Original Paper
Cell Death and Differentiation (2003) 10, 1005–1015. doi:10.1038/sj.cdd.4401271
Caspase-9 is activated in a cytochrome c-independent manner early during TNF
-induced apoptosis in murine cells
M A McDonnell1, D Wang1, S M Khan1, M G Vander Heiden2,4 and A Kelekar1,3
- 1Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis MN 55455, USA
- 2Pritzker School of Medicine, University of Chicago, Chicago, IL 60637, USA
- 3Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
Correspondence: A Kelekar, Tel: 612-625-3204; Fax: 612-625-1121; E-mail: ameeta@umn.edu
4Present address: Brigham and Women's Hospital, Department of Medicine, Boston, MA 02115
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 TNF
-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.
Keywords:
apoptosis, caspase-9, caspase-8, cytochrome c release, death receptors
Abbreviations:
TNF-, tumor necrosis factor-alpha; CHX, cycloheximide; cyt c, cytochrome c; DISC, death-inducing signaling complex
