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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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Amplification of GraB-induced apoptosis in an  in vitro cell free system. (A) GraB in increasing amounts was  added to S100 cytosol (○) or S100 cytosol plus mitochondria (•) and nuclei for 1 h as described in Materials and  Methods. Nuclei were stained by Hoechst dye and apoptosis  was quantitated. The data are the mean ± SE of three experiments. (B) GraB in increasing amounts was added to S100  cytosol and nuclei with increasing amounts of mitochondria  (0, 40, 130, and 400 μg/ml mitochondrial protein), then incubated for 1 h and apoptotic nuclei were enumerated as described in A. Other experiments (A, C, and  D) contained  100 μg/ml mitochondrial protein. (C) Recombinant GraB  (rGrB; •) or GraB containing an Ser203 to Ala mutation [rGrB(S-A); ○] were incubated with S100 cytosol, mitochondria, and nuclei as described in A.  (D) S100 caspase 3 processing after GraB treatment at increasing doses in the presence or absence of mitochondria (Mito). Incubations were carried out as  described in A and then cytosol was run on SDS-PAGE followed by Western blotting with anti–caspase 3 antibody. The dose of GraB increased from 0  (lanes 6 and 12) to 70 ng/ml (lanes 5 and 11), 200 ng/ml (lanes 4 and 10), 700 ng/ml (lanes 3 and 9), 2,000 ng/ml (lanes 2 and 8), and 6,000 ng/ml (lanes  1 and 7). The experiment was repeated three times with identical results.
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Figure 10: Amplification of GraB-induced apoptosis in an in vitro cell free system. (A) GraB in increasing amounts was added to S100 cytosol (○) or S100 cytosol plus mitochondria (•) and nuclei for 1 h as described in Materials and Methods. Nuclei were stained by Hoechst dye and apoptosis was quantitated. The data are the mean ± SE of three experiments. (B) GraB in increasing amounts was added to S100 cytosol and nuclei with increasing amounts of mitochondria (0, 40, 130, and 400 μg/ml mitochondrial protein), then incubated for 1 h and apoptotic nuclei were enumerated as described in A. Other experiments (A, C, and D) contained 100 μg/ml mitochondrial protein. (C) Recombinant GraB (rGrB; •) or GraB containing an Ser203 to Ala mutation [rGrB(S-A); ○] were incubated with S100 cytosol, mitochondria, and nuclei as described in A. (D) S100 caspase 3 processing after GraB treatment at increasing doses in the presence or absence of mitochondria (Mito). Incubations were carried out as described in A and then cytosol was run on SDS-PAGE followed by Western blotting with anti–caspase 3 antibody. The dose of GraB increased from 0 (lanes 6 and 12) to 70 ng/ml (lanes 5 and 11), 200 ng/ml (lanes 4 and 10), 700 ng/ml (lanes 3 and 9), 2,000 ng/ml (lanes 2 and 8), and 6,000 ng/ml (lanes 1 and 7). The experiment was repeated three times with identical results.

Mentions: We next directly determined the role of mitochondria in activating GraB apoptosis in an in vitro cell-free system. Previously we had shown that the simple addition of GraB without perforin to cell lysate initiates apoptosis of isolated nuclei and this is associated with the processing and activation of caspase 3 (14). We now reexamined this model using mitochondria-free S100 cytosol. The absence of cytochrome c on Western blotting confirmed that the S100 had no contaminating mitochondria. GraB-induced apoptotic activity is detectable without mitochondria (Fig. 10 A). However, the addition of an enriched mitochondrial fraction enhances apoptosis >15-fold with activity detectable at 120 ng/ml of GraB. These experiments were repeated using nuclei and S100 cytosol from several cellular sources, including HeLa, YAC-1 or U937 cells, and mitochondria from HeLa cells or rat liver with similar amplification of apoptosis in the presence of mitochondria. The level of amplification of GraB apoptosis by mitochondria was dependent on the amount of enriched mitochondrial fraction added to the reaction (Fig. 10 B). To confirm that the activity was due to the proteolytic activity of GraB in our purified preparations, we used recombinant GraB bearing an inactivating Ser203 to Ala mutation in the in vitro assay and found that it was unable to initiate apoptosis in the presence or absence of mitochondria in contrast to wild-type recombinant GraB (Fig. 10 C).


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)

Amplification of GraB-induced apoptosis in an  in vitro cell free system. (A) GraB in increasing amounts was  added to S100 cytosol (○) or S100 cytosol plus mitochondria (•) and nuclei for 1 h as described in Materials and  Methods. Nuclei were stained by Hoechst dye and apoptosis  was quantitated. The data are the mean ± SE of three experiments. (B) GraB in increasing amounts was added to S100  cytosol and nuclei with increasing amounts of mitochondria  (0, 40, 130, and 400 μg/ml mitochondrial protein), then incubated for 1 h and apoptotic nuclei were enumerated as described in A. Other experiments (A, C, and  D) contained  100 μg/ml mitochondrial protein. (C) Recombinant GraB  (rGrB; •) or GraB containing an Ser203 to Ala mutation [rGrB(S-A); ○] were incubated with S100 cytosol, mitochondria, and nuclei as described in A.  (D) S100 caspase 3 processing after GraB treatment at increasing doses in the presence or absence of mitochondria (Mito). Incubations were carried out as  described in A and then cytosol was run on SDS-PAGE followed by Western blotting with anti–caspase 3 antibody. The dose of GraB increased from 0  (lanes 6 and 12) to 70 ng/ml (lanes 5 and 11), 200 ng/ml (lanes 4 and 10), 700 ng/ml (lanes 3 and 9), 2,000 ng/ml (lanes 2 and 8), and 6,000 ng/ml (lanes  1 and 7). The experiment was repeated three times with identical results.
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Figure 10: Amplification of GraB-induced apoptosis in an in vitro cell free system. (A) GraB in increasing amounts was added to S100 cytosol (○) or S100 cytosol plus mitochondria (•) and nuclei for 1 h as described in Materials and Methods. Nuclei were stained by Hoechst dye and apoptosis was quantitated. The data are the mean ± SE of three experiments. (B) GraB in increasing amounts was added to S100 cytosol and nuclei with increasing amounts of mitochondria (0, 40, 130, and 400 μg/ml mitochondrial protein), then incubated for 1 h and apoptotic nuclei were enumerated as described in A. Other experiments (A, C, and D) contained 100 μg/ml mitochondrial protein. (C) Recombinant GraB (rGrB; •) or GraB containing an Ser203 to Ala mutation [rGrB(S-A); ○] were incubated with S100 cytosol, mitochondria, and nuclei as described in A. (D) S100 caspase 3 processing after GraB treatment at increasing doses in the presence or absence of mitochondria (Mito). Incubations were carried out as described in A and then cytosol was run on SDS-PAGE followed by Western blotting with anti–caspase 3 antibody. The dose of GraB increased from 0 (lanes 6 and 12) to 70 ng/ml (lanes 5 and 11), 200 ng/ml (lanes 4 and 10), 700 ng/ml (lanes 3 and 9), 2,000 ng/ml (lanes 2 and 8), and 6,000 ng/ml (lanes 1 and 7). The experiment was repeated three times with identical results.
Mentions: We next directly determined the role of mitochondria in activating GraB apoptosis in an in vitro cell-free system. Previously we had shown that the simple addition of GraB without perforin to cell lysate initiates apoptosis of isolated nuclei and this is associated with the processing and activation of caspase 3 (14). We now reexamined this model using mitochondria-free S100 cytosol. The absence of cytochrome c on Western blotting confirmed that the S100 had no contaminating mitochondria. GraB-induced apoptotic activity is detectable without mitochondria (Fig. 10 A). However, the addition of an enriched mitochondrial fraction enhances apoptosis >15-fold with activity detectable at 120 ng/ml of GraB. These experiments were repeated using nuclei and S100 cytosol from several cellular sources, including HeLa, YAC-1 or U937 cells, and mitochondria from HeLa cells or rat liver with similar amplification of apoptosis in the presence of mitochondria. The level of amplification of GraB apoptosis by mitochondria was dependent on the amount of enriched mitochondrial fraction added to the reaction (Fig. 10 B). To confirm that the activity was due to the proteolytic activity of GraB in our purified preparations, we used recombinant GraB bearing an inactivating Ser203 to Ala mutation in the in vitro assay and found that it was unable to initiate apoptosis in the presence or absence of mitochondria in contrast to wild-type recombinant GraB (Fig. 10 C).

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