Limits...
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.

Show MeSH

Related in: MedlinePlus

Restoration of apoptotic response to GrB after Bak gene transduction. (A) Bak expression, caspase-3 activation and PARP cleavage after adenoviral-mediated transduction of Bak. 24 h after infection of Bak-deficient cells with the indicated combinations of adenoviral vectors, cell extracts were assessed by immunoblotting for Bak expression. The membrane was stripped and reprobed for caspase-3 activation, stripped again, and reprobed with anti-PARP mAb. The p20 caspase-3 subunit in LacZ transduced cells represents the direct cleavage of caspase-3 by GrB. (B) Susceptibility of Bak-deficient cells to GrB 24 h after adenoviral transduction with Bak, but not with LacZ gene. The presence of apoptotic cells was assessed by annexin V staining of wild-type or Bak-deficient cells infected with the indicated combinations of adenoviral vectors and treated with GrB (1 μg/ml). Percentage of annexin V-positive cells is indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195982&req=5

fig3: Restoration of apoptotic response to GrB after Bak gene transduction. (A) Bak expression, caspase-3 activation and PARP cleavage after adenoviral-mediated transduction of Bak. 24 h after infection of Bak-deficient cells with the indicated combinations of adenoviral vectors, cell extracts were assessed by immunoblotting for Bak expression. The membrane was stripped and reprobed for caspase-3 activation, stripped again, and reprobed with anti-PARP mAb. The p20 caspase-3 subunit in LacZ transduced cells represents the direct cleavage of caspase-3 by GrB. (B) Susceptibility of Bak-deficient cells to GrB 24 h after adenoviral transduction with Bak, but not with LacZ gene. The presence of apoptotic cells was assessed by annexin V staining of wild-type or Bak-deficient cells infected with the indicated combinations of adenoviral vectors and treated with GrB (1 μg/ml). Percentage of annexin V-positive cells is indicated.

Mentions: Using a binary adenoviral vector system to avoid the toxic effects of Ad/Bak on the 293 packaging cells, we successfully produced large amounts of Ad/GT-Bak, whose gene product (Bak) was under the transcriptional control of the GT promoter and GV16 fusion protein (30, 31). The binary adenoviral LacZ vector system (Ad/GT-LacZ plus Ad/GV16) was used to determine transduction efficiency. As detected by immunoblotting, Bak expression was induced when Ad/GT-Bak plus Ad/GV16 were administered, but not when Ad/GT-LacZ plus Ad/GV16 were used (Fig. 3 A). High level expression of Bak has been reported to induce a rapid cell death (39). As assessed by flow cytometry of cells stained with FITC-conjugated annexin V, we also observed a substantial level of apoptotic cell death (∼35%) in both wild-type and Bak-deficient Jurkat cells 24 h after transduction with Ad/GT-Bak plus Ad/GV16 vectors, but not in mock infected cells (Fig. 3 B). Whereas Bak-deficient cells were resistant to GrB-mediated apoptosis, a significant increase in susceptibility (up to ∼90%) was detected in cells transduced with the Bak gene, but not in control cells transduced with LacZ (Fig. 3 B). Apoptosis was also confirmed by detection of active caspase-3 subunits and PARP cleavage in Bak-deficient cells infected with Bak, but not in mock-infected cells similarly treated (Fig. 3 A). Increased loss in prodomain caspase-3 or PARP was detected in cells infected with Bak and treated with GrB. Interestingly, the p20 subunit of caspase-3, but not the p19 or p17 subunits, was also detected in LacZ transduced cells treated with GrB. This subunit represents the direct cleavage of caspase-3 by GrB (6, 40). However, the partially activated caspase-3 was not sufficient to induce apoptosis, as assessed by annexin V staining (Fig. 3 B) or PARP cleavage (Fig. 3 A). These results confirm the role of Bak deficiency in the observed resistance of these cells to GrB/Ad-mediated apoptosis.


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)

Restoration of apoptotic response to GrB after Bak gene transduction. (A) Bak expression, caspase-3 activation and PARP cleavage after adenoviral-mediated transduction of Bak. 24 h after infection of Bak-deficient cells with the indicated combinations of adenoviral vectors, cell extracts were assessed by immunoblotting for Bak expression. The membrane was stripped and reprobed for caspase-3 activation, stripped again, and reprobed with anti-PARP mAb. The p20 caspase-3 subunit in LacZ transduced cells represents the direct cleavage of caspase-3 by GrB. (B) Susceptibility of Bak-deficient cells to GrB 24 h after adenoviral transduction with Bak, but not with LacZ gene. The presence of apoptotic cells was assessed by annexin V staining of wild-type or Bak-deficient cells infected with the indicated combinations of adenoviral vectors and treated with GrB (1 μg/ml). Percentage of annexin V-positive cells is indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195982&req=5

fig3: Restoration of apoptotic response to GrB after Bak gene transduction. (A) Bak expression, caspase-3 activation and PARP cleavage after adenoviral-mediated transduction of Bak. 24 h after infection of Bak-deficient cells with the indicated combinations of adenoviral vectors, cell extracts were assessed by immunoblotting for Bak expression. The membrane was stripped and reprobed for caspase-3 activation, stripped again, and reprobed with anti-PARP mAb. The p20 caspase-3 subunit in LacZ transduced cells represents the direct cleavage of caspase-3 by GrB. (B) Susceptibility of Bak-deficient cells to GrB 24 h after adenoviral transduction with Bak, but not with LacZ gene. The presence of apoptotic cells was assessed by annexin V staining of wild-type or Bak-deficient cells infected with the indicated combinations of adenoviral vectors and treated with GrB (1 μg/ml). Percentage of annexin V-positive cells is indicated.
Mentions: Using a binary adenoviral vector system to avoid the toxic effects of Ad/Bak on the 293 packaging cells, we successfully produced large amounts of Ad/GT-Bak, whose gene product (Bak) was under the transcriptional control of the GT promoter and GV16 fusion protein (30, 31). The binary adenoviral LacZ vector system (Ad/GT-LacZ plus Ad/GV16) was used to determine transduction efficiency. As detected by immunoblotting, Bak expression was induced when Ad/GT-Bak plus Ad/GV16 were administered, but not when Ad/GT-LacZ plus Ad/GV16 were used (Fig. 3 A). High level expression of Bak has been reported to induce a rapid cell death (39). As assessed by flow cytometry of cells stained with FITC-conjugated annexin V, we also observed a substantial level of apoptotic cell death (∼35%) in both wild-type and Bak-deficient Jurkat cells 24 h after transduction with Ad/GT-Bak plus Ad/GV16 vectors, but not in mock infected cells (Fig. 3 B). Whereas Bak-deficient cells were resistant to GrB-mediated apoptosis, a significant increase in susceptibility (up to ∼90%) was detected in cells transduced with the Bak gene, but not in control cells transduced with LacZ (Fig. 3 B). Apoptosis was also confirmed by detection of active caspase-3 subunits and PARP cleavage in Bak-deficient cells infected with Bak, but not in mock-infected cells similarly treated (Fig. 3 A). Increased loss in prodomain caspase-3 or PARP was detected in cells infected with Bak and treated with GrB. Interestingly, the p20 subunit of caspase-3, but not the p19 or p17 subunits, was also detected in LacZ transduced cells treated with GrB. This subunit represents the direct cleavage of caspase-3 by GrB (6, 40). However, the partially activated caspase-3 was not sufficient to induce apoptosis, as assessed by annexin V staining (Fig. 3 B) or PARP cleavage (Fig. 3 A). These results confirm the role of Bak deficiency in the observed resistance of these cells to GrB/Ad-mediated apoptosis.

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