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

Peter W. Mesner, Keith C. Bible, Luis M. Martins, Timothy J. Kottke, Srinivasa M. Srinivasula, Phyllis A. Svingen, Tamie J. Chilcote, Guriq S. Basi, Jay S. Tung, Stan Krajewski, John C. Reed, Emad S. Alnemri, William C. Earnshaw, Scott H. Kaufmann

Research output: Contribution to journalArticlepeer-review

65 Scopus citations


The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30 °C in the presence 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 dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5'-adenylyl methylenediphosphate, 5'-adenylyl imidodiphosphate, or 5'-adenylyl-O-(3- thiotriphosphate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn2+, aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin, N(α)-p-tosyl-L- phenylalanine chloromethyl ketone, N(α)-p-tosyl-L-lysine chloromethyl ketone, and N-(N(α)benzyloxycarbonylphenylalanyl) alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.

Original languageEnglish (US)
Pages (from-to)22635-22645
Number of pages11
JournalJournal of Biological Chemistry
Issue number32
StatePublished - Aug 6 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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