Wednesday, May 23, 2007

Involvement of protein kinase C- in DNA damage-induced apoptosis

Caspases are essential for the execution of cell death by apoptotic stimuli.1,2,3,4 The pathway of cell death varies depending on the cell type as well as the apoptotic stimuli. It is generally believed that binding of Fas ligand or tumor necrosis factor- (TNF) to their receptors causes activation of the initiator caspase-8 followed by the activation of a caspase cascade to execute cell death.2,5 In contrast, DNA damaging agents are known to induce release of mitochondrial Cytochrome c, which facilitates the interaction of apoptotic protease activating factor (Apaf-1) with procaspase-9 to initiate the activation of downstream effector caspases, such as caspases-3 or -7 to cause cell death.3 Both receptor-mediated and anticancer drug-induced apoptosis may, however, involve more than one pathway and there may be cross-talk between these two pathways.6,7,8

cis-Diamminedichloroplatinum(II) (cDDP or cisplatin) is one of the most important anticancer agents used for the treatment of solid tumors.9 Although the antitumor activity of cDDP is believed to be due to its interaction with chromosomal DNA, only a small fraction of cDDP actually interacts with DNA and inhibition of DNA replication cannot solely account for its biological activity.10 The efficacy of chemotherapeutic drugs not only depends on their ability to induce DNA damage but also on the cell's ability to detect and respond to DNA damage.11 cDDP, like other chemotherapeutic drugs, causes activation of caspases although the sequence of events that follow cDDP-induced DNA damage and lead to apoptosis remains to be unraveled.

We and others have shown that the PKC signal transduction pathway regulates cell death by cDDP.12,13,14,15,16,17 PKC is a family of 11 isozymes that are classified as the conventional PKCs (, I, II and ), novel PKCs (, , , and ), atypical PKCs ( and / ) and novel/atypical PKC.18 PKC is a substrate for caspase-3 and the catalytic fragment of PKC has been directly associated with apoptotic cell death.19,20 We have, however, demonstrated that downregulation of PKC that decreased the abundance of PKC catalytic fragment was associated with increased cellular sensitivity to cDDP.17 These results raise the possibility that PKC acts upstream of caspases to regulate cell death by cDDP. It is not known which PKC isozyme regulates activation of caspases and which step(s) of the cisplatin-induced cell death pathway is regulated by PKC.

Mitochondria play a pivotal role in the decision making process of a cell's life and death.21 It is believed that once Cytochrome c is released from mitochondria, cells are committed to die.22 An inability to induce release of Cytochrome c from mitochondria has been associated with cellular resistance to anticancer agents, including cDDP.23 In the present study, we have investigated how PKC regulates release of Cytochrome c and activation of caspases that emanate from mitochondria. Our results show that in HeLa cells, PKC was localized not only in the cytosol but also in the mitochondrial fraction and cDDP induced processing of both cytosolic and membrane-associated PKC. Rottlerin, a specific inhibitor of PKC, blocked cDDP-induced activation of caspases and proteolytic cleavage of PKC in both cytosolic and HM fractions but inhibited only late but not early release of Cytochrome c. Furthermore, inhibition of nPKC, but not of cPKCs, protected cells against cDDP-induced cell death. Taken together, these results demonstrate that PKC influences cDDP-induced cell death by acting at an early step of the mitochondrial cell death pathway that precedes activation of caspases.

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