Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.
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PMID:Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. 806 38

This study determined the occurrence of two molecular markers of apoptosis, chromosomal DNA strand breaks and oolemma phosphatidylserine redistribution, in >200 uninseminated and unfertilized human oocytes, and >800 newly ovulated and cultured mouse oocytes. DNA breaks were analysed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and phosphatidylserine by annexin V staining, with imaging by conventional epifluorescence and scanning laser confocal fluorescence microscopy. More than 300 intact and 500 fragmented mouse oocytes were examined at 24 h intervals during 6 days of culture in three different types of medium. For the human, 205 oocytes were examined at retrieval or at 24 h intervals during 7.5 days of culture in two types of medium. The perifollicular vascularity and the dissolved oxygen content of follicular fluid were determined for most of the follicles from which human oocytes were derived. The results demonstrate that TUNEL fluorescence of metaphase II (MII) chromosomes and annexin V staining of the oolemma in newly ovulated and cultured mouse and human oocytes are rare, and, when detected, are not spatially or temporally related. This finding also applied to mouse oocytes that fragmented during culture and exhibited morphological features that grossly resembled apoptotic body formation. In contrast, TUNEL but not annexin V staining occurred in the first polar body of a relatively high proportion of newly ovulated mouse oocytes, but was rarely detected in newly aspirated human oocytes. For the human, the occurrence of MII chromosomal TUNEL fluorescence was patient-specific and unrelated to perifollicular vascularity or dissolved oxygen content of the corresponding follicular fluid. The pattern of chromosomal TUNEL fluorescence observed in the first polar body and in the MII chromosomes of a very small number of mouse and human oocytes, especially after many days of culture, suggests that DNA strand breaks may not arise by apoptosis-associated endonuclease digestion. The results with these two markers suggest that it is premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects.
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PMID:DNA strand breaks and phosphatidylserine redistribution in newly ovulated and cultured mouse and human oocytes: occurrence and relationship to apoptosis. 964 66

Eosinophils, prominent cells in asthmatic inflammation, undergo apoptosis or programmed cell death following deprivation of contact with survival-promoting cytokines such as IL-5 and GM-CSF. The aim of this study was to assess a number of techniques for the quantification of apoptosis in human eosinophils cultured with or without IL-5 or GM-CSF and following staurosporine treatment. The relationship between apoptosis and necrosis in eosinophils was also determined. Eosinophils 'aged' in vitro for 48 h exhibited endonuclease DNA degradation, apoptotic morphology, increased red autofluorescence and externalisation of phosphatidylserine (PS) as assessed by binding of FITC-labelled annexin V. Annexin V-FITC binding was first detectable in eosinophils maintained at 37 degrees C for 5 h post-purification. This method proved to be the most sensitive marker of apoptosis. Morphological assessment of wet preparations of eosinophils by Kimura staining was found to be the next most-sensitive marker followed by increased red autofluorescence. The latter was a relatively insensitive method for the detection of apoptosis. At 5, 20 and 24 h of culture trypan blue exclusion indicated that eosinophil viability was high (85-90% viable cells). However, propidium iodide (PI) staining and flow cytometry revealed that, by 24 h, approximately 75% of cells had compromised membrane integrity. Eosinophils maintained in IL-5 or GM-CSF exhibited a non-apoptotic morphology and levels of annexin V-FITC binding and PI uptake similar to that of freshly isolated cells. Staurosporine (10(-5) M) treatment of eosinophils maintained in IL-5 or GM-CSF resulted in significant levels of apoptotic morphology at 2 h (23.8% +/- 6.9, p < 0.025) which was associated with negligible annexin binding. At 6 h post-staurosporine treatment significant annexin-FITC binding (38% +/- 1.5, p < 0.025) was observed compared with 93% +/- 1.2 of eosinophils displaying apoptotic morphology. Exclusion of PI demonstrated membrane integrity at all time points up to 6 h. Thus, eosinophils aged in vitro in the absence of viability-promoting cytokines exhibit evidence of both apoptosis and necrosis simultaneously. In contrast, staurosporine-treated eosinophils exhibited both membrane integrity and rapid apoptosis-associated morphological changes detected by single step Kimura staining which preceded externalisation of PS.
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PMID:A comparative study of different methods for the assessment of apoptosis and necrosis in human eosinophils. 977 85

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.
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PMID:Phospholipid rearrangement of apoptotic membrane does not depend on nuclear activity. 982 26

Pollutant particles induce apoptosis and inflammation, but the relationship between these two biological processes is not entirely clear. In this study, we compared the proapoptotic and proinflammatory effects of four particles: residual oil fly ash (ROFA), St. Louis particles SRM 1648 (SL), Chapel Hill PM10 (CHP), and Mount St. Helens dust (MSH). Human alveolar macrophages (AM) were incubated with these particles at 100 microg/ml. Cell death was assessed by annexin V (AV) expression, histone release, nuclear morphology, caspase 3-like activity and release of caspase 1 for apoptosis, and propidium iodide (PI) for necrosis, and inflammation was measured by interleukin (IL)-1beta and IL-6. We found that particle effects on these cell death measurements varied, and ROFA affected most (four out of five) endpoints, including nuclear morphological changes. CHP and SL also caused necrosis. For cytokine release, the potency was CHP > SL > ROFA > MSH. The proapoptotic and proinflammatory effects induced by the whole particles were unaltered after the particles were washed with water. The water-soluble fraction was relatively inactive, as were individual soluble metals (V, Ni, Fe). ROFA-induced nuclear fragmentation was associated with upregulation and mitochondrial release of apoptosis-inducing factor (AIF), a caspase-independent chromatin condensation factor, and upregulation of DNase II, a lysosomal acid endonuclease. These results indicate that the potential for particles to induce apoptosis does not correlate with their proinflammatory properties, although active components for both processes reside in the water-insoluble core. Both apoptosis and inflammatory endpoints should be included when the toxicity of different pollutant particles is assessed.
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PMID:Apoptotic and inflammatory effects induced by different particles in human alveolar macrophages. 1576 74

Renal cell carcinoma is the most common neoplasm occurring in the kidney and is largely resistant to current chemotherapy. Understanding the mechanisms involved in renal carcinoma cell death may lead to novel and more effective therapies. In Cak(i)-1 renal cancer cells, using phosphatidylserine externalization as a marker of apoptosis, the anti-cancer drugs 5-fluorouracil (5-FU), and its pro-drugs, doxifluridine (Dox) and floxuridine (Flox) proceeds via a caspase-dependent mechanism. In contrast, phosphatidylserine externalization produced by staurosporine in the renal cancer cell lines Cak(i)-1 and A-498 proceeds via a caspase-independent mechanism. That is, the pan caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) did not ameliorate annexin V binding, cell shrinkage or changes in nuclear morphology. Subsequent experiments were conducted to determine mediators of phosphatidylserine externalization, using annexin V binding, when caspases were inhibited. Prior treatment of A-498 cells with cathepsin B (CA74 methyl ester), cathespsin D (pepstatin A) or calpain inhibitors (calpeptin, E64d) in the presence or absence of ZVAD did not ameliorate annexin V binding. The endonuclease inhibitor aurintricarboxylic acid (ATA), phospholipase A(2) inhibitor bromoenol lactone (BEL), protein synthesis inhibitor cycloheximide (CH) and chloride channel blockers niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) all had no effect on staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. We also modulated sphingomyelin and the de novo pathways of ceramide synthesis and found no amelioration of staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. These results indicate that 5-FU, Dox and Flox induce externalization of phosphatidylserine during apoptosis in Cak(i)-1 renal cancer cells primarily through a caspase-dependent mechanism and that externalization of phosphatidylserine during apoptosis produced by staurosporine in the renal cancer cell line A-498 is independent of many of the common signaling pathways known to be involved in this process.
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PMID:Caspase-dependent and -independent induction of phosphatidylserine externalization during apoptosis in human renal carcinoma Cak(1)-1 and A-498 cells. 1709 91

Cisplatin is an anticancer drug currently used in the treatment of genital and head and neck tumors. Its use in these and other types of tumors is narrowed by onset of chemoresistance and severe undesired side effects, like as nephro- and ototoxicity, whose mechanisms of action are only partially understood. In the present study we investigated the effects of cisplatin (cis-dichlorodiaminoplatin, CDDP) on a cell line (OC-k3) developed from organs of Corti of transgenic mice. We observed at 48 h that cell death due to cisplatin was time and concentration-dependent. The cell death displayed some morphological hallmarks of apoptosis, including nuclear fragmentation into several large nuclear fragments, surrounded by a rearranged and thickened actin cytoskeleton. No DNA laddering was detected, suggesting absence of endonuclease activity, nor annexin V positivity, suggesting absence of phosphatidylserine externalization. Several molecules protected the cells against CDDP induced cytotoxicity, including methionine, suramin and PD98059. Methionine reduced CDDP-uptake, while suramin, a polycathionic compound a specifically binding external proteins, did not. This finding suggested that suramin could exert its protective effect by acting on an intracellular transduction pathway. We tested this hypothesis by studying the effect of suramin and PD98059, a MEK inhibitor, on the mitogen activated protein kinase (MAPK) cascade. After CDDP treatment, we found an increase of phosphorylation of extracellular regulated kinases (ERK)1/2, that could be inhibited by PD98059 and suramin. These data suggest that ERK pathways can play a role in mediating the cell death induction in presence of a CDDP challenge.
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PMID:Cisplatin cytotoxicity in organ of Corti-derived immortalized cells. 1724 13

Apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. Studies have suggested that abnormal Ref-1 levels and/or activities are associated with tumor progression and sensitivities to treatment, but no direct evidence has yet been published regarding the role of Ref-1 in malignant transformation. We utilized the well-documented tumor promotor-sensitive JB6 mouse epithelial cell model as well as new transformants [by ultraviolet light B (UVB), H2O2 or Cd] to study this phenomenon. Significant increases of reactive oxygen species (ROS) were observed in JB6P+ and all the transformants compared with promotor-resistant JB6P- cells. These increases were paralleled by a sustained elevation of Ref-1 expression. Further analysis exhibited a strong inverse correlation between oxidative DNA lesions [8-oxodeoxyguanosine (8-oxo-dG)] and Ref-1 levels in all JB6 cells. Notably, apoptosis occurred after knock-down of Ref-1 by small interfering RNA (siRNA)] demonstrated by a approximately 2-fold increase of Annexin V-positive JB6P+ cells. Ref-1 depletion also inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced anchorage-independent growth of JB6P+ by 40% and reduced the colony numbers of JB6P+/H2O2 and JB6P+/Cd cells. Mechanistic studies revealed that Ref-1 reduction was associated with an increase of intracellular ROS levels and a marked decrease of activator protein-1 (AP-1) transcription activities in JB6P+/H2O2 cells. This is the first report of the novel role of Ref-1 in cellular transformation. Based on the data presented here, we propose that induction of Ref-1, serving as an adaptive response to elevated ROS, plays a critical role in transformation and protects cells from excess ROS stresses through both DNA repair and activation of transcription factors such as activator protein-1.
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PMID:Redox effector factor-1, combined with reactive oxygen species, plays an important role in the transformation of JB6 cells. 1756 60

DNA fragmentation factor 40 (DFF40) is an endonuclease that acts downstream in the apoptotic cascade. The objective of this study was to generate a novel humanized chimeric protein with human DFF40 fused with GM-CSF for targeting acute myeloid leukemia (AML) cells. cDNA cloning of human DFF40 and GM-CSF was done and the chimeric gene GM-CSF-DFF40 was generated by overlap extension PCR. The fusion protein was expressed in E.coli, purified, refolded and characterized. In vitro cytotoxicity was evaluated on various AML cell lines. Treated cell lines were screened for various morphological and biochemical changes that are characteristic of apoptosis, by different assays like annexin V-FITC staining, TUNEL assay, JC-1 staining and immunocytochemistry of pro-apoptotic proteins and caspases. Cell cycle analysis of treated cells was done to quantify the percentage of apoptotic cells. The chimeric protein was found to be cytotoxic to AML cells in a dose and time dependent manner. Morphological changes such as formation of apoptotic bodies were revealed by microscopic examination of treated cells after staining. Immunocytochemical staining demonstrated biochemical changes such as changes in mitochondrial membrane potential, mitochondrial co-localization of Bax, cytochrome c release, presence of activated caspase-3 and DNA fragmentation. FACS analysis proved the presence of apoptotic cells following treatment. The chimeric protein GM-CSF-DFF40 was found to mediate targeted killing of AML cells by inducing apoptosis. Thus, this chimeric construct can act as a prospective candidate for targeted therapy of AML and other malignancies where GM-CSF receptor expression is upregulated.
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PMID:GM-CSF-DFF40: a novel humanized immunotoxin induces apoptosis in acute myeloid leukemia cells. 2352 88

It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.
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PMID:Induction of caspase-3-like activity in rice following release of cytochrome-f from the chloroplast and subsequent interaction with the ubiquitin-proteasome system. 2510 21


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