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)

NAD is hydrolyzed during incubation with isolated renal brush border membranes (BBM). The specific enzymatic mechanisms have not been identified apart from the activity of ADP-ribosyltransferase, which accounts for a very small proportion of the total hydrolysis. In the present study, an NAD-glycohydrolase (NGH) was identified in the renal BBM using the cyanide-addition assay to monitor hydrolysis of NAD at the nicotinamide-ribose bond. The production of nicotinamide and ADP-ribose, the expected reaction products, was determined by thin-layer chromatography. The NGH was enriched ninefold in the BBM fraction and accounted for 36% of the total rate of NAD hydrolysis by BBM enzymes at pH 7.4. Assay of NGH in sealed BBM vesicles subjected to osmotic shock indicated that about 23% of the NGH is exposed on the cytoplasmic surface of the BBM. The enzyme was inhibited by nicotinamide in vitro and also when the nicotinamide was administered in vivo, suggesting, indirectly, that the enzyme may play a role in mediating the effects of nicotinamide on BBM phosphate transport.
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PMID:NAD-glycohydrolase in renal brush border membranes. 241 52

Previous studies in our laboratory have shown that nitric oxide (NO) gas enhances NMDA-stimulated release of preloaded tritiated norepinephrine ([3H]NA) from rat brain slices in a dose-dependent, oxygen-sensitive, and cyclic GMP-independent manner. In this study we have attempted to determine the mechanism for the enhancement of neurotransmitter release seen with NO. No-enhanced transmitter release was not due to buffer acidification or generation of NO degradation products, since reducing buffer pH below 7.3 inhibited NMDA-stimulated [3H]NA release and nitrite or nitrate ions (3-100 microM) had no significant effect on release. Carbon monoxide (CO, 10-300 microM), another diatomic gas with properties similar to NO including heme binding and guanylate cyclase activation, had no significant effect on depolarization-induced [3H]NA release. The NO effect was probably not due to mono-ADP-ribosylation of cellular proteins, since the ADP-ribosyltransferase (ADPRT) inhibitors nicotinamide (10 microM-10 microM) and luminol (1 microM-1mM) did not diminish the enhancement of transmitter release seen with NO. The NA reuptake inhibitor desmethylimipramine (DMI, 10 nM-10 microM) neither mimicked nor blocked the effect of NO, suggesting that NO was not acting via inhibition or reversal of the NA transporter. Similar to NO, the metabolic inhibitors sodium azide (NaN3, 0.1-3 mM), potassium cyanide (KCN, 0.1-3 mM), and 2,4-dinitrophenol (2,4-DNP, 10-300 microM) also dose-dependently enhanced NMDA-stimulated [3H]NA release. These results suggest that NO may enhance neurotransmitter release by inhibiting cellular respiration and perhaps ultimately via altering calcium homeostasis.
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PMID:Mechanism for nitric oxide's enhancement of NMDA-stimulated [3H]norepinephrine release from rat hippocampal slices. 853 39

A panel of human B-lineage lymphoma cell lines differing in cancer drug-resistance status and Bcl-2/Bax expression were used to study the contribution of mitochondrial-based perturbations and regulation in differential induction of apoptosis. Mitochondrial dysfunction was induced in cells by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (mClCCP) and the respiratory chain inhibitor antimycin A. Cells were then assayed for early changes in MAP kinase signaling and subsequent induction of apoptosis. The cancer drug-resistant cell lines EW36 and CA46, overexpressing Bcl-2 and deficient in Bax, respectively, were both resistant to mitochondrial toxicant-induced cleavage of poly(ADP-ribose) polymerase (PARP) and morphologically detectable apoptotic cell death. In contrast, cancer drug-sensitive ST486 cell line, with low Bcl-2 expression, was sensitive to PARP cleavage and apoptosis engagement. Interestingly, mClCCP induced twofold more apoptosis than antimycin A in the ST486 cells. Exposure to the mitochondrial toxicants resulted in the early and preferential activation of the ERK and p38 MAP kinase pathways in only the drug-sensitive ST486 cell line, with mClCCP more potent than antimycin A. Specific inhibition of the p38 pathway augmented baseline and mClCCP-induced apoptosis. These results show that multi-drug-resistant and -sensitive B-lineage cells are also resistant and sensitive to compounds inducing mitochondrial dysfunction. The differential sensitivity to mitochondrial toxicant effects involved regulation by MAP kinases, since ERK and p38 were found to be preferentially activated only in the drug-sensitive B-lineage cells. Modulation of the p38 signaling pathway altered the sensitivity of cells to mitochondrial stress and may play a more general role in regulating the sensitivity of B-lineage cells to drugs and environmental toxicants.
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PMID:Differential induction of apoptosis and MAP kinase signaling by mitochondrial toxicants in drug-sensitive compared to drug-resistant B-lineage lymphoid cell lines. 1148 85

Disruption of mitochondrial electron transport and opening of the so-called mitochondrial permeability transition pores (PTPs) are early events in apoptotic cell death and may be caused by the uncoupler of mitochondrial oxidation and phosphorylation, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). We investigated the cellular toxicity of FCCP in HL60 and CCRF-CEM cells alone or in combination with the known apoptosis inducers such as inhibitor of serine/threonine protein kinases staurosporine (Sts) and protein kinase C inhibitor chelerythrine. FCCP induced apoptotic cell death in both cell lines in a dose-dependent manner, and we were able to demonstrate an appearance of caspase-3-dependent PARP cleavage fragments with Western blot and the appearance of large (15-50 kb) DNA fragments using pulsed-field gel electrophoresis. After 2 hr of incubation with Che or Sts more than half of the cells had died by apoptosis. We observed a statistically significant delay in Sts- and Che-induced apoptotic cell death in CCRF-CEM cells when the cells were preincubated with FCCP but not with zVAD-FMK: about 50% more cells survived after pre-treatment with FCCP, as compared to 1 hr treatment with Che alone (P<0.05), and 25% more cells were alive after 6 hr of treatment, as compared to 6 hr exposure to Sts alone (P<0.05). The protective effect of FCCP was, however, transient and lasted only 6 hr. Treatment with aurintricarboxylic acid completely prevented Che- and Sts-induced apoptotic cell death in CCRF-CEM and HL60 cells. Incubation with Che resulted in a drop in the intracellular ATP content, predominantly distinctive in HL60, and in NAD(+) content in CCRF-CEM cells. Both ATP and NAD(+) drop were prevented with ATA, but not with FCCP or zVAD. Our data suggest that treatment with uncouplers of oxidative phosphorylation can induce apoptotic cell death in haematopoietic cell lines. However, when used in combination with serine/threonine protein kinase inhibitors FCCP can even prevent apoptosis.
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PMID:Modulation of apoptosis by mitochondrial uncouplers: apoptosis-delaying features despite intrinsic cytotoxicity. 1185 98

Execution of apoptosis can involve activation of the caspase family of proteases. Recent studies show that caspase inhibition can switch the morphology of cell death from apoptotic to necrotic without altering the level of death among cell populations. In the present study, the effect of caspase inhibition on cortical (CX) cell death induced by cyanide was investigated. In primary cultured CX cells exposed to cyanide (400 microM), death was primarily apoptotic as indicated by positive TUNEL staining. Reactive oxygen species (ROS) generation and subsequent caspase activation mediated the apoptosis. Inhibition of the caspase cascade with zVAD-fmk switched the apoptotic response to necrotic cell death, as assessed by increased cellular efflux of LDH and propidium iodide uptake by the cells. The change in death mode was accompanied by a marked increase in poly (ADP-ribose) polymerase-1 (PARP-1) activity, reactive oxygen species (ROS) generation, a reduction in the mitochondrial membrane potential (Delta psi(m)), and reduced cellular ATP. Prior treatment of cells with 3-aminobenzamide (3-AB), a PARP-1 inhibitor, prevented the cells from undergoing necrosis and preserved intracellular ATP levels. These findings indicate that apoptosis and necrosis share common initiation pathways and caspase inhibition can switch the apoptotic response to necrosis. Inhibition of PARP-1 preserves cellular ATP levels and in turn blocks execution of the necrotic death pathway.
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PMID:Caspase inhibition switches the mode of cell death induced by cyanide by enhancing reactive oxygen species generation and PARP-1 activation. 1499 85

The alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) alters DNA and stimulates the activity of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme involved in DNA repair. The consumption of cellular NAD(+) by PARP-1 is accompanied by ATP depletion, mitochondrial depolarization and release of proapoptotic proteins, but whether a causal relationship exists among these events remains an open question. Most of cellular NAD(+) is stored in the mitochondrial matrix and becomes available for cytosolic and nuclear processes only after its release through the permeability transition pore (PTP), a voltage-gated inner membrane channel. Here we have explored whether MNNG affects mitochondrial function upstream of PARP-1 activation. We show that MNNG has a dual effect on isolated mitochondria. At relatively low concentrations (up to 0.1 mM), it selectively sensitizes the PTP to opening, while at higher concentrations (above 0.5 mM) it inhibits carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)-stimulated respiration. MNNG caused PTP opening and activation of the mitochondrial proapoptotic pathway in intact HeLa cells, which resulted in cell death that could be prevented by the PTP inhibitor CsA. We conclude that a key event in MNNG-dependent cell death is induction of PTP opening that occurs independently of PARP-1 activation.
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PMID:Induction of the mitochondrial permeability transition by the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Sorting cause and consequence of mitochondrial dysfunction. 1528 75

Mitochondrial dysfunction resulting from mitochondrial DNA (mtDNA) mutations and/or depletion has been correlated with cancer progression and drug resistance. To investigate the role of mtDNA in prostate cancer progression, we used LNCaP and PC-3 prostate carcinoma cells as experimental model. Compared to minimally invasive androgen-dependent LNCaP cells, highly invasive androgen-independent PC-3 cells, as well as androgen-independent DU145 and C4-2 cells, exhibited significantly reduced mtDNA content. In PC-3 cells, reduction of mtDNA was accompanied by decreased mitochondrial membrane potential (DeltaPsi(m)), increased migration onto the basement membrane protein laminin-1, reduced chemosensitivity to paclitaxel (IC(50)=110 nM vs. 22 nM) and decreased expression of poly(ADP-ribose) polymerase (PARP)-1. To investigate the relationship between mtDNA depletion and these phenotypic characteristics, we established mtDNA-depleted LNCaP cells [Rho(-)] by long-term exposure to ethidium bromide or treated wild-type LNCaP cells with a mitochondrial ionophore, carbonyl cyanide m-chlorophenylhydrazone. Both manipulations resulted in DeltaPsi(m) loss, acquisition of invasive cytology, increased motility onto laminin-1, reduced sensitivity to paclitaxel (IC(50)= approximately 100 nM) and approximately 75% reduction in PARP-1 protein levels, resembling PC-3 cells. Overall, these results provide novel evidence demonstrating that mtDNA depletion in early prostate carcinoma may contribute to the acquisition of a more invasive phenotype that is less sensitive to paclitaxel-induced apoptosis.
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PMID:Mitochondrial DNA depletion reduces PARP-1 levels and promotes progression of the neoplastic phenotype in prostate carcinoma. 1860 66

Constitutive ERK activation, superoxide dismutases (SOD) and p53 mutations are implicated in modulating tumor apoptotic response. We now investigated whether human melanoma survival in response to sodium nitroprusside (SNP) is modulated by: (a) stable introduction of a DN-mutant p53; (b) pharmacologically inhibiting ERK activation with UO126; (c) addition of exogenous SOD. Nitroprusside releases nitric oxide (NO) when intact, or acts in a NO-independent manner via iron and residual cyanide after light exposure (lex-SNP). When tested at 300 microM in 72 h treatments by cytometric live-dead assays, intact SNP caused a 50% lethality versus a 30% lethality induced by lex-SNP. No protection from SNP toxicity was seen when inhibiting the PI3-kinase pathway with LY294002 or c-Jun NH(2) kinase signaling with SP600125. However, pretreatment with UO126 protected from SNP-mediated cell death including counteracting apoptosis-associated Bax expression and PARP cleavage, plus reversing loss of Cu,Zn-SOD. Moreover, addition of exogenous SOD also protected cells from SNP toxicity. In spite of the greater earlier effects of intact SNP, cells treated with single doses of either intact or lex-SNP, revealed about a 90% mortality in longer 120 h treatments, and these were also counteracted by UO126 or exogenous SOD. This report is the first to show that: constitutive ERK activation characteristic of cancer cells, increases a nitroprusside-induced apoptosis modulated by SOD.
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PMID:ERK activation increases nitroprusside induced apoptosis in human melanoma cells irrespective of p53 status: role of superoxide dismutases. 1939 58

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we evaluated the in vitro effects of FCCP on the growth of Calu-6 lung cancer cells. FCCP inhibited the growth of Calu-6 cells with an IC(50) of approximately 6.64+/-1.84 microM at 72 h, as shown by MTT. DNA flow cytometric analysis indicated that FCCP induced G1 phase arrest below 20 microM of FCCP. Treatment with FCCP decreased the level of CDKs and cyclines in relation to G1 phase. In addition, FCCP not only increased the p27 level but also enhanced its binding with CDK4, which was associated with hypophosphorylation of Rb protein. While transfection of p27 siRNA inhibited G1 phase arrest in FCCP-treated cells, it did not enhance Rb phosphorylation. FCCP also efficiently induced apoptosis. The apoptotic process was accompanied with an increase in sub-G1 cells, annexin V staining cells, mitochondria membrane potential (MMP) loss and cleavage of PARP protein. All of the caspase inhibitors (caspase-3, -8, -9 and pan-caspase inhibitor) markedly rescued the Calu-6 cells from FCCP-induced cell death. However, knock down of p27 protein intensified FCCP-induced cell death. Moreover, FCCP induced the depletion of GSH content in Calu-6 cells, which was prevented by all of the caspase inhibitors. In summary, our results demonstrated that FCCP inhibits the growth of Calu-6 cells in vitro. The growth inhibitory effect of FCCP might be mediated by cell cycle arrest and apoptosis via decrease of CDKs and caspase activation, respectively. These findings now provide a better elucidation of the mechanisms involved in FCCP-induced growth inhibition in lung cancer.
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PMID:Effects of carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone on the growth inhibition in human pulmonary adenocarcinoma Calu-6 cells. 1981 88

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in mitochondria. This study evaluated the effects of FCCP on the growth of juxtaglomerular As4.1 cells in relation to the cell cycle and apoptosis. FCCP inhibited the growth of As4.1 cells with an IC(50) of approximately 10 muM at 48 hours. DNA flow cytometry indicated that FCCP did not induce the specific phase arrests of the cell cycle. This agent efficiently reduced mitochondrial membrane potential (MMP; DeltaPsi(m)) levels within 1 hour and dose-dependently induced loss of MMP (DeltaPsi(m)) at 48 h. FCCP also induced apoptosis in As4.1 cells, as evidenced by sub-G(1) cells and annexin V binding assay. This apoptotic process was accompanied by caspase-3 activation and PARP cleavage. Although caspase-3 inhibitor significantly reduced the activity of caspase-3, all of the caspase inhibitors tested in this study failed to rescue As4.1 cells from FCCP-induced cell death. In conclusion, this study demonstrated that FCCP, as a mitochondria-damaging agent, potently induces apoptosis in As4.1 juxtaglomerular cells via a caspase-independent manner.
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PMID:Carbonyl cyanide p-(trifluoromethoxy) phenylhydroazone induces caspase-independent apoptosis in As4.1 juxtaglomerular cells. 2068 24


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