Apoptosis and programmed cell death: a role in cerebral ischemia

Hypoxic-ischemic neuronal death has long been considered to represent necrosis, but it now appears that many brain neurons undergo apoptosis after either global or focal ischemic insults. Recent studies demonstrated: 1) DNA cleavage into oligonucleosome-sized fragments demonstrated by a typical ladder pattern; 2) early endonuclease activation, as demonstrated by the presence of high molecular weight DNA fragments (300 to 50 kbp); 3) chromatin condensation and apoptotic bodies formation; 4) activation of apoptosis-associated proteins. These results may indicate that apoptosis contributes to the development of the ischemic infarct and is probably substantially distinct from ischemia-triggered excitotoxicity, which tends to produce necrosis.     

Apoptosis: bcl-2 as a key protein for programmed cell death

The aim of the present paper is to present results of sperm morphology using an objective and manual technique by video image. Experiment 1:252 spermatozoa heads were measured in a microscope and in a monitor by each of three independent observers. The results allowed the calibration of an acetate overlay according to the WHO guideline and following the strict criteria. Experiment 2: 10 morphology slides from normal and abnormal patients were studied. These slides were evaluated by three independent observers, each counting at least 200 cells using the calibrate acetate overlay. In the first experiment the calculation of the regression out-put was: constant: 0.24, standard error of Yc: 0.04, R squared: 0.96, X coefficient: 0.36, and standard error of the coefficient: 0.03. In the second experiment, it can be seen that the differences among the operators are not statistically significant and therefore the experiment is independent from the operator. In conclusion, the methodology developed in this paper for the evaluation of morphology would be a good tool for the evaluation of human sperm morphology.     

Diverse molecular provocation of programmed cell death

The experimental study and manipulation of programmed cell death has been greatly assisted by the identification of genetic and pharmacological tools that can either induce or block cell lethality. This review discusses new insights into the molecular sensing of perturbations induced by such tools, as well as the possible consequences of this detection in determining cell survival.     

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.     

Apoptosis: regulation and relevance to toxicology

1. Apoptosis is a remarkably stereotyped morphological event across all tissues in response to a vast array of damaging agents. 2. Our very existence depends upon a willing exchange of old life for new: apoptotic cell death is our guardian and saviour from genetic damage. 3. There is a close link between cell proliferation and apoptosis: When a cell picks up the machinery to proliferate it also acquires an abort pathway--'better dead than wrong'. 4. A wide variety of highly conserved genes have been implicated in triggering apoptosis. 5. The release of DNA loops from the nuclear scaffold is a more crucial intracellular event than DNA 'laddering' in apoptotic cells. 6. The manipulation of apoptotic rates in many of the common diseases in man will be a major therapeutic strategy in the future.     

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.     

Apoptosis as a proinflammatory event: what can we learn from bacteria-induced cell death?

At least 2 genes, detectable by DNA methods, encode autosomal dominant polycystic kidney disease (ADPKD), which remains the most frequent and serious hereditary renal disease. PKD1 gene, localized on chromosome 16, responds for the clinical course in the majority of ADPKD patients, whereas PKD2 gene, localized on chromosome 4, is responsible for less than 10-15% of cases, with presumed milder phenotypic manifestations. To start the clinical and genetic correlation in patients with different genotypes (PKD1 vs. PKD2) in the Czech population, a pilot group of 88 patients with ADPKD was analysed. Families with PKD1 (n = 44) represented 95.6% and families with PKD2 (n = 2) 4.4% of all families investigated (n = 46). Our clinical analysis, yet based only on a limited number of PKD2 subjects, does not definitely support the concept of a milder phenotype and prognosis in PKD2 versus PKD1 patients, in terms of mean age of diagnosis (29 vs. 29 years), mean age at onset of arterial hypertension (33 vs. 33 years), more favourable renal function or ultrasound findings.     

Apoptosis signaling by death receptors

How do stimulus size and item number relate to the magnitude and direction of error on center estimation and line cancellation tests? How might this relationship inform theories concerning spatial neglect? These questions were addressed by testing twenty patients with right hemisphere lesions, eleven with left hemisphere lesions and eleven normal control subjects on multiple versions of center estimation and line cancellation tests. Patients who made large errors on these tests also demonstrated an optimal or pivotal stimulus value, i.e., a particular size center estimation test or number of lines on cancellation that either minimized error magnitude relative to other size stimuli (optimal) or marked the boundary between normal and abnormal performance (pivotal). Patients with right hemisphere lesions made increasingly greater errors on the center estimation test as stimuli were both larger and smaller than the optimal value, whereas those with left hemisphere lesions made greater errors as stimuli were smaller than a pivotal value. In normal subjects, the direction of errors on center estimation stimuli shifted from the right of true center to the left as stimuli decreased in size (i.e., the crossover effect). Right hemisphere lesions exaggerated this effect, whereas left hemisphere lesions diminished and possibly reversed the direction of crossover. Error direction did not change as a function of stimulus value on cancellation tests. The demonstration of optimal and pivotal stimulus values indicates that performances on center estimation and cancellation tests in neglect are only relative to the stimuli used. In light of other studies, our findings indicate that patients with spatial neglect grossly overestimate the size of small stimuli and underestimate the size of large stimuli, that crossover represents an "apparent" shift in error direction that actually results from normally occurring errors in size perception, and that the left hemisphere is specialized for one aspect of size estimation, whereas the right performs dual roles.     

The regulation of reactive oxygen species production during programmed cell death

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.     

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 after gamma irradiation. Is it an important cell death modality?

Apoptosis and necrosis are two different forms of cell death that can be induced by cytotoxic stress, such as ionizing radiation. We have studied the importance of apoptotic death induced after treatment with 6 Gy of gamma-irradiation in a panel of eight human tumour cell lines of different radiosensitivities. Three different techniques based on the detection of DNA fragmentation have been used, a qualitative one--DNA ladder formation --and two quantitative approaches--in situ tailing and comet assay. No statistically significant relationship between the two quantitative assays was found (r= 0.327, P = 0.159) so these methods seem to show different aspects of the process of cell death. The presence of the DNA ladder related well to the end-labelling method in that the least amount of end labelling was seen in samples in which necrotic degradation rather than apoptotic ladders were seen. However, as the results obtained by the comet assay are not in agreement with the DNA ladder experiments, we suggest that the distinction between the degraded DNA produced by apoptosis and necrosis may be difficult by this technique. Finally, although apoptosis has been proposed to be dependent on p53 functionality, and this may explain differences in cellular radiosensitivity, no statistically significant relationship was found between these parameters and apoptosis in the eight cell lines studied.     

Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury

The complex biochemical interactions following acute spinal cord injury have undergone considerable investigation recently. Progress has been made in discovering both primary and secondary injury cascades that combine to produce the ultimate neurologic insult. Traditionally, neuronal and supporting cell death following spinal cord injury have focused on necrotic death pathways resulting passively from the actual mechanical tissue damage and inflammatory processes which follow. However, the occurrence of programmed apoptotic cell death which is an actively mediated cellular process may occur following acute spinal cord injury and, if present, may play a role in the ultimate neurologic insult. In this study, we document a chronologically-specific course of apoptotic cell death by the TUNEL assay technique following an acute experimental spinal cord injury in the rat model. In this manner, apoptotic cell death following acute spinal cord injury may play a pivotal role in the secondary injury cascade which produces the ultimate neurologic insult and may allow potential for mediating neuronal survival via anti-apoptotic factors such as the protooncogene Bcl-2.     

The molecular mechanisms of 5-fluoro-2'-deoxyuridine induced cell death

The molecular mechanism of cell death induced by 5-Fluoro-2'-deoxyuridine (FUdR) was investigated. FUdR caused cell death to induce dNTP pool imbalance and following DNA double strand breaks in mouse mammary tumor FM3A cells. We isolated a new endonuclease from FUdR-treated cells, named endonuclease S, that played an important role in FUdR-induced cell death. Cells treated with FUdR showed intracellular acidification before cell death formation. We observed that the endonuclease S in acidic cells may lead the DNA fragmentation. On the other hand, we observed that protease inhibitors (such as TLCK, TPCK, PMSF, p-APMSF, Pefabloc SC and Z-Asp-CH2-DCB) blocked intracellular acidification, DNA fragmentation and FUdR-induced cell death. But the inhibitors did not affect dNTP pool imbalance in the cells. These results suggest that proteases act at the point of downstream of dNTP pool imbalance and upstream of the intracellular acidification.     

Cell death in the Schwann cell lineage and its regulation by neuregulin

The development of Schwann cells, the myelin-forming glial cells of the vertebrate peripheral nervous system, involves a neonatal phase of proliferation in which cells migrate along and segregate newly formed axons. Withdrawal from the cell cycle, around postnatal days 2-4 in rodents, initiates terminal differentiation to the myelinating state. During this time, Schwann cell number is subject to stringent regulation such that within the first postnatal week, axons and myelinating Schwann cells attain the one-to-one relationship characteristic of the mature nerve. The mechanisms that underly this developmental control remain largely undefined. In this report, we examine the role of apoptosis in the determination of postnatal Schwann cell number. We find that Schwann cells isolated from postnatal day 3 rat sciatic nerve undergo apoptosis in vitro upon serum withdrawal and that Schwann cell death can be prevented by beta forms of neuregulin (NRG-beta) but not by fibroblast growth factor 2 or platelet-derived growth factors AA and BB. This NRG-beta-mediated Schwann cell survival is apparently transduced through an ErbB2/ErbB3 receptor heterodimer. We also provide evidence that postnatal Schwann cells undergo developmentally regulated apoptosis in vivo. Together with other recent findings, these results suggest that Schwann cell apoptosis may play an important role in peripheral nerve development and that Schwann cell survival may be regulated by access to axonally derived NRG.     

Programmed cell death (apoptosis): molecular mechanisms and the role in biology and medicine

Over the recent years studies of the cell death (CD) were progressing with an outstanding speed. CD is found to play a key role in proliferation, differentiation, embryogenesis, morphogenesis and homeostatic processes. CD abnormalities significantly contribute into numerous human diseases as cancer, AIDS, degenerative pathologies of nervous system and developmental abnormalities. The elucidation of CD mechanisms may promote our understanding of pathogenesis of various diseases and facilitate search for their treatment.     

Proteases in renal cell death: calpains mediate cell death produced by diverse toxicants

In recent years, the combination of gel electrophoresis and mass spectrometry has developed into one of the most powerful approaches for the analysis of proteins. However, a number of gel electrophoresis-induced protein modifications have been described. Cysteine is the most endangered amino acid readily reacting with mercaptoethanol or free acrylamide. In the course of studies on glucan phosphorylases (E.C.2.4.1.1) from white potato (Solanum tuberosum L.) and the T cell receptor, we noticed that proteolytic peptides from these proteins can undergo an unexpected modification, giving rise to a mass increment of 14 Da. By post-source decay (PSD) analysis the modification was identified as methylation of the glutamic acid side chain carboxyl group. The methylation takes place during Coomassie blue staining of proteins if both trichloroacetic acid and methanol are present in the staining solution. Replacement of methanol by ethanol under otherwise unchanged conditions results in ethylation of the peptides. The in vitro alkylation was further studied by using synthetic peptides which contain, at different positions: glutamic acid, aspartic acid or the corresponding amides. The kinetic analysis of the observed reactions revealed that glutamic acid is preferentially methylated. The three other amino acid residues can be methylated but with a velocity at least one order of magnitude lower. Although these modifications complicate the interpretation of the spectra, they provide valuable structural information.