Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type-or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made.

Interleukin 1␤-converting enzyme-like proteases (caspases) are crucial components of cell death pathways. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the requirement for caspase-3 in apoptosis, we took advantage of the MCF-7 breast carcinoma cell line, which we show here has lost caspase-3 owing to a 47-base pair deletion within exon 3 of the CASP-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Although MCF-7 cells were still sensitive to tumor necrosis factor (TNF)-or staurosporineinduced apoptosis, no DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing. Introduction of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF-or staurosporine-induced apoptosis, it is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis.Apoptosis (programmed cell death) is typically accompanied by the activation of a class of death proteases (caspases) and widespread biochemical and morphological changes to the cell (1, 2). These changes almost invariably involve chromatin condensation and its margination at the nuclear periphery, extensive double-stranded DNA fragmentation, and cellular shrinkage and blebbing (3-5). However, apoptosis can also occur in the absence of DNA fragmentation (6 -9). Recently, it has been demonstrated that a caspase activates an endonuclease (CAD) 1 responsible for fragmentation of the DNA at the linker region between nucleosomes by specifically cleaving and inactivating ICAD (DFF45), the inhibitor of CAD (9 -11).There is evidence that caspases contribute to the drastic morphological changes of apoptosis by proteolysing and disabling a number of key substrates, including the structural proteins gelsolin, PAK2, focal adhesion kinase, and rabaptin-5 (12-15). The most commonly activated caspase (caspase-3) can mediate the limited proteolysis of these proteins, as well as the cleavage inactivation of DNA fragmentation factor (DFF45; ICAD) (9 -11). Because there are several caspase-3-like proteases (1, 2), it is not known if caspase-3 is required in vivo for breakdown of DNA or cleavage of any of the proteins involved in maintaining cellular architecture. Here we show that MCF-7 carcinoma cells, which can be killed by apoptotic stimuli without DNA fragmentation and many of the hallmarks of apoptosis (7), are devoid of caspase-3 owing to a functional deletion in the CASP-3 gene. This has enabled us to address the question of whether caspase-3 is essential for double-strande...

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-PK cs , gelsolin, and DFF-45, but not ␣-fodrin. In contrast, all these substrates including ␣-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 ␣-fodrin cleavage. In addition, tumor necrosis factor-or staurosporine-induced apoptosis of MCF-7 cells stably expressing pro-caspase-3 also resulted in ␣-fodrin cleavage. Although the specific caspase inhibitory peptides Z-VADfmk 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 ␣-fodrin, but dispensable for the cleavage of PARP, Rb, PAK2, DNA-PK cs , 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.