Yamakawa H, Ito Y, Naganawa T, Banno Y, Nakashima S, Yoshimura S, Sawada M, Nishimura Y, Nozawa Y, Sakai N
Department of Neurosurgery, Gifu University School of Medicine, Japan.
Neurol Res. 2000 Sep;22(6):556-64. doi: 10.1080/01616412.2000.11740718.
The treatment of PC12 cells with H2O2 (100-500 microM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DNA fragmentation observed as DNA ladder. H2O2-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PC12 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H2O2-treated PC12 cells, and that Bcl-2 inhibits H2O2-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9.
用H2O2（100 - 500微摩尔）处理PC12细胞会导致典型的凋亡变化，包括细胞核的碎片化和凝聚，以及作为DNA梯状条带观察到的DNA片段化。通过特定荧光底物肽的切割以及原半胱天冬酶-3和聚（ADP - 核糖）聚合酶的加工评估，H2O2诱导的凋亡与半胱天冬酶-3的激活相关。然而，未观察到通常位于半胱天冬酶-3上游的神经酰胺的形成。对半胱天冬酶-3相对特异的抑制肽z - DEVD - FMK和非选择性半胱天冬酶抑制剂z - VAD - FMK抑制了半胱天冬酶-3的激活和凋亡细胞死亡。然而，对半胱天冬酶-1相对特异的抑制剂Ac - YVKD和对半胱天冬酶-8/6的Ac - IETD并不影响凋亡细胞死亡的发生。作为半胱天冬酶-3的上游激活，通过蛋白质印迹法观察到细胞色素c释放并随后原半胱天冬酶-9的加工，尽管未观察到细胞内神经酰胺的形成。另一方面，在过表达Bcl - 2的PC12细胞中，凋亡细胞的数量显著减少，并且半胱天冬酶-9和-3的激活均被阻止。这些结果表明，细胞色素c和半胱天冬酶-9在H2O2处理的PC12细胞中启动执行半胱天冬酶-3的激活，并且Bcl - 2抑制H2O2诱导的细胞色素c从线粒体的释放以及随后原半胱天冬酶-9的蛋白水解加工。
