Generation of an oxidative stress precedes caspase activation during 7β‐hydroxycholesterol‐induced apoptosis in U937 cells

The oxysterol 7beta‐hydroxycholesterol (7β‐OH) has been shown to induce apoptosis in a number of cell lines. Though not fully elucidated, the mechanism through which this oxysterol induces cell death is thought to involve the generation of an oxidative stress leading to perturbation of the mitochondrion and release of cytochrome c into the cytosol. Cytochrome c together with Apaf‐1 causes activation of the initiator caspase, caspase‐9, which in turn activates caspase‐3 ultimately leading to the degradation of poly(ADP‐ribose) polymerase (PARP). The objective of the present study was to investigate the signalling pathway in 7β‐OH‐induced apoptosis in U937 cells, a human monocytic blood cell line known to undergo apoptosis upon treatment with 7β‐OH, over a time course of 48 h. Apoptosis was evident after 24 h incubation. Glutathione levels were decreased after 6 h and this was coupled with an increase in SOD activity. Through western blot analysis we examined expression of caspase‐3, ‐8, and ‐9 and cleavage of the caspase‐3 substrate PARP. The sequence proceeded with activation of caspase‐9 after 9 h, caspase‐3 at the 12 h timepoint, and cleavage of PARP after 24 h treatment with 7β‐OH. Caspase‐8 did not appear to play a major role in this particular apoptotic pathway. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:50–59, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20007