Anel A, Gamen S, Alava M A, Schmitt-Verhulst A M, Piñeiro A, Naval J
Departamento de Bioquimica y Biologia Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Int Immunol. 1996 Jul;8(7):1173-83. doi: 10.1093/intimm/8.7.1173.
The implication of oxidative damage and/or intact mitochondrial function in physiological Fas-based cytotoxicity has been tested using the cytolytic hybridoma d11S and the CD8(+) CTL clone KB5.C20, previously stimulated to express Fas ligand (FasL) on their surface, as effectors and U937 or U937-rho0 cells (depleted of mitochondrial DNA) as targets. Immobilized anti-Fas mAb, which induced death of U937 cells, inhibited the growth of U937-rho0 cells but without inducing cell death. By contrast, FasL-expressing effectors readily killed both targets, with induction of DNA fragmentation, in 20 h assays. These results demonstrate the lack of involvement of mitochondrial-derived free radicals and/or intact mitochondrial function in physiological Fas-based cytotoxicity. Supplementation of Fas-sensitive cells (Jurkat, U937, L1210Fas) with a polyunsaturated fatty acid, which induces cell death through the generation of lipid free radicals, resulted in the potentiation of Fas-based cytotoxicity. This potentiating effect, but not Fas-based cytotoxicity itself, was eliminated by the physiological antioxidant vitamin E. On the other hand, the IL-1beta-converting enzyme (ICE)-like protease tetrapeptide inhibitor Ac-YVAD-cmk partially inhibited Fas-based cytotoxicity, while the specific inhibitor of CPP32/Yama Ac-DEVD-CHO was a much more effective inhibitor of Fas-induced apoptosis. It was concluded that Fas-induced cytotoxicity was clearly dependent on ICE-like protease activation, and especially on that of CPP32 in Fas-sensitive cells, including mitochondrial DNA-depleted ones.
利用溶细胞杂交瘤d11S和CD8(+) CTL克隆KB5.C20（此前已被刺激在其表面表达Fas配体(FasL)）作为效应细胞，以及U937或U937-rho0细胞（线粒体DNA缺失）作为靶细胞，测试了氧化损伤和/或完整的线粒体功能在基于Fas的生理性细胞毒性中的作用。固定化抗Fas单克隆抗体可诱导U937细胞死亡，抑制U937-rho0细胞生长，但不诱导细胞死亡。相比之下，在20小时的实验中，表达FasL的效应细胞能轻易杀死这两种靶细胞，并诱导DNA片段化。这些结果表明，线粒体衍生的自由基和/或完整的线粒体功能不参与基于Fas的生理性细胞毒性。用一种多不饱和脂肪酸补充Fas敏感细胞（Jurkat、U937、L1210Fas），该脂肪酸通过产生脂质自由基诱导细胞死亡，导致基于Fas的细胞毒性增强。这种增强作用，但不是基于Fas的细胞毒性本身，被生理性抗氧化剂维生素E消除。另一方面，IL-1β转化酶(ICE)样蛋白酶四肽抑制剂Ac-YVAD-cmk部分抑制基于Fas的细胞毒性，而CPP32/Yama的特异性抑制剂Ac-DEVD-CHO是Fas诱导凋亡更有效的抑制剂。得出的结论是，Fas诱导的细胞毒性明显依赖于ICE样蛋白酶的激活，尤其是Fas敏感细胞（包括线粒体DNA缺失的细胞）中CPP32的激活。
