title: Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis.
abstract: Although the commonly activated death protease caspase-3 appears not to be essential for apoptosis during development except in the brain, it was not shown whether substrates known to be cleaved by caspase-3 are still proteolyzed in its absence. We have addressed this question with MCF-7 breast carcinoma cells that we recently showed lack caspase-3 owing to the functional deletion of the CASP-3 gene. Tumor necrosis factor- or staurosporine-induced apoptosis of caspase-3-deficient MCF-7 cells resulted in cleavage of the death substrates PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45, but not alpha-fodrin. In contrast, all these substrates including alpha-fodrin were cleaved in apoptotic HeLa cells expressing caspase-3. Introduction of CASP-3 cDNA, but not CASP-10 cDNA, into MCF-7 cells restored alpha-fodrin cleavage. In addition, tumor necrosis factor- or staurosporine-induced apoptosis of MCF-7 cells stably expressing pro-caspase-3 also resulted in alpha-fodrin cleavage. Although the specific caspase inhibitory peptides Z-VAD-fmk and Z-DEVD-fmk prevented apoptosis of MCF-7 cells, we were unable to detect activation of caspases 2 and 7, which are known to be inhibited by Z-DEVD-fmk. Together our results suggest that caspase-3 is essential for cleavage of alpha-fodrin, but dispensable for the cleavage of PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45 and imply that one or more caspases other than caspases 2, 3, and 7 is activated and plays a crucial role in the cleavage of these substrates in MCF-7 cells.
authors.affiliation: Institute of Molecular and Cell Biology, The National University of Singapore, 30 Medical Dr., Singapore 117609, Republic of Singapore. mcbrj@imcb.nus.edu.sg
authors.forename: R U
authors.initials: RU
authors.lastname: Jänicke
authors.forename: P
authors.initials: P
authors.lastname: Ng
authors.forename: M L
authors.initials: ML
authors.lastname: Sprengart
authors.forename: A G
authors.initials: AG
authors.lastname: Porter
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D013049:Spectrin
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D002352:Carrier Proteins; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D008840:Microfilament Proteins; D009842:Oligopeptides; D011065:Poly(ADP-ribose) Polymerases; D016160:Retinoblastoma Protein; D019311:Staurosporine; D014409:Tumor Necrosis Factor-alpha; D013049:Spectrin; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse