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.

Caspase-8 plays an essential role in apoptosis triggered by death receptors. Through the cleavage of Bid, a proapoptotic Bcl-2 member, it further activates the mitochondrial cytochrome c/Apaf-1 pathway. Because caspase-8 can be processed also by anticancer drugs independently of death receptors, we investigated its exact role and order in the caspase cascade. We show that in Jurkat cells either de®cient for caspase-8 or overexpressing its inhibitor c-FLIP apoptosis mediated by CD95, but not by anticancer drugs was inhibited. In the absence of active caspase-8, anticancer drugs still induced the processing of caspase-9, -3 and Bid, indicating that Bid cleavage does not require caspase-8. Overexpression of Bcl-x L prevented the processing of caspase-8 as well as caspase-9, -6 and Bid in response to drugs, but was less e ective in CD95-induced apoptosis. Similar responses were observed by overexpression of a dominant-negative caspase-9 mutant. To further determine the order of caspase-8 activation, we employed MCF7 cells lacking caspase-3. In contrast to caspase-9 that was cleaved in these cells, anticancer drugs induced caspase-8 activation only in caspase-3 transfected MCF7 cells. Thus, our data indicate that, unlike its proximal role in receptor signaling, in the mitochondrial pathway caspase-8 rather functions as an amplifying executioner caspase.

Interleukin 1beta‐converting enzyme‐like (ICE‐like) proteases are important mediators of apoptosis in diverse cell types and organisms. However, the role of these proteases in apoptosis cannot be satisfactorily explained on the basis of the physiological functions of their known substrates. Here we show that the C‐terminal 42 amino acid peptide of the retinoblastoma (Rb) protein, an important cell cycle regulator with a known anti‐apoptotic function, is specifically cleaved off by an ICE‐like protease in tumour necrosis factor (TNF)‐ and staurosporine‐induced apoptosis. Cleavage of Rb induced by TNF was blocked in vivo and in vitro by two specific inhibitors of ICE‐like proteases, and in vitro by a point mutation (Asp886 to Ala) within the ICE‐like protease cleavage site of Rb, (883)DEAD(886). An antibody raised against the C‐terminal 15 amino acid peptide of Rb recognized the full‐length but not the cleaved form of Rb. The extent of Rb cleavage correlated directly with TNF‐induced apoptosis in all tumour cell lines examined. Cleaved Rb bound cyclin D3 and inhibited the transcriptional activity of E2F‐1, but failed to bind to the regulatory protein MDM2, which has been implicated in apoptosis. As Rb suppresses cell death and its C‐terminus has important regulatory functions, our results suggest that Rb cleavage is an important event in apoptosis.

The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1b in lipopolysaccharide (LPS)-primed monocytes and macrophages, the latter depending on caspase-1 activation by an unknown mechanism. Here, we investigate the pathway that triggers cell death and activates caspase-1. We show that without LPS priming, nigericin alone triggered caspase-1 activation and IL-18 generation in THP-1 monocytic cells. Simultaneously, nigericin induced caspase-1-independent necrotic cell death, which was blocked by the cathepsin B inhibitor CA-074-Me and other cathepsin inhibitors. Cathepsin B activation after nigericin treatment was determined biochemically and corroborated by rapid lysosomal leakage and translocation of cathepsin B to the cytoplasm. IL-18 maturation was prevented by both caspase-1 and cathepsin B inhibitors in THP-1 cells, primary mouse macrophages and human blood monocytes. Moreover, IL-18 generation was reduced in THP-1 cells stably transformed either with cystatin A (an endogenous cathepsin inhibitor) or antisense cathepsin B cDNA. Collectively, our study establishes a critical role for cathepsin B in nigericin-induced caspase-1-dependent IL-18 maturation and caspase-1-independent necrosis.

Based on high sequence homology, there are six members in the caspase-1 subfamily: caspases 1, 4, 5, and 13 in humans and caspases 1, 11, and 12 in mice. Only caspase-1 is known to activate interleukin-1␤ and interleukin-18, and caspase-11 activates pro-caspase-1 in vivo. Almost nothing is known about caspases 4, 5, and 13. Here we report a sensitive and specific polymerase chain reaction system to analyze closely related genes.We employed this system to analyze the gene expression and regulation of human caspases 1, 4, 5, and 13, demonstrating that they have different expression patterns in normal tissues and cell lines. Interferon-␥ strongly induced CASP1 and CASP5 but not CASP4 or CASP13 gene expression in HT-29 colon carcinoma cells. In contrast to the mRNA, interferon-␥ up-regulated caspase-1 but not caspase-5 protein. In the monocytic cell line THP-1, CASP1 mRNA and caspase-1 protein are expressed constitutively, and their levels were not increased by lipopolysaccharide, whereas both CASP5 mRNA and caspase-5 protein were induced by lipopolysaccharide. Caspase-1 subfamily members displayed different in vitro activities toward pro-caspases 1 and 3 and pro-interleukin-1␤. Our results demonstrate that caspase-1 and caspase-5 levels are modulated by interferon-␥ and lipopolysaccharide, respectively, and suggest that caspase-1 subfamily members are differentially regulated and may have distinct functions.

Tissue reoxygenation following hypoxia is associated with ischemia-reperfusion injury (IRI) and may signal the development of ischemic preconditioning, an adaptive state that is protective against subsequent IRI. Here we used microarray RNA analysis of in vivo and in vitro models of IRI to delineate the underlying molecular mechanisms. Microarray analysis of renal tissue after ischemia-reperfusion revealed a number of highly up-regulated antioxidant genes including aldehyde dehydrogenases (ALDH1A1 and ALDH1A7), glutathione S-transferases (GSTM5, GSTA2 and GSTP1), and NAD(P)H quinone oxidoreductase (NQO1). The transcription factor NF-E2-related factor-2 (Nrf2), a master regulator of this antioxidant response, is also elevated in IRI. Furthermore, microarray analysis of renal epithelial cells exposed to hypoxia/reoxygenation identified Nrf2 to be up-regulated on reoxygenation. We also reveal a reoxygenation-specific nuclear accumulation of Nrf2 protein and subsequent activation of a NQO1 promoter reporter construct. Attenuating reactive oxygen species (ROS) in reoxygenation using the antioxidant N-acetyl cysteine results in inhibition of Nrf-2 activation. mRNA levels for Nrf2-dependent genes were detected in human liver biopsy 1 h after transplantation. These results indicate that reoxygenation-dependent Nrf-2 activity facilitates ischemic preconditioning through the induction of antioxidant gene expression and that ROS may be critical in signaling this event.