CASPASES MEDIATE 6-HYDROXYDOPAMINE-INDUCED APOPTOSIS BUT NOT NECROSISIN PC12 CELLS

The neurotoxin 6-hydroxydopamine (6-OHDA) induces apoptosis in the rat phaeochromocytoma cell line PC12. 6-OHDA-induced apoptosis is morphol ogically indistinguishable from serum deprivation-induced apoptosis. E xposure of PC12 cells to a low concentration of 6-OHDA (25 mu M) resul ts in apoptosis, whereas an increased concentration (50 mu M) results in a mixture of apoptosis and necrosis. We investigated the involvemen t of caspases in the apoptotic death of PC12 cells induced by 6-OHDA, using a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluor omethyl ketone (zVAD-fmk), and compared this with serum deprivation-in duced apoptosis, which is known to involve caspases. We show that zVAD -fmk (100 mu M) completely prevented the apoptotic morphology of chrom atin condensation induced by exposure to either 6-OHDA (25 and 50 mu M ) Or serum deprivation. Furthermore, cell lysates from 6-OHDA-treated cultures showed cleavage of a fluorogenic substrate for caspase-3-like proteases (caspase-2, 3, and 7), acetyl-Asp-Glu-Val-Asp-aminomethylco umarin, and this was inhibited by zVAD-fmk, However, although zVAD-fmk restored total cell viability to serum-deprived cells or cells expose d to 25 mu M 6-OHDA, the inhibitor did not restore viability to cells exposed to 50 mu M 6-OHDA, These data show the involvement of a caspas e-9-like protease in 6-OHDA-induced apoptosis and that caspase inhibit ion is sufficient to rescue PC12 cells from the apoptotic but not the necrotic component of 6-OHDA neurotoxicity.