Caspase-9 is activated in a cytochrome c-independent manner early during TNFalpha-induced apoptosis in murine cells

Cell Death Differ. 2003 Sep;10(9):1005-15. doi: 10.1038/sj.cdd.4401271.


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-x(L), fail to protect these cells against TNF-receptor-activated death. Bcl-x(L) expression delays, but does not totally block, the release of mitochondrial cytochrome c (cyt c) in these cells in response to TNFalpha-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-x(L) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Apoptosis*
  • Apoptotic Protease-Activating Factor 1
  • B-Lymphocytes / cytology
  • Caspase 8
  • Caspase 9
  • Caspases / chemistry
  • Caspases / metabolism*
  • Caspases / physiology
  • Cell Line, Tumor
  • Cytochromes c / metabolism*
  • Enzyme Activation
  • Kinetics
  • Mice
  • Mice, Knockout
  • Mitochondria / metabolism
  • Molecular Sequence Data
  • Proteins / genetics
  • Proto-Oncogene Proteins c-bcl-2 / physiology
  • RNA Interference
  • Receptors, Tumor Necrosis Factor / metabolism
  • Sequence Alignment
  • Stem Cells / cytology
  • Stem Cells / drug effects
  • Stem Cells / metabolism
  • Tumor Necrosis Factor-alpha / toxicity*
  • bcl-X Protein


  • Apaf1 protein, mouse
  • Apoptotic Protease-Activating Factor 1
  • Bcl2l1 protein, mouse
  • Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Receptors, Tumor Necrosis Factor
  • Tumor Necrosis Factor-alpha
  • bcl-X Protein
  • Cytochromes c
  • Casp8 protein, mouse
  • Casp9 protein, mouse
  • Caspase 8
  • Caspase 9
  • Caspases