Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation.
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.
Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, RCSI York House, York Street, Dublin 2, Ireland.
Christian T
CT
Hellwig
Barbara F
BF
Kohler
Anna-Kaisa
AK
Lehtivarjo
Heiko
H
Dussmann
Michael J
MJ
Courtney
Jochen H M
JH
Prehn
Markus
M
Rehm
D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism*; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology*; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D051023:BH3 Interacting Domain Death Agonist Protein; D011500:Protein Synthesis Inhibitors; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D053181:Caspase 8; D020169:Caspases