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J. Biol. Chem., Vol. 283, Issue 31, 21676-21685, August 1, 2008

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Real Time Analysis of Tumor Necrosis Factor-related Apoptosis-inducing Ligand/Cycloheximide-induced Caspase Activities during Apoptosis Initiation^*

Christian T. Hellwig, Barbara F. Kohler, Anna-Kaisa Lehtivarjo, Heiko Dussmann, Michael J. Courtney, Jochen H. M. Prehn, and Markus Rehm¹

From the Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, RCSI York House, York Street, Dublin 2, Ireland and the Department of Neurobiology, University of Kuopio, Kuopio 70210, Finland

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.

Received for publication, April 15, 2008 , and in revised form, June 2, 2008.

^* This work was supported by Academy of Finland Grant 203520 (to M. J. C.) and Science Foundation Ireland Grants 03/RP1/B344 (to J. H. M. P.) and 05/RFP/BIM056 and 07/RFP/BICF601 (to M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed. Tel.: 353-1-402-8563; Fax: 353-1-402-2447; E-mail: mrehm{at}rcsi.ie.

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