Caspase-3 Is Required for α-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-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.

MCF-7 breast carcinoma cells do not express caspase-3

“…Ten years ago, we demonstrated unambiguously that the lack of caspase-3 in these cells is caused by a 47-base pair deletion within exon 3 of the CASP-3 gene resulting in the skipping of this exon during premRNA splicing and introduction of a premature stop codon at position 42 that completely abrogates translation of the CASP-3 mRNA [17]. Although caspase-3-deficient MCF-7 cells are still sensitive to cell death induction by several stimuli including TNF, staurosporine [17,18] and various DNA damaging agents [19,20], death of these cells occurs in the absence of DNA fragmentation. In addition, the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing are also not evident in caspase-3-deficient MCF-7 cells.…”

Apoptotic mechanisms in T47D and MCF-7 human breast cancer cells

“…Caspase-3 has been shown to play a central role in key apoptotic events such as DNA fragmentation and membrane blebbing, and caspase-3 null mice die perinatally and display hypercellularity (Woo et al, 1998). MCF-7 breast cancer cells also lack caspase-3 protein, yet they remain responsive to many apoptotic stimuli suggesting functional redundancy within the caspase family (Janicke et al, 1998a). The present study was aimed at investigating the intracellular apoptotic signalling mechanisms in T47D (caspase-3 positive) and MCF-7 (caspase-3 negative) breast cancer cells in response to the general protein kinase inhibitor and apoptotic stimulus, staurosporine (STS).…”

Coupling caspase cleavage and ubiquitin–proteasome-dependent degradation of SSRP1 during apoptosis

“…Their combined molecular weight was approximately equal to the full-length protein (87 kDa), suggesting that SSRP1 is subjected to a single cleavage. Detection of PARP and DNA-PKcs, two known apoptotic targets, 25 showed a similar decrease of the full-length with appearance of shorter forms. In contrast, Spt16 levels did not decrease (data not shown).…”

“…25 Apoptosis was efficiently induced by DRB, UVC, MG132 and ALLN ( Figure 3b, lanes 2-5) as demonstrated by the cleavage of PARP. 25 However, SSRP1 was not reduced compared to untreated cells and no apoptotic fragments were detected ( Figure 3b, lanes 2-5). Taken together, these results suggest that SSRP1 is specifically cleaved by caspase 3 and/ or 7 in vivo.…”