Resistance to anti-IgM-induced apoptosis in a WEHI-231 subline is due to insufficient production of ceramide

We describe the properties of a physiological cell death (PCD)-resistant subline of WEHI-231 generated from the PCD-susceptible WEHI-231.7 JM cell line maintained in our laboratory. The PCD-resistant WEHI-231.7 JMRE subline was uniquely resistant to anti-immunoglobulin (Ig)M-induced PCD but not to irradiation and etoposide. In these sublines, we compared the expression of genes implicated in regulating PCD. Northern analysis of c-myc, c-fos, egr-1, Fas, p53 and retinoblastoma revealed similar basal levels of expression in all sublines tested and comparable responses to anti-IgM treatment. Similarly, the expression of bcl-2, bcl-x, bax and IL-1β converting enzyme did not correlate with susceptibility to anti-IgM-induced PCD. Next, we systematically studied signal transduction events including: tyrosine phosphorylation, Ca⁺⁺ flux, and ceramide production in the JM and JMRE sublines. The tyrosine phosphorylation patterns and the Ca⁺⁺ influx generated following sIgM engagement were very similar in the JM and JMRE sublines. In contrast, the generation of ceramide differed in the PCD-resistant and PCD-susceptible sublines. Ceramide is produced following cross-linking sIgM on WEHI-231.7 JM cells and causes PCD. Ceramide levels in anti-IgM-treated WEHI-231.7 JMRE cells are low and appear to be insufficient to induce PCD.     

Ceramide regulates SR protein phosphorylation during adenoviral infection

In this study, we show that adenoviral infection induced accumulation of the sphingolipid ceramide in a dose- and time-dependent manner. This accumulation preceded cell lysis, occurred in the absence of biochemical evidence of apoptosis, and was derived from de novo synthesis of ceramide. An adenovirus mutant that lacks the adenovirus death protein (ADP) produced ceramide accumulation in the absence of cell lysis. This suggested that ceramide accumulation was either driven by adenovirus or was a cellular stress response but was unlikely a result of cell death. The use of inhibitors of ceramide synthesis resulted in a significant delay in cell lysis, suggesting that ceramide was necessary for the lytic phase of the infection. Serine/arginine-rich (SR) proteins were dephosphorylated during the late phase of the viral cycle, and inhibitors of ceramide synthesis reversed this. These findings suggest that adenovirus utilizes the ceramide pathway to regulate SR proteins during infection.     

Time course of programmed cell death in Ciona intestinalis in relation to mitotic activity and MAPK signaling.

Programmed cell death (PCD) in the ascidian species Ciona intestinalis (Tunicata; Chordata) is investigated from early larvae to juvenile stages, by means of digoxigenin-based terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) technique. At first, PCD in the swimming larva affects trunk mesenchyme and central nervous system (CNS), then it participates extensively to metamorphosis, until it is restricted to developing organs of juveniles. Analysis of patterns of cell death and division in the larval CNS question old models on the genesis of the adult C. intestinalis brain. Upon performing immunochemical and functional assays for mitogen-activated protein kinase (MAPK) kinase kinase-1 (MEKK1), MAPK kinase 1/2 (MEK1/2), c-Jun NH2-terminal kinase (JNK), and dual phosphorylated extracellular regulated kinase 1/2 (dpERK1/2), the neurogenic competence of the larval brain appears to rely on a combinatorial regulation of PCD by the mitogen-activated protein kinase signaling cascade. These results show that, in tunicates, PCD consists of a multistep program implicated in growth and patterning with various roles.     

Dissociated ROS production and ceramide generation in sulfasalazine-induced cell death in Raw 264.7 cells

Sulfasalazine (SSZ) is a drug used in inflammatory bowel disease, whose precise mechanism of action remains to be clarified. Here, we report that incubation of Raw 264.7 cells with SSZ but not salicylates [acetylsalicylic acid (ASA), 4-aminosalicylic acid (4-ASA), and 5-ASA] causes a mixed apoptotic and necrotic form of cell death. In contrast to its metabolites, sulfapyridine and 5-ASA, SSZ exposure in Raw 264.7 cells resulted in a threefold increase in ceramide generation, as well as a robust production of reactive oxygen species (ROS). However, inhibition of ceramide production by fumonisin B1 failed to attenuate cell death. Preincubation with catalase, cyclosporin A (CsA), and bongkrekic acid attenuated ROS production. When dead cells were quantified for apoptotic versus necrotic cell death, catalase and N-acetylcysteine reproducibly attenuated apoptosis, whereas CsA, in addition to reducing apoptosis, was observed to dramatically enhance necrosis. In conclusion, the cell-death response induced by SSZ in Raw 264.7 cells involves ROS in the apoptotic limb but is independent of ceramide formation.     

The influences of sphingolipid metabolites on gentamicin-induced hair cell loss of the rat cochlea

Sphingolipid metabolites inducing ceramide, sphingosine, and sphingosine-1-phosphate (S1P) play important roles in the regulation of cell proliferation, survival, and death. Aminoglycoside antibiotics including gentamicin induce inner ear hair cell loss and sensorineural hearing loss. Apoptotic cell death is considered to play a key role in this injury. The present study was designed to investigate the possible involvement of ceramide and S1P in hair cell death due to gentamicin. In addition, the effects of other metabolites of ceramide, gangliosides GM1 (GM1) and GM3 (GM3), on gentamicin ototoxicity were also investigated. Basal turn organ of Corti explants from p3 to p5 rats were maintained in tissue culture and exposed to 20 or 35 μM gentamicin for 48 h. The effects of ceramide, S1P, GM1, and GM3 on gentamicin-induced hair cell loss were examined. Gentamicin-induced hair cell loss was increased by ceramide but was decreased by S1P. GM1 and GM3 exhibited protective effects against gentamicin-induced hair cell death at the limited concentrations. These results indicate that ceramide enhances gentamicin ototoxicity by promoting apoptotic hair cell death, and that S1P, GM1, and GM3 act as cochlear protectants. In conclusion, sphingolipid metabolites influence the apoptotic reaction of hair cells to gentamicin ototoxicity.     

Intertwined pathways of programmed cell death in immunity

Summary: Programmed cell death (PCD) occurs widely in species from every kingdom of life. It has been shown to be an integral aspect of development in multicellular organisms, and it is an essential component of the immune response to infectious agents. An analysis of the phylogenetic origin of PCD now shows that it evolved independently several times, and it is fundamental to basic cellular physiology. Undoubtedly, PCD pervades all life at every scale of analysis. These considerations provide a backdrop for understanding the complexity of intertwined, but independent, cell death programs that operate within the immune system. In particular, the contributions of apoptosis, autophagy, and necrosis in the resolution of an immune response are considered.     

Inhibitors of ubiquitin-dependent proteolysis can delay programmed cell death of adult intersegmental muscles in the moth Manduca sexta.

In the moth Manduca sexta, intersegmental muscles (ISMs) undergo rapid programmed cell death (PCD) within 48 hr of adult emergence. ISM PCD involves ubiquitin-dependent proteasomal degradation accompanied by the down-regulation of expression of actin genes and the up-regulation of degradative gene expression such as ubiquitin. Hemin chloride and N-acetyl-leu-leu-norleucinal (ALLN), both inhibitors of proteasomal activity, administered before adult emergence delayed PCD for up to 5 days in ISMs maintained from the larval stage, such as the dorsal internal medial muscle in abdominal segment 4 (DIM-A4). ISMs that developed during metamorphosis from respecified larval muscles such as the DIM-A2 were less dramatically affected. The increase in polyubiquitinated proteins and the decrease in actin mRNA expression accompanying maintained ISM PCD were delayed after inhibitor application. No changes were detected in respecified ISMs. These results reveal a regulatory role for proteasomal activity in an early stage of maintained ISM cell death.     

Mechanisms of neutrophil death in human immunodeficiency virus-infected patients: role of reactive oxygen species, caspases and map kinase pathways

Neutrophils from human immunodeficiency virus-positive (HIV+) patients have an increased susceptibility to undergo programmed cell death (PCD), which could explain neutropenia during advanced disease. In this work, key steps of PCD have been evaluated in neutrophils from HIV+ patients. The role of caspase-3, caspase-8, mitogen activated protein kinase (MAPK) and reactive oxygen species (ROS) was analysed. Spontaneous neutrophil death is dependent upon caspase-3 but independent of caspase-8, suggesting that the intrinsic pathway is involved as a pathogenic mechanism of PCD. Inhibition of ROS decreased spontaneous PCD and caspase-3 hydrolysis, connecting oxidative stress and caspase-3 activation with neutrophil PCD in HIV-infected patients. Additionally, an increased neutrophil death was observed in HIV+ patients, following inhibition of p38 MAPK, suggesting a role for p38 MAPK in cell survival during the disease. We conclude that oxidative stress secondary to HIV infection can accelerate neutrophil death.     

Death to flies: Drosophila as a model system to study programmed cell death

Programmed cell death (PCD) is essential for the removal of unwanted cells and is critical for both restricting cell numbers and for tissue patterning during development. Components of the cell death machinery are remarkably conserved through evolution, from worms to mammals. Central to the PCD process is the family of cysteine proteases, known as caspases, which are activated by death-inducing signals. Comparisons between C. elegans and mammalian PCD have shown that there is additional complexity in the regulation of PCD in mammals. The fruitfly, Drosophila melanogaster, is proving an ideal genetically tractable model organism, of intermediary complexity between C. elegans and mammals, in which to study the intricacies of PCD. Here, we review the literature on PCD during Drosophila development, highlighting the methods used in these studies.     

Programmed Cell Death in the Developing Nervous System

Virtually all cell populations in the vertebrate nervous system undergo massive "naturally-occurring" or "programmed" cell death (PCD) early in development. Initially neurons and glia are overproduced followed by the demise of approximately one-half of the original cell population. In this review we highlight current hypotheses regarding how large-scale PCD contributes to the construction of the developing nervous system. More germane to the theme of this symposium, we emphasize that the survival of cells during PCD depends critically on their ability to access "trophic" molecular signals derived primarily from interactions with other cells. Here we review the cell-cell interactions and molecular mechanisms that control neuronal and glial cell survival during PCD, and how the inability of such signals to suppress PCD may contribute to cell death in some diseases such as spinal muscular atrophy. Finally, by using neurotrophic factors (e.g. CNTF, GDNF) and genes that control the cell death cascade (e.g. Bcl-2) as examples, we underscore the importance of studying the mechanisms that control neuronal and glial cell survival during normal development as a means of identifying molecules that prevent pathology-induced cell death. Ultimately this line of investigation could reveal effective strategies for arresting neuronal and glial cell death induced by injury, disease, and/or aging in humans.     

Ceramide: does it matter for T cells?

Ceramide and its derivatives have been implicated in cellular activation, differentiation, survival and death. Although the main biological function of ceramide appears to be linked to its potency to induce cell death, its actual relevance as a regulator of cell death has been the subject of controversial discussions. Here, we discuss the significance of ceramide in T-cell biology, with special emphasis on the regulation of T-cell receptor expression, growth arrest, costimulation and death.     

Altered expression of insulin-like growth factor-1 and insulin like growth factor binding proteins-2 and 5 in the mouse mutant Hypodactyly (Hd) correlates with sites of apoptotic activity

Insulin-like growth factor-I (IGF-I) mediated signalling has been implicated to be of significant importance during vertebrate embryonic development. IGF-I signalling has also been shown to be modulated by a number of IGF binding proteins that are thought to act as either agonists or antagonists of IGF activity. IGF-I has been implicated in a number of cellular processes, including cell division and programmed cell death (apoptosis). We have used the mouse mutant Hypodactyly (Hd) as a tool to determine the role of IGF-I and two key IGF binding proteins (IGFBP-2 and IGFBP-5) during embryonic development. The Hd mutant is a good model with which to study developmental cascades, since it has a distinct phenotype in the limb where cellular and molecular circuits have been thoroughly investigated. The distinctive pointed limb buds observed in Hd mutant embryos have been shown to be the result of a massive increase in apoptosis. We show that all three genes, IGF-1, IGFBP-2 and IGFBP-5, display restricted expression patterns during limb development. Indeed, IGFBP-5 shows a remarkable similarity to the expression of Engrailed-1, which is the vertebrate homologue of the Drosophila selector gene Engrailed. We show that there is downregulation in the expression of IGFBP-2 in the entire apical ectodermal ridge (AER) in homozygous Hd/Hd limb buds, whereas IGFBP-5 is downregulated in specific regions in the mutant AER. IGF-I expression is downregulated in Hd limb buds in regions undergoing high levels of cell death, consistent with its proposed role as an anti-apoptotic factor, while IGFBP-5 is found at higher levels in regions of cell death, consistent with reports of its association with apoptosis in adult tissues. We propose that these three components of the IGF axis could be involved in the manifestation of the mutant phenotype in Hypodactyly, and that this is probably a result of their ability to regulate cell survival and cell death.     

Programmed cell death and AIDS: from hypothesis to experiment.

Here, Jean Claude Ameisen discusses new findings supporting the hypothesis that abnormal induction of programmed cell death (PCD) is relevant to AIDS pathogenesis. Recent evidence also suggests that the prevention of PCD is a factor in oncogenesis. Together, these ideas provide a framework for the reinterpretation of cell survival disorders in terms of PCD dysregulation, and suggest that in vivo control of cell signalling has wide-ranging therapeutic potential.     

Ceramide catabolism critically controls survival of human dendritic cells

The regulation of dendritic cell (DC) survival is crucial for the modulation of adaptive immunity. Ceramide is a lipid mediator of the stress response, which accumulates intracellularly during DC differentiation. We found that ceramide levels are tightly regulated in human DCs and that the pharmacological inhibition of enzymes responsible for ceramide catabolism, such as ceramidases and sphingosine kinases, sensitizes DCs to ceramide-induced cell death. It is important that inhibition of sphingosine kinases, during lipopolysaccharide stimulation, causes extensive ceramide accumulation and death of DCs. These data indicate that ceramide catabolism regulates survival of human DCs and reveal novel potential targets for the pharmacological manipulation of the immune response.     

Preventing chondrocyte programmed cell death caused by iatrogenic injury

Cartilage repair technology is advancing at a rapid pace. However, all techniques share a common weakness-unintentional chondrocyte cell death resulting from cartilage injury that occurs during preparation of the defect site. The loss of chondrocytes at the edge of host cartilage is likely to contribute to failed integration of regenerated tissue or grafts to the surrounding cartilage. Recent studies have demonstrated that "apoptosis", or programmed cell death (PCD), may be responsible for much of the cell death caused by cartilage injury. Theoretically, inhibitors of key pathways responsible for PCD could rescue chondrocytes and improve the results of cartilage repair surgery. The purpose of this study was to test the hypothesis that short-term, intra-articular PCD inhibitor treatment can limit chondrocyte death in vivo following simulated preparation of host cartilage for a repair procedure. A microcurette was used to create full-thickness articular cartilage injuries to the femoral condyles of adult New Zealand White rabbits. Animals received daily intra-articular injections either with a potent PCD inhibitor or with vehicle alone. Treatment with the inhibitor resulted in a significant reduction in the percentage of chondrocytes undergoing PCD compared to controls [treated=10.1+/-2.4%; controls=26.5+/-3.6%; (p=0.0013)]. These results provide proof of concept for the use of PCD inhibitors to enhance the results of cartilage repair surgeries.     

Mitochondria and programmed cell death in Parkinson's disease: apoptosis and beyond

Abstract Significance: Activation of mitochondrion-dependent programmed cell death (PCD) pathways is instrumental to the demise of substantia nigra pars compacta dopaminergic neurons in experimental mouse models of Parkinson's disease (PD). Supporting the relevance of these findings for PD, key molecular elements of this pathogenic cascade have also been demonstrated in postmortem brain samples of PD patients. Recent Advances and Critical Issues: Mounting evidence indicates that different morphological types of cell death co-exist in the brain of PD patients, all of which may result from the activation of common upstream PCD pathways. Indeed, contrary to initial views, it is now established that the deleterious effects of PCD pathways are not limited to mitochondrion-mediated caspase-dependent apoptosis but also involve caspase-independent nonapoptotic cell death, including necrosis. This notion may help reconcile the observation of both apoptotic and nonapoptotic dopaminergic cell death in postmortem PD samples. Future Directions: Potential neuroprotective strategies for PD should be aimed at targeting both apoptotic and nonapoptotic pathways, all of which may simultaneously occur in PD patients through activation of common upstream PCD pathways involving the mitochondria. Antioxid. Redox Signal. 16, 883-895.     

Transforming growth factor beta mediates apoptosis in the ganglion cell layer during all programmed cell death periods of the developing murine retina

Transforming growth factor beta (TGF-beta) is an extracellular signaling molecule known to mediate programmed cell death (PCD) in the developing retina. In the present study, we investigated the expression profiles and activity levels of TGF-beta ligand and TGF-beta receptors (TbetaR) during the successive physiological PCD periods of the developing postnatal mouse retina. The peak of TbetaR expression levels--revealed by Western Blots and MLEC assays--coincided with the main periods of postnatal (P) retinal murine PCD at P2, P9, and P15. Immunocytochemical studies showed that the localization of the TbetaRs is restricted to the ganglion cell layer. Application of a neutralizing anti-TGF-beta antibody to E15 and P9 retinal cultures resulted in a significant decrease in the number of TUNEL-positive neurons specifically in the ganglion cell or prospective ganglion cell layer. Treatment of P2 and P15 organotypic murine retinal wholemount cultures with exogenous recombinant TGF-beta significantly increased cell death levels. In the P15 retina, where PCD affects ganglion cells and photoreceptors, TGF-beta induced cell death of large retinal ganglion cells, whereas small ganglion cells and photoreceptor neurons remained unaffected. Our data indicate that TGF-beta mediated apoptosis during all postnatal retinal PCD phases specifically affects the fate of retinal ganglion cells.