Endocrine Abstracts (2002) 3 P239

Dexamethasone blocks mitochondrial targeting-induced apoptosis

CJ Newton1, D Bilko1, IP Adams2, J Ran1, V Green2 & SL Atkin2

1Molecular Pharmacology, Post Graduate Medical School, University of Hull, Hull, UK; 2Endocrinology and Diabetes, Post Graduate Medical School, University of Hull, Hull, UK.

The release of cytochrome c from mitochondria is often the trigger for the nuclear and cytoplasmic changes that characterise apoptotic cell death. Previously, we have shown that vascular endothelial cells of the line, EA.hy 926, undergo apoptosis in response to TNFalpha, camptothecin and oxidants and that this is blocked by the glucocorticoid, dexamethasone (Dex). As a tool to determine where in the apoptotic pathway Dex acts to block the apoptotic process, we have developed a means to target mitochondria and release cytochrome c. For this we have used the cationic compound, hexadecyl triphenyl phosphonium bromide (HDTPB). By electron microscopy, we have observed mitochondrial swelling within 2h of the addition of HDTPB (5microM) and by Western blotting studies, we show that this is associated with the release of cytochrome c. Morphological characteristics of apoptosis are observed some 8-10h later. For cells pre-exposed to 1microM Dex, the release of cytochrome c from mitochondria is still induced by HDTPB, but morphological signs of apoptosis are no longer apparent. After several days (3-5) treatment with HDTPB, most cells are dead. With Dex and the cation, many cells show large vacuoles but they still fail to display the morphological characteristics of apoptosis. As these observations indicate that Dex blocks the apoptotic pathway down -stream of changes in mitochondrial function, we have considered the role of inhibitory apoptotic proteins (IAPs). The IAP Survivin, is expressed in endothelial cells. Therefore, using Western blotting, we have tested the effect of Dex. No change in Survivin expression was noted in response to Dex, but a small increase in expression was noted in response to HDTPB.

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