Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Features of apoptotic cells measured by flow cytometry. 133 43

The cellular basis for the enhanced sensitivity to ionising radiation and some DNA damaging chemicals in ataxia-telangiectasia (AT) cells is not clearly understood. Abnormalities in cell-cycle traverse, chromosome stability and DNA synthesis patterns have suggested that a chromatin associated defect may be the primary lesion in AT. This study involves an attempt to define such an anomaly by the use of a vital DNA specific bis-benzimidazole dye (Hoechst 33342) and deoxyribonuclease II as probes for chromatin organisation in intact and permeabilised human cells respectively. Despite similar DNA binding characteristics (determined by flow cytometry) of Ho33342 in normal and AT transformed fibroblasts, the AT cells show: (i) enhanced cell killing and increased accumulation of cells in G2 phase of the cell-cycle [both biological responses being relatively resistant in AT cells to modification by an inhibitor of poly (ADP ribosyl)ation], (ii) no resistance of de novo DNA synthesis to Ho33342-induced inhibition, (iii) elevated levels of slow-rejoining ligand-induced DNA strand-breaks, and (iv) enhanced expression of chromatin regions accessible to an exogenously supplied endonuclease. The results are interpreted on the basis that a chromatin anomaly of enhanced nuclease susceptibility, involving a minor fraction of the genome, may be a controlling factor in the expression of the various in vivo and in vitro characteristics of AT cells.
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PMID:Relationship between a chromatin anomaly in ataxia-telangiectasia cells and enhanced sensitivity to DNA damage. 648 55

A murine fibrosarcoma clone, Gc-4 SD, grows depending on fetal calf serum. In MTT assay, protein-free cultivation resulted in a reduction of the viable cell number time-dependently. Electron-microscopic and flow-cytometric analyses revealed that the reduction in growth was accompanied by the appearance of apoptotic cells. However, no internucleosomal fragmentation was observed even after SI-nuclease treatment. On the other hand, pulse field gel electrophoresis revealed that cleavage of DNA into high-molecular-weight fragments estimated as 50 to 150 kilobase pairs (kbp), with a peak of 100 kbp, was found in the serum-deprived cells. Large fragments disappeared from the DNA extracts when the smaller cells with high blue fluorescence with Hoechst 33342 were removed by flow cytometry, suggesting direct correlation between the large DNA fragmentation and apoptosis. The addition of aurintricarboxylic acid neither abolished the large DNA fragmentation nor inhibited the reduction in the number of viable cells. Both cycloheximide and actinomycin D enhanced the reduction in the number of viable cells as well as the large DNA fragmentation. These results suggest that apoptosis of a fibrosarcoma induced by protein-free culture involves a specific endogenous endonuclease, which may be distinct from and independent of the ATA-sensitive endonuclease producing internucleosomal DNA fragmentation.
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PMID:Apoptosis of a fibrosarcoma induced by protein-free culture involves DNA cleavage to large fragments but not internucleosomal fragmentation. 762 95

DNA fragmentation is a common biochemical hallmark of apoptosis. It is catalyzed by endogenous Ca2+, Mg(2+)-dependent endonuclease(s). Although the exact identity of the apoptotic endonuclease is still a matter of debate, a number of candidate nucleases have been proposed like NUC18, DNase II and DNase I. Relatively large amounts of nucleases are also expressed by mycoplasmas, cell wall-less bacteria of the class Mollicutes, which are found as contaminants in up to 45% of the continuous cell lines in current use. In order to clarify the effect of these pathogens on the investigation of apoptosis in cell culture systems, we looked for biochemical markers (DNA fragmentation, nuclease expression) and morphological changes characteristic of apoptosis (cell shrinkage, chromatin condensation, apoptotic bodies) in Mycoplasma hyorhinis-free and -infected cultures of the human pancreatic adenocarcinoma cell line PaTu 8902 and of mouse NIH 3T3 fibroblasts. For that purpose we employed cells cultured under standard conditions and cells exposed to the protein synthesis inhibitor cycloheximide, which is known to induce apoptosis in various cell systems. After exposure to cycloheximide only the mycoplasma-positive cells exhibited internucleosomal DNA degradation. In contrast, nuclease activities in the molecular range of 47 to 54 kDa were detected in cell homogenates and culture supernatants of infected cultures of both control and cycloheximide-treated cells, whereas mycoplasma-free cultures were nuclease-negative. The expression of the nucleases and the cycloheximide-induced DNA fragmentation were suppressed by the prokaryote-specific protein synthesis inhibitor chloramphenicol. Moreover, partially purified nucleases from supernatants of infected cells were able to cleave the DNA of isolated substrate nuclei at internucleosomal sites. These data indicate that DNA ladder formation in cell culture systems can also be caused by mycoplasmal nucleases which apparently penetrate the host cells after cycloheximide treatment or more generally after cellular stress. Therefore, internucleosomal DNA fragmentation in established cell lines has to be regarded with care, unless mycoplasmal infection can be excluded, or the existence of endogenous endonucleases can be proven. The presence of endonucleolytic activities of about 47 to 54 kDa molecular mass has now to be regarded as highly indicative of contaminations with M. hyorhinis. In contrast, the expression of an apoptotic morphology was not restricted to infected cells; in both mycoplasma-free and -contaminated cultures, cells with condensed chromatin were observed after staining with the DNA binding dye Hoechst 33342. Electron microscopic studies revealed that most of the cells containing compacted DNA were phagocytosed by unaffected fellow cells. Presumably because of the relatively long exposure (72 h) to cycloheximide we also observed secondary necrosis as indicated by the parallel occurrence of morphological characteristics of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling).
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PMID:Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases. 888 84

Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature T-cells, predominantly of the helper phenotype, that primarily invade the skin. Different photo- and chemotherapeutic treatments are known to be beneficial in early-stage CTCL. This observation has initiated prospective investigations into the efficacy of phototherapeutic regimens. The purpose of our study was to investigate the ability of medium-dose UVA1 phototherapy (60 J/cm2) to induce apoptosis (programmed cell death) in skin infiltrating T-cells of CTCL in vivo. We describe the results of three different staining methods for formalin-fixed, paraffin-embedded tissue sections. The in situ end-labeling (ISEL) procedure, nuclear staining using the DNA-binding fluorochrome Hoechst 33342, and immunohistochemistry using polyclonal antibodies against recombinant mouse deoxyribonuclease I (DNase I) demonstrated that UVA1 irradiation was able to induce marked apoptosis in CTCL. Thereby, ISEL and Hoechst staining clearly revealed DNA-condensation and nuclear fragmentation, accompanied by the formation of typical "apoptotic bodies". The accumulation of DNase I immunoreactivity in the cytoplasm of lymphocytes in UVA1 irradiated skin indicated that DNase I or DNase I-related endonucleases may have acted as apoptotic endonuclease(s) which were synthesized after UVA1 irradiation prior to their apoptotic elimination.
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PMID:UVA1 irradiation induces deoxyribonuclease dependent apoptosis in cutaneous T-cell lymphoma in vivo. 1113 31

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was attenuated by Zn2+ (modulator of Ca2+ dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the 'apoptotic' type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5-3 h) of various pharmacological agents viz., Trolox (antioxidant), EGTA (Ca2+ modulator) and aurintricarboxylic acid (ATA; Ca2+/Mg2+ dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox also increased the LD(50) of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.
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PMID:Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo. 1127 22

Apoptosis is commonly associated with DNA digestion, but it remains controversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-dependent endonuclease in cell lysates, has been used frequently to suggest this is the endonuclease involved. However, zinc has many other effects on cells, and here it is shown that zinc also prevents many upstream events in apoptosis. These studies were performed in human ML-1 cells following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all of these events. An upstream event in apoptosis is activation of ICE/CED-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event even further upstream in apoptosis; zinc was also found to inhibit Rb dephosphorylation. Therefore, zinc must protect cells at a very early step in the apoptotic pathway, and not as a direct inhibitor of an endonuclease.
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PMID:Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease. 1646 18

Through the use of a scanning electronic microscope, it was found that alveolar macrophages treated with 10 micro M of methylmercury for 24 h showed a decrease of surface microvilli, and those treated with 15 micro M of methylmercury underwent deformity and subsequent cell death. To investigate their death patterns, DNA was aspirated from alveolar macrophages and analyzed by electrophoresis. It was discovered that the DNA ladder phenomenon became more obvious as the methylmercury increased in concentration. When 5 mM EGTA was used to eliminate calcium ions, a decrease of the ladder phenomenon was observed. Zinc at 1 mM had a similar inhibitory effect. Moreover, an apoptosis peak was observed on flow cytometry analysis of DNA stained with propidium iodide. Alveolar macrophages stained with Hoechst 33342 demonstrated apoptotic bodies induced by methylmercury. The above data indicate that methylmercury can induce a typical apoptosis in alveolar macrophages. Continuing onto the study of the mechanism of apoptosis as induced by methylmercury in alveolar macrophages, it was discovered that methylmercury could increase the intracellular calcium ion concentration and decrease the pH in alveolar macrophages. To find out which endonuclease was responsible for the methylmercury-induced DNA fragmentation of alveolar macrophages, the nuclear proteins of alveolar macrophages was aspirated and tested under different pH values and in conditions with or without calcium ions, and it was discovered that the endonuclease was calcium dependent without relations to pH values.
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PMID:Methylmercury induces alveolar macrophages apoptosis. 1856 66