Apoptosis, an evolutionarily conserved form of cell suicide, requires specialized machinery. The central component of this machinery is a proteolytic system involving a family of proteases called caspases. These enzymes participate in a cascade that is triggered in response to proapoptotic signals and culminates in cleavage of a set of proteins, resulting in disassembly of the cell. Understanding caspase regulation is intimately linked to the ability to rationally manipulate apoptosis for therapeutic gain.

The protease responsible for the cleavage of poly(ADP-ribose) polymerase and necessary for apoptosis has been purified and characterized. This enzyme, named apopain, is composed of two subunits of relative molecular mass (M(r)) 17K and 12K that are derived from a common proenzyme identified as CPP32. This proenzyme is related to interleukin-1 beta-converting enzyme (ICE) and CED-3, the product of a gene required for programmed cell death in Caenorhabditis elegans. A potent peptide aldehyde inhibitor has been developed and shown to prevent apoptotic events in vitro, suggesting that apopain/CPP32 is important for the initiation of apoptotic cell death.

Recent studies suggest that proteases of the interleukin 1-beta-converting enzyme (ICE)/ced-3 family are involved in initiating the active phase of apoptosis. Here we identify a novel protease resembling ICE (prICE) that is active in a cell-free system that reproduces the morphological and biochemical events of apoptosis. prICE cleaves the nuclear enzyme poly(ADP-ribose) polymerase (PARP) at a tetrapeptide sequence identical to one of two ICE sites in pro-interleukin-1-beta. However, prICE does not cleave purified pro-interleukin-1-beta, and purified ICE does not cleave PARP, indicating that the two activities are distinct. Inhibition of prICE abolishes all manifestations of apoptosis in the extracts including morphological changes, cleavage of PARP and production of an oligonucleosomal ladder. These studies suggest that prICE might be pivotal in initiating the active phase of apoptosis in vitro and in intact cells.

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.

A current view is that cytotoxic stress, such as DNA damage, induces apoptosis by regulating the permeability of mitochondria. Mitochondria sequester several proteins that, if released, kill by activating caspases, the proteases that disassemble the cell. Cytokines activate caspases in a different way, by assembling receptor complexes that activate caspases directly; in this case, the subsequent mitochondrial permeabilization accelerates cell disassembly by amplifying caspase activity. We found that cytotoxic stress causes activation of caspase-2, and that this caspase is required for the permeabilization of mitochondria. Therefore, we argue that cytokine-induced and stress-induced apoptosis act through conceptually similar pathways in which mitochondria are amplifiers of caspase activity rather than initiators of caspase activation.

The idea that conversion of glucose to ATP is an attractive target for cancer therapy has been supported in part by the observation that glucose deprivation induces apoptosis in rodent cells transduced with the proto-oncogene MYC, but not in the parental line. Here, we found that depletion of glucose killed normal human cells irrespective of induced MYC activity and by a mechanism different from apoptosis. However, depletion of glutamine, another major nutrient consumed by cancer cells, induced apoptosis depending on MYC activity. This apoptosis was preceded by depletion of the Krebs cycle intermediates, was prevented by two Krebs cycle substrates, but was unrelated to ATP synthesis or several other reported consequences of glutamine starvation. Our results suggest that the fate of normal human cells should be considered in evaluating nutrient deprivation as a strategy for cancer therapy, and that understanding how glutamine metabolism is linked to cell viability might provide new approaches for treatment of cancer.

To identify human proteins that bind to the Smac and caspase-9 binding pocket on the baculoviral inhibitor of apoptosis protein (IAP) repeat 3 (BIR3) domain of human XIAP, we used BIR3 as an affinity reagent, followed by elution with the BIR3 binding peptide AVPIA, microsequencing, and mass spectrometry. The mature serine protease Omi (also known as HtrA2) was identified as a mitochondrial direct BIR3-binding protein and a caspase activator. Like mature Smac (also known as Diablo), mature Omi contains a conserved IAP-binding motif (AVPS) at its N terminus, which is exposed after processing of its N-terminal mitochondrial targeting sequence upon import into the mitochondria. Mature Omi is released together with mature Smac from the mitochondria into the cytosol upon disruption of the outer mitochondrial membrane during apoptosis. Finally, mature Omi can induce apoptosis in human cells in a caspase-independent manner through its protease activity and in a caspase-dependent manner via its ability to disrupt caspase-IAP interaction. Our results provide clear evidence for the involvement of a mitochondrial serine protease in the apoptotic pathway, emphasizing the critical role of the mitochondria in cell death.

Although proteases related to the interleukin 1,8-converting enzyme (ICE) are known to be essential for apoptotic execution, the number of enzymes involved, their substrate specificities, and their specific roles in the characteristic biochemical and morphological changes of apoptosis are currently unknown. These questions were addressed using cloned recombinant ICE-related proteases (IRPs) and a cellfree model system for apoptosis (S/M extracts A key question in cell death research is whether the apoptotic cascade is driven by the action of a single interleukin 1l3-converting enzyme (ICE)-related protease (IRP) (1-7) or by multiple IRPs acting in concert (8). In the nematode Caenorhabditis elegans, a single IRP is required for all developmental cell deaths (1, 9). In contrast, cDNA cloning experiments show that at least seven IRP mRNAs are expressed in a single human cell type (4, 7, 10, 11), raising the possibility that multiple IRPs might be required for completion of apoptosis in vertebrates. The individual roles of these multiple IRPs during apoptosis are currently unclear.To begin to address this question, we have compared proteolytic cleavage of two apoptotic substrates by cloned IRPs expressed in Escherichia coli and by cell-free extracts (named S/M extracts, prepared from chicken DU249 hepatoma cells committed to apoptosis by an S-phase aphidocolin block and subsequently collected in M phase) (12, 13). Exogenous nuclei incubated in S/M extracts recapitulate nuclear apoptotic events, including endonucleolytic cleavage of DNA, chromatin condensation, and fragmentation of the nucleus (12). Incubation of nuclei or purified poly(ADP-ribose) polymerase (PARP) in S/M extracts results in rapid, quantitative cleavage of the PARP to a 85-kDa fragment indistinguishable from that observed in a wide variety of apoptotic cells (13)(14)(15). This cleavage occurs at a conserved DEVDUG sequence and is mediated by an enzyme with substrate recognition properties and inhibitor sensitivity similar to ICE. We termed this proteolytic activity prICE [protease(s) resembling ICE (13)]. Subsequent investigations have shown that the cloned human IRPs CPP32, Mch2a, and Mch3a as well as the C. elegans IRP CED-3 all cleave PARP (5,7,11,16,17). ICE itself can also cleave a PARP subfragment when added in considerable excess (18); however, at near physiological levels, it does not cleave full-length native PARP (5, 13).Although PARP was the first apoptosis-specific IRP substrate to be identified, the physiological significance of PARP cleavage in apoptosis is presently unknown (for review, see ref.15). In contrast, cleavage of the nuclear lamins is a proteolytic event that appears to be required for completion of nuclear reorganization during apoptosis. Lamin A is cleaved in S/M extracts (8) to fragments that are indistinguishable from those produced in cells undergoing apoptosis (8,(19)(20)(21). The inhibitor profile of the lamin protease suggests that lamin cleavage depends upon the activity of an IRP distinct from the PARPcle...