25-hydroxycholesterol activates a cytochrome c release-mediated caspase cascade

We have previously shown that 25-hydroxy-cholesterol (25-OHC) treated CHO-K 1 cells could be used as a model to investigate the signaling pathway of ap optosis induced by oxidized LDL in vascular cells. In the present study, we examine the execution phase of the apoptotic pathway in CHO-K1 cell death induced by 25-OHC. Oxysterol-induced apoptosis in CHO-K1 was accompanied by caspase activation and was preceded by mitochondrial cytochrome c release. The addition of a competitive caspase-3 inhibitor, Ac-DEVD-CHO, prevented 25-OHC-induced apoptotic cell death. Furthermore, immunoblot analysis showe d that 25-OHC treatment induced the degradation of poly(ADP-ribose) polymer ase (PARP)-a substrate for caspase 3 and a key enzyme involved in genome su rveillance and DNA repair. Thus, we could demonstrate in CHO-K1 cells that 25-OHC activates the apoptotic machinery through induction of the release o f cytochrome c from mitochodria into the cytosol and activation of a typica l caspase cascade. (C) 2000 Academic Press.