Gorman A M, Bonfoco E, Zhivotovsky B, Orrenius S, Ceccatelli S
Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Eur J Neurosci. 1999 Mar;11(3):1067-72. doi: 10.1046/j.1460-9568.1999.00512.x.
The microtubule-disrupting agent colchicine is known to be neurotoxic toward certain neuronal populations including cerebellar granule cells (CGCs). In this study we investigated the involvement of cytochrome c release and caspase-3 activation during colchicine-induced CGC apoptosis. Treatment of rat CGCs with 1 micrometer colchicine (for up to 24 h) caused high molecular weight DNA fragmentation and nuclear condensation. An involvement of group II caspases (which includes caspase-3) was demonstrated by the proteolytic degradation of poly(ADP-ribose) polymerase (PARP) after 18 h exposure to colchicine. Colchicine induced a time-dependent increase in Ac-Asp-Glu-Val-Asp-alpha-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity in CGCs, which was blocked with a specific, peptide-based, aldehyde inhibitor of group II caspases, i. e. DEVD-CHO. We also observed a time-dependent proteolysis of caspase-3 as judged by the appearance of p17 which is one of the subunits of active caspase-3. Activation of caspase-3 during colchicine-induced apoptosis may be mediated by cytochrome c since there was a close correlation between the time courses of cytochrome c release from the mitochondria and of caspase-3 activation. Furthermore, colchicine-induced apoptosis, as assessed by propidium iodide visualization of the nuclei, could be blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl ketone.
已知破坏微管的药物秋水仙碱对包括小脑颗粒细胞（CGCs）在内的某些神经元群体具有神经毒性。在本研究中，我们调查了秋水仙碱诱导CGC凋亡过程中细胞色素c释放和半胱天冬酶-3激活的情况。用1微摩尔秋水仙碱处理大鼠CGCs（长达24小时）会导致高分子量DNA片段化和核浓缩。在暴露于秋水仙碱18小时后，聚（ADP-核糖）聚合酶（PARP）的蛋白水解降解证明了II组半胱天冬酶（包括半胱天冬酶-3）的参与。秋水仙碱诱导CGCs中Ac-Asp-Glu-Val-Asp-α-（4-甲基-香豆素-7-酰胺）（DEVD-MCA）切割活性随时间增加，这被II组半胱天冬酶的特异性肽基醛抑制剂即DEVD-CHO所阻断。我们还观察到半胱天冬酶-3随时间的蛋白水解，这可通过活性半胱天冬酶-3的亚基之一p17的出现来判断。秋水仙碱诱导凋亡过程中半胱天冬酶-3的激活可能由细胞色素c介导，因为线粒体中细胞色素c释放的时间进程与半胱天冬酶-3激活的时间进程之间存在密切相关性。此外，通过碘化丙啶对细胞核的可视化评估，秋水仙碱诱导的凋亡可被半胱天冬酶抑制剂苄氧羰基-Val-Ala-Asp（O-甲基）氟甲基酮阻断。
