¹Department of Molecular Biology & Immunology, University of North Texas Health Science Center, Fort Worth, Texas, 76107, USA

²Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, Texas, 76107, USA

Correspondence to: A Basu, Department of Molecular Biology & Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, USA. Tel: 817-735-2487; Fax: 817-735-2118; E-mail: abasu@hsc.unt.edu

Edited by S Martin

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 PKC. Rottlerin, a selective inhibitor of novel PKC, blocked activation of caspases, proteolytic activation of PKC and cell death induced by cDDP. In contrast, Gö 6976, an inhibitor of conventional PKC and I, did not prevent cDDP-induced caspase activation and cDDP cytotoxicity. In HeLa cells, PKC 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 PKC. Rottlerin attenuated late but not early release of Cytochrome c by cDDP. It, however, inhibited activation of caspases and proteolytic cleavage of PKC 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 PKC inhibitor acts at an early stage of the cDDP-induced cell death pathway that precedes caspase activation. Cell Death and Differentiation (2001) 8, 899-908

cisplatin; protein kinase C; caspases; apoptosis; Cytochrome c

Ac-DEVD-AFC, Acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarine; Apaf-1, apoptotic protease activating factors; BIM, bisindolylmaleimide; cDDP, cis-diamminedichloroplatinum(II); CF, catalytic fragment; Cox-II, cytochrome oxidase subunit II; HM, heavy membrane; MBP, myelin basic protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PDBu, phorbol 12,13-dibutyrate; PKC, protein kinase C; aPKC, atypical PKC; cPKC, conventional PKC; nPKC, novel PKC; PS, phosphatidylserine; Rot, rottlerin; TNF, tumor necrosis factor-; TPA, 12-O-tetradecanoylphorbol 13-acetate