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May 30, 2000The Journal of Experimental MedicineOpen Access

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

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Authors

AKAkira KomoriyaOncoImmune (United States)BPBeverly Z. PackardOncoImmune (United States)MBMartin J. BrownDublin City University

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Implication

In vitro study reveals stimulus-dependent caspase activation sequences in mouse thymocytes, indicating that downstream apoptotic cascades depend on the trigger.

Key Points

  • Investigate the real-time activation order of downstream caspases in intact apoptotic cells exposed to different apoptotic stimuli using cell-permeable fluorogenic substrates.
  • Synthesized cell-permeable fluorogenic substrates containing optimal recognition motifs for caspases 1, 3/7, 6, 8, and 9.
  • Treated mouse thymocytes in vitro with dexamethasone or anti-Fas antibody and evaluated temporal caspase activity using flow cytometry and confocal microscopy.
  • Assessed the inhibitory effect of the antiapoptotic agent Z-VAD[OMe]-FMK against already activated intracellular caspases.
  • Dexamethasone induced caspase activation in the sequential order of LEHDase, WEHDase, VEIDase, IETDase, and DEVDase, with confocal microscopy confirming VEIDase preceded DEVDase in every apoptotic cell.
  • Anti-Fas stimulation initiated activation with IETDase, and DEVDase activation preceded VEIDase.
  • Z-VAD[OMe]-FMK produced no significant reductions in caspase activities after their intracellular activation.

Cite This Study

Komoriya et al. (2000) studied this question.

synapsesocial.com/papers/6a6f5bb15d37378ac1dc6cdahttps://doi.org/10.1084/jem.191.11.1819
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