Showing papers in "Cell Death & Differentiation in 2003"

Tumor necrosis factor signaling.

Abstract: A single mouse click on the topic tumor necrosis factor (TNF) in PubMed reveals about 50 000 articles providing one or the other information about this pleiotropic cytokine or its relatives. This demonstrates the enormous scientific and clinical interest in elucidating the biology of a molecule (or rather a large family of molecules), which began now almost 30 years ago with the description of a cytokine able to exert antitumoral effects in mouse models. Although our understanding of the multiple functions of TNF in vivo and of the respective underlying mechanisms at a cellular and molecular level has made enormous progress since then, new aspects are steadily uncovered and it appears that still much needs to be learned before we can conclude that we have a full comprehension of TNF biology. This review shortly covers some general aspects of this fascinating molecule and then concentrates on the molecular mechanisms of TNF signal transduction. In particular, the multiple facets of crosstalk between the various signalling pathways engaged by TNF will be addressed. Cell Death and Differentiation (2003) 10, 45–65. doi:10.1038/ sj.cdd.4401189

The CD95(APO-1/Fas) DISC and beyond

Abstract: CD95 (APO-1/Fas) is a prototype death receptor characterized by the presence of an 80 amino acid death domain in its cytoplasmic tail. This domain is essential for the recruitment of a number of signaling components upon activation by either agonistic anti-CD95 antibodies or cognate CD95 ligand that initiate apoptosis. The complex of proteins that forms upon triggering of CD95 is called the death-inducting signaling complex (DISC). The DISC consists of an adaptor protein and initiator caspases and is essential for induction of apoptosis. A number of proteins have been reported to regulate formation or activity of the DISC. This review discusses recent developments in this area of death receptor research.

Decision making by p53: life, death and cancer.

Abstract: The p53 tumor-suppressor plays a critical role in the prevention of human cancer. In the absence of cellular stress, the p53 protein is maintained at low steady-state levels and exerts very little, if any, effect on cell fate. However, in response to various types of stress, p53 becomes activated; this is reflected in elevated protein levels, as well as augmented biochemical capabilities. As a consequence of p53 activation, cells can undergo marked phenotypic changes, ranging from increased DNA repair to senescence and apoptosis. This review deals with the mechanisms that underlie the apoptotic activities of p53, as well as the complex interactions between p53 and central regulatory signaling networks. In p53-mediated apoptosis, the major role is played by the ability of p53 to transactivate specific target genes. The choice of particular subsets of target genes, dictated by covalent p53 modifications and protein-protein interactions, can make the difference between life and apoptotic death of a cell. In addition, transcriptional repression of antiapoptotic genes, as well as transcription-independent activities of p53, can also contribute to the apoptotic effects of p53. Regarding the crosstalk between p53 and signaling networks, this review focuses on the interplay between p53 and two pivotal regulatory proteins: beta-catenin and Akt/PKB. Both proteins can regulate p53 as well as be regulated by it. In addition, p53 interacts with the GSK-3beta kinase, which serves as a link between Akt and beta-catenin. This review discusses how the functional balance between these different interactions might dictate the likelihood of a given cell to become cancerous or be eliminated from the replicative pool, resulting in suppression of cancer.

Many cuts to ruin: a comprehensive update of caspase substrates

Abstract: Apoptotic cell death is executed by the caspase-mediated cleavage of various vital proteins. Elucidating the consequences of this endoproteolytic cleavage is crucial for our understanding of cell death and other biological processes. Many caspase substrates are just cleaved as bystanders, because they happen to contain a caspase cleavage site in their sequence. Several targets, however, have a discrete function in propagation of the cell death process. Many structural and regulatory proteins are inactivated by caspases, while other substrates can be activated. In most cases, the consequences of this gain-of-function are poorly understood. Caspase substrates can regulate the key morphological changes in apoptosis. Several caspase substrates also act as transducers and amplifiers that determine the apoptotic threshold and cell fate. This review summarizes the known caspase substrates comprising a bewildering list of more than 280 different proteins. We highlight some recent aspects inferred by the cleavage of certain proteins in apoptosis. We also discuss emerging themes of caspase cleavage in other forms of cell death and, in particular, in apparently unrelated processes, such as cell cycle regulation and cellular differentiation.

Apo2L/TRAIL and its death and decoy receptors.

Abstract: Apo2 ligand or tumour necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is one of the several members of the tumour necrosis factor (TNF) gene superfamily that induce apoptosis through engagement of death receptors (DRs). Apo2L/TRAIL interacts with an unusually complex receptor system of two DRs and three decoys. This protein has garnered intense interest as a potential candidate for cancer therapy because as a trimer it selectively induces apoptosis in many transformed cells but not in normal cells. While much of the early characterisation of Apo2L/TRAIL and its receptors relied on overexpression studies, recent work using untransfected cells has clarified how endogenous proteins transmit apoptotic signals from this ligand. In this review, we focus on the apoptotic signalling pathways stimulated by Apo2L/TRAIL and summarise what is known about its physiological role.

Dynamics of mitochondrial morphology in healthy cells and during apoptosis

Abstract: Mitochondria exist as dynamic networks that often change shape and subcellular distribution. The number and morphology of mitochondria within a cell are controlled by precisely regulated rates of organelle fusion and fission. Recent reports have described dramatic alterations in mitochondrial morphology during the early stages of apoptotic cell death, a fragmentation of the network and the remodeling of the cristae. Surprisingly, proteins discovered to control mitochondrial morphology appear to also participate in apoptosis and proteins associated with the regulation of apoptosis have been shown to affect mitochondrial ultrastructure. In this review the recent progress in understanding the mechanisms governing mitochondrial morphology and the latest advances connecting the regulation of mitochondrial morphology with programmed cell death are discussed.

Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis

Abstract: During apoptosis, the mitochondrial membrane potential (MMP) decreases, but it is not known how this relates to the apoptotic process. It was recently suggested that cytochrome c is compartmentalized in closed cristal regions and therefore, matrix remodeling is required to attain complete cytochrome c release from the mitochondria. In this work we show that, at the onset of apoptosis, changes in MMP control matrix remodeling prior to cytochrome c release. Early after growth factor withdrawal the MMP declines and the matrix condenses. Both phenomena are reversed by adding oxidizable substrates. In mitochondria isolated from healthy cells, matrix condensation can be induced by either denying oxidizable substrates or by protonophores that dissipate the membrane potential. Matrix remodeling to the condensed state results in cristal unfolding and exposes cytochrome c to the intermembrane space facilitating its release from the mitochondria during apoptosis. In contrast, when a transmembrane potential is generated due to either electron transport or a pH gradient formed by acidifying the medium, mitochondria maintain an orthodox configuration in which most cytochrome c is sequestered in the cristae and is resistant to release by agents that disrupt the mitochondrial outer membrane.

Degradation of chromosomal DNA during apoptosis

Abstract: Apoptosis is often accompanied by degradation of chromosomal DNA. CAD, caspase-activated DNase, was identified in 1998 as a DNase that is responsible for this process. In the last several years, mice deficient in the CAD system have been generated. Studies with these mice indicated that apoptotic DNA degradation occurs in two different systems. In one, the DNA fragmentation is carried out by CAD in the dying cells and in the other, by lysosomal DNase II after the dying cells are phagocytosed. Several other endonucleases have also been suggested as candidate effectors for the apoptotic degradation of chromosomal DNA. In this review, we will discuss the mechanism and role of DNA degradation during apoptosis.

Nitric oxide: NO apoptosis or turning it ON?

Abstract: Nitric oxide (NO) is known for its diverse activities throughout biology Among signaling qualities, NO affects cellular decisions of life and death either by turning on apoptotic pathways or by shutting them off Although copious reports support both notions, the dichotomy of NO actions remains unsolved Proapoptotic pathways of NO are compatible with established signaling circuits appreciated for mitochondria-dependent roads of death, with some emphasis on the involvement of the tumor suppressor p53 as a target during cell death execution Antiapoptotic actions of NO are numerous, ranging from an immediate interference with proapoptotic signaling cascades to long-lasting effects based on expression of cell protective proteins with some interest on the ability of NO-redox species to block caspases by S-nitrosylation/S-nitrosation Summarizing emerging concepts to understand p53 accumulation on the one hand while proposing inhibition of procaspase processing on the other may help to define the pro- versus antiapoptotic roles of NO

Cation channels trigger apoptotic death of erythrocytes

Abstract: Erythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca(2+)-ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850 mOsm for 24 h, to tert-butyl-hydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca(2+) and mimicked by ionomycin (1 microM). Osmotic shrinkage and oxidative stress activate Ca(2+)-permeable cation channels and increase cytosolic Ca(2+) concentration. The channels are inhibited by amiloride (1 mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca(2+)-permeable cation channels in erythrocytes, thus increasing cytosolic Ca(2+) activity and triggering erythrocyte apoptosis.

M-CSF, TNFalpha and RANK ligand promote osteoclast survival by signaling through mTOR/S6 kinase.

Abstract: Multinucleated bone-resorbing osteoclasts (Ocl) are cells of hematopoietic origin that play a major role in osteoporosis pathophysiology. Ocl survival and activity require M-CSF and RANK ligand (RANKL). M-CSF signals to Akt, while RANKL, like TNFalpha, activates NF-kappaB. We show here that although these are separate pathways in the Ocl, signaling of all three cytokines converges on mammalian target of rapamycin (mTOR) as part of their antiapoptotic action. Accordingly, rapamycin blocks M-CSF- and RANKL-dependent Ocl survival inducing apoptosis, and suppresses in vitro bone resorption proportional to the reduction in Ocl number. The cytokine signaling intermediates for mTOR/ribosomal protein S6 kinase (S6K) activation include phosphatidylinositol-3 kinase, Akt, Erks and geranylgeranylated proteins. Inhibitors of these intermediates suppress cytokine activation of S6K and induce Ocl apoptosis. mTOR regulates protein translation acting via S6K, 4E-BP1 and S6. We find that inhibition of translation by other mechanisms also induces Ocl apoptosis, demonstrating that Ocl survival is highly sensitive to continuous de novo protein synthesis. This study thus identifies mTOR/S6K as an essential signaling pathway engaged in the stimulation of cell survival in osteoclasts.

Regulation of p53 responses by post-translational modifications.

Abstract: The p53 gene is the most commonly mutated tumor suppressor gene in human cancers. It has been well established that p53 plays multiple tumor suppression roles in cells upon introduction of stresses: cell cycle G1 and G2 arrest or apoptosis, in part depending on the cell types. Structural and functional analyses of p53 have shown that p53 is a transcription factor with a sequence-specific DNA-binding domain in the central region and a transcriptional activation domain at the N-terminal. Three additional domains, including a nuclear localization signal, a tetramerization domain and an extreme C-terminal regulatory domain, are present in the C-terminal of p53. The transcriptional activities of p53 are essential for its tumor suppression functions. p21, a downstream target of p53 and a universal inhibitor of cyclin-dependent kinases, plays a major role in mediating p53-dependent cell cycle G1 arrest. 2–4 While earlier studies reached conflicting conclusions on the requirement of p53 transcriptional activities in p53-dependent apoptosis, recent analysis of knockin mice and cells, in which two missense mutations encoding Gln and Ser in place of Leu25 and Trp26 were introduced into the endogenous p53 gene, indicated that p53 transcriptional activities are essential for p53-dependent apoptosis. Therefore, identification of genes whose expression is either activated or repressed by p53 will be essential in order to understand the mechanism of p53-dependent tumor suppression. In response to DNA damage and other cellular stresses, the protein levels of p53 are greatly upregulated and its activities induced. It had been postulated that p53 was present in an inactive form in normal cells in the absence of stress. In this context, the extreme C-terminal of p53 was proposed a role in negatively regulating the sequence-specific DNA binding of p53. However, evidence provided by two recent studies argued against such a model and indicated that p53 could bind to its sequence-specific DNA-binding site in cells in the absence of stress. Therefore, it remains to be elucidated how p53 activities are induced after introduction of stress. The induction of p53 protein levels post stresses is regulated posttranslationally and mainly because of increased protein stability. MDM2, a transcriptional target of p53, plays a major role in the regulation of p53 stability. MDM2 interacts with the N-terminal of p53 and this interaction could inhibit p53 transcriptional activity. In addition, MDM2 acts as the E3 ligase for p53 and thus promotes p53 ubiquitination and degradation. Therefore, MDM2-p53 interaction represents a negative autoregulatory mechanism to regulate p53 stability and activity. Accumulating evidence has indicated that post-translational modifications of p53 and MDM2, including phosphorylation, acetylation and sumoylation, could modulate their interaction and thus p53 stability and activity.

Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation

Abstract: Small cell lung cancer cell lines were resistant to FasL and TRAIL-induced apoptosis, which could be explained by an absence of Fas and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 could be detected. Analysis of SCLC tumors revealed reduced levels of Fas, TRAIL-R1 and caspase-8 mRNA compared to non-small cell lung cancer (NSCLC) tumors. Methylation-specific PCR demonstrated methylation of CpG islands of the Fas, TRAIL-R1 and caspase-8 genes in SCLC cell lines and tumor samples, whereas NSCLC samples were not methylated. Cotreatment of SCLC cells with the demethylating agent 5'-aza-2-deoxycytidine and IFNgamma partially restored Fas, TRAIL-R1 and caspase-8 expression and increased sensitivity to FasL and TRAIL-induced death. These results suggest that SCLC cells are highly resistant to apoptosis mediated by death receptors and that this resistance can be reduced by a combination of demethylation and treatment with IFNgamma.

Autophagic programmed cell death in Drosophila.

Abstract: Autophagic programmed cell death occurs during the development of diverse animal groups, but the mechanisms that control this genetically regulated form of cell killing are poorly understood. Genetic studies of bulk protein degradation in yeast have provided important advances in our understanding of autophagy, and recent investigations of Drosophila autophagic cell death suggest that some of these mechanisms may be conserved. In Drosophila, several steroid-regulated genes that encode transcription regulators are required for autophagic cell death. These transcription regulators appear to activate a large number of genes that play a more direct role in cell killing, including genes that function in apoptosis such as caspases. While caspase function is required for autophagic cell death during Drosophila development, genes encoding proteins that are similar to the yeast autophagy regulators are also induced in dying salivary glands. Furthermore, numerous noncaspase proteases, cytoplasmic organizing factors, signaling molecules, and unknown factors are expressed in interesting patterns during autophagic cell death. This article reviews the current knowledge of the regulation of autophagic programmed cell death during development of Drosophila.

Autoimmune lymphoproliferative syndromes: genetic defects of apoptosis pathways.

Abstract: Human and mouse natural mutants presenting with lymphoproliferative syndrome and autoimmunity (ALPS) have enlightened the role of the Fas and FasL in lymphocyte cell death and peripheral tolerance. Further study of the genetic basis of the human pathology led to the identification of apoptosis signaling defect, and pointed out to the crucial role of caspase-10 in the process of apoptosis induction. In contrast, the absence of lymproliferation in engineered mutants of 'death pathways' suggests that additional events are necessary to recapitulate the overt phenotype of ALPS patients or MRL/lpr mice. Moreover, these models highlight the roles of Fas and associated molecules, such as FADD and caspase-8, in lymphocyte development or activation. This review will discuss the main findings provided by the study of mouse models and human conditions.

Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine.

Abstract: There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 muM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.

Transcriptional repression mediated by the p53 tumour suppressor.

Abstract: The survival and well-being of multicellular organisms is dependent on appropriate cellular responses to a myriad of external and internal signals. Accordingly, critical cellular regulators exist to integrate these signals and coordinate reactions to them. One such ‘master’ regulator is the p53 tumour suppressor protein. Upon exposure to stress stimuli such as DNA damage, hypoxia, oncogene activation, or nucleotide depletion, p53 becomes activated and promotes cell cycle arrest and apoptosis. The ability of p53 to control cell growth in this manner is considered important for its function as a tumour suppressor. p53 is a transcription factor that binds to DNA in a sequence-specific manner to activate transcription of target genes. The consensus DNA-binding sequence for p53 consists of two repeats of the 10 bp motif 50PuPuPuC(A/T)(A/T)GPyPyPy-30 separated by 0–13 bp. Mutated p53 alleles typically found in tumours encode defective products no longer capable of binding to DNA or activating transcription. There is now compelling evidence that the transcriptional activity of p53 is required for its growth suppressing and tumour suppressing activities. For instance, p21, GADD45, and 14-3-3s represent three wellcharacterized p53 target genes that are involved in mediating cell cycle arrest by p53. The pathway through which p53 promotes apoptosis is less well understood, but is believed to involve transcriptional regulation of a different subset of genes as well as transcription-independent functions of p53, possibly reflecting distinct mechanisms of p53 action in different cell types. p53 activates transcription of a variety of apoptosis-associated genes including Bax, PUMA, Pidd, Killer/DR5, Fas/APO-1, Noxa, p53AIP1, and Ei24/PIG8. Most studies have focused on the transactivation function of p53 because of the strong association between transactivation and tumour suppression. However, p53 is also able to repress transcription from various promoters and emerging evidence indicates that transcriptional repression by p53 is important for its ability to promote apoptosis. The ability of p53 to repress transcription at various viral and cellular promoters has been known for some time, but the underlying mechanism and functional consequences of transcriptional repression have remained largely unexplored. Until more recently, the simplistic view had been that p53 activates transcription from genes that contain a p53-binding site and that p53 has a general repressive effect on promoters that lack a p53-binding site, possibly by sequestering components of the basal transcriptional machinery.

Hepatitis C and liver fibrosis.

Abstract: Chronic hepatitis C progresses to cirrhosis within 20 years in an estimated 20-30% of patients, while running a relatively uneventful course in most others. Certain HCV proteins, such as core and NS5A, can induce derangement of lipid metabolism or alter signal transduction of infected hepatocytes which leads to the production of reactive oxygen radicals and profibrogenic mediators, in particular TGF-beta1. TGF-beta1 is the strongest known inducer of fibrogenesis in the effector cells of hepatic fibrosis, i.e. activated hepatic stellate cells and myofibroblasts. However, fibrogenesis proceeds only when additional profibrogenic stimuli are present, e.g. alcohol exposure, metabolic disorders such as non-alcoholic steatohepatitis, or coinfections with HIV or Schistosoma mansoni that skew the immune response towards a Th2 T cell reaction. Furthermore, profibrogenic polymorphisms in genes that are relevant during fibrogenesis have been disclosed. This knowledge will make it possible to identify those patients who are most likely to progress and who need antiviral or antifibrotic therapies most urgently. However, even the best available treatment, the combination of pegylated interferon and ribavirin, which is costly and fraught with side effects, eradicates HCV in only 50% of patients. While the suggestive antifibrotic effect of interferons (IF-gamma>alpha,beta), irrespective of viral elimination, has to be proven in randomised prospective studies, additional, well tolerated and cost-effective antifibrotic therapies have to be developed. The combination of cytokine strategies, e.g. inhibition of the key profibrogenic mediator TGF-beta, with other potential antifibrotic agents appears promising. Such adjunctive agents could be silymarin, sho-saiko-to, halofuginone, phosphodiesterase inhibitors, and endothelin-A-receptor or angiotensin antagonists. Furthermore, drug targeting to the fibrogenic effector cells appears feasible. Together with the evolving validation of serological markers of hepatic fibrogenesis and fibrolysis an effective and individualised treatment of liver fibrosis is anticipated.

Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity

Abstract: We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 microM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 microM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.

Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain.

Abstract: A single episode of ethanol intoxication triggers widespread apoptotic neurodegeneration in the infant rat or mouse brain. The cell death process occurs over a 6–16 h period following ethanol administration, is accompanied by a robust display of caspase-3 enzyme activation, and meets ultrastructural criteria for apoptosis. Two apoptotic pathways (intrinsic and extrinsic) have been described, either of which may culminate in the activation of caspase-3. The intrinsic pathway is regulated by Bax and Bcl-XL and involves Bax-induced mitochondrial dysfunction and release of cytochrome c as antecedent events leading to caspase-3 activation. Activation of caspase-8 is a key event preceding caspase-3 activation in the extrinsic pathway. In the present study, following ethanol administration to infant mice, we found no change in activated caspase-8, which suggests that the extrinsic pathway is not involved in ethanol-induced apoptosis. We also found that ethanol triggers robust caspase-3 activation and apoptotic neurodegeneration in C57BL/6 wildtype mice, but induces neither phenomenon in homozygous Bax-deficient mice. Therefore, it appears that ethanol-induced neuroapoptosis is an intrinsic pathway-mediated phenomenon involving Bax-induced disruption of mitochondrial membranes and cytochrome c release as early events leading to caspase-3 activation.

Hepatitis C virus biology.

Abstract: Hepatitis C virus infection represents a major problem of public health with around 350 millions of chronically infected individuals worldwide. The frequent evolution towards severe liver disease and cancer are the main features of HCV chronic infection. Antiviral therapies, mainly based on the combination of IFN and ribavirin can only assure a long term eradication of the virus in less than half of treated patients. The mechanisms underlying HCV pathogenesis and persistence in the host are still largely unknown and the efforts made by researchers in the understanding the viral biology have been hampered by the absence of a reliable in vitro and in vivo system reproducing HCV infection. The present review will mainly focus on viral pathogenetic mechanisms based on the interaction of HCV proteins (especially core, NS3 and NS5A) with host cellular signaling transduction pathways regulating cell growth and viability and on the strategies developed by the virus to persist in the host and escape to antiviral therapy. Past and recent data obtained in this field with different experimental approaches will be discussed.

The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release, caspase activation, and mitochondrial dysfunction.

Abstract: Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (Δψm). Exogenous ROS and caspase-3 induced Δψm drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased Δψm, and GSH, events associated with caspase activation and cytochrome c release.

Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin

Abstract: The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1β 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.

A new quantitative assay for cytochrome c release in apoptotic cells.

Abstract: 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.

The endocannabinoid system, anandamide and the regulation of mammalian cell apoptosis

Abstract: Endocannabinoids are a new class of lipid mediators, which include amides, esters and ethers of long-chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors able to mimic several pharmacological effects of Delta-9-tetrahydrocannabinol, the active principle of Cannabis sativa preparations like hashish and marijuana. The pathways leading to the synthesis and release of AEA and 2-AG from neuronal and non-neuronal cells are still rather uncertain. Instead, it is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter, followed by intracellular degradation by a fatty acid amide hydrolase. Together with AEA and congeners these proteins form the 'endocannabinoid system'. Here, the involvement of AEA in apoptosis and the underlying signal transduction pathways will be reviewed, along with the metabolic routes and the molecular targets of this endocannabinoid. Also, recent findings on the apoptotic potential of AEA for neuronal cell differentiation and brain development will be discussed.

Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency

Abstract: Apoptotic and necrotic blebs elicited by H2O2 were compared in terms of dynamics, structure and underlying biochemistry in HeLa cells and Clone 9 cells. Apoptotic blebs appeared in a few minutes and required micromolar peroxide concentrations. Necrotic blebs appeared much later, prior to cell permeabilization, and required millimolar peroxide concentrations. Strikingly, necrotic blebs grew at a constant rate, which was unaffected throughout successive cycles of budding and detachment. At 1 μm diameter, the necks of necrotic and apoptotic blebs were almost identical. ATP depletion was discarded as a major factor for both types of bleb. Inhibition of ROCK-I, MLCK and p38MAPK strongly decreased apoptotic blebbing but had no effect on necrotic blebbing. Taken together, these data suggest the existence of a novel structure of fixed dimensions at the neck of both types of plasma membrane blebs in epithelial cells. However, necrotic blebs can be distinguished from apoptotic blebs in their susceptibility to actomyosin kinase inhibition.

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions.

Abstract: Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.

Apoptosis in hepatitis C virus infection.

Abstract: Infection with hepatitis C virus (HCV) is characterized by inflammatory liver damage and a long viral persistence associated with an increased risk of developing hepatocellular carcinoma. Both in liver damage and in oncogenesis a disturbance of apoptosis has been implicated, although the underlying mechanisms in these apparently opposite processes are incompletely understood. HCV-triggered liver injury is mediated mainly by host immune mechanisms and eventually by direct cytopathic effects of HCV. Recent data shows that caspase activation, either triggered by death ligands, other cytokines, granzyme B or HCV proteins, is considerably upregulated in HCV-infected liver. Interestingly, caspase activation appears to correlate closely with the inflammatory response. Data about the role of single HCV proteins, either in cultured cells or transgenic animals models, however, are contradictory, as both pro- and anti-apoptotic effects have been observed. Nevertheless, apoptosis induction upon HCV infection may critically contribute to liver damage, while inhibition of apoptosis may result in HCV persistence and development of hepatocellular carcinoma.

Signaling and transcriptional control of Fas ligand gene expression

Abstract: Fas ligand (FasL), a member of the tumor necrosis factor family, initiates apoptosis by binding to its surface receptor Fas. As a consequence, there is sequential activation of caspases and the release of cytochrome c from the mitochondria, with additional caspase activation followed by cellular degradation and death. Recent studies have shed important insight into the molecular mechanisms controlling FasL gene expression at the level of transcription. Nuclear factors such as nuclear factor in activated T cells, nuclear factor-kappa B, specificity protein-1, early growth response factor, interferon regulatory factor, c-Myc and the forkhead transcriptional regulator, alone or cooperatively, activate FasL expression. These factors are often coexpressed with FasL in pathophysiologic settings including human atherosclerotic lesions. Here, we review these important advances in our understanding of the signaling and transcriptional mechanisms controlling FasL gene expression.

Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy.

Abstract: Inactivation of p53 has been shown to correlate with poor prognosis and drug resistance in malignant tumors. Nevertheless, few reports have directly shown such effects in primary tumor cells. Here, we investigated the p53 mutational status in 138 B-CLL samples and compared these findings with drug and gamma-irradiation sensitivity profiles. p53 mutations resulted not only in a shorter survival but, notably also in selective resistance to alkylating agents, fludarabine and gamma-irradiation. In contrast, no such effect was observed for vincristine, anthracyclines and glucocorticoids. Thus, these latter compounds induce cell death at least in part by p53-independent pathways. Interestingly, p53 mutations clustered in patients who had received prior chemotherapy. In fact, we show for the first time that treatment with DNA-damaging alkylating agents correlates with occurrence of p53 mutations in a clinical setting. This finding may explain at least to some extent the development of resistance to second-line anticancer chemotherapy.