Investigation of events committing cells to death revealed that a concealed NH2-terminal epitope of the pro-apoptotic protein Bak became exposed in vivo before apoptosis. This occurred after treatment of human Jurkat or CEM-C7A T-lymphoma cells with the mechanistically disparate agents staurosporine, etoposide or dexamethasone. The rapid, up to 10-fold increase in Bak-associated immunofluorescence was measured with epitope-specific monoclonal antibodies using flow cytometry and microscopy. In contrast, using a polyclonal antibody to Bak, immunofluorescence was detected both before and after treatment. There were no differences in Bak protein content nor in subcellular location before or after treatment. Immunofluorescence showed Bcl-xL and Bak were largely associated with mitochondria and in untreated cells they coimmunoprecipitated in the presence of nonioinic detergent. This association was significantly decreased after cell perturbation suggesting that Bcl-xL dissociation from Bak occurred on exposure of Bak's NH2 terminus. Multiple forms of Bak protein were observed by two dimensional electrophoresis but these were unchanged by inducers of apoptosis. This indicated that integration of cellular damage signals did not take place directly on the Bak protein. Release of proteins, including Bcl-xL, from Bak is suggested to be an important event in commitment to death.

Caspases are cysteine proteases involved in apoptosis and cytokine maturation. In erythroblasts, keratinocytes, and lens epithelial cells undergoing differentiation, enucleation has been regarded as a caspase-mediated incomplete apoptotic process. Here, we show that several caspases are activated in human peripheral blood monocytes whose differentiation into macrophages is induced by macrophage colony-stimulating factor (M-CSF). This activation is not associated with cell death and cannot be detected in monocytes undergoing dendritic cell differentiation in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanisms and consequences of caspase activation were further studied in U937 human monocytic cells undergoing phorbol ester-induced differentiation into macrophages. Differentiation-associated caspase activation involves the release of cytochrome c from the mitochondria and leads to the cleavage of the protein acinus while the poly(ADP-ribose)polymerase remains uncleaved. Inhibition of caspases by either exposure to the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-DL -Asp-fluoromethylketone (z-VAD-fmk) or expression of the p35 baculovirus inhibitory protein or overexpression of Bcl-2 inhibits the differentiation process. In addition, z-VAD-fmk amplifies the differentiation-associated production of radical oxygen species in both phorbol ester-differentiated U937 cells and M-CSF-treated monocytes, shifting the differentiation process to nonapoptotic cell death. Altogether, these results indicate that caspase activation specifically contributes to the differentiation of monocytes into macrophages, in the absence of cell death. IntroductionA family of cysteine proteases known as caspases plays a central role in many forms of apoptosis. 1 These enzymes are synthesized as inactive zymogens that must be cleaved after conserved aspartate residues to be activated. Two main pathways were shown to trigger caspase activation in cells undergoing apoptosis. 2 Schematically, the intrinsic pathway involves the disruption of the outer mitochondrial membrane barrier function, thus permitting the release of proapoptotic molecules from the mitochondria to the cytosol. One of these molecules is cytochrome c, which, once in the cytosol, oligomerizes the adaptor molecule Apaf-1 to recruit and activate the initiator caspase-9. In turn, caspase-9 cleaves and activates downstream effector enzymes such as caspase-3. The extrinsic pathway to cell death involves plasma membrane death receptors. In response to their engagement, these receptors trimerize and recruit the adaptor molecule Fas-associated death domain protein (FADD), which, in turn, interacts with and activates an initiator enzyme, usually caspase-8. This enzyme, either directly or through the previously described mitochondrial pathway, activates downstream effector enzymes including caspase-3. 3 In both pathways, effector caspases trigger the limited proteolytic cleavage of intracellular structural and regulatory p...

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Inhibitors of apoptosis proteins (IAPs) are a conserved family of proteins identified in species ranging from virus, yeasts, nematodes, fishes, flies and mammals. The common structural feature is the presence of at least one Baculovirus IAP Repeat (BIR) domain. Hence, IAPs are also known as BIR-containing proteins (BIRCs). Most of them display anti-apoptotic properties when overexpressed. In drosophila, IAPs are sufficient and necessary to promote cell survival through a direct regulation of apoptotic proteases called caspases. In mammals, BIRC4/XIAP, the most studied IAP member can directly inhibit the activity of caspase-3, 7 and 9. However, this activity is not conserved in other IAPs and physiological relevancies of such anti-caspase activities are still discussed. A detailed analysis of IAP-deficient mice or derived cells, deletion experiments performed in drosophila and zebrafish, or research of protein partners have revealed the importance of IAPs in adaptive response to cellular stress, in cell proliferation, differentiation, signaling, motility and in immune response. This review discusses recent data that help understanding of cellular functions of IAPs.

The inhibitors of apoptosis (IAPs) constitute a family of proteins involved in the regulation of various cellular processes, including cell death, immune and inflammatory responses, cell proliferation, cell differentiation, and cell motility. There is accumulating evidence supporting IAP-targeting in tumors: IAPs regulate various cellular processes that contribute to tumor development, such as cell death, cell proliferation, and cell migration; their expression is increased in a number of human tumor samples, and IAP overexpression has been correlated with tumor growth, and poor prognosis or low response to treatment; and IAP expression can be rapidly induced in response to chemotherapy or radiotherapy because of the presence of an internal ribosome entry site (IRES)-dependent mechanism of translation initiation, which could contribute to resistance to antitumor therapy. The development of IAP antagonists is an important challenge and was subject to intense research over the past decade. Six molecules are currently in clinical trials. This review focuses on the role of IAPs in tumors and the development of IAP-targeting molecules for anticancer therapy.

Inhibitors of Apoptosis (IAPs) are a family of proteins with various biological functions including regulation of innate immunity and inflammation, cell proliferation, cell migration and apoptosis. They are characterized by the presence of at least one N-terminal baculoviral IAP repeat (BIR) domain involved in protein-protein interaction. Most of them also contain a C-terminal RING domain conferring an E3-ubiquitin ligase activity. In drosophila, IAPs are essential to ensure cell survival, preventing the uncontrolled activation of the apoptotic protease caspases. In mammals, IAPs can also regulate apoptosis through controlling caspase activity and caspase-activating platform formation. Mammalian IAPs, mainly X-linked IAP (XIAP) and cellular IAPs (cIAPs) appeared to be important determinants of the response of cells to endogenous or exogenous cellular injuries, able to convert the survival signal into a cell death-inducing signal. This review highlights the role of IAP in regulating apoptosis in Drosophila and Mammals.

We investigated the role of proteases in the pathway that leads from specific DNA damage induced by etoposide (VP‐16), a topoisomerase II inhibitor, to apoptotic DNA fragmentation in the U937 human leukemic cell line. In a reconstituted cell‐free system, Triton‐soluble extracts from VP‐16‐treated cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This effect was inhibited by the tetrapeptide Ac‐DEVD‐CHO, a competitive inhibitor of the interleukin‐1 beta‐converting enzyme (ICE)‐related protease CPP32, but was not influenced by Ac‐YVAD‐CHO and Ac‐YVAD‐CMK, two specific inhibitors of ICE. The three tetrapeptides inhibited Fas‐mediated apoptotic DNA fragmentation in the cell‐free system. Internucleosomal DNA fragmentation, triggered by either VP‐16 or an anti‐Fas antibody, was associated with proteolytic cleavage of the poly(ADP‐ribose)polymerase (PARP), a decrease in the level of 32 kDa CPP32 proenzyme and the appearance of the CPP32 p17 active subunit. Conversely, the expression of Ich‐1L, another ICE‐like protease, remained stable in apoptotic U937 cells. Several cysteine and serine protease inhibitors prevented apoptotic DNA fragmentation by acting either upstream or downstream of the DEVD‐sensitive protease(s) activation and PARP cleavage. We conclude that a DEVD‐sensitive step, which could involve CPP32, plays a central role in the proteolytic pathway that mediates apoptotic DNA fragmentation in VP‐16‐treated leukemic cells at the crossing with Fas‐mediated pathway.

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