Pu Yongmei, Luo Kathy Q, Chang Donald C
Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Biochem Biophys Res Commun. 2002 Dec 20;299(5):762-9. doi: 10.1016/s0006-291x(02)02722-5.
We showed previously that a cytosolic Ca(2+) signal is involved in regulating UV-induced apoptosis in HeLa cells. In this study, we found evidence that this Ca(2+) signal occurs upstream of the release of cytochrome c from mitochondria. First, when we abolished Ca(2+) increases by injecting BAPTA or heparin into UV-treated HeLa cells, cytochrome c release was either blocked or severely delayed. Second, using a living cell imaging technique, we observed a series of transient Ca(2+) increases (typically lasting about 40-60s) in many apoptotic cells induced by either UV- or TNFalpha-treatment. Third, using GFP-tagged cytochrome c, we found that the Ca(2+) spikes appear in a time window before cytochrome c was released. Finally, by fixing the TNFalpha-treated cell at the time when it started to display Ca(2+) spikes, we examined the distribution of its endogenous cytochrome c using immunostaining. We found that cytochrome c was not yet released from mitochondria. These findings suggest the existence of certain apoptotic pathways, in which an early Ca(2+) signal is activated upstream of cytochrome c release.
我们之前表明，胞质Ca(2+)信号参与调控HeLa细胞中紫外线诱导的细胞凋亡。在本研究中，我们发现证据表明该Ca(2+)信号在线粒体细胞色素c释放的上游发生。首先，当我们通过向紫外线处理的HeLa细胞中注射BAPTA或肝素消除[Ca(2+)]i增加时，细胞色素c的释放被阻断或严重延迟。其次，使用活细胞成像技术，我们在紫外线或TNFα处理诱导的许多凋亡细胞中观察到一系列短暂的[Ca(2+)]i增加（通常持续约40 - 60秒）。第三，使用绿色荧光蛋白标记的细胞色素c，我们发现Ca(2+)峰值出现在细胞色素c释放之前的一个时间窗口内。最后，通过在TNFα处理的细胞开始显示Ca(2+)峰值时固定细胞，我们使用免疫染色检查其内源性细胞色素c的分布。我们发现细胞色素c尚未从线粒体中释放。这些发现表明存在某些凋亡途径，其中早期Ca(2+)信号在细胞色素c释放的上游被激活。
