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Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.

Wang GQ, Wieckowski E, Goldstein LA, Gastman BR, Rabinovitz A, Gambotto A, Li S, Fang B, Yin XM, Rabinowich H - J. Exp. Med. (2001)

Bottom Line: Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB.Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added.These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.

ABSTRACT
Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

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Resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis. (A) and (B) Flow cytometry analysis of staining by annexin V of Jurkat cells treated with Ad (10 PFU/ml), GrB (1 μg/ml), and a combination of GrB and Ad for 2 h (A) or 24 h (B) at 30°C. The data are means ± SD of results obtained in five independent experiments. The asterisks indicate a statistically significant difference between wild-type and Bak-deficient cells (P < 0.05, Mann-Whitney U). (C) GrB-mediated cleavage of PARP and DFF45/ICAD in wild-type, but not in Bak-deficient cells. Wild-type and Bak-deficient cells were treated with Ad, GrB, or a combination of GrB and Ad, as described previously. The cell extracts were resolved on SDS/PAGE and immunoblotted with anti-PARP mAb or anti-DFF45/ICAD Ab. (D) and (E) Lack of mitochondrial apoptotic events in Bak-deficient Jurkat cells treated with GrB. After 2 h of treatment with GrB and Ad, as described previously, the cells were assessed by flow cytometry for mitochondrial staining with CMXRos or NAO. Staining with CMXRos (100 nM) served to assess changes in mitochondria permeability transition; staining with NAO (100 nM) served to assess loss in mitochondrial cardiolipin. (F) and (G) Susceptibility of Bak-deficient Jurkat cells to TRAIL or taxol. Wild-type or Bak-deficient Jurkat cells were treated with TRAIL (100 ng/ml) or taxol (10 μg/ml) for 16 h. The cells were then analyzed by flow cytometry for staining by annexin V or propidium iodide. Percentage of apoptotic cells are indicated for TRAIL.
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fig2: Resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis. (A) and (B) Flow cytometry analysis of staining by annexin V of Jurkat cells treated with Ad (10 PFU/ml), GrB (1 μg/ml), and a combination of GrB and Ad for 2 h (A) or 24 h (B) at 30°C. The data are means ± SD of results obtained in five independent experiments. The asterisks indicate a statistically significant difference between wild-type and Bak-deficient cells (P < 0.05, Mann-Whitney U). (C) GrB-mediated cleavage of PARP and DFF45/ICAD in wild-type, but not in Bak-deficient cells. Wild-type and Bak-deficient cells were treated with Ad, GrB, or a combination of GrB and Ad, as described previously. The cell extracts were resolved on SDS/PAGE and immunoblotted with anti-PARP mAb or anti-DFF45/ICAD Ab. (D) and (E) Lack of mitochondrial apoptotic events in Bak-deficient Jurkat cells treated with GrB. After 2 h of treatment with GrB and Ad, as described previously, the cells were assessed by flow cytometry for mitochondrial staining with CMXRos or NAO. Staining with CMXRos (100 nM) served to assess changes in mitochondria permeability transition; staining with NAO (100 nM) served to assess loss in mitochondrial cardiolipin. (F) and (G) Susceptibility of Bak-deficient Jurkat cells to TRAIL or taxol. Wild-type or Bak-deficient Jurkat cells were treated with TRAIL (100 ng/ml) or taxol (10 μg/ml) for 16 h. The cells were then analyzed by flow cytometry for staining by annexin V or propidium iodide. Percentage of apoptotic cells are indicated for TRAIL.

Mentions: Resistance of Bak-deficient Cells to GrB-mediated Apoptosis. To study the susceptibility of Bak-deficient cells to GrB-mediated cell death we used a CTL-free system, wherein purified GrB was added in combination with a replication-deficient Ad to the target cell suspension (5, 11, 35). Although GrB enters the cell independently, via its surface receptor (36), efficient apoptosis does not occur in the absence of perforin or a substituting Ad. As assessed by flow cytometric analysis of cells stained with annexin V, treatment of Jurkat cells for 2 h with a combination of GrB (1 μg/ml) and Ad (10–100 pfu/ml) induced apoptosis in wild-type, but not in Bak-deficient Jurkat cells (Fig. 2 A). After GrB/Ad treatment for 24 h, ∼20% of Bak-deficient cells were annexin V-positive, as compared with ∼80% of wild-type cells (Fig. 2 B). Similar results (data not shown) were obtained in cells treated with GrB and LAK extracts that served as a source of perforin (37). These findings suggest that despite the ability of GrB to initiate apoptosis at multiple sites by direct cleavage of caspases or other apoptosis effector proteins, the major apoptotic mechanism of GrB is blocked in our Jurkat Bak-deficient cells. Wild-type cell apoptosis induced by 2-h treatment with GrB/Ad was associated with cleavage of PARP and loss in DFF45/ICAD expression (Fig. 2 C). In similarly treated Bak-deficient cells, no cleavage of PARP or loss in DFF45/ICAD was detected. To assess mitochondrial alterations associated with GrB-mediated apoptosis, we used two fluorescent dyes, which target different mitochondrial components. After 2 h of treatment with GrB/Ad, the cells were stained with the cationic lipophilic dye, chloromethyl X-rosamine (CMXRos), to measure disruption of the mitochondrial transmembrane potential. GrB/Ad treated wild-type, but not Bak-deficient cells, demonstrated reduction in incorporation of this dye (Fig. 2 D). We also assessed mitochondrial changes by nonyl acrydine orange (NAO), which interacts specifically with nonoxidized cardiolipin, a lipid that is exclusively localized in the inner mitochondrial membrane (38). Loss in cardiolipin was detected in wild-type Jurkat cells treated with GrB/Ad, but not in Bak-deficient cells (Fig. 2 E). These findings suggest that mitochondrial apoptotic events induced in wild-type Jurkat cells by GrB were blocked in our Bak-deficient cells. Despite their differential susceptibility to GrB-mediated apoptosis both wild-type and Bak-deficient Jurkat cells demonstrated sensitivity to apoptosis induced by either TRAIL (Fig. 2 F) or taxol (Fig. 2 G). TRAIL-mediated apoptosis is usually enhanced by a mitochondrial apoptotic loop (1), but may also proceed via a caspase-dependent, mitochondria-independent cascade. In accordance with this model, Bak-deficient cells were TRAIL-sensitive, albeit at a decreased level relative to wild-type Jurkat cells. The reduced susceptibility of Bak-deficient Jurkat cells to TRAIL may relate to the block in the mitochondrial apoptotic loop in these cells (Fig. 2 F). Wild-type and Bak-deficient Jurkat cells were similarly susceptible to taxol (Fig. 2 G), suggesting that Bak was not involved in the mechanism of cytotoxicity mediated by this anticancer drug. These results suggest that Bak-deficiency in Jurkat cells has a significant inhibitory effect on GrB-mediated cytotoxicity, executed mainly by a mitochondrial apoptotic cascade.


Resistance to granzyme B-mediated cytochrome c release in Bak-deficient cells.

Wang GQ, Wieckowski E, Goldstein LA, Gastman BR, Rabinovitz A, Gambotto A, Li S, Fang B, Yin XM, Rabinowich H - J. Exp. Med. (2001)

Resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis. (A) and (B) Flow cytometry analysis of staining by annexin V of Jurkat cells treated with Ad (10 PFU/ml), GrB (1 μg/ml), and a combination of GrB and Ad for 2 h (A) or 24 h (B) at 30°C. The data are means ± SD of results obtained in five independent experiments. The asterisks indicate a statistically significant difference between wild-type and Bak-deficient cells (P < 0.05, Mann-Whitney U). (C) GrB-mediated cleavage of PARP and DFF45/ICAD in wild-type, but not in Bak-deficient cells. Wild-type and Bak-deficient cells were treated with Ad, GrB, or a combination of GrB and Ad, as described previously. The cell extracts were resolved on SDS/PAGE and immunoblotted with anti-PARP mAb or anti-DFF45/ICAD Ab. (D) and (E) Lack of mitochondrial apoptotic events in Bak-deficient Jurkat cells treated with GrB. After 2 h of treatment with GrB and Ad, as described previously, the cells were assessed by flow cytometry for mitochondrial staining with CMXRos or NAO. Staining with CMXRos (100 nM) served to assess changes in mitochondria permeability transition; staining with NAO (100 nM) served to assess loss in mitochondrial cardiolipin. (F) and (G) Susceptibility of Bak-deficient Jurkat cells to TRAIL or taxol. Wild-type or Bak-deficient Jurkat cells were treated with TRAIL (100 ng/ml) or taxol (10 μg/ml) for 16 h. The cells were then analyzed by flow cytometry for staining by annexin V or propidium iodide. Percentage of apoptotic cells are indicated for TRAIL.
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fig2: Resistance of Bak-deficient Jurkat cells to GrB-mediated apoptosis. (A) and (B) Flow cytometry analysis of staining by annexin V of Jurkat cells treated with Ad (10 PFU/ml), GrB (1 μg/ml), and a combination of GrB and Ad for 2 h (A) or 24 h (B) at 30°C. The data are means ± SD of results obtained in five independent experiments. The asterisks indicate a statistically significant difference between wild-type and Bak-deficient cells (P < 0.05, Mann-Whitney U). (C) GrB-mediated cleavage of PARP and DFF45/ICAD in wild-type, but not in Bak-deficient cells. Wild-type and Bak-deficient cells were treated with Ad, GrB, or a combination of GrB and Ad, as described previously. The cell extracts were resolved on SDS/PAGE and immunoblotted with anti-PARP mAb or anti-DFF45/ICAD Ab. (D) and (E) Lack of mitochondrial apoptotic events in Bak-deficient Jurkat cells treated with GrB. After 2 h of treatment with GrB and Ad, as described previously, the cells were assessed by flow cytometry for mitochondrial staining with CMXRos or NAO. Staining with CMXRos (100 nM) served to assess changes in mitochondria permeability transition; staining with NAO (100 nM) served to assess loss in mitochondrial cardiolipin. (F) and (G) Susceptibility of Bak-deficient Jurkat cells to TRAIL or taxol. Wild-type or Bak-deficient Jurkat cells were treated with TRAIL (100 ng/ml) or taxol (10 μg/ml) for 16 h. The cells were then analyzed by flow cytometry for staining by annexin V or propidium iodide. Percentage of apoptotic cells are indicated for TRAIL.
Mentions: Resistance of Bak-deficient Cells to GrB-mediated Apoptosis. To study the susceptibility of Bak-deficient cells to GrB-mediated cell death we used a CTL-free system, wherein purified GrB was added in combination with a replication-deficient Ad to the target cell suspension (5, 11, 35). Although GrB enters the cell independently, via its surface receptor (36), efficient apoptosis does not occur in the absence of perforin or a substituting Ad. As assessed by flow cytometric analysis of cells stained with annexin V, treatment of Jurkat cells for 2 h with a combination of GrB (1 μg/ml) and Ad (10–100 pfu/ml) induced apoptosis in wild-type, but not in Bak-deficient Jurkat cells (Fig. 2 A). After GrB/Ad treatment for 24 h, ∼20% of Bak-deficient cells were annexin V-positive, as compared with ∼80% of wild-type cells (Fig. 2 B). Similar results (data not shown) were obtained in cells treated with GrB and LAK extracts that served as a source of perforin (37). These findings suggest that despite the ability of GrB to initiate apoptosis at multiple sites by direct cleavage of caspases or other apoptosis effector proteins, the major apoptotic mechanism of GrB is blocked in our Jurkat Bak-deficient cells. Wild-type cell apoptosis induced by 2-h treatment with GrB/Ad was associated with cleavage of PARP and loss in DFF45/ICAD expression (Fig. 2 C). In similarly treated Bak-deficient cells, no cleavage of PARP or loss in DFF45/ICAD was detected. To assess mitochondrial alterations associated with GrB-mediated apoptosis, we used two fluorescent dyes, which target different mitochondrial components. After 2 h of treatment with GrB/Ad, the cells were stained with the cationic lipophilic dye, chloromethyl X-rosamine (CMXRos), to measure disruption of the mitochondrial transmembrane potential. GrB/Ad treated wild-type, but not Bak-deficient cells, demonstrated reduction in incorporation of this dye (Fig. 2 D). We also assessed mitochondrial changes by nonyl acrydine orange (NAO), which interacts specifically with nonoxidized cardiolipin, a lipid that is exclusively localized in the inner mitochondrial membrane (38). Loss in cardiolipin was detected in wild-type Jurkat cells treated with GrB/Ad, but not in Bak-deficient cells (Fig. 2 E). These findings suggest that mitochondrial apoptotic events induced in wild-type Jurkat cells by GrB were blocked in our Bak-deficient cells. Despite their differential susceptibility to GrB-mediated apoptosis both wild-type and Bak-deficient Jurkat cells demonstrated sensitivity to apoptosis induced by either TRAIL (Fig. 2 F) or taxol (Fig. 2 G). TRAIL-mediated apoptosis is usually enhanced by a mitochondrial apoptotic loop (1), but may also proceed via a caspase-dependent, mitochondria-independent cascade. In accordance with this model, Bak-deficient cells were TRAIL-sensitive, albeit at a decreased level relative to wild-type Jurkat cells. The reduced susceptibility of Bak-deficient Jurkat cells to TRAIL may relate to the block in the mitochondrial apoptotic loop in these cells (Fig. 2 F). Wild-type and Bak-deficient Jurkat cells were similarly susceptible to taxol (Fig. 2 G), suggesting that Bak was not involved in the mechanism of cytotoxicity mediated by this anticancer drug. These results suggest that Bak-deficiency in Jurkat cells has a significant inhibitory effect on GrB-mediated cytotoxicity, executed mainly by a mitochondrial apoptotic cascade.

Bottom Line: Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB.Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added.These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.

ABSTRACT
Granzyme B (GrB), a serine protease with substrate specificity similar to the caspase family, is a major component of granule-mediated cytotoxicity of T lymphocytes. Although GrB can directly activate caspases, it induces apoptosis predominantly via Bid cleavage, mitochondrial outer membrane permeabilization, and cytochrome c release. To study the molecular regulators for GrB-mediated mitochondrial apoptotic events, we used a CTL-free cytotoxicity system, wherein target cells are treated with purified GrB and replication-deficient adenovirus (Ad). We report here that the Bcl-2 proapoptotic family member, Bak, plays a dominant role in GrB-mediated mitochondrial apoptotic events. A variant of Jurkat cells, deficient in Bak expression, was resistant to GrB/Ad-mediated apoptosis, as determined by lack of membranous phosphatidylserine exposure, lack of DNA breaks, lack of mitochondrial outer membrane permeabilization, and unchanged expression of inner mitochondrial membrane cardiolipin. The resistance of Bak-deficient cells to GrB/Ad cytotoxicity was reversed by transduction of the Bak gene into these cells. The requirement for both Bid and Bak, was further demonstrated in a cell-free system using purified mitochondria and S-100 cytosol. Purified mitochondria from Bid knockout mice, but not from Bax knockout mice, failed to release cytochrome c in response to autologous S-100 and GrB. Also, Bak-deficient mitochondria did not release cytochrome c in response to GrB-treated cytosol unless recombinant Bak protein was added. These results are the first to report a role for Bak in GrB-mediated mitochondrial apoptosis. This study demonstrates that GrB-cleaved Bid, which differs in size and site of cleavage from caspase-8-cleaved Bid, utilizes Bak for cytochrome c release, and therefore, suggests that deficiency in Bak may serve as a mechanism of immune evasion for tumor or viral infected cells.

Show MeSH
Related in: MedlinePlus