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The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.

Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, Fassy F, Reed JC, Kroemer G - J. Exp. Med. (1997)

Bottom Line: Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release.These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation.In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-UPR420, B.P.8, F-94801 Villejuif, France.

ABSTRACT
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

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Recombinant purified ICE is sufficient to induce  PT, as well as the release of an  apoptosis-inducing factor from  mitochondria. Purified liver mitochondria were treated with CFS  buffer only (graph 1), purified  recombinant human ICE (graphs  2–4), the prooxidant t-BHP  (graphs 5 and 6), and/or different protease inhibitors (Ac-YVAD.cmk, graphs 3 and 6 or  Ac-DEVD.CHO, graph 4). These  reagents were added together to  the mitochondria and the following parameters were assessed: large  amplitude swelling (A), ΔΨm  (DiOC6(3) staining, 30 min after  addition of the reagents) (B), and  release of AIF (C). Arrows in A  indicate addition of the indicated  combination of reagents or  buffer only (Control). The dotted line in graph B 1 indicates  the negative control of DiOC6(3)  staining obtained in the presence of the ΔΨm-dissipating reagent mClCCP. For the determination of AIF release (C), mitochondria were centrifuged (1.5 × 10−5 g, 1 h) after 5 min of treatment, and the supernatant was incubated for 30 min with purified HeLa nuclei, followed by determination of their DNA content using the flurochrome propidium iodide, as  described in Materials and Methods. Percentages detail the percentage of nuclei exhibiting an apparent subdiploidy.
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Figure 3: Recombinant purified ICE is sufficient to induce PT, as well as the release of an apoptosis-inducing factor from mitochondria. Purified liver mitochondria were treated with CFS buffer only (graph 1), purified recombinant human ICE (graphs 2–4), the prooxidant t-BHP (graphs 5 and 6), and/or different protease inhibitors (Ac-YVAD.cmk, graphs 3 and 6 or Ac-DEVD.CHO, graph 4). These reagents were added together to the mitochondria and the following parameters were assessed: large amplitude swelling (A), ΔΨm (DiOC6(3) staining, 30 min after addition of the reagents) (B), and release of AIF (C). Arrows in A indicate addition of the indicated combination of reagents or buffer only (Control). The dotted line in graph B 1 indicates the negative control of DiOC6(3) staining obtained in the presence of the ΔΨm-dissipating reagent mClCCP. For the determination of AIF release (C), mitochondria were centrifuged (1.5 × 10−5 g, 1 h) after 5 min of treatment, and the supernatant was incubated for 30 min with purified HeLa nuclei, followed by determination of their DNA content using the flurochrome propidium iodide, as described in Materials and Methods. Percentages detail the percentage of nuclei exhibiting an apparent subdiploidy.

Mentions: In accordance with the data obtained with cytosolic extracts, recombinant purified ICE suffices to induce both large amplitude swelling and dissipation of the ΔΨm in isolated mitochondria in vitro (Fig. 3, A and B). This effect of ICE is rapid (<30 s) and can be neutralized by Ac-YVAD.cmk and Ac-YVAD.CHO, but not by AcDEVD.CHO, indicating that it relies on the enzymatic activity of ICE. In contrast, Ac-YVAD.cmk does not interfere with t-BHP-induced PT, thus excluding that this modified tetrapeptide might prevent PT in a nonspecific fashion (Fig. 3, A and B). The use of additional inhibitors underscores the different mechanisms involved in ICE– and t-BHP–triggered PT. For example, cyclosporin A, bongkrekic acid, monochlorobiman (9), and the calpain inhibitor Cbz-LLT. CHN2 (17) all inhibited the t-BHP– but not the ICE–induced PT in vitro (Table 1). Other proteases besides ICE, such as trypsin and proteinase K, also induce PT in isolated mitochondria (not shown), in accord with previous observations that microinjection of such proteases induces apoptosis in cells (40).


The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis.

Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, Fassy F, Reed JC, Kroemer G - J. Exp. Med. (1997)

Recombinant purified ICE is sufficient to induce  PT, as well as the release of an  apoptosis-inducing factor from  mitochondria. Purified liver mitochondria were treated with CFS  buffer only (graph 1), purified  recombinant human ICE (graphs  2–4), the prooxidant t-BHP  (graphs 5 and 6), and/or different protease inhibitors (Ac-YVAD.cmk, graphs 3 and 6 or  Ac-DEVD.CHO, graph 4). These  reagents were added together to  the mitochondria and the following parameters were assessed: large  amplitude swelling (A), ΔΨm  (DiOC6(3) staining, 30 min after  addition of the reagents) (B), and  release of AIF (C). Arrows in A  indicate addition of the indicated  combination of reagents or  buffer only (Control). The dotted line in graph B 1 indicates  the negative control of DiOC6(3)  staining obtained in the presence of the ΔΨm-dissipating reagent mClCCP. For the determination of AIF release (C), mitochondria were centrifuged (1.5 × 10−5 g, 1 h) after 5 min of treatment, and the supernatant was incubated for 30 min with purified HeLa nuclei, followed by determination of their DNA content using the flurochrome propidium iodide, as  described in Materials and Methods. Percentages detail the percentage of nuclei exhibiting an apparent subdiploidy.
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Related In: Results  -  Collection

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Figure 3: Recombinant purified ICE is sufficient to induce PT, as well as the release of an apoptosis-inducing factor from mitochondria. Purified liver mitochondria were treated with CFS buffer only (graph 1), purified recombinant human ICE (graphs 2–4), the prooxidant t-BHP (graphs 5 and 6), and/or different protease inhibitors (Ac-YVAD.cmk, graphs 3 and 6 or Ac-DEVD.CHO, graph 4). These reagents were added together to the mitochondria and the following parameters were assessed: large amplitude swelling (A), ΔΨm (DiOC6(3) staining, 30 min after addition of the reagents) (B), and release of AIF (C). Arrows in A indicate addition of the indicated combination of reagents or buffer only (Control). The dotted line in graph B 1 indicates the negative control of DiOC6(3) staining obtained in the presence of the ΔΨm-dissipating reagent mClCCP. For the determination of AIF release (C), mitochondria were centrifuged (1.5 × 10−5 g, 1 h) after 5 min of treatment, and the supernatant was incubated for 30 min with purified HeLa nuclei, followed by determination of their DNA content using the flurochrome propidium iodide, as described in Materials and Methods. Percentages detail the percentage of nuclei exhibiting an apparent subdiploidy.
Mentions: In accordance with the data obtained with cytosolic extracts, recombinant purified ICE suffices to induce both large amplitude swelling and dissipation of the ΔΨm in isolated mitochondria in vitro (Fig. 3, A and B). This effect of ICE is rapid (<30 s) and can be neutralized by Ac-YVAD.cmk and Ac-YVAD.CHO, but not by AcDEVD.CHO, indicating that it relies on the enzymatic activity of ICE. In contrast, Ac-YVAD.cmk does not interfere with t-BHP-induced PT, thus excluding that this modified tetrapeptide might prevent PT in a nonspecific fashion (Fig. 3, A and B). The use of additional inhibitors underscores the different mechanisms involved in ICE– and t-BHP–triggered PT. For example, cyclosporin A, bongkrekic acid, monochlorobiman (9), and the calpain inhibitor Cbz-LLT. CHN2 (17) all inhibited the t-BHP– but not the ICE–induced PT in vitro (Table 1). Other proteases besides ICE, such as trypsin and proteinase K, also induce PT in isolated mitochondria (not shown), in accord with previous observations that microinjection of such proteases induces apoptosis in cells (40).

Bottom Line: Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release.These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation.In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique-UPR420, B.P.8, F-94801 Villejuif, France.

ABSTRACT
According to current understanding, cytoplasmic events including activation of protease cascades and mitochondrial permeability transition (PT) participate in the control of nuclear apoptosis. However, the relationship between protease activation and PT has remained elusive. When apoptosis is induced by cross-linking of the Fas/APO-1/CD95 receptor, activation of interleukin-1beta converting enzyme (ICE; caspase 1) or ICE-like enzymes precedes the disruption of the mitochondrial inner transmembrane potential (DeltaPsim). In contrast, cytosolic CPP32/ Yama/Apopain/caspase 3 activation, plasma membrane phosphatidyl serine exposure, and nuclear apoptosis only occur in cells in which the DeltaPsim is fully disrupted. Transfection with the cowpox protease inhibitor crmA or culture in the presence of the synthetic ICE-specific inhibitor Ac-YVAD.cmk both prevent the DeltaPsim collapse and subsequent apoptosis. Cytosols from anti-Fas-treated human lymphoma cells accumulate an activity that induces PT in isolated mitochondria in vitro and that is neutralized by crmA or Ac-YVAD.cmk. Recombinant purified ICE suffices to cause isolated mitochondria to undergo PT-like large amplitude swelling and to disrupt their DeltaPsim. In addition, ICE-treated mitochondria release an apoptosis-inducing factor (AIF) that induces apoptotic changes (chromatin condensation and oligonucleosomal DNA fragmentation) in isolated nuclei in vitro. AIF is a protease (or protease activator) that can be inhibited by the broad spectrum apoptosis inhibitor Z-VAD.fmk and that causes the proteolytical activation of CPP32. Although Bcl-2 is a highly efficient inhibitor of mitochondrial alterations (large amplitude swelling + DeltaPsim collapse + release of AIF) induced by prooxidants or cytosols from ceramide-treated cells, it has no effect on the ICE-induced mitochondrial PT and AIF release. These data connect a protease activation pathway with the mitochondrial phase of apoptosis regulation. In addition, they provide a plausible explanation of why Bcl-2 fails to interfere with Fas-triggered apoptosis in most cell types, yet prevents ceramide- and prooxidant-induced apoptosis.

Show MeSH
Related in: MedlinePlus