Kanazawa Takashi, Kono Takuo, Watanabe Masahiko, Matsushima-Hibiya Yuko, Nakano Tsuyoshi, Koyama Kotaro, Tanaka Noriaki, Sugimura Takashi, Wakabayashi Keiji
Cancer Prevention Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, 104-0045, Tokyo, Japan.
Biochem Biophys Res Commun. 2002 Aug 9;296(1):20-5. doi: 10.1016/s0006-291x(02)00815-x.
Pierisin-1, a 98-kDa protein that induces apoptosis in mammalian cell lines, is capable of being incorporated into cells where it ADP-ribosylates guanine residues in DNA. To investigate the apoptotic pathway induced by this unique protein, the bcl-2 gene was transfected into HeLa cells. Cy2-fluorescent pierisin-1 was incorporated into the resultant cells expressing Bcl-2 protein and ADP-ribosylated dG was detected to almost the same extent as in parent cells. However, bcl-2-transfected HeLa cells did not display apoptotic morphological changes, PARP cleavage, and DNA fragmentation, indicating acquisition of resistance. In parent HeLa cells, activation of caspase-9 and release of cytochrome c were observed after 8h treatment with 0.5ng/ml pierisin-1. Caspase substrate assays revealed further cleavage of Ac-DEVD-pNA, Ac-VDVAD-pNA, and Ac-VEID-pNA, suggesting activation of caspase-2, -3, and -6 in pierisin-1-treated HeLa cells. The caspase-3 inhibitor, Ac-DEVD-CHO, was also found to inhibit apoptosis. In contrast, this caspase activation was not observed in bcl-2-transfected HeLa cells. Our results thus indicate that pierisin-1-induced apoptosis is mediated primarily via a mitochondrial pathway involving Bcl-2 and caspases.
Pierisin-1是一种能在哺乳动物细胞系中诱导凋亡的98 kDa蛋白质，它能够进入细胞并在其中对DNA中的鸟嘌呤残基进行ADP核糖基化修饰。为了研究这种独特蛋白质所诱导的凋亡途径，将bcl-2基因转染到HeLa细胞中。Cy2荧光标记的Pierisin-1进入表达Bcl-2蛋白的所得细胞中，并且检测到ADP核糖基化的dG的程度与亲代细胞几乎相同。然而，转染了bcl-2的HeLa细胞未表现出凋亡形态变化、PARP裂解和DNA片段化，表明获得了抗性。在亲代HeLa细胞中，用0.5 ng/ml Pierisin-1处理8小时后观察到caspase-9的激活和细胞色素c的释放。Caspase底物分析显示Ac-DEVD-pNA、Ac-VDVAD-pNA和Ac-VEID-pNA进一步裂解，表明在Pierisin-1处理的HeLa细胞中caspase-2、-3和-6被激活。还发现caspase-3抑制剂Ac-DEVD-CHO可抑制凋亡。相比之下，在转染了bcl-2的HeLa细胞中未观察到这种caspase激活。因此，我们的结果表明，Pierisin-1诱导的凋亡主要通过涉及Bcl-2和caspases的线粒体途径介导。
