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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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Effect of Bcl-2 on  the AIF release triggered by cytosols from ceramide- or Fas-stimulated cells. Cytosols (107  cells/100 μl CFS buffer) were  prepared from washed (three  times) cells which were either  left untreated (control) or treated  with C2 ceramide (50 μM) or anti-Fas during 30 min. These cytosols (5  μl) were added to 25 μl CFS buffer only or CFS buffer containing mitochondria (50 μg protein) from control CEM-C7-H2 cells (Co. mito) or  from Bcl-2–transfected cells (Bcl-2 mito), followed by an incubation step  of 30 min at 37°C. The supernatants of these cultures (14,000 g, 10 min,  4°C) were added to purified HeLa nuclei (3 × 104 nuclei in 10 μl CFS  buffer). After 90 min of incubation at 37°C, nuclei were stained with PI  and analyzed for the frequency of hypoploid events. One experiment out  of two yielding similar results is shown. Independent control experiments  indicate that C2 ceramide itself does not induce PT in isolated mitochondria at a dose up to 50 μM (not shown).
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Figure 5: Effect of Bcl-2 on the AIF release triggered by cytosols from ceramide- or Fas-stimulated cells. Cytosols (107 cells/100 μl CFS buffer) were prepared from washed (three times) cells which were either left untreated (control) or treated with C2 ceramide (50 μM) or anti-Fas during 30 min. These cytosols (5 μl) were added to 25 μl CFS buffer only or CFS buffer containing mitochondria (50 μg protein) from control CEM-C7-H2 cells (Co. mito) or from Bcl-2–transfected cells (Bcl-2 mito), followed by an incubation step of 30 min at 37°C. The supernatants of these cultures (14,000 g, 10 min, 4°C) were added to purified HeLa nuclei (3 × 104 nuclei in 10 μl CFS buffer). After 90 min of incubation at 37°C, nuclei were stained with PI and analyzed for the frequency of hypoploid events. One experiment out of two yielding similar results is shown. Independent control experiments indicate that C2 ceramide itself does not induce PT in isolated mitochondria at a dose up to 50 μM (not shown).

Mentions: As outlined in the Introduction, Bcl-2 is incapable of suppressing the Fas-induced apoptosis in a number of different models (26–28). This applies also to CEM-C7-H2 lymphoma cells (Fig. 1 C, see above). Since mitochondrial hyperexpression of Bcl-2 prevents the induction of PT by different substances, including prooxidants (9, 11; Fig. 4), we tested whether it would also interfere with ICE-induced PT. Mitochondria isolated from Bcl-2–transfected cells manifest large amplitude swelling when treated with recombinant ICE, exactly as do control mitochondria from cells not hyperexpressing Bcl-2. In addition, Bcl-2 hyperexpression does not prevent the mitochondrial release of AIF induced by ICE, although it does suppress the t-BHP–induced PT and release of AIF (Fig. 4). This dichotomy in the Bcl-2–mediated regulation of PT, inhibition of prooxidant-induced PT and failure to prevent ICE-induced PT, was observed in human CEM-C7-H2 cells transfected with tetracycline-repressable bcl-2 construct (Fig. 4), as well as in murine 2B4.11 T cell hybridoma cell lines stably transfected with the human bcl-2 gene (not shown). Thus, Bcl-2 fails to neutralize the effects of ICE on mitochondria in vitro, consistent with its inability to prevent ICE-dependent apoptosis in cells. Since Bcl-2 prevents ceramide-induced apoptosis and ΔΨm disruption (Fig. 1 C), we investigated the AIF release of Bcl-2 hyperexpressing mitochondria treated with cytosolic extracts from cells that have been treated during a short interval (30 min) with either ceramide or anti-Fas. Control mitochondria readily release AIF upon incubation with such cytosols (Fig. 5). Bcl-2–hyperexpressing mitochondria demonstrate a selective protection against yet unidentified ceramide-elicited PT inducers, yet release AIF upon incubation with ICE-containing cytosols from anti-Fas–treated cells (Fig. 5). These results are compatible with the hypothesis that Bcl-2 prevents ceramide-induced apoptosis at the level of mitochondria.


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)

Effect of Bcl-2 on  the AIF release triggered by cytosols from ceramide- or Fas-stimulated cells. Cytosols (107  cells/100 μl CFS buffer) were  prepared from washed (three  times) cells which were either  left untreated (control) or treated  with C2 ceramide (50 μM) or anti-Fas during 30 min. These cytosols (5  μl) were added to 25 μl CFS buffer only or CFS buffer containing mitochondria (50 μg protein) from control CEM-C7-H2 cells (Co. mito) or  from Bcl-2–transfected cells (Bcl-2 mito), followed by an incubation step  of 30 min at 37°C. The supernatants of these cultures (14,000 g, 10 min,  4°C) were added to purified HeLa nuclei (3 × 104 nuclei in 10 μl CFS  buffer). After 90 min of incubation at 37°C, nuclei were stained with PI  and analyzed for the frequency of hypoploid events. One experiment out  of two yielding similar results is shown. Independent control experiments  indicate that C2 ceramide itself does not induce PT in isolated mitochondria at a dose up to 50 μM (not shown).
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Related In: Results  -  Collection

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Figure 5: Effect of Bcl-2 on the AIF release triggered by cytosols from ceramide- or Fas-stimulated cells. Cytosols (107 cells/100 μl CFS buffer) were prepared from washed (three times) cells which were either left untreated (control) or treated with C2 ceramide (50 μM) or anti-Fas during 30 min. These cytosols (5 μl) were added to 25 μl CFS buffer only or CFS buffer containing mitochondria (50 μg protein) from control CEM-C7-H2 cells (Co. mito) or from Bcl-2–transfected cells (Bcl-2 mito), followed by an incubation step of 30 min at 37°C. The supernatants of these cultures (14,000 g, 10 min, 4°C) were added to purified HeLa nuclei (3 × 104 nuclei in 10 μl CFS buffer). After 90 min of incubation at 37°C, nuclei were stained with PI and analyzed for the frequency of hypoploid events. One experiment out of two yielding similar results is shown. Independent control experiments indicate that C2 ceramide itself does not induce PT in isolated mitochondria at a dose up to 50 μM (not shown).
Mentions: As outlined in the Introduction, Bcl-2 is incapable of suppressing the Fas-induced apoptosis in a number of different models (26–28). This applies also to CEM-C7-H2 lymphoma cells (Fig. 1 C, see above). Since mitochondrial hyperexpression of Bcl-2 prevents the induction of PT by different substances, including prooxidants (9, 11; Fig. 4), we tested whether it would also interfere with ICE-induced PT. Mitochondria isolated from Bcl-2–transfected cells manifest large amplitude swelling when treated with recombinant ICE, exactly as do control mitochondria from cells not hyperexpressing Bcl-2. In addition, Bcl-2 hyperexpression does not prevent the mitochondrial release of AIF induced by ICE, although it does suppress the t-BHP–induced PT and release of AIF (Fig. 4). This dichotomy in the Bcl-2–mediated regulation of PT, inhibition of prooxidant-induced PT and failure to prevent ICE-induced PT, was observed in human CEM-C7-H2 cells transfected with tetracycline-repressable bcl-2 construct (Fig. 4), as well as in murine 2B4.11 T cell hybridoma cell lines stably transfected with the human bcl-2 gene (not shown). Thus, Bcl-2 fails to neutralize the effects of ICE on mitochondria in vitro, consistent with its inability to prevent ICE-dependent apoptosis in cells. Since Bcl-2 prevents ceramide-induced apoptosis and ΔΨm disruption (Fig. 1 C), we investigated the AIF release of Bcl-2 hyperexpressing mitochondria treated with cytosolic extracts from cells that have been treated during a short interval (30 min) with either ceramide or anti-Fas. Control mitochondria readily release AIF upon incubation with such cytosols (Fig. 5). Bcl-2–hyperexpressing mitochondria demonstrate a selective protection against yet unidentified ceramide-elicited PT inducers, yet release AIF upon incubation with ICE-containing cytosols from anti-Fas–treated cells (Fig. 5). These results are compatible with the hypothesis that Bcl-2 prevents ceramide-induced apoptosis at the level of mitochondria.

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