Abstract
FL5.12 pro-B lymphoma cells utilize the mitochondrial pathway to apoptosis in response to tumor necrosis factor (TNF) receptor occupation, yet high levels of the Bcl-2 family antiapoptotic protein, Bcl-xL, fail to protect these cells against TNF-receptor-activated death. Bcl-xL expression delays, but does not totally block, the release of mitochondrial cytochrome c (cyt c) in these cells in response to TNFα-induced apoptosis and caspase-9 is processed prior to mitochondrial cyt c release under these circumstances. Early processing of caspase-9 also occurred in Apaf-1 knockout murine fibroblasts in response to TNF-receptor occupation. A caspase-9-specific inhibitor was more effective in delaying the progression of apoptosis in the FL5.12 Bcl-xL cells than was an inhibitor specific to caspase-3. Furthermore, downregulation of caspase-9 levels by RNA interference resulted in partial protection of these cells against TNF-receptor-activated apoptosis, indicating that caspase-9 activation contributed to early amplification of the caspase cascade. Consistent with this, proteolytic processing of caspase-9 was observed prior to processing by caspase-3, suggesting that caspase-3 was not responsible for early caspase-9 activation. We show that murine caspase-9 is efficiently processed by active caspase-8 at SEPD, the motif at which caspase-9 autoprocesses following its recruitment to the apoptosome. Our results suggest that, in addition to processing procaspase-3 and the BH3 protein Bid, active caspase-8 can cleave and activate procaspase-9 in response to TNF receptor crosslinking in murine cells.
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Abbreviations
- TNF-α:
-
tumor necrosis factor-alpha
- CHX:
-
cycloheximide
- cyt c:
-
cytochrome c
- DISC:
-
death-inducing signaling complex
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Acknowledgements
The authors are grateful to Craig Thompson for stimulating discussions and for a critical reading of the manuscript. We also thank Manuel Melendez for excellent technical assistance. This work was supported by a grant from the Leukemia Research Fund.
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McDonnell, M., Wang, D., Khan, S. et al. Caspase-9 is activated in a cytochrome c-independent manner early during TNFα-induced apoptosis in murine cells. Cell Death Differ 10, 1005–1015 (2003). https://doi.org/10.1038/sj.cdd.4401271
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DOI: https://doi.org/10.1038/sj.cdd.4401271
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