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Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase

Christian T Hellwig1, Barbara F Kohler, Anna-Kaisa Lehtivarjo

  • 1Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, RCSI York House, York Street, Dublin 2, Ireland.

The Journal of Biological Chemistry
|June 5, 2008
PubMed

Insights

Apoptosis initiation involves a threshold mechanism where caspase-8 activation, triggered by TRAIL receptor pathway signaling, leads to cell death only after a specific level of substrate cleavage, regardless of initial drug dose.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Effector caspase activation in apoptosis is often an all-or-none response.
  • Understanding the signaling dynamics of apoptosis initiation is crucial for cell death research.

Purpose of the Study:

  • Investigate signaling dynamics during apoptosis initiation via the TRAIL receptor pathway.
  • Determine how drug exposure variability translates into cell death execution pathways.
  • Elucidate the kinetic control mechanisms of apoptosis.

Main Methods:

  • Utilized fluorescence resonance energy transfer (FRET) imaging in living HeLa cells.
  • Monitored caspase-8 activation and activity dynamics.
  • Assessed mitochondrial outer membrane permeabilization.
  • Examined the role of Bid in apoptosis signaling.

Main Results:

  • Demonstrated dose-dependent caspase-8 activation and activity following TRAIL/cycloheximide addition.
  • Caspase-8 activation occurred 45-600 minutes post-treatment, with activity persisting for 15-60 minutes.
  • Mitochondrial outer membrane permeabilization was consistently induced upon 10% FRET substrate cleavage.
  • Bid depletion prolonged caspase-8 activity without immediate cell death.

Conclusions:

  • Apoptosis initiation via the TRAIL pathway involves a threshold mechanism controlling execution.
  • A specific level of substrate cleavage by caspase-8 is required to trigger mitochondrial outer membrane permeabilization.
  • Intracellular signaling kinetics play a critical role in the decision to execute cell death.

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