Abstract
We examined the temporal and causal relationship between Smac/DIABLO release, cytochrome c (cyt-c) release, and caspase activation at the single cell level during apoptosis. Cells treated with the broad-spectrum caspase inhibitor z-VAD-fmk, caspase-3 (Casp-3)-deficient MCF-7 cells, as well as Bax-deficient DU-145 cells released Smac/DIABLO and cyt-c in response to proapoptotic agents. Real-time confocal imaging of MCF-7 cells stably expressing Smac/DIABLO-yellow fluorescent protein (YFP) revealed that the average duration of Smac/DIABLO-YFP release was greater than that of cyt-c-green fluorescent protein (GFP). However, there was no significant difference in the time to the onset of release, and both cyt-c-GFP and Smac/DIABLO-YFP release coincided with mitochondrial membrane potential depolarization. We also observed no significant differences in the Smac/DIABLO-YFP release kinetics when z-VAD-fmk-sensitive caspases were inhibited or Casp-3 was reintroduced. Simultaneous measurement of DEVDase activation and Smac/DIABLO-YFP release demonstrated that DEVDase activation occurred within 10 min of release, even in the absence of Casp-3.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Apoptosis / drug effects
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Apoptosis / physiology*
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Apoptosis Regulatory Proteins
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Bacterial Proteins
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Carrier Proteins / drug effects
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Carrier Proteins / metabolism*
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Caspase 3
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Caspase Inhibitors
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Caspases / deficiency
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Caspases / genetics
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Caspases / metabolism
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Cell Line
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Cytochrome c Group / drug effects
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Cytochrome c Group / metabolism
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Enzyme Inhibitors / pharmacology
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Eukaryotic Cells / cytology
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Eukaryotic Cells / drug effects
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Eukaryotic Cells / metabolism*
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Green Fluorescent Proteins
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HeLa Cells
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Humans
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Intracellular Signaling Peptides and Proteins
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Luminescent Proteins
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Membrane Potentials / drug effects
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Membrane Potentials / physiology
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Mitochondria / drug effects
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Mitochondria / metabolism
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Mitochondrial Proteins / drug effects
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Mitochondrial Proteins / metabolism*
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Peptide Hydrolases / drug effects
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Peptide Hydrolases / metabolism
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Proto-Oncogene Proteins / deficiency
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins c-bcl-2*
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Time Factors
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bcl-2-Associated X Protein
Substances
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Apoptosis Regulatory Proteins
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BAX protein, human
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Bacterial Proteins
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Carrier Proteins
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Caspase Inhibitors
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Cytochrome c Group
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DIABLO protein, human
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Enzyme Inhibitors
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Intracellular Signaling Peptides and Proteins
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Luminescent Proteins
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Mitochondrial Proteins
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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bcl-2-Associated X Protein
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yellow fluorescent protein, Bacteria
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Green Fluorescent Proteins
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Peptide Hydrolases
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CASP3 protein, human
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Caspase 3
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Caspases
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DEVDase