Sci. STKE, 20 June 2000
Apoptosis In Vivo Measurement of Caspase Activity
At least nine separate caspases have been implicated in the activation of apoptosis. Although methods exist to identify which caspases are required in specific apoptotic pathways, these methods largely involve in vitro analysis of cell lysates. Also, whereas some caspases have been assigned positions within the hierarchy of apoptotic signaling, the assignment of a few caspases remains less certain. Komoriya et al. have developed a method for assessing caspase activation in intact thymocytes, with cell permeable caspase substrates. The authors incubated thymocytes with fluorogenic peptides that contained specific caspase cleavage sequences, and determined which caspases were activated under Fas- or dexamethasone (Dex)-mediated conditions. Dex-treated cells quickly activated caspases in a sharply ordered cascade, whereas Fas-directed apoptosis led to slower caspase activation and a less sharply defined hierarchy of activation. Also, thymocytes treated with Dex activated caspases 3 before caspase 6, in contrast to reports elsewhere. However, this result was confirmed by confocal microscopy showing that caspase 3 activation occurred before and concomitantly with caspase 6 activation; no caspase 6 activation was observed before caspase 3 activation, under these conditions. The authors also showed that the generic caspase inhibitor ZVAD-FMK blocked Dex-directed apoptosis, but only if administered before Dex. This suggests that ZVAD-FMK may inhibit an upstream activator of the caspase cascade. Thus, a new method now exists for intracellular assessment of the apoptosis cascade without perturbing cell integrity.
Komoriya, A., Packard, B.Z., Brown, M.J., Wu, M.-L., and Henkart, P.A. (2000) Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates. J. Exp. Med. 191: 1819-1828. [Abstract] [Full Text]
Citation: In Vivo Measurement of Caspase Activity. Sci. STKE 2000, tw1 (2000).
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