CASPASE-3 IS REQUIRED FOR ALPHA-FODRIN CLEAVAGE BUT DISPENSABLE FOR CLEAVAGE OF OTHER DEATH SUBSTRATES IN APOPTOSIS

Although the commonly activated death protease caspase-3 appears not t o be essential for apoptosis during development except in the brain, i t was not shown whether substrates known tea he cleaved by caspase-3 a re still proteolyzed in its absence, We have addressed this question w ith 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, DN A-PKcs, gelsolin, and DFF-45, but not alpha-fodrin. In contrast, all t hese 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-os staurosporine-induced apoptosis of MCF-7 cel ls stably expressing pso-caspase-3 also resulted in alpha-fodrin cleav age. 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 tee be inhibited by Z -DEVD-fmk, Together our results suggest that caspase-3 is essential fo r cleavage of Lu-fodrin, but dispensable for the cleavage of PARP, Rb, PAK2, DNA-PKcs, gelsolin, and DFF-45 and imply that one or more caspa ses other theta caspases 2, 3, and 7 is activated and plays a crucial role in the cleavage of these substrates in MCF-7 cells.