Assessment of caspase activities in intact apoptotic thymocytes using cell-permeable fluorogenic caspase substrates

To detect caspase activities in intact apoptotic cells at the single cell, level, cell-permeable fluorogenic caspase substrates were synthesized incor porating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitr o treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. De xamethasone induced the following order of appearance of caspase activities as judged by now cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDas e. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamine d using confocal microscopy. The VEIDase activity appeared before DEVDase i n every apoptotic cell treated with dexamethasone. Tn contrast, anti-Fas st imulation altered this sequence: IETDase was the first measurable caspase a ctivity and DEVDase preceded VEIDase. In an attempt to determine the intrac ellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl- aspartyl(beta-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examin ed its ability to inhibit previously activated intracellular caspases. Howe ver, no significant reductions of these activities were observed. These flu orogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase ac tivation is dependent on the apoptotic stimulus.