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Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.

Sutton VR, Davis JE, Cancilla M, Johnstone RW, Ruefli AA, Sedelies K, Browne KA, Trapani JA - J. Exp. Med. (2000)

Bottom Line: However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis.These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2.Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia.

ABSTRACT
The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

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Loss of mitochondrial membrane depolarization in response to granzyme B is independent of caspases. Loss of rhodamine 123 (Rh123) accumulation in Jurkat cells after exposure to granzyme B (GrB, 60 nM) and sublytic PLO (P) for 2 h. The same cell samples were also analyzed for DNA fragmentation by TUNEL and for changes in forward and side scatter characteristics by flow cytometry at 2 h. Before commencement of the experiment, the cells were preincubated with the caspase inhibitors z-DEVD-fmk or z-VAD-fmk, or with the control compound z-FA-fmk (each 100 μM) for 30 min at 37°C. The numerals indicate the percentage of rhodamine-positive or -negative cells, or TUNEL-positive cells, to the nearest integer.
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Figure 10: Loss of mitochondrial membrane depolarization in response to granzyme B is independent of caspases. Loss of rhodamine 123 (Rh123) accumulation in Jurkat cells after exposure to granzyme B (GrB, 60 nM) and sublytic PLO (P) for 2 h. The same cell samples were also analyzed for DNA fragmentation by TUNEL and for changes in forward and side scatter characteristics by flow cytometry at 2 h. Before commencement of the experiment, the cells were preincubated with the caspase inhibitors z-DEVD-fmk or z-VAD-fmk, or with the control compound z-FA-fmk (each 100 μM) for 30 min at 37°C. The numerals indicate the percentage of rhodamine-positive or -negative cells, or TUNEL-positive cells, to the nearest integer.

Mentions: As Bid is processed directly by granzyme B, we predicted that cleaved Bid should be capable of inducing mitochondrial perturbation without a requirement for caspase activation. Therefore, we measured changes in mitochondrial membrane potential under conditions in which caspases were either active or inhibited by fmk compounds (Fig. 10). Consistent with our previous results 18, preincubation of Jurkat cells in z-VAD-fmk or DEVD-fmk reduced DNA fragmentation in response to granzyme B and porin to background levels, confirming that caspases had been blocked. However, neither z-VAD-fmk nor DEVD-fmk had a significant influence on the loss of mitochondrial membrane potential (measured as flux of rhodamine 123 from the cells) in the same cell samples compared with the control inhibitor z-FA-fmk, an inhibitor of chymotrypsin-like proteases. Consistent with our findings that Bcl-2 can abrogate apoptotic changes in response to granzyme B and lead to long-term cell survival, Bcl-2 overexpression in Jurkat cells also inhibited the loss of mitochondrial membrane potential 24. Thus, prior caspase activation is not necessary for mitochondrial perturbation triggered by granzyme B, and this function can be achieved by cleaved Bid, independently of caspases.


Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation.

Sutton VR, Davis JE, Cancilla M, Johnstone RW, Ruefli AA, Sedelies K, Browne KA, Trapani JA - J. Exp. Med. (2000)

Loss of mitochondrial membrane depolarization in response to granzyme B is independent of caspases. Loss of rhodamine 123 (Rh123) accumulation in Jurkat cells after exposure to granzyme B (GrB, 60 nM) and sublytic PLO (P) for 2 h. The same cell samples were also analyzed for DNA fragmentation by TUNEL and for changes in forward and side scatter characteristics by flow cytometry at 2 h. Before commencement of the experiment, the cells were preincubated with the caspase inhibitors z-DEVD-fmk or z-VAD-fmk, or with the control compound z-FA-fmk (each 100 μM) for 30 min at 37°C. The numerals indicate the percentage of rhodamine-positive or -negative cells, or TUNEL-positive cells, to the nearest integer.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193191&req=5

Figure 10: Loss of mitochondrial membrane depolarization in response to granzyme B is independent of caspases. Loss of rhodamine 123 (Rh123) accumulation in Jurkat cells after exposure to granzyme B (GrB, 60 nM) and sublytic PLO (P) for 2 h. The same cell samples were also analyzed for DNA fragmentation by TUNEL and for changes in forward and side scatter characteristics by flow cytometry at 2 h. Before commencement of the experiment, the cells were preincubated with the caspase inhibitors z-DEVD-fmk or z-VAD-fmk, or with the control compound z-FA-fmk (each 100 μM) for 30 min at 37°C. The numerals indicate the percentage of rhodamine-positive or -negative cells, or TUNEL-positive cells, to the nearest integer.
Mentions: As Bid is processed directly by granzyme B, we predicted that cleaved Bid should be capable of inducing mitochondrial perturbation without a requirement for caspase activation. Therefore, we measured changes in mitochondrial membrane potential under conditions in which caspases were either active or inhibited by fmk compounds (Fig. 10). Consistent with our previous results 18, preincubation of Jurkat cells in z-VAD-fmk or DEVD-fmk reduced DNA fragmentation in response to granzyme B and porin to background levels, confirming that caspases had been blocked. However, neither z-VAD-fmk nor DEVD-fmk had a significant influence on the loss of mitochondrial membrane potential (measured as flux of rhodamine 123 from the cells) in the same cell samples compared with the control inhibitor z-FA-fmk, an inhibitor of chymotrypsin-like proteases. Consistent with our findings that Bcl-2 can abrogate apoptotic changes in response to granzyme B and lead to long-term cell survival, Bcl-2 overexpression in Jurkat cells also inhibited the loss of mitochondrial membrane potential 24. Thus, prior caspase activation is not necessary for mitochondrial perturbation triggered by granzyme B, and this function can be achieved by cleaved Bid, independently of caspases.

Bottom Line: However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis.These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2.Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia.

ABSTRACT
The essential upstream steps in granzyme B-mediated apoptosis remain undefined. Herein, we show that granzyme B triggers the mitochondrial apoptotic pathway through direct cleavage of Bid; however, cleavage of procaspases was stalled when mitochondrial disruption was blocked by Bcl-2. The sensitivity of granzyme B-resistant Bcl-2-overexpressing FDC-P1 cells was restored by coexpression of wild-type Bid, or Bid with a mutation of its caspase-8 cleavage site, and both types of Bid were cleaved. However, Bid with a mutated granzyme B cleavage site remained intact and did not restore apoptosis. Bid with a mutation preventing its interaction with Bcl-2 was cleaved but also failed to restore apoptosis. Rapid Bid cleavage by granzyme B (<2 min) was not delayed by Bcl-2 overexpression. These results clearly placed Bid cleavage upstream of mitochondrial Bcl-2. In granzyme B-treated Jurkat cells, endogenous Bid cleavage and loss of mitochondrial membrane depolarization occurred despite caspase inactivation with z-Val-Ala-Asp-fluoromethylketone or Asp-Glu-Val-Asp-fluoromethylketone. Initial partial processing of procaspase-3 and -8 was observed irrespective of Bcl-2 overexpression; however, later processing was completely abolished by Bcl-2. Overall, our results indicate that mitochondrial perturbation by Bid is necessary to achieve a lethal threshold of caspase activity and cell death due to granzyme B.

Show MeSH
Related in: MedlinePlus