Characterization of caspase processing and activation in HL-60 cell cytosol under cell-free conditions - Nucleotide requirement and inhibitor profile

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblott ing revealed that incubation of HL-60 cytosol at 30 degrees C in the presen ce of cytochrome c and ATP (or dATP) resulted in activation of procaspases- 3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitro and in situ differed in charge and abundance. ATP and d ATP levels in intact HL-60 cells were higher than required for caspase acti vation in vitro and did not change before caspase activation in situ. Repla cement of ATP with the poorly hydrolyzable analogs 5'-adenylyl methylenedip hosphate, 5'-adenylyl imidodiphosphate, or 5'-adenylyl-O-(3-thiotriphosphat e) slowed caspase activation in vitro, suggesting that ATP hydrolysis is re quired. Caspase activation in vitro was insensitive to phosphatase and kina se inhibitors (okadaic acid, staurosporine, and genistein) but was inhibite d by Zn2+, aurintricarboxylic acid, and various protease inhibitors, includ ing 3,4-dichloroisocoumarin, N-alpha-p-tosyl-L-phenylalanine chloromethyl k etone, N-alpha-p-tosyl-L-Iysine chloromethyl ketone, and N-(N-alpha-benzylo xycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope a ntiserum confirmed that these agents inhibited caspase-9 activation. Collec tively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptos is by other means.