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Real-time single cell analysis of Smac/DIABLO release during apoptosis.

Rehm M, Düssmann H, Prehn JH - J. Cell Biol. (2003)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Clinical Research, University Münster Clinics, Münster, Germany.

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.

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Comparison of Smac/DIABLO and cyt-c release during apoptosis: effect of Casp-3 and z-VAD-fmk–sensitive caspases. (A) MCF-7/Casp-3 cells and MCF-7 cells were treated with 3 μM STS or 3 μM STS plus 200 μM of the broad-spectrum caspase inhibitor z-VAD-fmk for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results. (B) Immunofluorescence analysis showing the redistribution of cyt-c and Smac/DIABLO during apoptosis. Cells were treated with 3 μM STS, 3 μM STS plus 200 μM z-VAD-fmk or 200 ng/ml, and 1 μg/ml TNF-α/CHX for 6 h. Control cells received vehicle (DMSO). Arrows indicate cells that show a cyt-c and Smac/DIABLO redistribution in response to the agents. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (C) Quantification of cells showing cyt-c or Smac/DIABLO release as judged by immunofluorescence analysis. MCF-7/Casp-3 cells (indicated as Casp-3 +) were treated with 3 μM STS. MCF-7 cells (indicated as Casp-3 −) were treated with 3 μM STS in the presence or absence of 200 μM z-VAD-fmk. Data were collected from n = 200–300 cells per treatment in 11–14 randomly selected image frames from n = 3 independent experiments. There was no statistically significant difference between the three treatment groups or between cyt-c and Smac/DIABLO release at any time point investigated. Error bars equal SEM.
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fig1: Comparison of Smac/DIABLO and cyt-c release during apoptosis: effect of Casp-3 and z-VAD-fmk–sensitive caspases. (A) MCF-7/Casp-3 cells and MCF-7 cells were treated with 3 μM STS or 3 μM STS plus 200 μM of the broad-spectrum caspase inhibitor z-VAD-fmk for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results. (B) Immunofluorescence analysis showing the redistribution of cyt-c and Smac/DIABLO during apoptosis. Cells were treated with 3 μM STS, 3 μM STS plus 200 μM z-VAD-fmk or 200 ng/ml, and 1 μg/ml TNF-α/CHX for 6 h. Control cells received vehicle (DMSO). Arrows indicate cells that show a cyt-c and Smac/DIABLO redistribution in response to the agents. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (C) Quantification of cells showing cyt-c or Smac/DIABLO release as judged by immunofluorescence analysis. MCF-7/Casp-3 cells (indicated as Casp-3 +) were treated with 3 μM STS. MCF-7 cells (indicated as Casp-3 −) were treated with 3 μM STS in the presence or absence of 200 μM z-VAD-fmk. Data were collected from n = 200–300 cells per treatment in 11–14 randomly selected image frames from n = 3 independent experiments. There was no statistically significant difference between the three treatment groups or between cyt-c and Smac/DIABLO release at any time point investigated. Error bars equal SEM.

Mentions: We investigated the process of Smac/DIABLO and cyt-c release during apoptosis in Casp-3–deficient MCF-7 cells and MCF-7 cells stably transfected with Casp-3 (MCF-7/Casp-3; Janicke et al., 1998). Exposure of MCF-7/Casp-3 cells to proapoptotic agents such as staurosporine (STS), etoposide (Eto), or TNF-α plus cycloheximide (TNF-α/CHX) results in an efficient activation of effector caspases that is followed in ∼30 min by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation (Janicke et al., 1998; Luetjens et al., 2001; Rehm et al., 2002). In contrast, due to the lack of Casp-3, effector caspase activation sets in slower and less efficient in MCF-7 cells and is largely mediated by caspase-7 (Cuvillier et al., 2001; Liang et al., 2001; Rehm et al., 2002). Cell shrinkage is delayed by ∼120 min, and cell death sets in without oligonucleosomal DNA fragmentation (Janicke et al., 1998; Rehm et al., 2002). We activated the mitochondrial apoptosis pathway in both cell types by addition of 3 μM of the protein kinase inhibitor STS. Selective plasma membrane permeabilization and subsequent immunoblotting revealed that cyt-c and Smac/DIABLO were both released from mitochondria and accumulated in the cytosol after 4 h of STS treatment, independent of the presence or absence of Casp-3 (Fig. 1 A). Similar results were obtained in MCF-7 and MCF-7/Casp-3 cells after treatment with submaximal STS concentrations (0.1 μM) or after stimulation of death receptors with TNF-α/CHX (unpublished data).


Real-time single cell analysis of Smac/DIABLO release during apoptosis.

Rehm M, Düssmann H, Prehn JH - J. Cell Biol. (2003)

Comparison of Smac/DIABLO and cyt-c release during apoptosis: effect of Casp-3 and z-VAD-fmk–sensitive caspases. (A) MCF-7/Casp-3 cells and MCF-7 cells were treated with 3 μM STS or 3 μM STS plus 200 μM of the broad-spectrum caspase inhibitor z-VAD-fmk for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results. (B) Immunofluorescence analysis showing the redistribution of cyt-c and Smac/DIABLO during apoptosis. Cells were treated with 3 μM STS, 3 μM STS plus 200 μM z-VAD-fmk or 200 ng/ml, and 1 μg/ml TNF-α/CHX for 6 h. Control cells received vehicle (DMSO). Arrows indicate cells that show a cyt-c and Smac/DIABLO redistribution in response to the agents. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (C) Quantification of cells showing cyt-c or Smac/DIABLO release as judged by immunofluorescence analysis. MCF-7/Casp-3 cells (indicated as Casp-3 +) were treated with 3 μM STS. MCF-7 cells (indicated as Casp-3 −) were treated with 3 μM STS in the presence or absence of 200 μM z-VAD-fmk. Data were collected from n = 200–300 cells per treatment in 11–14 randomly selected image frames from n = 3 independent experiments. There was no statistically significant difference between the three treatment groups or between cyt-c and Smac/DIABLO release at any time point investigated. Error bars equal SEM.
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Related In: Results  -  Collection

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fig1: Comparison of Smac/DIABLO and cyt-c release during apoptosis: effect of Casp-3 and z-VAD-fmk–sensitive caspases. (A) MCF-7/Casp-3 cells and MCF-7 cells were treated with 3 μM STS or 3 μM STS plus 200 μM of the broad-spectrum caspase inhibitor z-VAD-fmk for the indicated time periods. Release of Smac/DIABLO and cyt-c from the mitochondria-containing pellet fractions into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. Experiments were repeated twice with similar results. (B) Immunofluorescence analysis showing the redistribution of cyt-c and Smac/DIABLO during apoptosis. Cells were treated with 3 μM STS, 3 μM STS plus 200 μM z-VAD-fmk or 200 ng/ml, and 1 μg/ml TNF-α/CHX for 6 h. Control cells received vehicle (DMSO). Arrows indicate cells that show a cyt-c and Smac/DIABLO redistribution in response to the agents. Nuclear morphology was detected by Hoechst staining. Bar, 10 μm. (C) Quantification of cells showing cyt-c or Smac/DIABLO release as judged by immunofluorescence analysis. MCF-7/Casp-3 cells (indicated as Casp-3 +) were treated with 3 μM STS. MCF-7 cells (indicated as Casp-3 −) were treated with 3 μM STS in the presence or absence of 200 μM z-VAD-fmk. Data were collected from n = 200–300 cells per treatment in 11–14 randomly selected image frames from n = 3 independent experiments. There was no statistically significant difference between the three treatment groups or between cyt-c and Smac/DIABLO release at any time point investigated. Error bars equal SEM.
Mentions: We investigated the process of Smac/DIABLO and cyt-c release during apoptosis in Casp-3–deficient MCF-7 cells and MCF-7 cells stably transfected with Casp-3 (MCF-7/Casp-3; Janicke et al., 1998). Exposure of MCF-7/Casp-3 cells to proapoptotic agents such as staurosporine (STS), etoposide (Eto), or TNF-α plus cycloheximide (TNF-α/CHX) results in an efficient activation of effector caspases that is followed in ∼30 min by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation (Janicke et al., 1998; Luetjens et al., 2001; Rehm et al., 2002). In contrast, due to the lack of Casp-3, effector caspase activation sets in slower and less efficient in MCF-7 cells and is largely mediated by caspase-7 (Cuvillier et al., 2001; Liang et al., 2001; Rehm et al., 2002). Cell shrinkage is delayed by ∼120 min, and cell death sets in without oligonucleosomal DNA fragmentation (Janicke et al., 1998; Rehm et al., 2002). We activated the mitochondrial apoptosis pathway in both cell types by addition of 3 μM of the protein kinase inhibitor STS. Selective plasma membrane permeabilization and subsequent immunoblotting revealed that cyt-c and Smac/DIABLO were both released from mitochondria and accumulated in the cytosol after 4 h of STS treatment, independent of the presence or absence of Casp-3 (Fig. 1 A). Similar results were obtained in MCF-7 and MCF-7/Casp-3 cells after treatment with submaximal STS concentrations (0.1 μM) or after stimulation of death receptors with TNF-α/CHX (unpublished data).

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Center for Clinical Research, University Münster Clinics, Münster, Germany.

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.

Show MeSH
Related in: MedlinePlus