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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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Effect of caspase and proteasome inhibition on the release of mitochondrial proteins. (A) Western blot analysis of Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h in combination with 200 μM z-VAD-fmk and/or 1 μM of the proteasome inhibitor lactacystin. Release of Smac/DIABLO and cyt-c from mitochondria containing pellet fraction into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. α-Tubulin and porin served as control for equal loading of the samples. Experiment was repeated twice with similar results. (B) Western blot analysis of the cytosol and mitochondria-containing pellet fraction of HeLa D98 cells exposed to 3 μM STS for 8 h, either in the absence or presence of z-VAD-fmk or lactacystin. Experiment was repeated with similar results. (C) Western blot analysis demonstrating the processing of caspase-2, -9, and -7 in Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h and its inhibition by 200 μM z-VAD-fmk. α-Tubulin served as control for equal sample loading.
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fig2: Effect of caspase and proteasome inhibition on the release of mitochondrial proteins. (A) Western blot analysis of Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h in combination with 200 μM z-VAD-fmk and/or 1 μM of the proteasome inhibitor lactacystin. Release of Smac/DIABLO and cyt-c from mitochondria containing pellet fraction into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. α-Tubulin and porin served as control for equal loading of the samples. Experiment was repeated twice with similar results. (B) Western blot analysis of the cytosol and mitochondria-containing pellet fraction of HeLa D98 cells exposed to 3 μM STS for 8 h, either in the absence or presence of z-VAD-fmk or lactacystin. Experiment was repeated with similar results. (C) Western blot analysis demonstrating the processing of caspase-2, -9, and -7 in Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h and its inhibition by 200 μM z-VAD-fmk. α-Tubulin served as control for equal sample loading.

Mentions: It has been reported previously that Smac/DIABLO is subject to proteasomal degradation (MacFarlane et al., 2002; Hu and Yang, 2003). To investigate whether z-VAD-fmk may promote the degradation of Smac/DIABLO after its release, we treated MCF-7 cells for 8 h with 3 μM STS in the presence or absence of 200 μM z-VAD-fmk or 1 μM of the proteasome inhibitor lactacystin (Fig. 2 A). STS-only treatment induced the cytosolic accumulation of Smac/DIABLO and cyt-c, whereas STS plus z-VAD-fmk–treated cultures showed a reduced accumulation of both proteins in the cytosolic fraction, despite complete release from mitochondria. When cells were treated with lactacystin, high amounts of Smac/DIABLO and cyt-c reappeared in the cytosolic fraction of STS plus z-VAD-fmk treated cells. A higher cytosolic content of Smac/DIABLO was also observed in STS plus lactacystin- versus STS-only– treated cultures, suggesting that Smac/DIABLO is also partially degraded when caspase activation is not blocked. Similar effects were observed in HeLa D98 cells (Fig. 2 B). z-VAD-fmk–insensitive release of Smac/DIABLO was also observed in MCF-7 cells treated with the topoisomerase II inhibitor Eto (unpublished data).


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

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

Effect of caspase and proteasome inhibition on the release of mitochondrial proteins. (A) Western blot analysis of Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h in combination with 200 μM z-VAD-fmk and/or 1 μM of the proteasome inhibitor lactacystin. Release of Smac/DIABLO and cyt-c from mitochondria containing pellet fraction into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. α-Tubulin and porin served as control for equal loading of the samples. Experiment was repeated twice with similar results. (B) Western blot analysis of the cytosol and mitochondria-containing pellet fraction of HeLa D98 cells exposed to 3 μM STS for 8 h, either in the absence or presence of z-VAD-fmk or lactacystin. Experiment was repeated with similar results. (C) Western blot analysis demonstrating the processing of caspase-2, -9, and -7 in Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h and its inhibition by 200 μM z-VAD-fmk. α-Tubulin served as control for equal sample loading.
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Related In: Results  -  Collection

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fig2: Effect of caspase and proteasome inhibition on the release of mitochondrial proteins. (A) Western blot analysis of Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h in combination with 200 μM z-VAD-fmk and/or 1 μM of the proteasome inhibitor lactacystin. Release of Smac/DIABLO and cyt-c from mitochondria containing pellet fraction into the cytosol was analyzed by Western blotting. Controls were treated with DMSO. α-Tubulin and porin served as control for equal loading of the samples. Experiment was repeated twice with similar results. (B) Western blot analysis of the cytosol and mitochondria-containing pellet fraction of HeLa D98 cells exposed to 3 μM STS for 8 h, either in the absence or presence of z-VAD-fmk or lactacystin. Experiment was repeated with similar results. (C) Western blot analysis demonstrating the processing of caspase-2, -9, and -7 in Casp-3–deficient MCF-7 cells treated with 3 μM STS for 8 h and its inhibition by 200 μM z-VAD-fmk. α-Tubulin served as control for equal sample loading.
Mentions: It has been reported previously that Smac/DIABLO is subject to proteasomal degradation (MacFarlane et al., 2002; Hu and Yang, 2003). To investigate whether z-VAD-fmk may promote the degradation of Smac/DIABLO after its release, we treated MCF-7 cells for 8 h with 3 μM STS in the presence or absence of 200 μM z-VAD-fmk or 1 μM of the proteasome inhibitor lactacystin (Fig. 2 A). STS-only treatment induced the cytosolic accumulation of Smac/DIABLO and cyt-c, whereas STS plus z-VAD-fmk–treated cultures showed a reduced accumulation of both proteins in the cytosolic fraction, despite complete release from mitochondria. When cells were treated with lactacystin, high amounts of Smac/DIABLO and cyt-c reappeared in the cytosolic fraction of STS plus z-VAD-fmk treated cells. A higher cytosolic content of Smac/DIABLO was also observed in STS plus lactacystin- versus STS-only– treated cultures, suggesting that Smac/DIABLO is also partially degraded when caspase activation is not blocked. Similar effects were observed in HeLa D98 cells (Fig. 2 B). z-VAD-fmk–insensitive release of Smac/DIABLO was also observed in MCF-7 cells treated with the topoisomerase II inhibitor Eto (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