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:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme activated by DNA damage. Activated PARP cleaves NAD(+) into nicotinamide and (ADP-ribose) and polymerizes the latter on nuclear acceptor proteins. Over-activation of PARP by reactive oxygen and nitrogen intermediates represents a pathogenetic factor in various forms of inflammation, shock, and reperfusion injury. Using a novel commercially available substrate, 6-biotin-17-nicotinamide-adenine-dinucleotide (bio-NAD(+)), we have developed three applications, enzyme cytochemistry, enzyme histochemistry, and cell ELISA, to detect the activation of PARP in oxidatively stressed cells and tissues. With the novel assay we were able to detect basal and hydrogen peroxide-induced PARP activity in J774 macrophages. We also observed that mitotic cells display remarkably elevated PARP activity. Hydrogen peroxide-induced PARP activation could also be detected in wild-type peritoneal macrophages but not in macrophages from PARP-deficient mice. Application of hydrogen peroxide to the skin of mice also induced bio-NAD(+) incorporation in the keratinocyte nuclei. Hydrogen peroxide-induced PARP activation and its inhibition by pharmacological PARP inhibitors could be detected in J774 cells with the ELISA assay that showed good correlation with the traditional [(3)H]-NAD incorporation method. The bio-NAD(+) assays represent sensitive, specific, and non-radioactive alternatives for detection of PARP activation.
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PMID:Detection of poly(ADP-ribose) polymerase activation in oxidatively stressed cells and tissues using biotinylated NAD substrate. 1174 98

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Poly (ADP-ribose) (PAR) is formed upon activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), and therefore was suggested as a new marker of apoptosis. Since DNA of epidermal cells represents a well-known chromophore for UVB irradiation, and UVB is known to generate H2O2 in keratinocytes, we hypothesized that PAR is a very sensitive marker of UVB- and H2O2-induced apoptosis in keratinocytes. In order to test this hypothesis, human immortalized keratinocytes (HaCaT) were UVB-irradiated or treated with H2O2, and subsequently apoptosis was identified by comparing conventional parameters such as morphological analysis, DNA laddering, and TUNEL assay, with PAR formation. Both, UVB and H2O2 treatment induced PAR formation in HaCaT cells in a dose-dependent manner, and its formation was detected as early as 4 h after irradiation, and at lower UVB doses (10 mJ/cm2) than observed by DNA laddering and the TUNEL assay. In conclusion, the detection of PAR formation is a very sensitive and early method for the identification of apoptotic cells in UVB-induced apoptosis of human keratinocytes.
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PMID:Detection of poly(ADP-ribose) by immunocytochemistry: a sensitive new method for the early identification of UVB- and H2O2-induced apoptosis in keratinocytes. 1203 59

Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and that AIF is necessary for PARP-1-dependent cell death. N-methyl-N'-nitro-N-nitrosoguanidine, H2O2, and N-methyl-d-aspartate induce AIF translocation and cell death, which is prevented by PARP inhibitors or genetic knockout of PARP-1, but is caspase independent. Microinjection of an antibody to AIF protects against PARP-1-dependent cytotoxicity. These data support a model in which PARP-1 activation signals AIF release from mitochondria, resulting in a caspase-independent pathway of programmed cell death.
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PMID:Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. 1211 11

Oxidative stress plays an important role in the development of ischemia/reperfusion (I/R)-induced apoptosis of hepatocytes. We aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1,000 micro M). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250 micro M. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked H2O2-induced procaspase-3 proteolysis and PARP cleavage without changing Bax cleavage, but partially attenuated H2O2-induced apoptosis. Calpeptin, a calpain inhibitor, did not inhibit caspase-3 activation, Bax cleavage or apoptosis. Our results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes exposed to H2O2, but not independent upon calpain. Molecular targeting of Bax cleavage may allow the development of strategies to prevent hepatic I/R injury.
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PMID:Bax cleavage implicates caspase-dependent H2O2-induced apoptosis of hepatocytes. 1257 42

This study was undertaken in order to examine the roles of lipid peroxidation and poly (ADP-ribose) polymerase (PARP) activation in oxidant-induced renal cell death. Opossum kidney cell cultures were used as the renal epithelial cell model, and an inorganic hydroperoxide H2O2 and an organic hydroperoxide t-butylhydroperoxide were employed as model oxidants. Cell death by both oxidants could be prevented by thiols (dithiothreitol and glutathione), iron chelators (deferoxamine and phenanthroline), and hydroxyl radical scavengers (dimethylthiourea and pyruvate). Phenolic antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) and butylated hydroxyanisole had no effect on the H2O2-induced cell death. However, the t-butylhydroperoxide-induced cell death was effectively prevented by these antioxidants. The PARP inhibitor 3-aminobenzamide prevented the cell death induced by H2O2, but not cell death by t-butylhydroperoxide. The PARP activity was increased in cells exposed to H2O2 but not t-butylhydroperoxide. Unlike in opossum kidney cells, in rabbit renal cortical slices both oxidants H2O2 and t-butylhydroperoxide induced cell death through a lipid peroxidation-dependent and PARP-independent mechanism. Effects of DPPD and 3-aminobenzamide on H2O2-induced cell death in primary cultured rabbit proximal tubular cells were similar to those in opossum kidney cells. These results indicate that 1) the H2O2-induced cell death in cultured renal epithelial cells is associated with PARP activation but not lipid peroxidation, whereas the t-butylhydroperoxide-induced cell death is mediated by lipid peroxidation, and 2) the role of lipid peroxidation in H2O2 cytotoxicity may be different between freshly isolated renal tubular cells and cultured renal epithelial cells.
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PMID:Oxidant-induced cell death in renal epithelial cells: differential effects of inorganic and organic hydroperoxides. 1271 May 97

Poly(ADP-ribosyl)ation plays important roles in cellular DNA repair mechanisms as well as in cellular proliferation and genomic stability. Four carcinoma cell lines, LNCaP, PC-3, C4-2 and MatLu, and one prostate epithelial cell line, PNT1A, were tested using the H10 antibody to poly(ADP-ribose) by Western blotting and FACS analysis for basal and activated (10 mM H2O2) PARP activity. In both analyses, higher levels of basal polymer were present in the LNCaP cell line than in the prostate epithelial cell line or the MatLu cell an line. LNCaP cells demonstrated greater than 90% positive analyzed cells in FACS analysis both before and after treatment with H2O2. PNT1A epithelial cells demonstrated evidence of polymer presence only after treatment with H2O2. Microsatellite instability due to increased oxidative damage in tumor cells has been proposed as a means for accumulation of genetic changes. As oxidative stress can stimulate poly(ADP-ribose) reactions, an increased presence of the polymer may indicate a high level of oxidative damage occurring in the tumor cells, contributing to higher levels of genetic instability towards progression.
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PMID:Intrinsic presence of poly (ADP-ribose) is significantly increased in malignant prostate compared to benign prostate cell lines. 1282 Apr 12

Mistletoe lectin-II, a major component of Korean mistletoe (Viscum album var. coloratum) induces apoptotic death in cancer cells. In this study, we demonstrated that lectin-II induced the generation of pro-oxidants and thus resulted in the apoptotic death of human myeloleukemic U937 cells. We observed that lectin-II-induced apoptotic death was inhibited by antioxidants including reduced glutathione (GSH), N-acetylcysteine (NAC), ebselen, mnTBP, catalase and pyrrolidine dithiocarbamate (PDTC). GSH and NAC also abolished the apoptotic DNA ladder pattern fragmentation of U937 cells after lectin-II stimulation. Obviously, lectin-II treatment of cells resulted in a remarkable generation of intracellular hydrogen peroxide (H2O2) as an early event, which was monitored fluorimetrically using scopoletin-horse radish peroxidase (HRP) assay and peroxide-sensitive fluorescent probe, DCF-DA. In addition, antioxidants inhibited the activation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) as well as cytosolic release of cytochrome c by mistletoe lectin-II. Moreover, lectin-II-induced activation of caspase-9 and 3-like protease and cleavage of poly(ADP-ribose) polymerase (PARP) were inhibited by pretreatment of cells with thiol antioxidants, GSH and NAC. Taken together, these results suggest that Korean mistletoe lectin-II is a strong inducer of pro-oxidant generation such as H2O2, which mediates the JNK/SAPK activation, cytochrome c release, activation of caspase-9 and caspase 3-like protease, and PARP cleavage in human myeloleukemic U937 cells.
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PMID:Involvement of hydrogen peroxide in mistletoe lectin-II-induced apoptosis of myeloleukemic U937 cells. 1285 Feb 39

The loss of retinal pigment epithelium (RPE) with aging is related to age-related macular degeneration (AMD). This study was conducted to investigate the mechanism of hydrogen peroxide (H2O2) induced cell death in a human retinal pigment epithelial cell line, ARPE-19. Hydrogen peroxide was added at different concentrations to ARPE-19 cells and cultured. The cytotoxicity was assayed by mitochondrial function using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) testing. The patterns of cell damage were assessed using an acridine orange-ethidium bromide differential staining method, in situ end labeling (ISEL) assay and transmission electron microscopy (TEM). Catalase, a major antioxidant, was used to prevent cell death. The cleavage of procaspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by western blot analysis. Hydrogen peroxide significantly induced cell death in ARPE-19 cells, whereas pretreatment of the cells with catalase prevented cell death. Application of the ISEL assay and acridine orange/ethidium bromide staining demonstrated that the H2O2-induced cell death occurred by an apoptotic mechanism at lower concentrations of H2O2 (400, 500, 600 microM), whereas higher concentrations of H2O2 induced necrosis rather than apoptosis. Caspase 3 was associated with the apoptotic pathway in human RPE cell death. Western blot analysis confirmed caspase 3 activation and cleavage of substrate proteins in ARPE-19 cells treated with an H2O2 concentration of 600 microM. These results indicate that treatment with H2O2 induces apoptotic and necrotic cell death in ARPE-19, and that caspase 3 is associated with apoptotic cell death. Therefore, H2O2 may induce the destruction of RPE cells in AMD by the combined effects of apoptosis and necrosis.
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PMID:Hydrogen peroxide-induced cell death in a human retinal pigment epithelial cell line, ARPE-19. 1288 4

The molecular role of poly (ADP-ribose) polymerase-1 in DNA repair is unclear. Here, we show that the single-strand break repair protein XRCC1 is rapidly assembled into discrete nuclear foci after oxidative DNA damage at sites of poly (ADP-ribose) synthesis. Poly (ADP-ribose) synthesis peaks during a 10 min treatment with H2O2 and the appearance of XRCC1 foci peaks shortly afterwards. Both sites of poly (ADP-ribose) and XRCC1 foci decrease to background levels during subsequent incubation in drug-free medium, consistent with the rapidity of the single-strand break repair process. The formation of XRCC1 foci at sites of poly (ADP-ribose) was greatly reduced by mutation of the XRCC1 BRCT I domain that physically interacts with PARP-1. Moreover, we failed to detect XRCC1 foci in Adprt1-/- MEFs after treatment with H2O2. These data demonstrate that PARP-1 is required for the assembly or stability of XRCC1 nuclear foci after oxidative DNA damage and suggest that the formation of these foci is mediated via interaction with poly (ADP-ribose). These results support a model in which the rapid activation of PARP-1 at sites of DNA strand breakage facilitates DNA repair by recruiting the molecular scaffold protein, XRCC1.
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PMID:A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage. 1450 Aug 14

The effects of epigallocatechin gallate (EGCG) on the phosphoinositide 3-kinase (PI3K)/Akt and glycogen synthase kinase-3 (GSK-3) pathway during oxidative-stress-induced injury were studied using H2O2-treated PC12 cells, which were differentiated by nerve growth factor (NGF). Following 100 microM H2O2 exposure, the viability of differentiated PC12 cells (EGCG or z-VAD-fmk pretreated vs. not pretreated) was evaluated the number of viable cell with Trypan blue and 3,4,5-dimethylthiazol-2-yl (MTT). Additionally, expression of cytochrome c, caspase-3, poly(ADP-ribose) polymerase (PARP), PI3K/Akt and GSK-3 was examined using Western blot analyses. EGCG or z-VAD-fmk-pretreated PC12 cells showed an increase of viability compared to untreated PC12 cells, and pretreatment of PC12 cells with either agent induced a dose-dependent inhibition of caspase-3 activation and PARP cleavage. However, inhibition of cytochrome c release was only detected in EGCG-pretreated cells. Upon examination of the PI3K/Akt and GSK-3 upstream pathway, Western blots of EGCG pretreated cells showed decreased immunoreactivity (IR) of Akt and GSK-3 and increased IR of p85a PI3K, phosphorylated Akt and phosphorylated GSK-3. In contrast, no changes were seen in z-VAD-fmk-pretreated cells. These results show that EGCG affects the PI3K/Akt, GSK-3 pathway as well as downstream signaling, including the cytochrome c and caspase-3 pathways. Therefore, it is suggested that EGCG-mediated activation of PI3K/Akt and inhibition of GSK-3 could be a new potential therapeutic strategy for neurodegenerative diseases associated with oxidative injury.
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PMID:Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3. 1455 56


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