Lim Maria L R, Chen Baohong, Beart Philip M, Nagley Phillip
Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Victoria, Australia.
Exp Cell Res. 2006 Apr 15;312(7):1174-84. doi: 10.1016/j.yexcr.2006.01.026.
Redistribution of cytochrome c and Smac/DIABLO from mitochondria occurs during apoptosis, although the relative timing of their release is not well characterized. Double immunocytochemistry was utilized here to study quantitatively the patterns of release of cytochrome c and Smac/DIABLO from mitochondria in single cells. Human osteosarcoma cells and murine embryonic cortical neurons were analyzed during apoptosis induced by staurosporine. In osteosarcoma cells treated with staurosporine for 24 h, a substantial proportion of cells (36%) released cytochrome c from the mitochondria before Smac/DIABLO. In contrast, these proteins were released mostly concordantly in neurons; only a minority of cells (< or = 15%) released cytochrome c without Smac/DIABLO (or vice versa) from mitochondria. Patterns of release in either cell type were unaltered by addition of the caspase inhibitor, zVAD-fmk. The double immunocytochemistry procedure facilitated clear definition of the temporal release of cytochrome c and Smac/DIABLO from mitochondria in intact apoptotic cells, enabling us to demonstrate for the first time that their mutual redistribution during apoptosis varies between different cell types.
细胞凋亡过程中会发生细胞色素c和Smac/DIABLO从线粒体的重新分布，尽管它们释放的相对时间还没有得到很好的界定。在此利用双重免疫细胞化学法定量研究单细胞中线粒体释放细胞色素c和Smac/DIABLO的模式。在用星形孢菌素诱导凋亡的过程中，对人骨肉瘤细胞和小鼠胚胎皮质神经元进行了分析。在用星形孢菌素处理24小时的骨肉瘤细胞中，相当一部分细胞（36%）在Smac/DIABLO之前从线粒体释放细胞色素c。相比之下，这些蛋白质在神经元中大多是同时释放的；只有少数细胞（≤15%）从线粒体中释放细胞色素c而不释放Smac/DIABLO（或反之亦然）。添加半胱天冬酶抑制剂zVAD-fmk不会改变任何一种细胞类型中的释放模式。双重免疫细胞化学方法有助于清晰界定完整凋亡细胞中线粒体释放细胞色素c和Smac/DIABLO的时间，使我们首次证明它们在细胞凋亡过程中的相互重新分布在不同细胞类型之间存在差异。
