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)

The requirement for caspases (ICE-like proteases) were investigated in mediating apoptosis of WEHI7.2 mouse lymphoma cells in response to two death inducers with different mechanisms of action, the glucocorticoid hormone dexamethasone (DX) and the calcium-ATPase inhibitor thapsigargin (TG). Apoptosis induction by these agents followed different kinetics, and was closely correlated with in vivo activation of caspase-3 (CPP32/Yama/Apopain) and cleavage of the caspase target protein poly(ADP-ribose) polymerase (PARP). Caspase activation and PARP cleavage were inhibited by Bcl-2 overexpression. Cell extracts from DX- and TG-treated cells cleaved the in vitro synthesized baculovirus p35 ICE-like protease target, producing 25 and 10 kDa fragments. p35 cleavage was inhibited by mutating the active site aspartic acid to alanine, and by a panel of protease inhibitors that inhibit caspase-3-like proteases, including iodoacetamide, N-ethylmaleimide, and Ac-DEVD-cho. Treatment of cells in vivo with two cell permeant peptide fluoromethylketone inhibitors of caspase activity, Z-VAD-fmk and Z-DEVD-fmk, inhibited DX- and TG-induced apoptotic nuclear changes and maintained plasma membrane integrity, whereas the cathepsin inhibitor, Z-FA-fmk, and two calpain inhibitors failed to inhibit apoptosis. An unexpected observation was that due to the delayed time course of DX-induced apoptosis, optimal preservation of plasma membrane integrity was achieved by adding caspase inhibitors beginning 8 h after DX addition. In summary, the findings indicate that two diverse apoptosis-inducing signals converge into a common Bcl-2-regulated pathway that leads to caspase activation and apoptosis.
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PMID:Apoptosis induction by the glucocorticoid hormone dexamethasone and the calcium-ATPase inhibitor thapsigargin involves Bc1-2 regulated caspase activation. 970 90

Cholera toxin (CT) increases intestinal secretion of water and electrolytes and modulates the mucosal immune response by stimulating cellular synthesis of arachidonic acid (AA) metabolites (e.g., prostaglandin E2), as well as the intracellular second messenger cyclic AMP (cAMP). While much is known about the mechanism of CT stimulation of adenylate cyclase, the toxin's activation of phospholipase A2, which results in increased hydrolysis of AA from membrane phospholipids, is not well understood. To determine whether CT activation of AA metabolism requires CT's known enzymatic activity (i.e., ADP-ribosylation of GSalpha), we used native CT and a mutant CT protein (CT-2*) lacking ADP-ribose transferase activity in combination with S49 wild-type (WT) and S49 cyc- murine Theta (Th)1.2-positive lymphoma cells deficient in GSalpha. The experimental results showed that native CT stimulated the release of [3H[AA from S49 cyc- cells at a level similar to that for S49 WT cells, indicating that GSalpha is not essential for this process. Further, levels of cAMP in the CT-treated cyc- cells remained the same as those in the untreated control cells. The ADP-ribosyltransferase-deficient CT-2* protein, which was incapable of increasing synthesis of cAMP, displayed about the same capacity as CT to evoke the release of [3H]AA metabolites from both S49 WT and cyc- cells. We concluded that stimulation of arachidonate metabolism in S49 murine lymphoma cells by native CT does not require enzymatically functional CT, capable of catalyzing the ADP-ribosylation reaction. These results demonstrated for the first time that stimulation of adenylate cyclase by CT and stimulation of AA metabolism by CT are not necessarily coregulated. In addition, the B subunits purified from native CT and CT-2* both simulated the release of [3H]AA from S49 cyc- cells and murine monocyte/macrophage cells (RAW 264.7), suggesting a receptor-mediated cell activation process of potential importance in enhancing immune responses to vaccine components.
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PMID:Cholera toxin B subunit activates arachidonic acid metabolism. 991 92

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin alpha subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.
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PMID:Characterization of NAD:arginine ADP-ribosyltransferases. 1033 46

ART-1, a cell surface ADP-ribosyltransferase, is imbedded in the membrane by a glycosylphosphatidylinositol anchor. Function of this enzyme in mouse T lymphocytes is to transfer ADP-ribose groups from NAD to arginine residues, exposed on the extracellular domain of cell surface molecules. As a consequence, T cell responses are modulated. To explore the precise action of the enzyme, the T cell lymphoma EL-4 was transfected with the ART-1 gene, and its effects were examined. It is shown that ART-1 ADP-ribosylates distinct cell surface molecules, causing inhibition of T cell receptor signaling, concomitant to suppression of p56(lck) kinase activation. These effects are explained by failure of T cell receptors and co-receptors to associate into a contiguous and functional receptor cluster.
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PMID:A cell surface ADP-ribosyltransferase modulates T cell receptor association and signaling. 1036 66

In this study, we investigated the involvement of caspases in TGFbeta-induced apoptosis in human B cells. Our results show that TGFbeta-mediated nuclear fragmentation, observed in the Epstein-Barr virus-negative Burkitt's Lymphoma cell line BL41, was abolished in the presence of the tripeptide caspase inhibitor zVAD-fmk or the specific caspase-3 inhibitor DEVD-fmk. Other apoptotic manifestations such as cell shrinkage, surface phosphatidylserine expression and chromatin condensation were strongly inhibited by zVAD-fmk but only partially by DEVD-fmk. This suggests that other caspases in addition to caspase-3 control these apoptotis-associated features. Specific activation of caspase-3 during TGFbeta-induced apoptosis was demonstrated by the DEVD-fmk-sensitive expression of the active p17 subunit of caspase-3 and by in vivo cleavage of PARP. In addition, TGFbeta treatment of BL41 promoted the expression of both dephosphorylated and truncated forms of the retinoblastoma protein. Inhibition of caspase-3 activity abolished both nuclear fragmentation and expression of the truncated retinoblastoma protein, without modifying the G1 cell cycle arrest induced by TGFbeta. Our data thus demonstrate that TGFbeta-induced apoptosis of lymphoma B lymphocytes is dependent on caspase activation and involves caspase-dependent cleavage of the retinoblastoma protein.
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PMID:Role of caspases and possible involvement of retinoblastoma protein during TGFbeta-mediated apoptosis of human B lymphocytes. 1037 29

We have previously shown that malignant B cells from non-Hodgkin's lymphomas (NHL) are resistant to Fas-mediated apoptosis. To determine the mechanisms underlying this resistance, we analysed by Western blotting the expression of several apoptotic regulators, caspase 3, caspase 8, FADD and poly(ADP-ribose) polymerase (PARP) in fresh lymphoma cells, isolated from 16 B-NHL biopsy samples of different histological subtypes, and displaying variable levels of Fas expression. The profiles of expression of these apoptotic regulators were monitored in cell lysates at different times following Fas with or without CD40 stimulation. Expression of FADD and of the uncleaved forms of PARP, caspase 3 and caspase 8 were detected in all untreated NHL samples. Low levels of PARP cleavage were noted in three untreated samples. Fas stimulation alone induced neither significant apoptosis nor significant changes in the expression profiles of FADD, caspases 3 and 8 and PARP in the 16 samples, except for variations in FADD and caspase 8 expression levels in a minority of samples. Fas/CD40 co-stimulation induced apoptosis and cleavage of caspase 3, caspase 8 and PARP in the five NHLs tested; expression of FADD was not modified. Our results showed (1) that induction of apoptosis in B-NHLs by Fas/CD40 co-stimulation used the same caspase executioner machinery as the normal Fas pathway, and (2) that NHL cells which resisted Fas-mediated apoptosis displayed no defect in either expression or functionality of caspases 3 and 8, nor in FADD expression. The dysfunction underlying NHL resistance to apoptosis must therefore lie upstream of caspase 8, or could alternatively be influenced by anti-apoptotic regulators of the Bcl-2 family.
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PMID:FADD expression and caspase activation in B-cell lymphomas resistant to Fas-mediated apoptosis. 1046 53

4-Hydroxynonenal (HNE), a diffusible product of lipid peroxidation, has been suggested to be a key mediator of oxidative stress-induced cell death. In this study, we partially characterized the mechanism of HNE-mediated cytotoxicity. Incubation of human T lymphoma Jurkat cells with 20-50 microM HNE led to cell death accompanied by DNA fragmentation. Western blot analysis showed that HNE-treatment induced time- and dose-dependent activation of caspase-8, caspase-9 and caspase-3. HNE-induced caspase-3 processing was confirmed by a flow cytometric demonstration of increased catalytic activity on the substrate peptide. HNE treatment also led to remarkable cleavage of poly(ADP-ribose) polymerase (PARP), which was prevented by pretreatment of cells with DEVD-FMK as a caspase-3 inhibitor. The HNE-mediated activation of caspases, cleavage of PARP and DNA fragmentation were blocked by antioxidants cysteine, N-acety-L-cysteine and dithiothreitol, but not by two other HNE-reactive amino acids lysine and histidine, or by cystine, the oxidized form of cysteine. HNE rapidly decreased levels of intracellular reduced glutathione (GSH) and its oxidized form GSSG, and these were also attenuated by the reductants. Coincubation of Jurkat cells with a blocking anti-Fas antibody prevented Fas-induced but not HNE-induced activation of caspase-3. HNE also activated caspase-3 in K562 cells that do not express functional Fas. Our results thereby demonstrate that HNE triggers oxidative stress-linked apoptotic cell death through activation of the caspase cascade. The results also suggest a possible mechanism involving a direct scavenge of intracellular GSH by HNE.
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PMID:4-hydroxynonenal induces a cellular redox status-related activation of the caspase cascade for apoptotic cell death. 1065 56

Poly (ADP-ribose) polymerase (PARP) is involved in the cellular responses to genotoxic damage and its inhibition has been proposed as potentiating anticancer drug activity. Here, we evaluated the ability of the PARP inhibitor, 6(5H)-phenanthridinone, to modulate the antiproliferative activity of bleomycin, carmustin and doxorubicin in a murine (RDM4) and a human (U937) lymphoma cell lines. 6(5H)-phenanthridinone was shown to suppress PARP activity with the same potency in both cell lines. At 25 microM, this compound potentiated the activity of carmustin in RDM4 but not in U937 cells. In contrast, 6(5H)-phenanthridinone failed to affect the doxorubicin toxicity in murine lymphoma cells, whereas it prevented the cytotoxicity of this drug in the human cell line. Altogether, these findings indicated that 6(5H)-phenanthridinone modulates the cytotoxicity of anticancer agents differently according to the cell type and the drug. Therefore, this PARP inhibitor could be considered as the prototype of a new class of adjuncts in cancer chemotherapy.
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PMID:Modulation of the antiproliferative activity of anticancer drugs in hematopoietic tumor cell lines by the poly(ADP-ribose) polymerase inhibitor 6(5H)-phenanthridinone. 1106 48

The mechanism underlying the cancericidal activity of 3-m-bromoacetylamino benzoic acid ethyl ester (3-BAABE) was investigated. 3-BAABE exerted a strong cancericidal effect on human leukemia and lymphoma cells (IC(50) < 0.2 microgram/mL) and on cell lines of prostate, colon, ductal, and kidney cancer (IC(50) 0.8 to 0.88 microgram/mL). Multiple drug resistance (MDR) had no effect on the susceptibility of human lymphoma cells to 3-BAABE, since Daudi/MDR(20) and wild-type Daudi cells had a similar susceptibility to the cytotoxic effect of 3-BAABE. The cancericidal effect of 3-BAABE, which was not associated with changes in the cell cycle, was mediated by apoptosis. Thus, cells exposed to 3-BAABE displayed the DNA fragmentation ladder characteristic for apoptosis, associated with a marked increase of the activity of apoptosis effector caspases-3 and -6, which was followed by proteolytic cleavage of DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP). Exposure of tumor cells to 3-BAABE increased the activity of apical caspase-9, but had no effect on caspase-8. Complete inhibition of 3-BAABE-induced apoptosis was exerted by LEHD-FMK, a caspase-9 inhibitor. DEVD-FMK, a caspase-3 inhibitor, and VEID-FMK, a caspase-6 inhibitor, partially inhibited 3-BAABE-induced apoptosis, whereas exposure to IETD-FMK, a caspase-8 inhibitor, had no effect. The fragmentation and elevated activity of caspase-9 in 3-BAABE-treated cells and the fact that only an inhibitor of caspase-9 abrogated 3-BAABE-induced apoptosis indicate that 3-BAABE is a distinctive compound that elicits apoptosis through a pathway that is limited specifically to activation of apical caspase-9.
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PMID:3-m-bromoacetylamino benzoic acid ethyl ester: a new cancericidal agent that activates the apoptotic pathway through caspase-9. 1107 52

JP-8 induces apoptosis in rat lung epithelial cells, primary mouse T lymphocytes, Jurkat T lymphoma cells, and U937 monocytic cells (Stoica et al., 2001). Here, we have observed a different mechanism of cytotoxicity in human keratinocytes grown in culture as well as when grafted onto nude mice. At lower levels of JP-8 (80 microg/ml; 1 x 10(-4) dilution), sufficient to induce apoptosis in other cell types, including lung epithelial cells (Stoica et al., 2001), no apoptosis was observed. At higher levels (>200 microg/ml; 2.5 x 10(-4) dilution), JP-8 is cytotoxic to both primary and immortalized human keratinocytes, as evidenced by the metabolism of calcein, as well as by morphological changes such as cell rounding and cell detachment. There was no evidence of activation of caspases-3, -7, or -8 either by enzyme activity or immunoblot analysis, and the stable expression of a dominant-negative inhibitor of apoptosis (FADD-DN) did not increase the survival of keratinocytes to JP-8. The pattern of poly(ADP-ribose) polymerase (PARP) cleavage was also characteristic of necrosis. PARP has been also been implicated in necrosis via its ability to lower levels of ATP in damaged cells. However, fibroblasts derived from PARP-/- mice underwent necrotic cell death similar to those derived from PARP+/+ mice, indicating that the effects of JP-8 are independent of PARP. Immunoblot analysis further revealed that exposure of keratinocytes to the toxic higher levels of JP-8 markedly downregulates the expression of the prosurvival members of the Bcl-2 family, Bcl-2 and Bcl-x(L), and upregulates the expression of antisurvival members of this family, including Bad and Bak. Bcl-2 and Bcl-x(L) have been shown to preserve mitochondrial integrity and suppress cell death. In contrast, Bak and Bad both promote cell death by alteration of the mitochondrial membrane potential, in part by heterodimerization with and inactivation of Bcl-2 and Bcl-x(L), and either inducing necrosis or activating a downstream caspase program. High intrinsic levels of Bcl-2 and Bcl-x(L) may prevent apoptotic death of keratinocytes at lower levels of JP-8, while perturbation of the balance between pro- and antiapoptotic Bcl-2 family members at higher levels may ultimately play a role in necrotic cell death in human keratinocytes. Finally, when human keratinocytes were grafted to form a human epidermis on nude mice, treatment of these grafts with JP-8 revealed cytotoxicity and altered histology in vivo.
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PMID:Mechanisms of JP-8 jet fuel cell toxicity. II. Induction of necrosis in skin fibroblasts and keratinocytes and modulation of levels of Bcl-2 family members. 1122 86


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