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

: Hellwig CT, Kohler BF, Lehtivarjo AK, Dussmann H, Courtney MJ, Prehn JHM, Rehm M

Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

: 2008

: Journal of Biological Chemistry

: JOURNAL OF BIOLOGICAL CHEMISTRY

: J BIOL CHEM

: 283

: 31

: 21676

: 21685

: 10

: 0021-9258

: 1083-351X

DOI: https://doi.org/10.1074/jbc.M802889200

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.