Li F, Srinivasan A, Wang Y, Armstrong R C, Tomaselli K J, Fritz L C
IDUN Pharmaceuticals, Inc., La Jolla, California 92037, USA.
J Biol Chem. 1997 Nov 28;272(48):30299-305. doi: 10.1074/jbc.272.48.30299.
Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.
Bcl-xL是Bcl-2家族的一个抗凋亡成员，可抑制多种细胞类型中的程序性细胞死亡。最近的报道表明，细胞色素c在细胞凋亡过程中从线粒体释放，并且提示这种释放可能是促凋亡半胱天冬酶激活及随后细胞死亡的关键步骤。此外，已经证明Bcl-2可以阻止细胞色素c从触发凋亡的细胞的线粒体中释放。这导致了这样一种假说，即Bcl-2家族成员的抗凋亡作用具体是由于它们具有阻止细胞色素c释放从而防止随后细胞色素c依赖性半胱天冬酶激活的能力。在本报告中，我们使用显微注射技术来研究细胞色素c释放、凋亡诱导以及完整细胞中Bcl-xL活性之间的关系。我们证明，将细胞色素c显微注射到人肾293细胞的细胞质中会导致剂量依赖性的凋亡诱导。相反，缺乏可检测水平的半胱天冬酶3（CPP32）的MCF7乳腺癌细胞（稳定转染以表达Fas抗原CD95，记为MCF7F）对细胞色素c的显微注射完全耐药。然而，用编码前体半胱天冬酶3而非其他前体半胱天冬酶的表达质粒转染MCF7F细胞可恢复对细胞色素c的敏感性。尽管MCF7F细胞对细胞色素c显微注射不敏感，但它们在响应肿瘤坏死因子或抗Fas加放线菌酮时会以半胱天冬酶依赖性方式迅速发生凋亡，并且这些死亡受到Bcl-xL表达的强烈抑制。此外，细胞色素c的显微注射并不能克服Bcl-xL的这些抗凋亡作用。我们的结果支持这样一种观点，即细胞色素c释放到细胞质中可促进表达前体半胱天冬酶3的细胞中的凋亡过程，但细胞色素c释放并不足以在所有细胞中诱导死亡。重要的是，Bcl-xL在细胞色素c不敏感的MCF7F细胞中抑制细胞死亡的能力不能仅仅归因于对细胞色素c从线粒体释放的抑制。
