Send to

Choose Destination
J Biol Chem. 2008 Aug 1;283(31):21676-85. doi: 10.1074/jbc.M802889200. Epub 2008 Jun 3.

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

Author information

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


Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.

[Indexed for MEDLINE]
Free full text

Supplemental Content

Full text links

Icon for HighWire
Loading ...
Support Center