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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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Chronology and  cause effect relationship between activation of ICE (or  ICE-like) protease(s) and ΔΨm  disruption. (A) Chronology of  the activation of ICE, ΔΨm disruption, and nuclear DNA fragmentation in human CEM-C7.H2 lymphoma cells subjected  to Fas cross-linking. The frequency of ΔΨmlow cells and of  cells exhibiting DNA strand  breaks were determined by double staining with the potential-sensitive dye CMXRos and  TdT-catalyzed FITC-dUTP incorporation (TUNEL method),  as described in Materials and  Methods. Note that the  TUNEL+ population is actually  a subset of CMXRoslow cells (see  B). Activation of ICE (-like)  protease(s) was determined by a  fluorogenic substrate containing  the ICE cleavage site YVAD  (filled symbols), the maximum activity being defined as 100%.  Similarly, the activation of  CPP32 (-like) protease(s) was  determined by means of a fluorogenic substrate containing the  cleavage site DEVD (open symbols). (B) Temporal relationship  between Fas-induced ΔΨm disruption and CPP32 cleavage, as  well as DEVDase activation.  CEM-C7.H2 cells were cultured  during 120 min in the presence  of anti-Fas antibody, followed by  staining with the ΔΨm-sensitive  dye DiOC6(3) plus Annexin V (revealed by phycoerythrin). Cells were then separated in the cytofluorometer into cells with a normal ΔΨm  (DiOC6(3)high Annexin V−) or cells with a DiOC6(3)low Annexin V− or DiOC6(3)low Annexin V+ phenotype (sorting according to Windows), followed  by determination of CPP32 cleavage using Western blots (lane 1, unstimulated control cells; lane 2, nonseparated Fas-stimulated cells; lane 3), purified  DiOC6(3)high cells; lane 4, purified DiOC6(3)low Annexin V− cells; lane 5, purified DiOC6(3)low Annexin V+ cells, 8 × 105 cells/lane). Alternatively, cytosols from these cell populations were tested for DEVDase activity in vitro as in A (C) Determination of ΔΨm disruption and DNA strand breaks in different cells. CEM-C7.H2 lymphoma cell stably transfected with a Neomycin selection vector (Neo) only (fluorescence displays 1–4), with the crmA  cowpox protease inhibitor (graphs 5 and 6), or with a Bcl-2–expressing construct negatively regulated by doxycyclin (graphs 7–12). Cells were either  pretreated with doxycyclin (10 ng/ml, 48 h before starting of the experiment) to repress Bcl-2 expression (Bcl-2−, graphs 7–9) or left untreated (Bcl-2+,  graphs 10–12), and then subjected to apoptosis induction with C2 ceramide (50 μM; graphs 9 and 12), anti-Fas (graphs 3, 4, 6, 8, and 11) and/or the  ICE inhibitor Ac-YVAD.cmk (50 μM, all during 4 h; graph 4), followed by double staining with CMXRos and the TUNEL method. Neo control cells  were treated during 15 min with 100 μM of the protonophore mClCCP, providing a negative control for the CMXRos staining (graph 2). Numbers indicate the percentage of cells in each quadrant. Results are representative for three independent experiments.
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Figure 1: Chronology and cause effect relationship between activation of ICE (or ICE-like) protease(s) and ΔΨm disruption. (A) Chronology of the activation of ICE, ΔΨm disruption, and nuclear DNA fragmentation in human CEM-C7.H2 lymphoma cells subjected to Fas cross-linking. The frequency of ΔΨmlow cells and of cells exhibiting DNA strand breaks were determined by double staining with the potential-sensitive dye CMXRos and TdT-catalyzed FITC-dUTP incorporation (TUNEL method), as described in Materials and Methods. Note that the TUNEL+ population is actually a subset of CMXRoslow cells (see B). Activation of ICE (-like) protease(s) was determined by a fluorogenic substrate containing the ICE cleavage site YVAD (filled symbols), the maximum activity being defined as 100%. Similarly, the activation of CPP32 (-like) protease(s) was determined by means of a fluorogenic substrate containing the cleavage site DEVD (open symbols). (B) Temporal relationship between Fas-induced ΔΨm disruption and CPP32 cleavage, as well as DEVDase activation. CEM-C7.H2 cells were cultured during 120 min in the presence of anti-Fas antibody, followed by staining with the ΔΨm-sensitive dye DiOC6(3) plus Annexin V (revealed by phycoerythrin). Cells were then separated in the cytofluorometer into cells with a normal ΔΨm (DiOC6(3)high Annexin V−) or cells with a DiOC6(3)low Annexin V− or DiOC6(3)low Annexin V+ phenotype (sorting according to Windows), followed by determination of CPP32 cleavage using Western blots (lane 1, unstimulated control cells; lane 2, nonseparated Fas-stimulated cells; lane 3), purified DiOC6(3)high cells; lane 4, purified DiOC6(3)low Annexin V− cells; lane 5, purified DiOC6(3)low Annexin V+ cells, 8 × 105 cells/lane). Alternatively, cytosols from these cell populations were tested for DEVDase activity in vitro as in A (C) Determination of ΔΨm disruption and DNA strand breaks in different cells. CEM-C7.H2 lymphoma cell stably transfected with a Neomycin selection vector (Neo) only (fluorescence displays 1–4), with the crmA cowpox protease inhibitor (graphs 5 and 6), or with a Bcl-2–expressing construct negatively regulated by doxycyclin (graphs 7–12). Cells were either pretreated with doxycyclin (10 ng/ml, 48 h before starting of the experiment) to repress Bcl-2 expression (Bcl-2−, graphs 7–9) or left untreated (Bcl-2+, graphs 10–12), and then subjected to apoptosis induction with C2 ceramide (50 μM; graphs 9 and 12), anti-Fas (graphs 3, 4, 6, 8, and 11) and/or the ICE inhibitor Ac-YVAD.cmk (50 μM, all during 4 h; graph 4), followed by double staining with CMXRos and the TUNEL method. Neo control cells were treated during 15 min with 100 μM of the protonophore mClCCP, providing a negative control for the CMXRos staining (graph 2). Numbers indicate the percentage of cells in each quadrant. Results are representative for three independent experiments.

Mentions: Human CEM-C7.H2 lymphoma cells can be induced to undergo apoptosis by cross-linking of Fas. As shown in Fig. 1 A, cells manifest a rapid activation of protease(s) capable of cleaving a fluorogenic substrate containing the tetrapeptide YVAD. As described (22, 25), activation of ICE-like proteases is a rapid process that peaks 15–30 min after Fas cross-linking. It thus precedes the Fas-induced ΔΨm disruption, as quantified by means of the ΔΨm-sensitive dye CMXRos. This ΔΨm collapse affects only a minor fraction of the cells beginning at 30 min after Fas ligation. An important fraction of cells (∼ 40%) exhibits a disrupted ΔΨm about 2 h after Fas cross-linking, when DEVDase activity is also significantly augmented. To further investigate the relationship between Fas-induced ΔΨm disruption and activation of CPP32, CEM-C7.H2 cells were stimulated during 2 h by Fas cross-linking, followed by staining with the ΔΨm-sensitive dye DiOC6(3) as well as Annexin V (which measures the aberrant phosphatidyl serine exposure on the outer plasma membrane leaflet) and cytofluorometric purification of cells with a still normal ΔΨm (DiOC6(3)high) as well as cells with a disrupted ΔΨm (DiOC6(3)low) that are either in an early stage of the apoptotic process (Annexin V−) or in an advanced stage (Annexin V+) (Fig. 1 B). Only ΔΨmlow cells have cleaved the CPP32 precursor to yield CPP32 fragments (p21 and p17) and exhibit DEVDase activity (Fig. 1 B). This is observed for both ΔΨmlow Annexin V− and ΔΨmlow Annexin V+ cells, indicating that CPP32/DEVDase activation occurs concomitant with (or shortly after) the ΔΨm disruption. In contrast, ΔΨmhigh cells behave like unstimulated control cells and lack any detectable CPP32 cleavage or DEVDase activation (Fig. 1 B). Thus, CPP32 is only activated in cells whose ΔΨm is disrupted. Similar results have been obtained in other models of apoptosis induction, including ceramide-induced cell death (not shown). As in other models of apoptosis induction (4, 6, 9, 12, 31), the Fas-induced ΔΨm collapse precedes nuclear chromatinolysis as identified with the TUNEL technique (Fig. 1 A). Thus, cells that have disrupted their ΔΨm (CMXRoslow cells) can be subdivided into TUNEL+ and TUNEL− populations, whereas TUNEL+ cells uniformly possess a ΔΨmlow (CMXRoslow) phenotype (Fig. 1 C). These findings place ΔΨm disruption upstream of nuclear apoptosis.


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)

Chronology and  cause effect relationship between activation of ICE (or  ICE-like) protease(s) and ΔΨm  disruption. (A) Chronology of  the activation of ICE, ΔΨm disruption, and nuclear DNA fragmentation in human CEM-C7.H2 lymphoma cells subjected  to Fas cross-linking. The frequency of ΔΨmlow cells and of  cells exhibiting DNA strand  breaks were determined by double staining with the potential-sensitive dye CMXRos and  TdT-catalyzed FITC-dUTP incorporation (TUNEL method),  as described in Materials and  Methods. Note that the  TUNEL+ population is actually  a subset of CMXRoslow cells (see  B). Activation of ICE (-like)  protease(s) was determined by a  fluorogenic substrate containing  the ICE cleavage site YVAD  (filled symbols), the maximum activity being defined as 100%.  Similarly, the activation of  CPP32 (-like) protease(s) was  determined by means of a fluorogenic substrate containing the  cleavage site DEVD (open symbols). (B) Temporal relationship  between Fas-induced ΔΨm disruption and CPP32 cleavage, as  well as DEVDase activation.  CEM-C7.H2 cells were cultured  during 120 min in the presence  of anti-Fas antibody, followed by  staining with the ΔΨm-sensitive  dye DiOC6(3) plus Annexin V (revealed by phycoerythrin). Cells were then separated in the cytofluorometer into cells with a normal ΔΨm  (DiOC6(3)high Annexin V−) or cells with a DiOC6(3)low Annexin V− or DiOC6(3)low Annexin V+ phenotype (sorting according to Windows), followed  by determination of CPP32 cleavage using Western blots (lane 1, unstimulated control cells; lane 2, nonseparated Fas-stimulated cells; lane 3), purified  DiOC6(3)high cells; lane 4, purified DiOC6(3)low Annexin V− cells; lane 5, purified DiOC6(3)low Annexin V+ cells, 8 × 105 cells/lane). Alternatively, cytosols from these cell populations were tested for DEVDase activity in vitro as in A (C) Determination of ΔΨm disruption and DNA strand breaks in different cells. CEM-C7.H2 lymphoma cell stably transfected with a Neomycin selection vector (Neo) only (fluorescence displays 1–4), with the crmA  cowpox protease inhibitor (graphs 5 and 6), or with a Bcl-2–expressing construct negatively regulated by doxycyclin (graphs 7–12). Cells were either  pretreated with doxycyclin (10 ng/ml, 48 h before starting of the experiment) to repress Bcl-2 expression (Bcl-2−, graphs 7–9) or left untreated (Bcl-2+,  graphs 10–12), and then subjected to apoptosis induction with C2 ceramide (50 μM; graphs 9 and 12), anti-Fas (graphs 3, 4, 6, 8, and 11) and/or the  ICE inhibitor Ac-YVAD.cmk (50 μM, all during 4 h; graph 4), followed by double staining with CMXRos and the TUNEL method. Neo control cells  were treated during 15 min with 100 μM of the protonophore mClCCP, providing a negative control for the CMXRos staining (graph 2). Numbers indicate the percentage of cells in each quadrant. Results are representative for three independent experiments.
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Figure 1: Chronology and cause effect relationship between activation of ICE (or ICE-like) protease(s) and ΔΨm disruption. (A) Chronology of the activation of ICE, ΔΨm disruption, and nuclear DNA fragmentation in human CEM-C7.H2 lymphoma cells subjected to Fas cross-linking. The frequency of ΔΨmlow cells and of cells exhibiting DNA strand breaks were determined by double staining with the potential-sensitive dye CMXRos and TdT-catalyzed FITC-dUTP incorporation (TUNEL method), as described in Materials and Methods. Note that the TUNEL+ population is actually a subset of CMXRoslow cells (see B). Activation of ICE (-like) protease(s) was determined by a fluorogenic substrate containing the ICE cleavage site YVAD (filled symbols), the maximum activity being defined as 100%. Similarly, the activation of CPP32 (-like) protease(s) was determined by means of a fluorogenic substrate containing the cleavage site DEVD (open symbols). (B) Temporal relationship between Fas-induced ΔΨm disruption and CPP32 cleavage, as well as DEVDase activation. CEM-C7.H2 cells were cultured during 120 min in the presence of anti-Fas antibody, followed by staining with the ΔΨm-sensitive dye DiOC6(3) plus Annexin V (revealed by phycoerythrin). Cells were then separated in the cytofluorometer into cells with a normal ΔΨm (DiOC6(3)high Annexin V−) or cells with a DiOC6(3)low Annexin V− or DiOC6(3)low Annexin V+ phenotype (sorting according to Windows), followed by determination of CPP32 cleavage using Western blots (lane 1, unstimulated control cells; lane 2, nonseparated Fas-stimulated cells; lane 3), purified DiOC6(3)high cells; lane 4, purified DiOC6(3)low Annexin V− cells; lane 5, purified DiOC6(3)low Annexin V+ cells, 8 × 105 cells/lane). Alternatively, cytosols from these cell populations were tested for DEVDase activity in vitro as in A (C) Determination of ΔΨm disruption and DNA strand breaks in different cells. CEM-C7.H2 lymphoma cell stably transfected with a Neomycin selection vector (Neo) only (fluorescence displays 1–4), with the crmA cowpox protease inhibitor (graphs 5 and 6), or with a Bcl-2–expressing construct negatively regulated by doxycyclin (graphs 7–12). Cells were either pretreated with doxycyclin (10 ng/ml, 48 h before starting of the experiment) to repress Bcl-2 expression (Bcl-2−, graphs 7–9) or left untreated (Bcl-2+, graphs 10–12), and then subjected to apoptosis induction with C2 ceramide (50 μM; graphs 9 and 12), anti-Fas (graphs 3, 4, 6, 8, and 11) and/or the ICE inhibitor Ac-YVAD.cmk (50 μM, all during 4 h; graph 4), followed by double staining with CMXRos and the TUNEL method. Neo control cells were treated during 15 min with 100 μM of the protonophore mClCCP, providing a negative control for the CMXRos staining (graph 2). Numbers indicate the percentage of cells in each quadrant. Results are representative for three independent experiments.
Mentions: Human CEM-C7.H2 lymphoma cells can be induced to undergo apoptosis by cross-linking of Fas. As shown in Fig. 1 A, cells manifest a rapid activation of protease(s) capable of cleaving a fluorogenic substrate containing the tetrapeptide YVAD. As described (22, 25), activation of ICE-like proteases is a rapid process that peaks 15–30 min after Fas cross-linking. It thus precedes the Fas-induced ΔΨm disruption, as quantified by means of the ΔΨm-sensitive dye CMXRos. This ΔΨm collapse affects only a minor fraction of the cells beginning at 30 min after Fas ligation. An important fraction of cells (∼ 40%) exhibits a disrupted ΔΨm about 2 h after Fas cross-linking, when DEVDase activity is also significantly augmented. To further investigate the relationship between Fas-induced ΔΨm disruption and activation of CPP32, CEM-C7.H2 cells were stimulated during 2 h by Fas cross-linking, followed by staining with the ΔΨm-sensitive dye DiOC6(3) as well as Annexin V (which measures the aberrant phosphatidyl serine exposure on the outer plasma membrane leaflet) and cytofluorometric purification of cells with a still normal ΔΨm (DiOC6(3)high) as well as cells with a disrupted ΔΨm (DiOC6(3)low) that are either in an early stage of the apoptotic process (Annexin V−) or in an advanced stage (Annexin V+) (Fig. 1 B). Only ΔΨmlow cells have cleaved the CPP32 precursor to yield CPP32 fragments (p21 and p17) and exhibit DEVDase activity (Fig. 1 B). This is observed for both ΔΨmlow Annexin V− and ΔΨmlow Annexin V+ cells, indicating that CPP32/DEVDase activation occurs concomitant with (or shortly after) the ΔΨm disruption. In contrast, ΔΨmhigh cells behave like unstimulated control cells and lack any detectable CPP32 cleavage or DEVDase activation (Fig. 1 B). Thus, CPP32 is only activated in cells whose ΔΨm is disrupted. Similar results have been obtained in other models of apoptosis induction, including ceramide-induced cell death (not shown). As in other models of apoptosis induction (4, 6, 9, 12, 31), the Fas-induced ΔΨm collapse precedes nuclear chromatinolysis as identified with the TUNEL technique (Fig. 1 A). Thus, cells that have disrupted their ΔΨm (CMXRoslow cells) can be subdivided into TUNEL+ and TUNEL− populations, whereas TUNEL+ cells uniformly possess a ΔΨmlow (CMXRoslow) phenotype (Fig. 1 C). These findings place ΔΨm disruption upstream of nuclear apoptosis.

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