Nitric oxide-mediated inhibition of androgen receptor activity: possible implications for prostate cancer progression.

Chronic inflammation increases the risk of cancer and many cancers, including prostate cancer, arise at sites of chronic inflammation. Inducible nitric oxide synthase (iNOS) is an enzyme dominantly expressed during inflammatory reactions. Although synthesis of high amounts of nitric oxide (NO) by iNOS has been demonstrated in pathophysiological processes, such as acute or chronic inflammation, autoimmune diseases or tumorigenesis, the role of iNOS activity in most of these diseases is poorly understood. Analysing prostate cancer biopsies by immunohistochemistry we found iNOS protein expression in tumor cells strongly paralleled by nitrotyrosine suggesting that iNOS is fully active. In vitro, NO inhibits androgen receptor-dependent promoter activity and prostate specific antigen production as well as DNA-binding activity of the androgen receptor (AR) in a concentration-dependent manner. Inhibition of the activity of androgen receptor-dependent reporter constructs is neither owing to diminished AR protein levels nor owing to an inhibition of its nuclear import. In addition, NO inhibits the proliferation of androgen receptor-positive prostate cancer cells significantly more efficiently than proliferation of androgen receptor-negative prostate cancer cells. In summary, our findings suggest that intratumoral iNOS activity favors development of prostate cancer cells that are able to proliferate androgen receptor-independently, thereby promoting prostate tumor progression.

Pancreatic islet cells are highly susceptible towards the cytotoxic effects of chemically generated nitric oxide.

To compare the sensitivity of different mammalian cell types towards the cytotoxic action of nitric oxide, freshly isolated rat pancreatic islet cells, hepatocytes, resident and activated macrophages, cultured aortic endothelial cells and two murine tumor cell lines were tested for susceptibility towards exogenous nitric oxide. As sources for nitric oxide nitroprusside, S-nitroso-N-acetyl-penicillamine and the sydnonimine-derivative SIN-1 were used. These generate nitric oxide by different mechanisms and kinetics. Among the cell types tested we found large differences in their susceptibility towards the three nitric oxide donors. Islet cells were by far the most sensitive of the investigated cells and were completely lysed by all three nitric oxide donors. Hepatocytes and endothelial cells were sensitive towards nitroprusside but relatively resistant towards toxicity of SIN-1 and S-nitroso-N-acetyl-penicillamine. Activated and resident macrophages were lysed by SIN-1, whereas high concentrations of nitroprusside and S-nitroso-N-acetyl-penicillamine led to partial cell lysis only. The tumor cell lines were both lysed by SIN-1 but showed differences in their sensitivity towards S-nitroso-N-acetyl-penicillamine. Nitric oxide, which is produced in large amounts during infection and inflammation, may play an important role in the destruction of islet cells during insulitis leading to insulin-dependent diabetes mellitus.

Even after UVA-exposure will nitric oxide protect cells from reactive oxygen intermediate-mediated apoptosis and necrosis.

Reactive oxygen species (ROS) play a pivotal role in UVA-induced cell damage. As expression of the inducible nitric oxide synthase (iNOS) is a normal response of human skin to UV radiation we examined the role of nitric oxide (NO) as a protective agent during or even after UVA1- or ROS-exposure against apoptosis or necrosis of rat endothelial cells. When added during or up to 2 h subsequent to UVA1 or ROS exposure the NO-donor S-nitroso-cysteine (SNOC) at concentrations from 100-1000 microM significantly protects from both apoptosis as well as necrosis. The NO-mediated protection strongly correlates with complete inhibition of lipid peroxidation (sixfold increase of malonedialdehyde formation in untreated versus 1.2-fold with 1 mM SNOC). NO-mediated protection of membrane function was also shown by the inhibition of cytochrome c leakage in UVA1 treated cells, a process not accompanied by alterations in Bax and Bcl-2 protein levels. Thus, the experiments presented demonstrate that NO exposure during or even after a ROS-mediated toxic insult fully protects from apoptosis or necrosis by maintaining membrane integrity and function.

Cysteine-Zn2+ complexes: unique molecular switches for inducible nitric oxide synthase-derived NO.

Nitric oxide (NO) in the low nanomolar range acts as a transcellular messenger molecule to initiate regulatory and physiological responses in nearby target cells via binding to the soluble guanylate cyclase heme moiety. Higher NO concentrations, as synthesized by the inducible NO synthase (iNOS) during inflammatory processes, show additional effects: NO may react with O2, yielding nitrogen oxides like N2O3 that are able to nitrosate thiols. A variety of proteins involved in very different functions of the cell contain cysteine-Zn2+ complexes. Effects of NO on different proteins containing cysteine-Zn2+ domains and playing essential roles during transcription, protein folding, and proteolysis are discussed. It is suggested that iNOS-derived NO acts as a signal molecule targeting cysteine-Zn2+ linkages, thus enabling cells to react toward nitrosative stress.

Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts.

Nitric oxide (NO) is known to exert cytotoxic and cytostatic effects in various cells and tissues. Although NO formation in human skin has been convincingly demonstrated, little is known about the NO-mediated effects in skin physiology and pathology. Here, we investigate the influence of NO on proliferation, differentiation, and apoptosis of primary cultures of normal human keratinocytes and fibroblasts. Four different NO donors at concentrations ranging from 0.01 to 5 mM were added every 12 h or 24 h to primary cultures of human keratinocytes and fibroblasts, and cells cultured for up to 3 d in the presence of these compounds. Cultures were examined for necrosis or apoptosis using trypan blue exclusion and in situ nick-translation. Cultures were also screened for the expression of the proliferation marker Ki67 and for an increase in cell numbers using neutral red staining. In addition, keratinocytes were stained for cytokeratin 6 expression to assess differentiation. We find that both keratinocytes and fibroblasts are highly resistant towards necrosis- or apoptosis-inducing effects of NO. In both cell types NO modulates cell growth, albeit in a cell-type specific pattern: cytostasis becomes significant in fibroblasts at concentrations of > or = 0.25 mM of the NO donor. In keratinocytes a biphasic effect is found with increased proliferation at low concentrations ranging from 0.01 to 0.25 mM and cytostasis at concentrations of > or = 0.5 mM. Conversely, expression of cytokeratin 6 is decreased at the lower NO donor concentrations and increased at higher concentrations as an indication of induction of differentiation at higher NO concentrations. Collectively, our results demonstrate that NO modulates proliferation and differentiation in human skin cells, a finding that will help to explain the pathophysiology of human skin diseases. Moreover, these findings suggest that NO generation in human skin diseases is not directly associated with local cell destruction, in contrast to findings in several other human diseases.

The effects of nitric oxide on the membrane potential and ionic currents of mouse pancreatic B cells.

Nitric oxide (NO) is considered to contribute to the impairment of B cell function in insulin-dependent diabetes mellitus. The effects of compounds that release NO were tested on the membrane potential and ionic currents of mouse pancreatic B cells using intracellular microelectrodes and the whole-cell patch-clamp technique. S-Nitrosocysteine led to a concentration-dependent reduction of electrical activity induced by 15 mM glucose. At a concentration of 1 mM, S-nitrosocysteine cause a hyperpolarization of the plasma membrane with complete suppression of electrical activity. In about half of the cells tested, electrical activity reappeared during treatment with S-nitroso-cysteine or after wash-out. However, in the other cells the hyperpolarization was followed by a slow depolarization and electrical activity did not reappear. The perforated-patch whole-cell K+ATP current first increased and subsequently decreased again during exposure to 1 mM S-nitroso-cysteine. With 0.1 and 0.01 mM S-nitroso-cysteine, only the rise of the current amplitude was observed. S-nitroso-cysteine (1 mM) almost completely abolished the current through voltage-dependent Ca2+ channels (measured with Ba2+ as charge carrier). Like S-nitroso-cysteine, 100 microM sodium-nitroprusside, another donor, evoked a marked hyperpolarization of the membrane potential that was at least in part reversible. To further ascertain that the effect of S-nitroso-cysteine was mediated by NO, we tested the decomposition products of S-nitroso-cysteine. Nitrite and denitrosylated S-nitroso-cystein (1 mM) did not alter electrical activity of B cells, whereas cysteine (1 mM) caused a slight depolarization. It is concluded that exogenous NO evokes rapid changes of B cell function by influencing the activity of ion channels.

Influence of nitric oxide on the intracellular reduced glutathione pool: different cellular capacities and strategies to encounter nitric oxide-mediated stress.

Different cell types exhibit huge differences towards the cytotoxic action of NO. In search for an explanation, we used subtoxic concentrations of the NO-donors S-nitrosocysteine (SNOC) for short-term challenge and of (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO) for longer periods of exposure, respectively, and subtoxic concentrations of the oxidant H2O2 to determine the impact on intracellular reduced glutathione (GSH) concentrations. We find that GSH concentrations are always decreased, but that different cell types show different responses. Incubation of the relatively NO-sensitive murine lymphocytes with both NO-donors, but not with H2O2, resulted in a nearly complete loss of intracellular GSH. Short-term NO-treatment of P815 mastocytoma cells, also sensitive to NO-mediated cell death, decreased GSH to a similar extent only if either glutathione reductase (GSHR) activity or y-glutamylcysteine synthetase (gammaGCS) activity were inhibited concomitantly by specific inhibitors. Long-term NO-treatment of P815 cells, however, resulted in a significant decrease of GSH that could be further enhanced by inhibiting gammaGCS activity. In contrast, neither short-term nor long-term NO-exposure nor H2O2-treatment affected intracellular GSH levels of L929 fibroblasts, which were previously shown to be extremely resistant towards NO, whereas concomitant gammaGCS inhibition, but not GSHR inhibition, completely decreased GSH concentrations. These results show that different cell types use different pathways trying to maintain glutathione concentrations to cope with nitrosative stress, and the overall capability to maintain a critical amount of GSH correlates with susceptibility to NO-induced cell death.

Cytotoxic action of IL-1 beta against pancreatic islets is mediated via nitric oxide formation and is inhibited by NG-monomethyl-L-arginine.

IL-1 beta has been previously shown to act as a cytotoxic agent in islets. Here we show by electron microscopy of alginate encapsulated islets, that islet cell lysis is induced by culturing islets for 24 or 48 h in the presence of IL-1 beta. The extent of lysis depends on the IL-1 beta concentration and is slightly enhanced by the addition of TNF-alpha. Cells can be protected from lysis by NG-monomethyl-L-arginine. Lysis is paralleled by an increase in nitrite concentration in culture supernatants of whole islets but not in supernatants of isolated endocrine cells. The results indicate that IL-1 beta toxicity occurs via inducing in non-endocrine islet cells the synthesis and release of nitric oxide, which has been shown earlier to be highly toxic for islet cells.

Nitric oxide mediates intracytoplasmic and intranuclear zinc release.

We previously described that NO. leads to destruction of ZnS clusters and release of Zn2+ from various proteins including zinc finger transcription factors. To assess the relevance in living cells, we investigated, whether exogenous NO. leads to an increase of cytoplasmic and nuclear free Zn2+. L929 cells, mouse splenocytes, or rat aorta endothelial cells were labeled with Zinquin-E, a Zn2+-specific fluorophore, and were treated with two different spontaneous NO donors, S-nitrosocysteine or DETA/NO. Both NO donors strongly increased the Zn2+-dependent fluorescence in the cellular cytosol and also in nuclei as compared to controls. NO-dependent Zn2+ release in splenocytes was quantitated by flow cytometry. These results show for the first time, that nitrosative stress mediates intracellular and intranuclear Zn2+ release which may be relevant in altering gene expression patterns.

Zinc finger proteins as molecular targets for nitric oxide-mediated gene regulation.

A multiplicity of biological functions have been ascribed to nitric oxide (NO). It plays a significant role as a signal as well as a cytotoxic effector molecule. NO may, however, also play regulatory and/or modulatory roles in biology. A growing body of evidence suggests that posttranslational modifications of transcription factors serve a regulating role on gene transcription, particularly after changes of the redox state of the cell. Zinc fingers are the most prevalent transcription factor DNA-binding motif. As NO is able to S-nitrosate thiols of zinc-sulfur clusters leading to reversible disruption of zinc finger structures, this provides a molecular mechanism to regulate the transcription of genes. Current knowledge about effects of NO on the cellular zinc homeostasis and on the gene-regulating activity of zinc finger transcription factors is reviewed.

Implications of inducible nitric oxide synthase expression and enzyme activity.

We summarize here our current knowledge about inducible nitric oxide synthase (NOS) activity in human diseases and disorders. As basic research discovers more and more effects of low or high concentrations of NO toward molecular and cellular targets, successful therapies involving inhibition of NO synthesis or application of NO to treat human diseases are still lacking. This is in part due to the fact that the impact of NO on cell function or death are complex and often even appear to be contradictory. NO may be cytotoxic but may also protect cells from a toxic insult; it is apoptosis-inducing but also exhibits prominent anti-apoptotic activity. NO is an antioxidant but may also compromise the cellular redox state via oxidation of thiols like glutathione. NO may activate specific signal transduction pathways but is also reported to inhibit exactly these, and NO may activate or inhibit gene transcription. The situation may even be more complicated, because NO, depending on its concentration, may react with oxygen or the superoxide anion radical to yield reactive species with a much broader chemical reaction spectrum than NO itself. Thus, the action of NO during inflammatory reactions has to be considered in the context of timing and duration of its synthesis as well as stages and specific events in inflammation.

Macrophage-generated nitric oxide as cytotoxic factor in destruction of alginate-encapsulated islets. Protection by arginine analogs and/or coencapsulated erythrocytes.

Isolated rat islets were microencapsulated in alginate beads of about 1.5 mm in diameter. These were cocultured with activated or resident peritoneal macrophages of syngeneic rats for 24 hr. Examination of the encapsulated islets by transmission electron microscopy showed that the islets were lysed by activated (80.0 +/- 12.8% of islets lysed), but not by resident, macrophages (17.5 +/- 12.2% lysis) despite encapsulation. Islet lysis was inhibited in a concentration-dependent manner by a specific nitric oxide-synthase inhibitor (0.5 mM NG-methyl-L-arginine: 5.9 +/- 3.9% lysis) in an L-arginine-reversible manner (0.5 mM NG-methyl-L-arginine + 10 mM L-arginine: 55.1 +/- 16.6% lysis). Incubation of encapsulated islets with 3 different nitric oxide-generating compounds also resulted in a concentration-dependent islet lysis. Coencapsulation of autologous erythrocytes was found to be an effective and easy way of protection from macrophage-mediated lysis. Protection was dependent upon the number of erythrocytes coencapsulated. This in vitro study demonstrates that nitric oxide secreted by activated macrophages is able to destroy islets despite encapsulation in alginate, and that both, inhibition of nitric oxide formation using enzyme inhibitors and scavenging of nitric oxide once formed exploiting the hemoglobin of autologous erythrocytes, protect encapsulated islets from destruction.

In situ nick-translation detects focal apoptosis in thymuses of glucocorticoid- and lipopolysaccharide-treated mice.

In this study we used in situ nick-translation to analyze apoptotic events in the thymus and in cultured thymocytes at the level of individual cell nuclei. In vitro nuclear DNA strand breaks were observed 3 hr after exposure of thymocytes to dexamethasone (Dex) in 30% of cells and increased to 78% after 15 hr. In sections of 10-day-old mouse thymus, single cells with DNA strand breaks were dispersed throughout the cortex and to a lesser degree in the medulla. In contrast, a large number of clusters of apoptotic cells were seen in the thymic cortex 3-18 hr after injection of Dex or lipopolysaccharide (LPS). After 48 hr apoptotic cells were no longer detectable. Positive signals correlated with the detection of DNA ladders of multimers of about 180 BP size on agarose gels. Electron microscopy confirmed the presence of apoptotic cell clusters and showed that apoptotic foci were located around capillaries in LPS-injected animals. We conclude that in situ nick translation is a suitable method to detect apoptotic nuclei in cultured cells and on cryostat sections. With this method we could demonstrate that in vivo spontaneous apoptosis occurs in single dispersed thymocytes, also including the medulla, whereas experimentally induced apoptosis affects cell clusters, possibly due to high local concentrations of apoptosis inducers.

Streptozotocin is not a spontaneous NO donor.

The reaction of streptozotocin with oxymyoglobin was analyzed and compared with results using various compounds that spontaneously generate nitric oxide in solution.

Inducible nitric oxide synthase-derived nitric oxide in gene regulation, cell death and cell survival.

Studies from many laboratories have demonstrated the complex role of NO in inflammatory processes. Prolonged exposure to NO shifts the cellular redox potential to a more oxidized state and this is critically regulated by intracellular levels of reduced glutathione. NO-mediated stress will alter gene expression patterns, and the number of genes known to be involved is steadily increasing. Indeed, due to its S-nitrosating activity in the presence of oxygen, NO can modify the activity of transcription factors containing zinc finger motifs or cysteines within the DNA-binding domain. In addition, we are faced with not only NO acting as a powerful inducer of apoptosis or of necrosis in some cells, but also representing an equally powerful protection from cell death in many instances. Some of these apparent discrepancies may be explained by different capacities of cells to cope with the stress of NO exposure. Here, we review our findings on the complex impact of NO on transcriptional regulation of genes, cell death and cell survival. These NO-mediated actions will contribute to a better understanding of the impact of inducible nitric oxide synthase (iNOS) enzyme activity during inflammatory reactions.

Suppression of low dose streptozotocin induced diabetes in mice by administration of a nitric oxide synthase inhibitor.

Nitric oxide has recently been identified as the primary toxic effector molecule in the lysis of islet cells by inflammatory macrophages. We show here that N-nitro-L-arginine-methylester (NAME), an inhibitor of endothelial and macrophage NO synthase partially suppresses diabetes development in the low dose streptozotocin induced diabetes model in C57BL/6J mice. Mean blood glucose levels were lower in the group receiving NAME throughout the observation period of 30d (p less than 0.05-0.001). Similar concentrations of NAME as expected in vivo were tested in vitro in macrophage-islet cell cocultures and were found to partially suppress NO production and islet cell lysis. We conclude that NO synthase activity is a pathogenetic factor in diabetes development.

Toxicity of chemically generated nitric oxide towards pancreatic islet cells can be prevented by nicotinamide.

Previous studies have indicated that nitric oxide is involved in the lysis of pancreatic islet cells by inflammatory macrophages. Here we show that the incubation of islet cells with chemical NO-donors leads to cell lysis in a concentration and time dependent way. Islet cell death could be prevented by nicotinamide and 3-aminobenzamide, which are known to inhibit ADP-ribosylation, while several scavengers of oxygen radicals, N-acetylcysteine, dihydrolipoic acid, dimethylthiourea and citiolone, provided no protection.