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J. Exp. Med. 191 (11): 1819-1828

Copyright © 2000 by the Rockefeller University Press.

Original Article

Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates

Akira Komoriyaa, Beverly Z. Packarda, Martin J. Brownb, Ming-Lei Wub, , and Pierre A. Henkartb

a OncoImmunin, Incorporated, Gaithersburg, Maryland 20877
b Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892
Experimental Immunology Branch, National Cancer Institute, Bldg. 10, Rm. 4B36, National Institutes of Health, Bethesda, MD 20892-1360.301-496-0887301-435-6404

ph8j{at}nih.gov

Abstract: To detect caspase activities in intact apoptotic cells at the single cell level, cell-permeable fluorogenic caspase substrates were synthesized incorporating the optimal peptide recognition motifs for caspases 1, 3/7, 6, 8, and 9. Caspase activities were then assessed at various times after in vitro treatment of mouse thymocytes with dexamethasone or anti-Fas antibody. Dexamethasone induced the following order of appearance of caspase activities as judged by flow cytometry: LEHDase, WEHDase, VEIDase, IETDase, and DEVDase. Since the relative order of caspases 3 (DEVDase) and 6 (VEIDase) in the cascade has been controversial, this caspase activation order was reexamined using confocal microscopy. The VEIDase activity appeared before DEVDase in every apoptotic cell treated with dexamethasone. In contrast, anti-Fas stimulation altered this sequence: IETDase was the first measurable caspase activity and DEVDase preceded VEIDase. In an attempt to determine the intracellular target of the potent antiapoptotic agent carbobenzoxy-valyl-alanyl-aspartyl(β-methyl ester)-fluoromethyl ketone (Z-VAD[OMe]-FMK), we examined its ability to inhibit previously activated intracellular caspases. However, no significant reductions of these activities were observed. These fluorogenic caspase substrates allow direct observation of the caspase cascade in intact apoptotic cells, showing that the order of downstream caspase activation is dependent on the apoptotic stimulus.

Key Words: apoptosis • caspase • PhiPhiLuxTM • thymocyte • lymphocyte

Abbreviations used in this paper: apaf-1, apoptotic protein activating factor 1; DTT, dithiothreitol; FADD, Fas-associated death domain protein; ICE, IL-1β–converting enzyme; PI, propidium iodide.

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