Hyperglycemia induces apoptosis in pre-implantation embryos through cell death effector pathways

Although perinatal mortality rates have improved for pregnant diabetic women because of insulin therapy and tight metabolic control, infants of diabetics still experience significantly higher rates of congenital malformations and spontaneous miscarriages compared with those of non-diabetic women. Our results here indicate that hyperglycemic conditions, either in vivo or in vitro, modulate the expression of an apoptosis regulatory gene as early as the pre-implantation blastocyst stage in the mouse. Apoptosis in the mammalian pre-implantation blastocyst is a normal process, thought to protect the early embryo by eliminating abnormal cells. Here we demonstrate that expression of Bax, a Bcl-2-like protein, is increased at the blastocyst stage in the presence of high concentrations of glucose, and that these changes correlate morphologically with increased DNA fragmentation. Expression of Bax and caspase are necessary for this in vitro glucose-induced apoptotic event, and ceramide is involved in mediating this embryotoxic effect of glucose. We also show that these apoptotic cellular changes can be prevented in vivo by treating hyperglycemic mice with insulin before and immediately after conception. These findings emphasize the importance of tight glycemic control in diabetic women at the earliest stages after conception.     

Signal transduction pathways that regulate cell survival and cell death

Apoptosis or programmed cell death (PCD) is a physiological process critical for organ development, tissue homeostasis and elimination of defective or potentially dangerous cells in complex organisms. Apoptosis permits cell death without a concomitant inflammatory response in the surrounding tissues. The process of apoptosis depends on the reception of multiple extracellular and intracellular signals, integration and amplification of these signals by second messengers and finally, activation of the death effector proteases. Defects in control of apoptotic pathways may contribute to a variety of diseases including cancer, autoimmune and neurodegenerative conditions and AIDS. While many components of the regulatory network controlling apoptosis have been defined, the mechanisms of action and patterns of interaction of these factors remain controversial. This article summarizes some of the known aspects of signaling pathways involved in apoptosis.     

A cell surface receptor defined by a mAb mediates a unique type of cell death similar to oncosis

Cell death is mediated by distinct pathways including apoptosis and oncosis in response to various death signals. To characterize molecules involved in cell death, a panel of mAbs was raised by immunizing mice with apoptotic cells. One of these antibodies, designated anti-Porimin (for pro-oncosis receptor inducing membrane injury), was found to directly induce a unique type of cell death in Jurkat cells. Anti-Porimin defines a 110-kDa cell surface receptor on Jurkat cells. Functionally, anti-Porimin alone rapidly mediates pore formation on the plasma membrane and induces cell death without participation of complement. Both the cellular expression and functional characteristics of the Porimin antigen indicate that it is distinct from the CD95 (Fas/Apo-1) and other cell receptors known to induce apoptosis. Anti-Porimin-mediated cell death was preceded by cell aggregation, formation of plasma membrane pores, and the appearance of membrane blebs. More important, these cells show neither DNA fragmentation nor apoptotic bodies, but display lethal damage of the cell membrane. Cell death by anti-Porimin is distinct from complement-dependent cytolysis or complement-independent apoptosis but is similar to that described for oncosis, a form of cell death accompanied by the membrane damage followed by karyolysis. The induction of cell death by anti-Porimin may represent a unique cell surface receptor-mediated pathway of cell death in the human lymphoid system.     

Fanconi anemia C protein acts at a switch between apoptosis and necrosis in mitomycin C-induced cell death

Deregulation of apoptosis seems to be a hallmark of the Fanconi anemia (FA) syndrome. In order to further define the role of the FA protein from complementation group C (FAC) in apoptosis, we characterized parameters modified during the mitomycin-C (MMC)-induced apoptotic program. It is shown that despite a higher level of cell death for FA compared to normal lymphoblasts after MMC treatment, FA cells do not display a marked DNA fragmentation. Furthermore, while playing a central role in MMC apoptosis of normal lymphoblasts, the activity of caspase-3-like proteases is altered in FA cells. Interestingly, the disruption of the mitochondrial transmembrane potential (Deltapsi), an early event that can lead to apoptotic or to necrotic death, is accomplished similarly in FA and in normal cells. Finally, it is shown that the overexpressed FAC protein inhibited the apoptotic steps, with the exception of the decrease of the Deltapsi. Altogether, our results indicate that the FAC protein acts at a step preceding the activation of the caspases and after the modification of the Deltapsi, a decision point at which cells can be pushed toward either apoptosis or necrosis and which, consequently, regulates the balance between the two modes of cell death.     

The tumor necrosis factor signaling complex: choosing a path toward cell death or cell proliferation

Signal transduction pathways which are initiated by the tumor necrosis factor (TNF) utilize receptors which are devoid of intrinsic catalytic activity. Recently identified two families of proteins that directly associate with the cytoplasmic domains of the TNF receptor family members, have partially bridged a molecular gap within the TNF-induced signaling pathways. Clearly, there are numerous alternate routes that originate from the TNF ligand-receptor assembly and terminate on the diverse cellular responses, including proliferation, differentiation, or death. This review focuses on recent advances characterizing the TNF ligand-receptor signaling network, which allow to better understand its participation in a life-death balance within the target cell.     

Cell renewal, cell differentiation and programmed cell death (apoptosis) in pilomatrixoma

Pilomatrixoma is a benign tumour of the cutaneous adnexa. Histologically, pilomatrixoma comprises masses of immature basophilic cells, small numbers of polygonal squamoid cells, few transitional cells, and clusters of 'shadow cells'. The mechanism leading to the formation of shadow cells is still unknown. Skin biopsy specimens of pilomatrixoma (n = 15) were studied histologically, immunohistologically, and by applying the in situ end-labelling technique. The basal layer of the basophilic cells induced most of the proliferating cells with high expression of bcl-2 and cytokeratin 19. The overlying basophilic cells showed a negligible mitotic activity, a high significant accumulation of p53 protein, and a heterogeneous, but progressive loss of bcl-2 and cytokeratin 19. They developed either into squamoid cells or into transitional cells. The squamoid cells were characterized as differentiated cells resembling mature keratinocytes of stratified mucosa. The transitional cells could be shown to represent apoptotic cells proceeding to shadow cells. The data suggest that apoptosis is the main mechanism leading to the development of the dead shadow cells and is most probably responsible for the banal biological behaviour of pilomatrixoma. Apart from that, pilomatrixoma represents a suitable biological model to study apoptosis in humans.     

Entamoeba histolytica induces host cell death in amebic liver abscess by a non-Fas-dependent, non-tumor necrosis factor alpha-dependent pathway of apoptosis

Amebic liver abscess is characterized by extensive areas of dead hepatocytes that form cavities surrounded by a thin rim of inflammatory cells and few Entamoeba histolytica trophozoites. E. histolytica produces pore-forming proteins and proteinases, but how trophozoites actually kill host cells has been unclear. Here, we report that E. histolytica induces apoptosis in both inflammatory cells and hepatocytes in a severe combined immunodeficient (SCID) mouse model of amebic liver abscess. By studying infection in C57/BL6.lpr and C57/BL6.gld mice, we found that E. histolytica-induced apoptosis does not require the Fas/Fas ligand pathway of apoptosis, and by using mice with a targeted deletion of the tumor necrosis factor receptor I gene, we have shown that E. histolytica-induced apoptosis is not mediated by tumor necrosis factor alpha. Our data indicate that apoptosis plays a prominent role in the host cell death seen in amebic liver abscess in a mouse model of disease and suggest that E. histolytica induces cell death without using two common pathways for apoptosis.     

Hormonal control of programmed cell death/apoptosis

Apoptosis or programmed cell death is a physiological form of cell death that occurs in embryonic development and during involution of organs. It is characterized by distinct biochemical and morphological changes such as DNA fragmentation, plasma membrane blebbing and cell volume shrinkage. Many hormones, cytokines and growth factors are known to act as general and/or tissue-specific survival factors preventing the onset of apoptosis. In addition, many hormones and growth factors are also capable of inducing or facilitating programmed cell death under physiological or pathological conditions, or both. Steroid hormones are potent regulators of apoptosis in steroid-dependent cell types and tissues such as the mammary gland, the prostate, the ovary and the testis. Growth factors such as epidermal growth factor, nerve growth factor, platelet-derived growth factor (PDGF) and insulin-like growth factor-I act as survival factors and inhibit apoptosis in a number of cell types such as haematopoietic cells, preovulatory follicles, the mammary gland, phaeochromocytoma cells and neurones. Conversely, apoptosis modulates the functioning and the functional integrity of many endocrine glands and of many cells that are capable of synthesizing and secreting hormones. In addition, exaggeration of the primarily natural process of apoptosis has a key role in the pathogenesis of diseases involving endocrine tissues. Most importantly, in autoimmune diseases such as autoimmune thyroid disease and type 1 diabetes mellitus, new data suggest that the immune system itself may not carry the final act of organ injury: rather, the target cells (i.e. thyrocytes and beta cells of the islets) commit suicide through apoptosis. The understanding of how hormones influence programmed cell death and, conversely, of how apoptosis affects endocrine glands, is central to further design strategies to prevent and treat diseases that affect endocrine tissues. This short review summarizes the available evidence showing where and how hormones control apoptosis and where and how programmed cell death exerts modulating effects upon hormonally active tissues.     

Is apoptosis a "programmed cell death"?]

The purpose of this study was to evaluate the results and complications of anterior cruciate ligament surgery in middle-aged patients. Forty-five consecutive patients over 40 years old (average age, 44.6 years) who had arthroscopically assisted anterior cruciate ligament reconstructions with midthird patellar tendon autografts were evaluated. The patients returned for interviews, physical examinations, radiographs, Biodex dynamometer strength testing, and KT-1000 arthrometer testing at an average of 37 months after their surgeries (range, 24 to 96 months). The mean Lysholm and Gillquist score was 91, which corresponds to symptoms only with vigorous activity. The overall scores from the International Knee Documentation Committee form were 29 (64%) normal or nearly normal and 2 (4%) severely abnormal. Side-to-side differences as determined by the KT-1000 arthrometer were < or = 3 mm in 31 of 40 patients (78%), between 3 and 5 mm in 4 patients (10%), and > 5 mm in 5 patients at 30 pounds of anterior displacement. Seventy-six percent of the patients (N = 34) returned to their preoperative activity levels. Three patients required repeat arthroscopic surgery for persistent knee pain and two patients had graft ruptures. This study shows that when middle-aged patients undergo surgery, their results can be successful and satisfying to a degree similar to those of younger patients.     

Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death

Infection of pigs with classical swine fever virus (CSFV), a member of the Flaviviridae family, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was to analyze mechanisms behind this CSFV-induced lymphopenia. To this end, the kinetics of leukocyte depletion, the appearance of apoptotic cells, and virus infection of leukocytes after infection of pigs with the virulent CSFV strain Brescia were analyzed. Depletion of B and T lymphocytes was noted as early as 1 day postinfection (p.i.). Circulating viable lymphocytes with reduced mitochondrial transmembrane potential--a particular early marker for apoptosis--were also detectable as early as 1 day p.i. When isolated peripheral blood mononuclear cells were cultured for 6 h, significantly more sub-G1 cells with reduced DNA content were detected among the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well as infection of leukocytes, was 3 days p.i. However, throughout the observation time of 1 week, <3% of the circulating leukocytes and no lymphocytes contained virus or viral antigen. Further analysis of the T lymphocytes from infected animals demonstrated an increase in CD49d, major histocompatibility complex class II, and Fas expression. An increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as apoptosis-inducing anti-Fas antibody to the cultures. Taken together, these results imply that activation-induced programmed cell death was the mechanism behind lymphopenia during classical swine fever.     

Neuroimmunodegeneration: do neurons and T cells use common pathways for cell death?

In syndromes of pediatric neuroimmunodegeneration (NID), certain neurons and T cells degenerate and disappear during early development at an accelerated rate without alerting the peripheral immune cells. Current studies of some of these NID syndromes suggest that the primary cause of neuronal and T cell death is an imbalanced cytokine signaling system with a dysfunctional redox status, and that the loss of T cells and neurons may be secondary to impaired functions of their accessory supportive cells. These dysfunctions include inappropriate production of developmental cytokines, inadequate secretion of reductants, and disregulation of excitotoxic amino acid metabolism. Two examples of pediatric NID in humans are ataxia telangiectasia and pediatric human immunodeficiency virus infection. An animal model is retrovirus-induced T and neuronal cell loss in neonatal mice infected with a neuroimmunopathogenic mutant, ts1, of the Moloney murine leukemia virus. Because both thymic and neuronal components share many growth factors and developmental signals, it is likely that disregulation of these signals would lead to concomitant dysfunction of neuronal and thymic cells. In this review, we focus on the pathogenic mechanisms involved in these developmental NID syndromes with the objective of identifying common pathogenic factors and pathways responsible for the concurrent losses of both neurons and T cells.     

Apoptosis and necrosis in toxicology: a continuum or distinct modes of cell death?

Mounting evidence indicates that apoptosis rather than necrosis predominates in many cytolethal toxic injuries. Associated cell death models of apoptosis and necrosis are either: (1) totally separate death modes, (2) a continuum whereby they are extremes of biochemically overlapping death pathways, or (3) essentially distinct processes with only limited molecular and cell biology overlap. We conclude that the current balance of evidence favours the third of these options. The established axiom that, even when considering the same toxicant, injury amplitude (dose) is a primary determinant of whether cells die via active cell death (apoptosis) or failure of homeostasis (necrosis) remains valid. Tissue selectivity of toxicants can stem from the apoptotic or necrotic thresholds at which different cells die, as well as targeting factors such as toxicokinetics, receptor recognition, bioactivation, and cell-specific lesions.     

Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis

Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca2+]C) and intramitochondrial ([Ca2+]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal; HNE) insult. STS and HNE each induced an early increase of [Ca2+]C followed by delayed increase of [Ca2+]M. Overexpression of Bcl-2 blocked the elevation of [Ca2+]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca2+]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca2+]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.     

Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death

Tumor necrosis factor-alpha (TNFalpha)-induced cell death involves a diverse array of mediators and regulators including proteases, reactive oxygen species, the sphingolipid ceramide, and Bcl-2. It is not known, however, if and how these components are connected. We have previously reported that GSH inhibits, in vitro, the neutral magnesium-dependent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNFalpha induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNFalpha-induced sphingomyelin hydrolysis and ceramide generation as well as cell death. Furthermore, no significant changes in GSH levels were observed in MCF7 cells treated with either bacterial SMase or ceramide, and GSH did not protect cells from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNFalpha signaling pathway. TNFalpha has been shown to activate at least two groups of caspases involved in the initiation and "execution" phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1beta-converting enzyme-like proteases but is upstream of the site of action of Bcl-2 and of the execution phase caspases. Taken together, these studies demonstrate a critical role for GSH in TNFalpha action and in connecting major components in the pathways leading to cell death.     

Apoptosis and necrosis: mechanisms of cell death induced by cyclosporine A in a renal proximal tubular cell line

BACKGROUND: The mechanisms of cyclosporine (CsA)-induced nephrotoxicity are not fully understood. While hemodynamic changes may be involved in vivo, there is also some evidence for tubular involvement. We previously showed direct toxicity of CsA in the LLC-PK1 renal tubular cell line. In the current study we examined mechanisms (apoptosis or necrosis) of cell death induced by CsA in the LLC-PK1 renal proximal tubular cell line. The possible role of the Fas (APO-1/CD95) antigen-Fas ligand system in the mediation of CsA-induced cell death was also investigated. METHODS: Cells were treated with CsA (0.42 nM to 83 microM) for 24 hours and alterations in DNA and protein synthesis and membrane integrity were examined. Flow cytometry was used to investigate: (i) alterations in the DNA content and cell cycle; (ii) the forward (FSC) and side (SSC) light scattering properties (indicators of cell size and granularity, respectively); (iii) the externalization of phosphatidylserine (PS) as a marker of early apoptosis using FITC-annexin V binding; and (iv) expression of the apoptotic Fas protein. DNA fragmentation in apoptotic cells was also determined by the TUNEL assay. RESULTS: CsA (all doses) caused a block in the G0/G1 phase of the cell cycle as indicated by a decrease in DNA synthesis and supported by an increase in the % of cells in the G0/G1 phase with concurrent decreases of those in the S and G2/M phases. The effect on protein synthesis appeared to be much less. Lower doses of CsA (4.2 nM) caused the appearance of a "sub-G0/G1" peak, indicative of reduced DNA content, on the DNA histogram that was paralleled by a reduction in cell size and an increased cell granularity and an increase in FITC-annexin V binding. DNA fragmentation was evident in these cells as assessed using the TUNEL assay. Higher doses of CsA increased cell size and decreased cell granularity and reduced membrane integrity. Expression of Fas, the cell surface molecule that stimulates apoptosis, was increased following low dose CsA exposure. CONCLUSIONS: These results indicate that CsA is directly toxic to LLC-PK1 cells with reduced DNA synthesis and cell cycle blockade. The mode of cell death, namely apoptosis or necrosis, is dose dependent. Fas may be an important mediator of CsA induced apoptosis in renal proximal tubular cells.