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Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis.

MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg AH - J. Exp. Med. (1999)

Bottom Line: Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release.Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria.We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9.

ABSTRACT
Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Deltapsi). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Deltapsi and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death. Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Deltapsi suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Deltapsi suppression, and apoptosis and cell death induced by GraB, GraK, or perforin. In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

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Inhibition of GraB-induced apoptosis and cytochrome c by caspase peptide inhibitors. (A) HeLa cells preincubated with peptide  caspase inhibitors zVAD-FMK, YVAD-CHO,  DEVD-CHO, or FA-FMK at the indicated concentrations were treated with GraB (2 μg/ml)  and perforin (80 ng/ml) for 2.5 h and the apoptotic nuclei were counted after Hoechst staining.  •, zVAD-FMK; ▾, YVAD-CHO; ▪, DEVD-CHO; ♦, FA-FMK. (B) HeLa cells treated with  GraB (2 μg/ml) and perforin (80 ng/ml) for 1.5 h  were preincubated in zVAD-FMK (40 μM; center panel), DEVD-CHO (100 μM; right panel),  or medium control (left panel) then stained as in  Fig. 5. The images show an overlay of the mitochondrial stains Mitotracker (green), cytochrome  c (red), and Hoechst (blue). Coincidence of cytochrome c and Mitotracker shows as yellow. Note that cells treated with DEVD-CHO have no evidence  of chromatin condensation (white arrows), yet cytochrome c no longer colocalizes with Mitotracker. Cells treated with YVAD-CHO (100 μM) were  identical to zVAD-FMK–treated cells (not shown). The concentrations of inhibitory peptide noted above inhibited apoptosis by 70% as determined in A.  (C) HeLa cells were treated with perforin alone (lanes 2 and 9) or GraB (2 μg/ml) and perforin (80 ng/ml; lanes 3–5, 9–11, and 14–16), staurosporine (1 μM;  lanes 6 and 12), or medium (lanes 1, 7, and 13) for 2.5 h. GraB- and perforin-treated cells were also preincubated in zVAD-FMK (40 μM; lanes 5, 11,  and 15), YVAD-CHO (100 μM; lane 16), or DEVD-CHO (100 μM; lanes 4 and 10) peptide inhibitors, control peptide FA-FMK (100 μM; lane 17),  or medium (lane 13) and cytochrome c in the S100 cytosolic fraction (lanes 7–17) and in the mitochondria-containing fractions assessed by Western blotting (lanes 1–6). The doses of each inhibitory peptide reduced apoptosis by 70% (see A).
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Figure 8: Inhibition of GraB-induced apoptosis and cytochrome c by caspase peptide inhibitors. (A) HeLa cells preincubated with peptide caspase inhibitors zVAD-FMK, YVAD-CHO, DEVD-CHO, or FA-FMK at the indicated concentrations were treated with GraB (2 μg/ml) and perforin (80 ng/ml) for 2.5 h and the apoptotic nuclei were counted after Hoechst staining. •, zVAD-FMK; ▾, YVAD-CHO; ▪, DEVD-CHO; ♦, FA-FMK. (B) HeLa cells treated with GraB (2 μg/ml) and perforin (80 ng/ml) for 1.5 h were preincubated in zVAD-FMK (40 μM; center panel), DEVD-CHO (100 μM; right panel), or medium control (left panel) then stained as in Fig. 5. The images show an overlay of the mitochondrial stains Mitotracker (green), cytochrome c (red), and Hoechst (blue). Coincidence of cytochrome c and Mitotracker shows as yellow. Note that cells treated with DEVD-CHO have no evidence of chromatin condensation (white arrows), yet cytochrome c no longer colocalizes with Mitotracker. Cells treated with YVAD-CHO (100 μM) were identical to zVAD-FMK–treated cells (not shown). The concentrations of inhibitory peptide noted above inhibited apoptosis by 70% as determined in A. (C) HeLa cells were treated with perforin alone (lanes 2 and 9) or GraB (2 μg/ml) and perforin (80 ng/ml; lanes 3–5, 9–11, and 14–16), staurosporine (1 μM; lanes 6 and 12), or medium (lanes 1, 7, and 13) for 2.5 h. GraB- and perforin-treated cells were also preincubated in zVAD-FMK (40 μM; lanes 5, 11, and 15), YVAD-CHO (100 μM; lane 16), or DEVD-CHO (100 μM; lanes 4 and 10) peptide inhibitors, control peptide FA-FMK (100 μM; lane 17), or medium (lane 13) and cytochrome c in the S100 cytosolic fraction (lanes 7–17) and in the mitochondria-containing fractions assessed by Western blotting (lanes 1–6). The doses of each inhibitory peptide reduced apoptosis by 70% (see A).

Mentions: Earlier work indicated that inhibition of caspases blocks GraB apoptosis (14, 17). Since GraB activates both mitochondrial cytochrome c release and Δψ suppression, we next examined the requirement for caspases in the induction of these mitochondrial changes. Target cells were first incubated with increasing amounts of the tetrapeptide caspase inhibitors that blocked all caspases (zVAD-FMK), or those related to caspase 1 (YVAD-CHO) or caspase 3 (DEVD-CHO), then treated with GraB and perforin to determine the dose of each that would inhibit apoptosis by 70% (Fig. 8 A). Using pretreatment with zVAD-FMK (40 μM) or YVAD-CHO (100 μM) under these conditions completely prevented cytochrome c release from mitochondria as detected by immunofluorescent localization (Fig. 8 B). However, cytochrome c release was not blocked by the addition of DEVD-CHO (100 μM) despite the nearly complete inhibition of apoptosis, as clearly shown by intact Hoechst-stained nuclei in cells with no mitochondrial cytochrome c staining.


Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis.

MacDonald G, Shi L, Vande Velde C, Lieberman J, Greenberg AH - J. Exp. Med. (1999)

Inhibition of GraB-induced apoptosis and cytochrome c by caspase peptide inhibitors. (A) HeLa cells preincubated with peptide  caspase inhibitors zVAD-FMK, YVAD-CHO,  DEVD-CHO, or FA-FMK at the indicated concentrations were treated with GraB (2 μg/ml)  and perforin (80 ng/ml) for 2.5 h and the apoptotic nuclei were counted after Hoechst staining.  •, zVAD-FMK; ▾, YVAD-CHO; ▪, DEVD-CHO; ♦, FA-FMK. (B) HeLa cells treated with  GraB (2 μg/ml) and perforin (80 ng/ml) for 1.5 h  were preincubated in zVAD-FMK (40 μM; center panel), DEVD-CHO (100 μM; right panel),  or medium control (left panel) then stained as in  Fig. 5. The images show an overlay of the mitochondrial stains Mitotracker (green), cytochrome  c (red), and Hoechst (blue). Coincidence of cytochrome c and Mitotracker shows as yellow. Note that cells treated with DEVD-CHO have no evidence  of chromatin condensation (white arrows), yet cytochrome c no longer colocalizes with Mitotracker. Cells treated with YVAD-CHO (100 μM) were  identical to zVAD-FMK–treated cells (not shown). The concentrations of inhibitory peptide noted above inhibited apoptosis by 70% as determined in A.  (C) HeLa cells were treated with perforin alone (lanes 2 and 9) or GraB (2 μg/ml) and perforin (80 ng/ml; lanes 3–5, 9–11, and 14–16), staurosporine (1 μM;  lanes 6 and 12), or medium (lanes 1, 7, and 13) for 2.5 h. GraB- and perforin-treated cells were also preincubated in zVAD-FMK (40 μM; lanes 5, 11,  and 15), YVAD-CHO (100 μM; lane 16), or DEVD-CHO (100 μM; lanes 4 and 10) peptide inhibitors, control peptide FA-FMK (100 μM; lane 17),  or medium (lane 13) and cytochrome c in the S100 cytosolic fraction (lanes 7–17) and in the mitochondria-containing fractions assessed by Western blotting (lanes 1–6). The doses of each inhibitory peptide reduced apoptosis by 70% (see A).
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Figure 8: Inhibition of GraB-induced apoptosis and cytochrome c by caspase peptide inhibitors. (A) HeLa cells preincubated with peptide caspase inhibitors zVAD-FMK, YVAD-CHO, DEVD-CHO, or FA-FMK at the indicated concentrations were treated with GraB (2 μg/ml) and perforin (80 ng/ml) for 2.5 h and the apoptotic nuclei were counted after Hoechst staining. •, zVAD-FMK; ▾, YVAD-CHO; ▪, DEVD-CHO; ♦, FA-FMK. (B) HeLa cells treated with GraB (2 μg/ml) and perforin (80 ng/ml) for 1.5 h were preincubated in zVAD-FMK (40 μM; center panel), DEVD-CHO (100 μM; right panel), or medium control (left panel) then stained as in Fig. 5. The images show an overlay of the mitochondrial stains Mitotracker (green), cytochrome c (red), and Hoechst (blue). Coincidence of cytochrome c and Mitotracker shows as yellow. Note that cells treated with DEVD-CHO have no evidence of chromatin condensation (white arrows), yet cytochrome c no longer colocalizes with Mitotracker. Cells treated with YVAD-CHO (100 μM) were identical to zVAD-FMK–treated cells (not shown). The concentrations of inhibitory peptide noted above inhibited apoptosis by 70% as determined in A. (C) HeLa cells were treated with perforin alone (lanes 2 and 9) or GraB (2 μg/ml) and perforin (80 ng/ml; lanes 3–5, 9–11, and 14–16), staurosporine (1 μM; lanes 6 and 12), or medium (lanes 1, 7, and 13) for 2.5 h. GraB- and perforin-treated cells were also preincubated in zVAD-FMK (40 μM; lanes 5, 11, and 15), YVAD-CHO (100 μM; lane 16), or DEVD-CHO (100 μM; lanes 4 and 10) peptide inhibitors, control peptide FA-FMK (100 μM; lane 17), or medium (lane 13) and cytochrome c in the S100 cytosolic fraction (lanes 7–17) and in the mitochondria-containing fractions assessed by Western blotting (lanes 1–6). The doses of each inhibitory peptide reduced apoptosis by 70% (see A).
Mentions: Earlier work indicated that inhibition of caspases blocks GraB apoptosis (14, 17). Since GraB activates both mitochondrial cytochrome c release and Δψ suppression, we next examined the requirement for caspases in the induction of these mitochondrial changes. Target cells were first incubated with increasing amounts of the tetrapeptide caspase inhibitors that blocked all caspases (zVAD-FMK), or those related to caspase 1 (YVAD-CHO) or caspase 3 (DEVD-CHO), then treated with GraB and perforin to determine the dose of each that would inhibit apoptosis by 70% (Fig. 8 A). Using pretreatment with zVAD-FMK (40 μM) or YVAD-CHO (100 μM) under these conditions completely prevented cytochrome c release from mitochondria as detected by immunofluorescent localization (Fig. 8 B). However, cytochrome c release was not blocked by the addition of DEVD-CHO (100 μM) despite the nearly complete inhibition of apoptosis, as clearly shown by intact Hoechst-stained nuclei in cells with no mitochondrial cytochrome c staining.

Bottom Line: Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release.Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria.We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba, Canada R3E OV9.

ABSTRACT
Granzyme B (GraB) is required for the efficient activation of apoptosis by cytotoxic T lymphocytes and natural killer cells. We find that GraB and perforin induce severe mitochondrial perturbation as evidenced by the release of cytochrome c into the cytosol and suppression of transmembrane potential (Deltapsi). The earliest mitochondrial event was the release of cytochrome c, which occurred at the same time as caspase 3 processing and consistently before the activation of apoptosis. Granzyme K/perforin or perforin treatment, both of which kill target cells efficiently but are poor activators of apoptosis in short-term assays, did not induce rapid cytochrome c release. However, they suppressed Deltapsi and increased reactive oxygen species generation, indicating that mitochondrial dysfunction is also associated with this nonapoptotic cell death. Pretreatment with peptide caspase inhibitors zVAD-FMK or YVAD-CHO prevented GraB apoptosis and cytochrome c release, whereas DEVD-CHO blocked apoptosis but did not prevent cytochrome c release, indicating that caspases act both up- and downstream of mitochondria. Of additional interest, Deltapsi suppression mediated by GraK or GraB and perforin was not affected by zVAD-FMK and thus was caspase independent. Overexpression of Bcl-2 and Bcl-XL suppressed caspase activation, mitochondrial cytochrome c release, Deltapsi suppression, and apoptosis and cell death induced by GraB, GraK, or perforin. In an in vitro cell free system, GraB activates nuclear apoptosis in S-100 cytosol at high doses, however the addition of mitochondria amplified GraB activity over 15-fold. GraB- induced caspase 3 processing to p17 in S-100 cytosol was increased only threefold in the presence of mitochondria, suggesting that another caspase(s) participates in the mitochondrial amplification of GraB apoptosis. We conclude that GraB-induced apoptosis is highly amplified by mitochondria in a caspase-dependent manner but that GraB can also initiate caspase 3 processing and apoptosis in the absence of mitochondria.

Show MeSH
Related in: MedlinePlus