Nigel J. Waterhouse

Tumor cell-selective cytotoxicity by targeting cell cycle checkpoints

Cell cycle checkpoints act to protect cells from external stresses and internal errors that would compromise the integrity of the cell. Checkpoints are often defective in cancer cells. Drugs that target checkpoint mechanisms should therefore be selective for tumor cells that are defective for the drug‐sensitive checkpoint. Histone deacetylase inhibitors typify this class of agents. They trigger a G2‐phase checkpoint response in normal cells but are cytotoxic in tumor cells in which this checkpoint is defective. In this study, we investigated the molecular basis of the tumor‐selective cytotoxicity of these drugs and demonstrated that it is due to the disruption of two cell cycle checkpoints. The first is the histone deacetylase inhibitor‐sensitive G2‐phase checkpoint, which is defective in drug‐sensitive cells and permits cells to enter an aberrant mitosis. The second is the drug‐dependent bypass of the mitotic spindle checkpoint that normally detects aberrant mitosis and blocks mitotic exit until the defect is rectified. The disruption of both checkpoints results in the premature exit of cells from an abortive mitosis followed by apoptosis. This study of histone deacetylase inhibitors demonstrates that drugs targeting cell cycle checkpoints can provide the selectivity and cytotoxicity desired in effective chemotherapeutic agents.

A new quantitative assay for cytochrome c release in apoptotic cells

Selective permeabilization of the mitochondrial outer membrane is an integral event in apoptosis induced by numerous stimuli. As a result, several proapoptotic proteins including cytochrome c are released from the mitochondrial intermembrane space to the cytoplasm. Cytochrome c then triggers activation of caspase proteases via the formation of a complex known as apoptosome. Current techniques to assay cytochrome c release rely on cellular fractionation followed by Western blotting, immunocytochemistry or the subcellular localization of GFP-tagged cytochrome c. These techniques have inherent problems that make it difficult to accurately quantitate the number of cells in which cytochrome c has translocated. In this letter, we highlight some of the problems associated with current methods to follow cytochrome c release (Table 1) and suggest an adaptation of current protocols to quantitate the number of cells with cytoplasmic cytochrome c in both adherent and nonadherent cell populations. Owing to the relative abundance of cytochrome c within cells (estimated between 0.5 and 5 mM in the intermembrane space), Western blotting of cellular fractions is a useful technique to observe gross changes in cytochrome c distribution within cells. It is of some concern however that, due to the abundance of cytochrome c, small changes in overall cellular distribution may appear substantial if only cytosolic fractions are assayed (Figure 1ai). Studies that assay both the mitochondrial and cytosolic fractions give a more representative analysis of the extent of cytochrome c release (Figure 1aii). Using Western blotting, it is often difficult to expose X-ray film such that the bands from control cell populations (often 5% apoptosis) and apoptotic populations (where most of the cells have cytoplasmic cytochrome c) are both within the linear range (i.e. quantifiable on a densitometer). It is therefore difficult to use Western blotting for accurate quantitative analysis. This problem is compounded by the fact that cytoplasmic cytochrome c may leak out of cells that have deviated to secondary necrosis (as often occurs several hours after the onset of apoptosis in long-term assays). It is also not ideal that Western blotting shows an averaged result from a population of cells. It is therefore not possible to determine whether all the cytochrome c is cytoplasmic in a small percentage of cells or all cells have partially redistributed their cytochrome c. Immunocytochemistry followed by fluorescence microscopy has been invaluable in quantitating the number of cells that have punctate or diffuse (released) distribution of cytochrome c. This data, however, is frequently not quantitated since counting the cells is laborious. Further, it is often difficult to determine whether cells in suspension or that have rounded up, have punctate or diffuse staining. Cells that have undergone secondary necrosis may have lost much of their staining and may be ignored, unless they are arbitrarily categorized as dead. Expression of GFP-tagged cytochrome has made it possible to follow cytochrome c redistribution within cells in real time. Using these cells in combination with selective permeabilization of the plasma membrane by digitonin, it is possible to use FACS analysis to rapidly determine the number of cells in which cyochrome c has translocated to the cytoplasm. This assay is based on the idea that permeabilization of cells will allow cytoplasmic cytochrome c-GFP to diffuse out of the cells. Cells with cytoplasmic cytochrome cGFP will therefore have less GFP fluorescence than cells with intact mitochondria. This method, however, is only useful for cells that express GFP-cytochrome c. We reasoned that this assay could be coupled with immunocytochemistry (outlined in Figure 1b and the method below) to quantitate the percentage of cells that have cytoplasmic cytochrome c in populations that do not express GFP-cytochrome c.

Granzyme M mediates a novel form of perforin-dependent cell death

Cell death is mediated by cytotoxic lymphocytes through various granule serine proteases released with perforin. The unique protease activity, restricted expression, and distinct gene locus of granzyme M suggested this enzyme might have a novel biological function or trigger a novel form of cell death. Herein, we demonstrate that in the presence of perforin, the protease activity of granzyme M rapidly and effectively induces target cell death. In contrast to granzyme B, cell death induced by granzyme M does not feature obvious DNA fragmentation, occurs independently of caspases, caspase activation, and perturbation of mitochondria and is not inhibited by overexpression of Bcl-2. These data raise the likelihood that granzyme M represents a third major and specialized perforin-dependent cell death pathway that plays a significant role in death mediated by NK cells.

HETERONUCLEAR RIBONUCLEOPROTEINS C1 AND C2, COMPONENTS OF THE SPLICEOSOME,ARE SPECIFIC TARGETS OF INTERLEUKIN 1BETA -CONVERTING ENZYME-LIKE PROTEASES IN APOPTOSIS

Apoptosis induced by a variety of agents results in the proteolytic cleavage of a number of cellular substrates by enzymes related to interleukin 1β-converting enzyme (ICE). A small number of substrates for these enzymes have been identified to date, including enzymes involved in DNA repair processes: poly(ADP-ribose) polymerase and DNA-dependent protein kinase. We describe here for the first time the specific cleavage of the heteronuclear ribonucleoproteins (hnRNPs) C1 and C2 in apoptotic cells induced to undergo apoptosis by a variety of stimuli, including ionizing radiation, etoposide, and ceramide. No cleavage was observed in cells that are resistant to apoptosis induced by ionizing radiation. Protease inhibitor data implicate the involvement of an ICE-like protease in the cleavage of hnRNP C. Using recombinant ICE-like proteases and purified hnRNP C proteins in vitro, we show that the C proteins are cleaved by Mch3α and CPP32 and, to a lesser extent, by Mch2α, but not by ICE, Nedd2, Tx, or the cytotoxic T-cell protease granzyme B. The results described here demonstrate that the hnRNP C proteins, abundant nuclear proteins thought to be involved in RNA splicing, belong to a critical set of protein substrates that are cleaved by ICE-like proteases during apoptosis.

p53 triggers apoptosis in oncogene-expressing fibroblasts by the induction of Noxa and mitochondrial Bax translocation.

AbstractThe mechanism of p53-dependent apoptosis is still only partly defined. Using early-passage embryonic fibroblasts (MEF) from wild-type (wt), p53−/− and bax−/− mice, we observe a p53-dependent translocation of Bax to the mitochondria and a release of mitochondrial Cytochrome c during stress-induced apoptosis. These events proceed independent of zVAD-inhibitable caspase activation, are not prevented by dominant negative FADD (DN-FADD), but are negatively regulated by Mdm-2. Bcl-xL expression prevents the release of mitochondrial Cytochrome c and apoptosis, but not Bax translocation. At a single-cell level, enforced expression of p53 is sufficient to induce Bax translocation and Cytochrome c release. Real-time RT-PCR analysis reveals a significant induction of RNA expression of Noxa and Bax in p53+/+, but not in p53−/− MEF. Noxa protein expression becomes detectable prior to Bax translocation, and downregulation of endogenous Noxa by RNA interference protects wt MEF against p53-dependent apoptosis. Hence, in oncogene-expressing MEF p53 induces apoptosis by BH3 protein-dependent caspase activation.

Asymmetric Cell Division of T Cells upon Antigen Presentation Uses Multiple Conserved Mechanisms

Asymmetric cell division is a potential means by which cell fate choices during an immune response are orchestrated. Defining the molecular mechanisms that underlie asymmetric division of T cells is paramount for determining the role of this process in the generation of effector and memory T cell subsets. In other cell types, asymmetric cell division is regulated by conserved polarity protein complexes that control the localization of cell fate determinants and spindle orientation during division. We have developed a tractable, in vitro model of naive CD8+ T cells undergoing initial division while attached to dendritic cells during Ag presentation to investigate whether similar mechanisms might regulate asymmetric division of T cells. Using this system, we show that direct interactions with APCs provide the cue for polarization of T cells. Interestingly, the immunological synapse disseminates before division even though the T cells retain contact with the APC. The cue from the APC is translated into polarization of cell fate determinants via the polarity network of the Par3 and Scribble complexes, and orientation of the mitotic spindle during division is orchestrated by the partner of inscuteable/G protein complex. These findings suggest that T cells have selectively adapted a number of evolutionarily conserved mechanisms to generate diversity through asymmetric cell division.

The MUC13 cell-surface mucin protects against intestinal inflammation by inhibiting epithelial cell apoptosis

Background and Aims The MUC13 transmembrane mucin is highly and constitutively expressed in the small and large intestine. Although MUC13 polymorphisms have been associated with human inflammatory bowel diseases and susceptibility to Escherichia coli infection in pigs, the biological functions of MUC13 are unknown. This study aimed to explore whether MUC13 modulates intestinal inflammation. Methods Muc13−/− mice were generated, phenotyped and challenged with the colitis-inducing agent, dextran sodium sulphate (DSS). Colitis was assessed by clinical symptoms and intestinal histopathology. Intestinal epithelial cell apoptosis and proliferation, macrophage infiltration and cytokine production were also quantified. Apoptosis of human LS513 intestinal epithelial cells in response to apoptotic agents, including DSS, was also measured, following knockdown of MUC13 with siRNA. Results Muc13−/− mice were viable, fertile and developed normally, with no spontaneous intestinal pathology except mild focal neutrophilic inflammation in the small and large intestines of old mice. In response to DSS challenge, Muc13−/− mice developed more severe acute colitis, as reflected by increased weight loss, rectal bleeding, diarrhoea and histological colitis scores compared with wild-type mice. Increased numbers of F4/80+ macrophages in inflamed mucosa of Muc13−/− mice were accompanied by increased expression of intestinal IL-1β and TNFα mRNA. Muc13−/− mice had significantly increased intestinal epithelial cell apoptosis within 3 days of DSS exposure. LS513 cells were more susceptible to DSS, actinomycin-D, ultraviolet irradiation and TRAIL-induced apoptosis when MUC13 was knocked down by siRNA. Conclusions These novel findings indicate a protective role for Muc13 in the colonic epithelium by inhibiting toxin-induced apoptosis and have important implications for intestinal infections, inflammatory diseases and the development of intestinal cancer.

Caspase-mediated Cleavage of the Ubiquitin-protein Ligase Nedd4 during Apoptosis

The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, γ-radiation, tumor necrosis factor-α, C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237↓ sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.

Role of Bid-induced mitochondrial outer membrane permeabilization in granzyme B-induced apoptosis

Cytotoxic lymphocytes (CL) induce death of their targets by granule exocytosis. During this process, enzymes contained within cytotoxic granules (granzymes) are delivered to the target cell where the enzymes trigger the cell death by cleaving specific substrates. Granzyme B is the only granzyme that has been shown to induce cell death by apoptosis, but the exact pathway by which this is achieved has been the subject of hot debate. Furthermore, several other death‐inducing granzymes have been identified; therefore, the exact contribution of granzyme B to CL‐induced death is unclear. In this study, we discuss our recent findings on granzyme B‐induced cell death and discuss the potential relevance of this pathway to CL‐induced death of viral‐infected and transformed cells.

Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis.

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C–null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8+ cytotoxic T lymphocyte (CTL) raised in cathepsin C–null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C–null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.