J].J Pathol,2003,200(3):327-335.
[12]Thomas RK,Kallenborn A,Wickenhauser C,et al.Constitutive expression of c-FLIP in Hodgkin and Reed-Sternberg cells[J].Am J Pathol,2002,160(4):1521-1528.
[13]Chen F,Guo J,Zhang Y,et al.Knockdown of c-FLIP enhanced AD5-10 anti-death receptor 5 monoclonal antibody-induced apoptosis in human lung cancer cells[J].Cancer Sci,2009,100(5):940-947.
[14]杨奎忠，杜庆聪，项继舜，黄凤昌．食管癌组织中c-FLIP蛋白的表达及意义[J]．山东医药，2007，47(3)：77-78．
[15]van Houdt IS,Muris JJ,Hesselink AT,et al.Expression of c-FLIP is primarily detected in diffuse large B-cell lymphoma and Hodgkin's lymphoma and correlates with lack of caspase 8 activation[J].Histopathology,2007,51(6):778-784.
[16]Valnet-Rabier MB,Challier B,Thiebault S,et al.c-Flip protein expression in Burkitt's lymphomas is associated with a poor clinical outcome[J].Br J Haematol,2005,128(6):767-773.
[17]陈红霞，刘元姣，黎辉，等．cFLIP基因在子宫内膜腺癌组织中的表达[J]．癌症，2004，23(3)：254-258．
[18]Kim SY,Song SY,Kim MS,et al.Immunohistochemical analysis of Fas and FLIP in prostate cancers[J].APMIS,2009,117(1):28-33.
[19]Hammond ME,Fitzgibbons PL,Compton CC,et al.College of American Pathologists Conference ⅩⅩⅩⅤ:solid tumor prognostic factors-which,how and so what? Summary Document and recommendations for implementation.Cancer Committeee and Conference Participants[J].Arch Pathol Lab Med,2000,124:958-965.
[20]Crowe JP,Gordon NH,Hubay CA,et al.Estrogen receptor determination and long term survival of patients with carcinoma of the breast[J].Surg Gynecol Obstet,1991,173:273-278.
[21]MacFarlane M.TRAIL-induced signaling and apoptosis[J].Toxicol Lett,2003,139(2-3):89-97.
[22]Romaswamy M,Efimova EV,Martinez O,et al.IG20 (MADD splice variant-5), a proapoptotic protein, interacts with DR4/DR5 and enhanced TRAIL-induced apoptosis by increasing recruitment of FADD and caspase-8 to the DISC[J].Oncogene, 2004,23(36):6083-6094.
[23] Sanlioglu AD, Korcum AF, Pestereli E, et al. TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma[J]. Int J Radiation Oncology Biol Phys, 2007,69(3):716-723.
[24] Day TW, Huang S, Safa AR. c-FLIP knockdown induces ligand-independent DR5-, FADD-, caspase-8-, and caspase-9-dependent apoptosis in breast cancer cells.Biochem Pharmacol, 2008,76(12): 1694-704.
[25] Kazhdan I, Marciniak RA. Death receptor 4 (DR4) efficiently kills breast cancer cells irrespective of their sensitivity to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) [J]. Cancer Gene Ther, 2004,11(10):691-698.
[26] Guseva NV, Rokhlin OW, Taghiyev AF, et al. Unique resistance of breast carcinoma cell line T47D to TRAIL but not anti-Fas is linked to p43cFLIP(L) [J].Breast Cancer Res Treat, 2008,107(3):349-357.
[27] Hyer ML, Croxton R, Krajewska M, et al. Synthetic triterpenoids cooperate with tumor necrosis factor-related apoptosis-inducing ligand to induce apoptosis of breast cancer cells[J]. Cancer Res, 2005,65(11):4799-4808.
[28] Kim IK, Jung YK, Noh DY, et al. Functional screening of genes suppressing TRAIL-induced apoptosis: distinct inhibitory activities of Bcl-XL and Bcl-2[J]. Br J Cancer, 2003,88(6):910-917.
[29] Kim S, Lee TJ, Leem J,et al. Sanguinarine-induced apoptosis: generation of ROS, down-regulation of Bcl-2, c-FLIP, and synergy with TRAIL[J]. J Cell Biochem, 2008,104(3):895-907.
[30] Park C, Moon DO, Ryu CH, et al. Beta-sitosterol sensitizes MDA-MB-231 cells to TRAIL-induced apoptosis[J]. Acta Pharmacol Sin, 2008,29(3):341-348.
[31] Zhang Y, Yoshida T, Zhang B. TRAIL induces endocytosis of its death receptors in MDA-MB-231 breast cancer cells[J]. Cancer Biol Ther, 2008,8(10).[Epub ahead of print]
[32] Yu JW, Shi Y. FLIP and the death effector domain family[J]. Oncogene, 2008,27:6216-6227.
[33] Zang FL, Sun BC, Zhao XL, et al. Critical role for c-FLIP_L on Fas resistance in colon carcinoma cell line HT-29[J]. Cell Biol Inter, 2008,32:329-336.
[34] Zhang N, Hopkins K, He YW. The long isoform of cellular FLIP is essencial for T lymphocyte proliferation through an NF-κB-independent pathway[J]. J Immuno,2008,180:5506-5511.
[35] Chang DW, Xing Z, Pan Y, et al. c-FLIP_L is a dual function regulator for caspase-8 activation and CD95-mediated apoptosis[J]. EMBO, 2002,21(14):3704-3714.
[36] Krueger A, Baumann S, Krammer PH, et al. FLICE-inhibitory proteins: regulations of death receptor-mediated apoptosis[J]. Mol Cell Bio, 2001, 21(24):8247-8254.
[37] Kurbanov BM, Fecker LF, Geilen CC, et al. Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-kappaB but is related to downregulation of initiator caspases and DR4[J]. Oncogene, 2007,26(23):3364-3377.
[38] Boatright KM, Deis C, Denault JB, et al. Activation of caspases-8 and -10 by FLIP(L)[J]. Biochem J, 2004;382(Pt 2):651-657.
[39] Micheau O, Thome M, Schneider P, et al. The long form of FLIP is an activator of caspase-8 at the Fas death-inducing signaling complex[J]. J Biol Chem, 2002;277 (47): 45162-45171.
[40] Dohrman A, Kataoka T, Cuenin S, et al. Cellular FLIP (long form) regulates CD8~+ T cell activation through caspase-8-dependent NF-kappa B activation[J]. J Immunol, 2005,174(9):5270-5278.
[41] Mitsiades CS, Poulaki V, Fanourakis G, et al. Fas signaling in thyroid carcinomas is diverted from apoptosis to proliferation. Clin Cancer Res[J]. 2006, 12(12):3705-3712.
[42] Lee TJ, Lee JT, Park JW, et al. Acquired TRAIL resistance in human breast cancer cells are caused by the sustained cFLIP(L) and XIAP protein levels and ERK activation[J]. Biochem Biophys Res Commun, 2006,351(4):1024-1030.
[43] Thai le M, Labrinidis A, Hay S, et al. Apo21/Tumor necrosis factor-related apoptosis-inducing ligand prevents breast cancer-induced bone destruction in a mouse model. Cancer Res, 2006,66(10):5363-70.
[1]Krammer PH.CD95's deadly mission in the immune system[J].Nature,2000,407(6805):789-795.Review.
[2]Thome M,Schneider P,Hofmann K,et al.Viral FLICE inhibitory proteins (FLIP) prevent apoptosis induced by death receptors[J].Nature,1997,386(6624):517-521.
[3]Irmler M,Thome M,Hahne M,et al.Inhibition of death receptor signals by celluar FLIP[J].Nature,1997,388(6624):190-195.
[4]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 the CD95 (Fas,APO-1) receptor complex[J].Cell Death Differ,1998,5(4):271-288.
[5]Schmitz IS,Kirchhoff PH,Krammer PH.Regulation of death receptor-mediated apoptosis pathways[J].Int J Biochem Cell Biol,2000,32:1123-1136.
[6] Oehme I, Neumann F, Bosser S, et al. Transgenic overexpression of the caspase-8 inhibitor FLIP(short) leads to impaired T cell proliferation and an increased memory T cell pool after staphylococcal enterotoxin B injection[J]. Eur J Immunol, 2005,35(4):1240-1249.
[7] Boatright KM, Deis C, Denault JB, et al. Activation of caspases-8 and -10 by FLIP(L) [J]. Biochem J, 2004,382(Pt 2):651-657.
[8] Dohrman A, Russell JQ, Cuenin S, et al. Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase-8 [J]. J Immunol, 2005,175(l):311-318.
[9] Goltsev YV, Kovalenko E, Arnold, et al. CASH, a novel caspase homologue with death effector domain[J]. J Biol Chem, 1997,272:19641-19644.
[10] Shu HB, Halpin DR, Goeddel DV. Casper is a FADD- and caspase-related inducer of apoptosis [J]. Immunity, 1997,6:751 -763.
[11] Srinivasula SM, Ahmad M, Ottilie S, et al. FLAME-1, a novel FADD-like anti-apoptotic molecule that regulates Fas/TNFR1-induced apoptosis[J]. J Biol Chem,1997,272:18542-18545.
[12] Han DK, Chaudhary PM, Wright ME, et al. MRIT, a novel death-effector domain-containing protein, interacts with caspases and BclX_L and initiates cell death[J]. Proc Natl Acad Sci USA, 1997,94:11333-11338.
[13] Hu S, Vincenz C, Ni J, et al. I-FLICE, a novel inhibitor of tumor necrosis factor receptor-1- and CD-95-induced apoptosis[J]. J Biol Chem, 1997,272:17255-17257.
[14] Kataoka T, Budd RC, Holler N, et al. The caspase 8 inhibitor FLIP promotes activation of NF-kB and Erk signaling pathways[J]. Curr Biol, 2000,10:640-648.
[15] Scaffidi C, Schmitz I, Krammer PH, et al. The role of c-FLIP in modulation of CD95-induced apoptosis[J]. J Biol Chem, 1999,274:1514-1548.
[16] Krueger A, Schmitz I, Baumann S, et al. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase 8 activation at the CD95 death- inducing signaling complex[J]. J Biol Chem, 2001,276:20633-20640.
[17] Earnshaw WC, Martins LM. Mammalian caspases: structure, activation, substrates, and functuions during apoptosis[J]. Annu Rev Biochem, 1999,68:383-424.
[18] Muzio M, Stockwell BR, Stennicke HR, et al. An induced proximity model for caspase 8 activation[J]. J Biol Chem, 1998,273:2926-2930.
[19] Zeiss CJ. The apoptosis-necrosis continuum: insights from genetically altered mice[J]. Vet Pathol, 2003,40(5):481-495. Review.
[20] 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-kappaB signaling pathway[J]. Mol Cell Biol, 2004,24(7):2627-2636.
[21] Kim YY, Park BJ, Seo GJ, et al. Long form of cellular FLICE-inhibitory protein interacts with Daxx and prevents Fas-induced JNK activation[J]. Biochem Biophys Res Commun, 2003,312(2):426-433.
[22] Wang X, Wang Y, Zhang J, et al. FLIP protects against hypoxia/reoxygenation- induced endothelial cell apoptosis by inhibiting Bax activation [J]. Mol Cell Biol,2005, 25(11):4742-4751.
[23] Susanne M, Lens A. The caspase 8 inhibitor c-FLIPL modulates T-cell receptor-induced proliferation but not activation-induced cell death oflymphocytes[J]. Mol Cell Biol, 2002,22(15):5419-5433.
[24] Grambihler A, Higuchi H, Bronk SF, et al. cFLIP-L inhibits p38 MAPK activation: an additional anti-apoptotic mechanism in bile acid-mediated apoptosis[J].J Biol Chem, 2003,278(29):26831-26837.
[25] Nakagiri S, Murakami A, Takada S, et al. Viral FLIP enhances Wnt signaling downstream of stabilized beta-catenin, leading to control of cell growth[J]. Mol Cell Biol, 2005,25(21):9249-9258.
[26] Cudrici C, Niculescu F, Jensen T, et al. C5b-9 terminal complex protects oligodendrocytes from apoptotic cell death by inhibiting caspase-8 processing and upregulating FLIP[J]. J Immunol, 2006,176(5):3173-3180.
[27] Yeh JH, Hsu SC, Han SH, et al. Mitogen-activated protein kinase kinase antagonized fas-associated death domain protein-mediated apoptosis by induced FLICE-inhibitory protein expression[J]. J Exp Med, 1998,188:1795-1802.
[28] Schlapbach R, Spanaus KS, Malipiero U, et al. TGF-beta induces the expression of the FLICE-inhibitory protein and inhibits Fas-mediated apoptosis of microglia[J].Eur J Immunol, 2000,30:3680-3688.
[29] Holmstr(?)m TH, Schmitz I, S(?)derstr(?)m TS, et al. MAPK/ERK signaling in activated T cells inhibits CD95/Fas-mediated apoptosis downstream of DISC assembly[J], EMBO J, 2000,19:5418-5428.
[30] Panka DJ, Mano T, Suhara T, et al. Phosphatidylinositol 3-kinase/Akt activity regulates c-FLIP expression in tumor cells[J]. J Biol Chem, 2001,276:6893-6896.
[31] Kreuz SD, Siegmund, et al. NF-kB inducers upregulate c-FLIP, a cycloheximide-sensitive inhibitor of death receptor signaling[J]. Mol Cell Biol, 2001,21:3964-3973.
[32] Micheau O, Lens S, Gaide O, et al. NF-kB signals induce the expression of c-FLIP[J]. Mol Cell Biol, 2001,21:5299-5305.
[33] Ricci MS, Jin Z, Dews M, et al. Direct repression of FLIP expression by c-myc is a major determinant of TRAIL sensitivity [J]. Mol Cell Biol,2004,24(19):8541-8555.
[34] Zhou XD, Yu JP, Chen HX, et al. Expression of cellular FLICE-inhibitory protein and its association with p53 mutation in colon cancer[J]. World J Gastroenterol, 2005,11 (16):2482-2485.
[35] Wang Q, Wang X, Zhou Y, et al. PKCdelta-mediated regulation of FLIP expression in human colon cancer cells[J]. Int J Cancer, 2006,118(2):326-334.
[36] Toh Y, Pencil SD, Nicolson GL. A novel candidate metastasis asscoiate gene,MTA1, diferentially expressed in highly metastatic mammary adenocarcinoma cell lines[J]. J Biol Chem, 1994,269(37):22958-22963.
[37] Okamoto K, Fujisawa J, Reth M, et al. Human T-cell leukemia virus T type-Ⅰ oncoprotein tax inhibits Fas-mediated apoptosis by inducing cellular FLIP through activation of NF kappaB[J]. Genes Cells, 2006,11(2):177-191.
[38] Horie R, Watanabe M, Okamura T, et al. DHMEQ, a new NF-kappaB inhibitor, induces apoptosis and enhances fludarabine effects on chronic lymphocytic leukemia cells[J]. Leukemia, 2006,20(5):800-806.
[39] Pencil SD, Toh Y, Nicolson GL. Candidate metastasis-associated gene of the rat 13762 NF mammary adenoearcinoma[J]. Breast Cancer Res Treat, 1993,25(2): 165-174.
[40] Toh Y, Kuninaka S, Endo K, et al. Molecular analysis of a candidate metastasis associated gene, MTA1: possible interaction with histone descetylase[J]. J Exp Clin Cancer Res, 2000,19(1): 105-111.
[41] Moil T, Doi R, Toyoda E, et al. Regulation of the resistance to TRAIL-induced apoptosis as a new strategy for pancreatic cancer[J]. Surgery, 2005,138(1):71-77.
[42] Bullani RR, Huard B, Viard-Leveugle I, et al. Selective expression of FLIP in malignant melanocytic skin lesions[J]. J Invest Dermatol, 2001,117(2):360-364.
[43] Penelope K, Athirm G, Cerassimos V, et al. c-FLIP expression in bladder urothelial carcinomas: its role in resistance to Fas-mediated apoptosis and clinicopathologic correlations [J]. Urology, 2004,63(1):1198-1204.
[44] Xiao C, Yang BF, Asadi N, et al. Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cell[J]. J Biol Chem,2002,277(28):25020-25025.
[45] Zhou XD, Yu JP, Liu J, et al. Overexpression of cellular FLICE-inhibitory protein (FLIP) in gastric adenocarcinoma[J]. Clin Sci (Lond), 2004,106(4): 3 97-405.
[46] Takeda K, Hayakawa Y, Smyth MJ, et al. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in NK cell mediated and IFN-gamma-dependent suppression of subcutaneous tumor growth[J]. Cell Immunol, 2001,214(2):194-200.
[47] French LE, Tschopp J. Defective death receptor signaling as a cause of tumor immune escapes [J]. Semin Cancer Biol, 2002,12(1):51-55.
[48] Thomas PK, Kallenbom A, Wickenhauser C, et al. Constitutive expression of c-FLIP in Hodgkin and Reed-Stemberg cells[J]. Am J Pathol, 2002,160(4): 1521-1528.
[49] Song JH, Song DK, Herlyn M, et al. Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1 beta-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells[J]. Clin Cancer Res, 2003,9(11):4255-4266.
[50] Abedi, Mohammad R, Qiu Q, et al. Possible role of FLICE-like inhibitory protein(FLIP) in chemoresistant ovarian cancer cells in vitron[J]. Oncogene,2004,23(42): 6997-7004.
[51]. Iwase M, Watanabe H, Kondo G, et al.Enhanced susceptibiligy of oral squamous cell carcinoma cell lines to FAS-mediated apoptosis by cisplatin and 5fluorouracil[J].Int J Cancer,2003,106(4):619-625.
[52] Eggert A, Grotzer MA, Zuzak TJ, et al. Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression[J]. Cancer Res,2001,61(4):1314-1319
[53] Fulda S, Kufer MU, Meyer E, et al. Sensitization for death receptor- or drug-induced apoptosis by re-expression of caspase-8 through demethylation or gene transfer[J]. Oncogene, 2001,20(41):5865-5877.
[54] Harada K, Toyooka S, Shivapurkar N, et al. Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines[J]. Cancer Res, 2002,62(20):5897-5901.
[55] Takita J, Yaog HW, Chen YY, et al. Allelic imbalance on chomosome 2q and alterations of the caspase-8 gene in neuroblastoma[J]. Oncogene, 2001,20(32):4424-4432.
[56] Poulaki V, Mitsiades CS, McMullan C, et al. Human retinoblastoma cells are resistant to apoptosis induced by death receptors: role of caspase-8 gene silencing[J].Invest Ophthalmol Vis Sci, 2005,46(1):358-366.
[57] Yoshikawa H, Nakajima Y, Tasakal K. Enhanced expression of fas-associated death domain-like IL-1-convening enzyme(FLICE)-inhibitory protein induces resistance to fas-mediated apoptosis in activated mast cells[J]. J Immunol, 2000,165(11): 6262-6269.
[58] Wu W, Rinaldi L, Former KA, et al. Cellular FLIP long form-transgenic mice manifest a Th2 cytokine bias and enhanced allergic airway inflammation[J]. J Immunol, 2004,172(8):4724-4732.
[59] Wei Y, Chen K, Sharp GC, et al. FLIP and FasI expression by inflammatory cells vs thyrocytes can be predictive of chronic inflammation or resolution of autoimmune ethyroiditis[J]. Clin Immunol, 2003,108(3):221-233.
[60] Tepper CG, Seldin MF. Modulation of caspase-8 and FLICE-inhibitory protein expression as a potential mechanism of Epstein-Barr virus tumorigenesis in Burkitt's lymphoma[J]. Blood, 1999,94(5): 1772-1737.
[61] Catrina AI, Ulfgren AK, Lindblad S, et al. Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium[J]. Ann Rheum Dis,2002,61(10): 934-936.
[62] Perlman H, Liu H, Georganas C, et al. Differential expression pattern of the antiapoptotic proteins, Bcl-2 and FLIP, in experimental arthritis[J]. Arthritis Rheum,2001,44(12):2899-2908.
[63] Kobayashi T, Okamoto K, Kobata T, et al. Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor alpha and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules[J]. J Immunol, 2000,43(5):1106-1114.
[64] Engidawork E, Gulesserian T, Yoo BC, et al. Alteration of caspases and apoptosis related proteins in brains of patients with Alzheimer s disease[J]. Biochem Biophys Res Commun, 2001,281(1):84-93.
[65] Zhao M, Cribbs DH, Anderson AJ, et al. The induction of TNFalpha death domain signaling pathway in Alzheimer s disease brain[J]. Neurochem Res,2003,28(2): 307-318.
[66] Kishimoto H, Sprent J. A defect in central tolerance in NOD mice[J]. Nat immunol, 2001,2(11): 1025-1031.
[67] Arreaza G, Salojin K, Yang W, et al. Deficient activation and resistance to activation-induced apoptosis of CD8 T cells is associated with defective peripheral tolerance in nonobese diabetic mice[J]. Clin Immunol, 2003,107(2): 103-115.
[68] Maedler K, Fontana A, Ris F, et al. FLIP switches Fas-mediated glucose signaling in human pancreatic beta cells from apoptosis to cell replication[J]. Proc Natl Acad Sci USA, 2002,99(12):8236-8241.
[69] Steenbergen C, Afshari CA, Petranka JG, et al .Alterations in apoptotic signaling in human idiopathic cardiomyopathic hearts in failure[J]. Am J Physiol Heart Circ Physiol, 2003,284(1): 268-276.
[70] The Swiss HIV Cohort Study. Persistent apoptosis in HIV-1-infected individuals receiving potent antiretroviral therapy is associated with poor recovery of CD4 T lymphocytes[J]. J Acquir Immune Defic Syndr, 2004,36(2):671-685.
[71] Imanishi T, McBride J, Ho Q, et al. Expression of cellular FLICE-inhibitory protein in human coronary arteries and in a rat vascular injury model[J]. Am J Pathol, 2000,156(1):125-137.
[72] Yeh WC, Itie A, Elia AJ, et al. Requirement for caspase (c-FLIP) in regulation of death receptor induced apoptosis and embryonic development[J]. Immunity, 2000,12(6): 633-642.
[73] Davidson SM, Stephanou A, Latehman DS. FLIP protects eardiomyocytes from apoptosis induced by simulated ischemia/reoxygenation, as demonstrated by short hair pin-induced(shRNA)silencing of FLIP Mrna[J]. J Mol Cell Cardiol, 2003,35(11):1359-1364.