Basu A, Woolard M D, Johnson C L
Department of Molecular Biology & Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Cell Death Differ. 2001 Sep;8(9):899-908. doi: 10.1038/sj.cdd.4400885.
We have previously shown that the protein kinase C (PKC) signal transduction pathway regulates cell death by the DNA damaging agent cis-diamminedichloroplatinum(II) (cDDP). In the present study we have investigated how PKC influences the sequence of events that are triggered by cDDP-induced DNA damage. cDDP caused activation of caspases-8, -9, -3, -7 and cleavage of PKCdelta. Rottlerin, a selective inhibitor of novel PKCdelta, blocked activation of caspases, proteolytic activation of PKCdelta and cell death induced by cDDP. In contrast, Gö 6976, an inhibitor of conventional PKCalpha and betaI, did not prevent cDDP-induced caspase activation and cDDP cytotoxicity. In HeLa cells, PKCdelta was distributed both in the cytosol and heavy membrane (HM) fraction containing mitochondria. While caspase-8 was primarily cytosolic, a small amount of caspases-9, -7 and -3 could be detected in the HM fraction. cDDP caused a time-dependent increase in Cytochrome c release from the mitochondria and processing of both cytosolic and membrane-associated caspases, as well as proteolytic cleavage of PKCdelta. Rottlerin attenuated late but not early release of Cytochrome c by cDDP. It, however, inhibited activation of caspases and proteolytic cleavage of PKCdelta in both cytosolic and HM fractions. The antiapoptotic effect of rottlerin was evident when it was added together with or following cDDP addition but not when added after cDDP was removed from the medium. Thus, the PKCdelta inhibitor acts at an early stage of the cDDP-induced cell death pathway that precedes caspase activation.
我们之前已经表明，蛋白激酶C（PKC）信号转导通路可调节由DNA损伤剂顺二氯二氨铂（II）（cDDP）诱导的细胞死亡。在本研究中，我们探究了PKC如何影响由cDDP诱导的DNA损伤所引发的一系列事件。cDDP可导致半胱天冬酶-8、-9、-3、-7的激活以及PKCδ的裂解。Rottlerin是新型PKCδ的选择性抑制剂，它可阻断cDDP诱导的半胱天冬酶激活、PKCδ的蛋白水解激活以及细胞死亡。相比之下，常规PKCα和βI的抑制剂Gö 6976并不能阻止cDDP诱导的半胱天冬酶激活和cDDP的细胞毒性。在HeLa细胞中，PKCδ分布于细胞质和含有线粒体的重膜（HM）部分。虽然半胱天冬酶-8主要位于细胞质中，但在HM部分可检测到少量的半胱天冬酶-9、-7和-3。cDDP导致细胞色素c从线粒体中释放以及细胞质和膜相关半胱天冬酶的加工呈时间依赖性增加，同时也导致PKCδ的蛋白水解裂解。Rottlerin可减弱cDDP诱导的细胞色素c的晚期而非早期释放。然而，它可抑制细胞质和HM部分中半胱天冬酶的激活以及PKCδ的蛋白水解裂解。当Rottlerin与cDDP一起添加或在cDDP添加后添加时，其抗凋亡作用明显，但在从培养基中去除cDDP后添加则无此作用。因此，PKCδ抑制剂作用于cDDP诱导的细胞死亡途径中半胱天冬酶激活之前的早期阶段。
