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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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(A) Bcl-2 inhibits the release of cytochrome c into  the cytosol after GraB and perforin treatment. Rat-1 or Rat-1/ Bcl-2 cells were incubated with GraB (2 μg/ml) and perforin  (80 ng/ml) or perforin alone for 1.5 h and then cells were  dounced and S100 cytosol was prepared by high speed centrifugation as described in Materials and Methods. Cytosol fractions  were run on SDS-PAGE and Western blotted with anti–cytochrome c antibody. The experiment was performed twice with  identical results. (B) Cytochrome c release and apoptosis with increasing concentrations of GraB and perforin (80 ng/ml) in  U937, U937/Bcl-2, and U937/Bcl-XL. Cells were counted for  apoptotic nuclei by Hoechst staining and for mitochondrial cytochrome c by immunofluorescence as described in Fig. 5. Results were essentially identical in three experiments taken after 3  or 4 h of incubation. (C) Western blot of cytochrome c release  and (D) caspase 3 processing after GraB and perforin treatment.  At the indicated times after treatment of HeLa cells with GraB  and perforin S100, mitochondrial fractions were prepared as described in Materials and Methods and then Western blotted for  either cytochrome c or caspase 3.
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Figure 7: (A) Bcl-2 inhibits the release of cytochrome c into the cytosol after GraB and perforin treatment. Rat-1 or Rat-1/ Bcl-2 cells were incubated with GraB (2 μg/ml) and perforin (80 ng/ml) or perforin alone for 1.5 h and then cells were dounced and S100 cytosol was prepared by high speed centrifugation as described in Materials and Methods. Cytosol fractions were run on SDS-PAGE and Western blotted with anti–cytochrome c antibody. The experiment was performed twice with identical results. (B) Cytochrome c release and apoptosis with increasing concentrations of GraB and perforin (80 ng/ml) in U937, U937/Bcl-2, and U937/Bcl-XL. Cells were counted for apoptotic nuclei by Hoechst staining and for mitochondrial cytochrome c by immunofluorescence as described in Fig. 5. Results were essentially identical in three experiments taken after 3 or 4 h of incubation. (C) Western blot of cytochrome c release and (D) caspase 3 processing after GraB and perforin treatment. At the indicated times after treatment of HeLa cells with GraB and perforin S100, mitochondrial fractions were prepared as described in Materials and Methods and then Western blotted for either cytochrome c or caspase 3.

Mentions: We next confirmed these observations by Western blotting cytochrome c released into the S100 cytosol. After treatment with GraB and perforin, cytosolic cytochrome c levels increased and this was not seen in cells over expressing Bcl-2 (Fig. 7 A). The release of cytochrome c in U937 cells was detected at lower doses of GraB than were able to induce apoptosis (Fig. 7 B), and Bcl-2 and Bcl-XL over expression were equally effective in suppressing cytochrome c release (Fig. 7 B). A time course showed that cytochrome c appeared in S100 cytosol as early as 15 min after GraB and perforin treatment but not with perforin treatment alone (Fig. 7 C). The cleavage of caspase 3 was also detected within 15 min before significant numbers of apoptotic cells were detected (Fig. 7 D).


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)

(A) Bcl-2 inhibits the release of cytochrome c into  the cytosol after GraB and perforin treatment. Rat-1 or Rat-1/ Bcl-2 cells were incubated with GraB (2 μg/ml) and perforin  (80 ng/ml) or perforin alone for 1.5 h and then cells were  dounced and S100 cytosol was prepared by high speed centrifugation as described in Materials and Methods. Cytosol fractions  were run on SDS-PAGE and Western blotted with anti–cytochrome c antibody. The experiment was performed twice with  identical results. (B) Cytochrome c release and apoptosis with increasing concentrations of GraB and perforin (80 ng/ml) in  U937, U937/Bcl-2, and U937/Bcl-XL. Cells were counted for  apoptotic nuclei by Hoechst staining and for mitochondrial cytochrome c by immunofluorescence as described in Fig. 5. Results were essentially identical in three experiments taken after 3  or 4 h of incubation. (C) Western blot of cytochrome c release  and (D) caspase 3 processing after GraB and perforin treatment.  At the indicated times after treatment of HeLa cells with GraB  and perforin S100, mitochondrial fractions were prepared as described in Materials and Methods and then Western blotted for  either cytochrome c or caspase 3.
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Related In: Results  -  Collection

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Figure 7: (A) Bcl-2 inhibits the release of cytochrome c into the cytosol after GraB and perforin treatment. Rat-1 or Rat-1/ Bcl-2 cells were incubated with GraB (2 μg/ml) and perforin (80 ng/ml) or perforin alone for 1.5 h and then cells were dounced and S100 cytosol was prepared by high speed centrifugation as described in Materials and Methods. Cytosol fractions were run on SDS-PAGE and Western blotted with anti–cytochrome c antibody. The experiment was performed twice with identical results. (B) Cytochrome c release and apoptosis with increasing concentrations of GraB and perforin (80 ng/ml) in U937, U937/Bcl-2, and U937/Bcl-XL. Cells were counted for apoptotic nuclei by Hoechst staining and for mitochondrial cytochrome c by immunofluorescence as described in Fig. 5. Results were essentially identical in three experiments taken after 3 or 4 h of incubation. (C) Western blot of cytochrome c release and (D) caspase 3 processing after GraB and perforin treatment. At the indicated times after treatment of HeLa cells with GraB and perforin S100, mitochondrial fractions were prepared as described in Materials and Methods and then Western blotted for either cytochrome c or caspase 3.
Mentions: We next confirmed these observations by Western blotting cytochrome c released into the S100 cytosol. After treatment with GraB and perforin, cytosolic cytochrome c levels increased and this was not seen in cells over expressing Bcl-2 (Fig. 7 A). The release of cytochrome c in U937 cells was detected at lower doses of GraB than were able to induce apoptosis (Fig. 7 B), and Bcl-2 and Bcl-XL over expression were equally effective in suppressing cytochrome c release (Fig. 7 B). A time course showed that cytochrome c appeared in S100 cytosol as early as 15 min after GraB and perforin treatment but not with perforin treatment alone (Fig. 7 C). The cleavage of caspase 3 was also detected within 15 min before significant numbers of apoptotic cells were detected (Fig. 7 D).

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