Gwyn T. Williams

Molecular regulation of apoptosis: Genetic controls on cell death

Cell death is now a very lively field. The study of active cellular self-destruction through apoptosis (Wyllie et al., 1980) has become very much part of the mainstream in cell biology, particularly in immunology, developmental biology, and oncology. The significance of the process is increasingly recognized both in physiological regulation and in pathological situations (reviewed by Williams et al., 1992). As the subject has been largely neglected for a long time, particularly at the molecular level, there is a lot of catching up to do. There are few, if any, other generally important aspects of cell behavior where there is so much fundamental work still to be done, which makes this a very interesting field to watch. It is now widely accepted that apoptosis is a genedirected process and can be seen, alongside more familiar gene-directed processes like differentiation, as part of the repertoire available to the cell to respond to external and internal stimuli (Figure 1). Until recently, most of the information available on the genetics of programed cell death was derived from studies on the nematode Caenorhabditis elegans (reviewed by Ellis et al., 1991) but now important information about the intracellular molecular signals involved in stimulation and suppression of apoptosis in mammalian cells is also emerging. Intracellular Inducers of Apoptosis The external signals that lead to apoptosis are probably as varied as those that lead to differentiation and proliferation (reviewed by Williams et al., 1992) and can include the withdrawal of extracellular signals as well as their appearance. Indeed, dependenceon essential survival factors for suppression of apoptosis appears to be very widespread (reviewed by Raff, 1992). In hemopoietic stem cells, the primary function of colony-stimulating factors appears to be suppression of apoptosis (Williams et al., 1990) and this effect allows an intrinsically determined pathway of differentiation to be followed (Fairbairn et al., 1993). In contrast, the stimulation of some cell surface molecules, such as the tumor necrosis factor receptor and the APO-l antigen (now known to be identical to the Fas antigen), can often induce cell death by apoptosis, although this is not always the outcome (Mapara et al., 1993). Several different second messenger systems have been associated with induction of apoptosis, and the final response varies with the cell type and the other signals being received. Consequently, in addition to genes exclusively involved in active cell death, there are likely to be some Minireview

Programmed cell death: Apoptosis and oncogenesis

The focus of molecular oncology is likely to remain on the control of proliferation, since this is where many oncogenes appear to act. However, there is now compelling evidence that the other side of equation, the rate of cell death, must also be considered. The growing appreciation of the importance of apoptosis and its regulation should lead to fundamental advances in the understanding and potentially also the treatment of cancer.

GAS5, a non-protein-coding RNA, controls apoptosis and is downregulated in breast cancer

Effective control of both cell survival and cell proliferation is critical to the prevention of oncogenesis and to successful cancer therapy. Using functional expression cloning, we have identified GAS5 (growth arrest-specific transcript 5) as critical to the control of mammalian apoptosis and cell population growth. GAS5 transcripts are subject to complex post-transcriptional processing and some, but not all, GAS5 transcripts sensitize mammalian cells to apoptosis inducers. We have found that, in some cell lines, GAS5 expression induces growth arrest and apoptosis independently of other stimuli. GAS5 transcript levels were significantly reduced in breast cancer samples relative to adjacent unaffected normal breast epithelial tissues. The GAS5 gene has no significant protein-coding potential but expression encodes small nucleolar RNAs (snoRNAs) in its introns. Taken together with the recent demonstration of tumor suppressor characteristics in the related snoRNA U50, our observations suggest that such snoRNAs form a novel family of genes controlling oncogenesis and sensitivity to therapy in cancer.

Apoptosis in the malaria protozoan, Plasmodium berghei: a possible mechanism for limiting intensity of infection in the mosquito

Death by apoptosis regulates cell numbers in metazoan tissues and it is mediated by activation of caspases and results in characteristic morphological and biochemical changes. We report here that the malaria protozoan, Plasmodium berghei, exhibits features typical of metazoan apoptotic cells including condensation of chromatin, fragmentation of the nuclear DNA and movement of phosphatidylserine from the inner to the outer lamellae of the cell membrane. In addition, proteins with caspase-like activity were identified in the cytoplasm of the ookinete suggesting that the cellular mechanism of cell death may be similar to that of multicellular eukaryotes. Our data show that more than 50% of the mosquito midgut stages of the parasite die naturally by apoptosis before gut invasion. Cell death was prevented by a caspase inhibitor, treatment resulting in a doubling of parasite intensity. All these features also occur in vitro. Cell suicide thus plays a major and hitherto unrecognised role in controlling parasite populations and could be a novel target for malaria control strategies.

Long non-coding RNA GAS5 regulates apoptosis in prostate cancer cell lines

While the role of small non-coding RNAs, such as miRNAs, in apoptosis control is well established, long non-coding RNAs (lncRNAs) have received less attention. Growth Arrest-Specific 5 (GAS5) encodes multiple snoRNAs within its introns, while exonic sequences produce lncRNA which can act as a riborepressor of the glucocorticoid and related receptors. GAS5 negatively regulates the survival of lymphoid and breast cells, and is aberrantly expressed in several cancers. Although cellular GAS5 levels decline as prostate cancer cells acquire castration-resistance, the influence of GAS5 on prostate cell survival has not been determined. To address this question, prostate cell lines were transfected with GAS5-encoding plasmids or GAS5 siRNAs, and cell survival was assessed. Basal apoptosis increased, and cell survival decreased, after transfection of 22Rv1 cells with plasmids encoding GAS5 transcripts, including mature GAS5 lncRNA. Similar effects were observed in PC-3 cells. In stable clones of 22Rv1, cell death correlated strongly with cellular GAS5 levels. Induction of 22Rv1 cell death by UV-C irradiation and chemotherapeutic drugs was augmented in cells transiently transfected with GAS5 constructs, and attenuated following down-regulation of GAS5 expression. Again, in these experiments, cell death was strongly correlated with cellular GAS5 levels. Thus, GAS5 promotes the apoptosis of prostate cells, and exonic sequence, i.e. GAS5 lncRNA, is sufficient to mediate this activity. Abnormally low levels of GAS5 expression may therefore reduce the effectiveness of chemotherapeutic agents. Although several lncRNAs have recently been shown to control cell survival, this is the first report of a death-promoting lncRNA in prostate cells.

Growth arrest in human T-cells is controlled by the non-coding RNA growth-arrest-specific transcript 5 (GAS5)

The control of growth of lymphocyte populations is crucial to the physiological regulation of the immune system, and to the prevention of both leukaemic and autoimmune disease. This control is mediated through modulation of the cell cycle and regulation of cell death. During log-phase growth the rate of proliferation is high and there is a low rate of cell death. As the population density increases, the cell cycle is extended and apoptosis becomes more frequent as the population enters growth arrest. Here, we show that growth-arrest-specific transcript 5 (GAS5) plays an essential role in normal growth arrest in both T-cell lines and non-transformed lymphocytes. Overexpression of GAS5 causes both an increase in apoptosis and a reduction in the rate of progression through the cell-cycle. Consistent with this, downregulation of endogenous GAS5 inhibits apoptosis and maintains a more rapid cell cycle, indicating that GAS5 expression is both necessary and sufficient for normal growth arrest in T-cell lines as well as human peripheral blood T-cells. Control of apoptosis and the cell cycle by GAS5 has significant consequences for disease pathogenesis, because independent studies have already identified GAS5 as an important candidate gene in the development of autoimmune disease.

Are snoRNAs and snoRNA host genes new players in cancer

Small nucleolar RNAs (snoRNAs) have long been considered important but unglamorous elements in the production of the protein synthesis machinery of the cell. Recently, however, several independent lines of evidence have indicated that these non-coding RNAs might have crucial roles in controlling cell behaviour, and snoRNA dysfunction could consequently contribute to oncogenesis in previously unsuspected ways.

CASPASE ACTIVITY IS REQUIRED FOR COMMITMENT TO FAS-MEDIATED APOPTOSIS

Recognition of the widespread importance of apoptosis has been one of the most significant changes in the biomedical sciences in the past decade. The molecular processes controlling and executing cell death through apoptosis are, however, still poorly understood. The ICE (Interleukin‐1β Converting Enzyme) family—recently named the caspases for cysteine aspartate‐specific proteases—plays a central role in apoptosis and may well constitute part of the conserved core mechanism of the process. Potentially, these proteases may be of great significance, both in the pathology associated with failure of apoptosis and also as targets for therapeutic intervention where apoptosis occurs inappropriately, e.g. in degenerative disease and AIDS. However, this is only likely if caspase activity is required before commitment to mammalian cell death. Here, we have used both peptide inhibitors and crmA transfection to inhibit these proteases in intact cells. Our experiments show that selective inhibition of some caspases protects human T cells (Jurkat and CEM‐C7) from Fas‐induced apoptosis, dramatically increasing their survival (up to 320‐fold) in a colony‐forming assay. This suggests that dysfunction of some, but not all, caspases could indeed play a crucial part in the development of some cancers and autoimmune disease, and also that these proteases could be appropriate molecular targets for preventing apoptosis in degenerative disease.

Apoptosis: final control point in cell biology.

The discovery of apoptosis, a widespread and morphologically distinct form of physiological cell death, has had an extraordinary impact on cell biology. The importance of apoptosis stems from its active nature and its potential for controlling biological systems. The growing appreciation of the significance of this process has stimulated intense investigation into the molecular mechanisms involved and into its fundamental implications for developmental biology, immunology and oncology.

Evidence for the involvement of cGMP and protein kinase G in nitric oxide-induced apoptosis in the pancreatic B-cell line, HIT-T15

Intracellular production of nitric oxide (NO) is thought to mediate the pancreatic B‐cell‐directed cytotoxicity of cytokines in insulin‐dependent diabetes mellitus, and recent evidence has indicated that this may involve induction of apoptosis. A primary effect of NO is to activate soluble guanylyl cyclase leading to increased cGMP levels and this effect has been demonstrated in pancreatic B‐cells, although no intracellular function has been defined for islet cGMP. Here we demonstrate that the NO donor, GSNO, induces apoptosis in the pancreatic B‐cell line HIT‐T15 in a dose‐ and time‐dependent manner. This response was significantly attenuated by micromolar concentrations of a specific inhibitor of soluble guanylyl cyclase, ODQ, and both 8‐bromo cGMP (100 μM) and dibutyryl cGMP (300 μM) were able to fully relieve this inhibition. In addition, incubation of HIT‐T15 cells with each cGMP analogue directly promoted cell death in the absence of ODQ. KT5823, a potent and highly selective inhibitor of cGMP‐dependent protein kinase (PKG), abolished the induction of cell death in HIT cells in response to either GSNO or cGMP analogues. This effect was dose‐dependent over the concentration range of 10–250 nM. Overall, these data provide evidence that the activation of apoptosis in HIT‐T15 cells by NO donors is secondary to a rise in cGMP and suggest that the pathway controlling cell death involves activation of PKG.