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M Lepoivre

Publications and source records attributed to M Lepoivre.

At least 19 recordsLinked to original sources
PubMed1996

Protection by glutathione against the antiproliferative effects of nitric oxide. Dependence on kinetics of no release.

Pretreatment by L-buthionine sulfoximine (BSO), which inactivates gamma-glutamylcysteine synthetase and, therefore, inhibits glutathione (GSH) synthesis, greatly increased the sensitivity of tumor cells to the antiproliferative effects of several NO-donating compounds. The sensitization that resulted from depletion of cellular GSH pools was observed in tumor cells exhibiting different degrees of resistance to NO. In contrast, GSH depletion of tumor target cells did not affect their sensitivity to the cytostatic activity of activated macrophages and other NO-producing cells (EMT6 cells treated by interferon gamma and LPS). The kinetics for NO generation is a parameter that may differentiate NO-producing cells and short-lived NO donors. To study the relationship between the magnitude of NO fluxes and the increased toxicity on BSO-pretreated cells, two NO-releasing zwitterions derived from polyamines (NONOates) with different half-lives were selected. NO fluxes as a function of time were simulated, according to the donor half-life and initial concentration, and antiproliferative effects on control and BSO-treated cells were compared. GSH depletion increased the sensitivity of tumor cells in the case of the less stable NO donor only. We, thus, propose that intracellular GSH is specifically protective against high fluxes of NO.

Animals↗
PubMed1996

Kinetic modelling of the nitric oxide gradient generated in vitro by adherent cells expressing inducible nitric oxide synthase.

Inducible nitric oxide (NO) synthase produces a long-lasting NO flux which can exert cytotoxic effects on target cells. A prerequisite for the understanding of the molecular basis of NO action is quantitative data on the availability of this small neutral radical molecule at both the spatial and temporal levels. The limits of NO availability depend on the respective rates of NO production, diffusion and autoxidation by molecular oxygen. Kinetic modeling of these processes has been performed for a widely used experimental system consisting of a monolayer of adherent cells cultured in vitro for hours in unstirred culture medium. It appears that: (i) the maximal NO concentration in the culture is in the immediate vicinity of the monolayer, where target cells will sediment; (ii) the steady-state NO concentration in this area is lower than 4 to 5 microM; and (iii) measurements of nitrite/nitrate or citrulline accumulation in the bulk cell medium culture during a given time period significantly underestimate (by a factor of up to 3 to 4) the true rate of NO synthesis at the level of the producer cell. This rate can be, nevertheless, easily estimated from the rate of production of the stable NO synthase products.

Cell Adhesion↗
PubMed1995

Inhibition of ribonucleotide reductase by nitric oxide derived from thionitrites: reversible modifications of both subunits.

Thionitrites are spontaneous nitric oxide (NO) donors in neutral aqueous solutions. Consequently, they inhibit ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis, from Escherichia coli and murine adenocarcinoma TA3 cells. They also inhibit tumor cell proliferation. Reaction of thionitrites with protein R1, the large subunit, results in the nitrosation of cysteines, as shown from the formation of a chromophore with a characteristic absorption at 340 nm. EPR spectroscopy both on whole murine R2-overexpressing L1210 cells and on the pure protein showed that the tyrosyl radical of protein R2, the small subunit, reversibly couples to the NO radical, presumably leading to nitrosotyrosine adducts. Both molecular events might be at the origin of the inhibition of ribonucleotide reductase by NO, since a number of cysteines and the tyrosyl radical are essential for catalysis. These results identify NO donors as a new class of inhibitors of ribonucleotide reductase with potential applications as anticancer or antiviral chemotherapy agents.

Adenocarcinoma↗
PubMed1994

N omega-hydroxyl-L-arginine, an intermediate in the L-arginine to nitric oxide pathway, is a strong inhibitor of liver and macrophage arginase.

N omega-Hydroxy-L-arginine (L-NOHA) is a potent inhibitor of the hydrolysis of L-arginine (L-Arg) to L-ornithine (L-Orn) catalyzed by purified bovine liver arginase (BLA). It appears as one of the most powerful arginase inhibitors reported so far (Ki = 150 microM). The other products of NO synthase are either without effect (NO2-, NO3-) or much weaker inhibitors (L-citrulline (L-Cit) and NO) of BLA. Products derived from a possible hydrolysis of L-Arg (L-Orn and urea) or of L-NOHA (L-Cit, hydroxyurea and hydroxylamine) are also inactive toward BLA at concentrations up to 2 mM. The configuration of L-NOHA is important as D-NOHA is much less active, and its free -COOH and alpha-NH2 functions are required for recognition of BLA. L-NOHA is also a potent inhibitor of the arginase activity of rat liver homogenates and of murine macrophages (IC50 of 150 and 450 microM, respectively). These remarkable properties of L-NOHA could play a role in the modulation of the biosynthesis of the biological mediator NO by increasing local L-Arg concentrations.

Animals↗
PubMed1994

Quenching of the tyrosyl free radical of ribonucleotide reductase by nitric oxide. Relationship to cytostasis induced in tumor cells by cytotoxic macrophages.

Nitric oxide (NO) synthesized by macrophages inhibits tumor cell replication. NO also inhibits ribonucleotide reductase, an enzyme essential for DNA synthesis, probably by quenching the catalytically active tyrosyl free radical of its R2 subunit. The role of this inhibition in NO-mediated cytostasis was thus evaluated. After a 4-h coculture with macrophages, quenching of the radical was demonstrated by electron paramagnetic resonance spectroscopy in transfected L1210-R2 cells over-expressing the R2 protein. Pronounced cytostasis was simultaneously observed. A NO synthase inhibitor greatly reduced both phenomena. Target cells withdrawn from macrophages partially recovered from cytostasis and radical loss within 90 min. Deoxyribonucleosides added to by-pass ribonucleotide reductase inhibition efficiently reversed cytostasis of K-562 cells. After a 24-h coculture, the quenched tyrosyl radical still reappeared in L1210-R2 cells withdrawn from macrophages, but DNA synthesis did not resume. Moreover, deoxyribonucleosides marginally reversed overnight cytostasis of K-562 cells mediated by macrophages but were efficient against cytostasis induced by hydroxyurea, a ribonucleotide reductase inhibitor. Autocrine cytostasis observed early in TA3-H2 cells committed to produce NO was closely correlated with quenching of the tyrosyl radical but not with formation of dinitrosyl-iron complexes. We thus propose that NO-dependent cytostasis begins with a rapid and reversible inhibition of ribonucleotide reductase, progressively reinforced by other, long-lasting antiproliferative effects.

Amino Acid Oxidoreductases↗
PubMed1993

N omega-hydroxy-L-arginine, a reactional intermediate in nitric oxide biosynthesis, induces cytostasis in human and murine tumor cells.

Conversion of L-arginine to L-citrulline and nitric oxide (NO) by NO synthase induced in the murine EMT-6 cells resulted in the release of a large amount of the stable reactional intermediate N omega-hydroxy-L-arginine into the extracellular medium. We have prepared [3H]N omega-hydroxy-L-arginine biosynthetically, and shown that, after its uptake, this molecule can induce cytostasis in NO synthase-deficient P-815 and U-937 tumor cells. This long-lived intermediate could behave as a supplier of NO or other toxic molecules in cell-cell interactions.

Adenocarcinoma↗
PubMed1993

Differential regulation of inducible nitric oxide synthase by fibroblast growth factors and transforming growth factor beta in bovine retinal pigmented epithelial cells: inverse correlation with cellular proliferation.

Bovine retinal pigmented epithelial (RPE) cells express, after activation with interferon gamma (IFN-gamma) and lipopolysaccharide (LPS), an inducible nitric oxide synthase (NOS). Experiments were done to investigate the effects of the transforming growth factor beta 1, epidermal growth factor, and fibroblast growth factors (FGFs), which are abundant in the retina, on NOS activity. Transforming growth factor beta 1 slightly increases the production of nitrite, an oxidation product of NO, induced by LPS plus IFN-gamma, whereas acidic and basic FGFs markedly inhibit the nitrite release due to LPS/IFN-gamma in a concentration-dependent manner, and epidermal growth factor did not modify LPS/IFN-gamma-induced NOS activity. The growth factors alone did not stimulate nitrite release. We have attempted to elucidate the mechanism of FGF inhibition. Results with heparin, suramin, and tyrphostin suggest involvement of the high-affinity receptor for FGF in its inhibition of LPS/IFN-gamma-stimulated NOS activity. Continued stimulation of RPE cells with LPS/IFN-gamma was essential for the induction of NO synthesis, and maximal inhibition was obtained when FGF was present during stimulation with LPS/IFN-gamma, suggesting that FGF inhibits NOS induction. Furthermore, an antiproliferative action of NO was demonstrated by an inverse correlation between the amounts of nitrite or citrulline produced in response to different stimuli (LPS/IFN-gamma or LPS/IFN-gamma with growth factors) and the level of cellular proliferation. Similar inhibition of growth was obtained when RPE cells were incubated with an NO donor, sydnonimide. Because NO acts as a cytotoxic compound in the retina, FGF, by inhibiting the induction of NOS in RPE cells, may have beneficial effects in protecting the retina from cytokine and endotoxin-mediated tissue damage.

Amino Acid Oxidoreductases↗
PubMed1993

EPR characterization of molecular targets for NO in mammalian cells and organelles.

Nitric oxide is synthesized in mammalian cells from L-arginine or from pharmaceutical drugs. It forms paramagnetic complexes with some metalloproteins, inhibiting key enzymes in DNA synthesis, mitochondrial respiration, iron metabolism, etc. This article reviews how electron paramagnetic resonance spectroscopy helps to detect unambiguously such specific molecular targets for NO in mammalian whole cells and organelles. EPR has also been used for the detection of spin adducts of free NO by spin-trapping methods.

Animals↗
PubMed1992

Early loss of the tyrosyl radical in ribonucleotide reductase of adenocarcinoma cells producing nitric oxide.

Nitric oxide (NO) has been previously shown to inhibit crude preparations of ribonucleotide reductase, a key enzyme in DNA synthesis, and to destroy the essential tyrosyl free radical in pure recombinant R2 subunit of the enzyme. In R2-overexpressing TA3 cells, a decrease in the tyrosyl radical was observed by whole-cell EPR spectroscopy, as soon as 4 h after NO synthase induction by immunological stimuli. Complete loss of the tyrosyl EPR signal occurred after 7 h in cells cultured at a high density. Disappearance of the tyrosyl radical was prevented by N omega-nitro-L-arginine, a specific inhibitor of NO synthesis, and by oxyhemoglobin, which reacts rapidly with NO. It was reproduced by S-nitrosoglutathione, a NO-releasing molecule. Stable end products of NO synthase metabolism did not affect the radical. Immunoblot analysis of the R2 subunit indicated that expression of the protein was not influenced by NO synthase activity. These results establish that NO, or a labile product of NO synthase, induces the disappearance of the R2-centered tyrosyl radical. Since the radical is necessary for ribonucleotide reductase activity, its destruction by NO would contribute markedly to the antiproliferative action exerted by macrophage-type NO synthase.

Adenocarcinoma↗
PubMed1992

8-Azidoadenosine and ribonucleotide reductase.

Inhibitors of ribonucleotide reductase are potential antiproliferative agents, since they deplete cells from DNA precursors. Substrate nucleoside analogues, carrying azido groups at the base moiety, are shown to have strong cytostatic properties, as measured by the inhibition of the incorporation of thymidine into DNA. One compound, 8-azidoadenosine, inhibits CDP reduction in cytosolic extracts from cancer cells. The corresponding diphosphate behaves as a substrate for ribonucleotide reductase while the triphosphate is an allosteric effector.

Adenosine↗
PubMed1992

Lipopolysaccharide and cytokines induce a macrophage-type of nitric oxide synthase in bovine retinal pigmented epithelial cells.

The present study demonstrates that bovine retinal pigmented epithelial cells, which are neuroectodermal in origin, produce nitric oxide (NO) upon treatment with interferon-gamma in the presence of lipopolysaccharide or tumor necrosis factor-alpha. NO production was measured by the accumulation of the stable endproduct NO2-. The biosynthesis of NO requires an induction period of approximately 12 hours and continues for at least 96 hours. The synthesis was abolished by the stereoselective inhibitors of NO synthase, NG-monomethyl-L-arginine and NG-nitro-L-arginine-benzylester. Cycloheximide and dexamethasone blocked cytokine-induced NO production. The results indicate that endotoxin and cytokines are capable of inducing NO synthase of the macrophage type, in retinal pigmented epithelial cells.

Amino Acid Oxidoreductases↗
PubMed1991

Inactivation of ribonucleotide reductase by nitric oxide.

Ribonucleotide reductase has been demonstrated to be inhibited by NO synthase product(s). The experiments reported here show that nitric oxide generated from sodium nitroprusside, S-nitrosoglutathione and the sydnonimine SIN-1 inhibits ribonucleotide reductase activity present in cytosolic extracts of TA3 mammary tumor cells. Stable derivatives of these nitric oxide donors were either inactive or much less inhibitory. EPR experiments show that the tyrosyl radical of the small subunit of E. Coli or mammalian ribonucleotide reductase is efficiently scavenged by these NO donors.

Animals↗
PubMed1991

High-performance liquid chromatographic analysis of the unusual pathway of oxidation of L-arginine to citrulline and nitric oxide in mammalian cells.

A very unusual pathway of the oxidation of L-arginine to citrulline and nitric oxide has been discovered recently in cytotoxic macrophages. In an attempt to detect molecules generated through this metabolic pathway, a fast radio high-performance liquid chromatographic method was developed to analyse the whole set of radiolabelled L-arginine-derived metabolites produced by mammalian cells after appropriate induction. A new intermediate which might be NG-hydroxy-L-arginine was found.

Adenocarcinoma↗
PubMed1990

Production of an arginine-derived relaxing factor induced by IFN-gamma plus endotoxin in murine adenocarcinoma EMT 6 cells.

Treatment of EMT 6 mammary adenocarcinoma cells with Interferon-gamma (IFN-gamma, 10 U.ml-1) plus endotoxin lipopolysaccharide (LPS, 100 ng.ml-1) induces concomitantly a growth arrest and production of citrulline and nitrite from L-arginine. A similar L-arginine-dependent metabolism is responsible for the vascular smooth muscle relaxing effect of stimulated endothelial cells. We therefore investigated the ability of EMT 6 cells to induce the relaxation of endothelium-denuded rat aortic rings precontracted with noradrenaline (1 microM). Pretreatment of EMT 6 cells with IFN-gamma + LPS increased their relaxing potency by 5-10 times. The relaxin effects of control and treated EMT 6 cells were entirely counteracted by NG-monomethyl-L-arginine (300 microM), a specific inhibitor of nitrite and citrulline production from L-arginine, and by methylene blue (10 microM) and LY 83583 (10 microM), two inhibitors of NOo-induced activation of guanylate cyclase. The effect of NG-monomethyl-L-arginine was reversed by L- but not D-arginine (1 mM). It is concluded that IFN-gamma + LPS increase the production of a relaxing factor in EMT 6 cells through the L-arginine-NOo-synthase pathway.

Adenocarcinoma↗
PubMed1990

Alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells.

The murine adenocarcinoma cell line TA 3 synthesized nitrite from L-arginine upon stimulation with gamma-interferon (IFN-gamma) associated with tumor necrosis factor (TNF), and/or bacterial lipopolysaccharide (LPS), but not with IFN-gamma, TNF, or LPS added separately. Induction of the NO2(-)-generating activity caused an inhibition of DNA synthesis in TA 3 cells. This inhibition was prevented by the L-arginine analog N omega-nitro-L-arginine, which inhibited under the same conditions nitrite production by TA 3 cells. The TA 3 M2 subclone, selected for enhanced ribonucleotide reductase activity, was found to be less sensitive than the wild phenotype TA 3 WT to the cytostatic activity mediated by the NO2(-)-generating system. Cytosolic preparations from TA 3 M2 cells treated for 24 or 48 h with IFN-gamma, TNF, and LPS exhibited a reduced ribonucleotide reductase activity, compared to untreated control cells. No reduction in ribonucleotide reductase activity was observed when N omega-nitro-L-arginine was added to treated cells. Addition of L-arginine, NADPH, and tetrahydrobiopterin into cytosolic extracts from 24-h treated TA 3 M2 cells triggered the synthesis of metabolic products from the NO2(-)-generating pathway. This resulted in a dramatic inhibition of the residual ribonucleotide reductase activity present in the extracts. The inhibition was reversed by NG-monomethyl-L-arginine, another specific inhibitor of the NO2(-)-generating activity. No L-arginine-dependent inhibition of ribonucleotide reductase activity was observed using extracts from untreated cells that did not express NO2(-)-generating activity. These results demonstrate that, in an acellular preparation, molecules derived from the NO2(-)-generating pathway exert an inhibitory effect on the ribonucleotide reductase enzyme. This negative action might explain the inhibition of DNA synthesis induced in adenocarcinoma cells by the NO2(-)-generating pathway.

Adenocarcinoma↗
PubMed1989

Antiproliferative activity of gamma-interferon combined with lipopolysaccharide on murine adenocarcinoma: dependence on an L-arginine metabolism with production of nitrite and citrulline.

Growth of the murine adenocarcinoma EMT6 was moderately inhibited by up to 32 units/ml of gamma-interferon (IFN-gamma). However, EMT6 growth was blocked by as low as 2 U/ml of IFN-gamma, when added with lipopolysaccharide. In the same time, the combination IFN-gamma + lipopolysaccharide induced synergistically the production of nitrite and citrulline by EMT6 cells. Synthesis of the two products was correlated with IFN-gamma concentrations. It required exogenous L-arginine and was inhibited by a methylated L-arginine derivative, NG-monomethyl-L-arginine. Inhibition was specific since urea synthesis was not reduced by NG-monomethyl-L-arginine. The L-arginine-dependent pathway was involved in EMT6 cytostasis mediated by IFN-gamma + lipopolysaccharide because cytostasis expression required L-arginine and was inhibited by NG-monomethyl-L-arginine.

Adenocarcinoma↗
PubMed1988

Cytostatic product(s) released by activated macrophages, unrelated to interleukin 1, tumor necrosis factor alpha, and interferon-alpha/beta.

Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.

Animals↗