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GO terms associated with upregulated genes bearing new ∙NO-induced H3K9ac peaks around promoter regions

Supplementary Materials and Methods. Figure S1. Nitric oxide induces significant changes in histone posttranslational modifications. Figure S2. NO-mediated gene expression changes are consistent with the induction of an oncogenic phenotype. Figure S3. Nitric oxide alters the distribution patterns of H3K9ac and H3K9me2 across the genome. Figure S4. NO-upregulated genes with a new promoter-associated H3K9ac peak are involved in critical tumorigenic pathways. Table S1. NO-mediated changes in global levels of histone acetylation. Table S2. List of NO-regulated miRNAs (1.5 Fold Change cut-off). Table S3. Control ChIP-PCR primer sequences. Table S4. List of transcription factors with binding motifs found exclusively around promoters of upregulated genes with a new NO-induced H3K9ac peak.

Significance: Cancer is a complex disease, which not only involves the tumor but its microenvironment comprising different immune cells as well. Nitric oxide (NO) plays specific roles within tumor cells and the microenvironment and determines the rate of cancer progression, therapy efficacy, and patient prognosis. Recent Advances: Key understanding of the processes leading to dysregulated NO flux within the tumor microenvironment over the past decade has provided better understanding of the dichotomous role of NO in cancer and its importance in shaping the immune landscape. It is becoming increasingly evident that nitric oxide synthase 2 (NOS2)-mediated NO/reactive nitrogen oxide species (RNS) are heavily involved in cancer progression and metastasis in different types of tumor. More recent studies have found that NO from NOS2(+) macrophages is required for cancer immunotherapy to be effective. Critical Issues: NO/RNS, unlike other molecules, are unique in their ability to target a plethora of oncogenic pathways during cancer progression. In this review, we subcategorize the different levels of NO produced by cells and shed light on the context-dependent temporal effects on cancer signaling and metabolic shift in the tumor microenvironment. Future Directions: Understanding the source of NO and its spaciotemporal profile within the tumor microenvironment could help improve efficacy of cancer immunotherapies by improving tumor infiltration of immune cells for better tumor clearance.

Nitrite represents an endocrine reserve of bioavailable nitric oxide (NO) that mediates a number of physiological responses including conferral of cytoprotection after ischemia/reperfusion (I/R). It has long been known that nitrite can react with non-heme iron to form dinitrosyliron complexes (DNIC). However, it remains unclear how quickly nitrite-dependent DNIC form in vivo, whether formation kinetics differ from that of NO-dependent DNIC, and whether DNIC play a role in the cytoprotective effects of nitrite. Here we demonstrate that chronic but not acute nitrite supplementation increases DNIC concentration in the liver and kidney of mice. Although DNIC have been purported to have antioxidant properties, we show that the accumulation of DNIC in vivo is not associated with nitrite-dependent cytoprotection after hepatic I/R. Further, our data in an isolated mitochondrial model of anoxia/reoxygenation show that while NO and nitrite demonstrate similar S-nitrosothiol formation kinetics, DNIC formation is significantly greater with NO and associated with mitochondrial dysfunction as well as inhibition of aconitase activity. These data are the first to directly compare mitochondrial DNIC formation by NO and nitrite. This study suggests that nitrite-dependent DNIC formation is a physiological consequence of dietary nitrite. The data presented herein implicate mitochondrial DNIC formation as a potential mechanism underlying the differential cytoprotective effects of nitrite and NO after I/R, and suggest that DNIC formation is potentially responsible for the cytotoxic effects observed at high NO concentrations., Graphical abstract fx1, Highlights • Dietary nitrite results in DNIC formation in many tissues, most notably the liver. • Nitrite-dependent DNIC accumulate within the mitochondrion. • NO generates greater DNIC formation in the mitochondrion than nitrite. • At high concentrations of NO DNIC formation is associated with mitochondrial injury.

Mechanical ventilation (MV) with supraphysiological levels of oxygen (hyperoxia) is a life-saving therapy for the management of patients with respiratory distress. However, a significant number of patients on MV develop ventilator-associated pneumonia (VAP). Previously, we have reported that prolonged exposure to hyperoxia impairs the capacity of macrophages to phagocytize Pseudomonas aeruginosa (PA), which can contribute to the compromised innate immunity in VAP. In this study, we show that the high mortality rate in mice subjected to hyperoxia and PA infection was accompanied by a significant decrease in the airway levels of nitric oxide (NO). Decreased NO levels were found to be, in part, due to a significant reduction in NO release by macrophages upon exposure to PA lipopolysaccharide (LPS). Based on these findings, we postulated that NO supplementation should restore hyperoxia-compromised innate immunity and decrease mortality by increasing the clearance of PA under hyperoxic conditions. To test this hypothesis, cultured macrophages were exposed to hyperoxia (95% O2) in the presence or absence of the NO donor, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate/D-NO). Interestingly, D-NO (up to 37.5 µM) significantly attenuated hyperoxia-compromised macrophage migratory, phagocytic, and bactericidal function. To determine whether the administration of exogenous NO enhances the host defense in bacteria clearance, C57BL/6 mice were exposed to hyperoxia (99% O2) and intranasally inoculated with PA in the presence or absence of D-NO. D-NO (300 µM–800 µM) significantly increased the survival of mice inoculated with PA under hyperoxic conditions, and significantly decreased bacterial loads in the lung and attenuated lung injury. These results suggest the NO donor, D-NO, can improve the clinical outcomes in VAP by augmenting the innate immunity in bacterial clearance. Thus, provided these results can be extrapolated to humans, NO supplementation may represent a potential therapeutic strategy for preventing and treating patients with VAP.

The metabolic requirements and functions of cancer and normal tissues are vastly different. Due to the rapid growth of cancer cells in the tumor microenvironment, distorted vasculature is commonly observed, which creates harsh environments that require rigorous and constantly evolving cellular adaption. A common hallmark of aggressive and therapeutically resistant tumors is hypoxia and hypoxia-induced stress markers. However, recent studies have identified alterations in a wide spectrum of metabolic pathways that dictate tumor behavior and response to therapy. Accordingly, it is becoming clear that metabolic processes are not uniform throughout the tumor microenvironment. Metabolic processes differ and are cell type specific where various factors promote metabolic heterogeneity within the tumor microenvironment. Furthermore, within the tumor, these metabolically distinct cell types can organize to form cellular neighborhoods that serve to establish a pro-tumor milieu in which distant and spatially distinct cellular neighborhoods can communicate via signaling metabolites from stroma, immune and tumor cells. In this review, we will discuss how biochemical interactions of various metabolic pathways influence cancer and immune microenvironments, as well as associated mechanisms that lead to good or poor clinical outcomes.

The prolonged exposure to hyperoxia can compromise macrophage functions and contribute to the development of ventilator-associated pneumonia. High levels of extracellular high-mobility group box-1 (HMGB1) in the airways of mice exposed to hyperoxia can directly cause macrophage dysfunction. Hence, inhibition of the release of nuclear HMGB1 into the extracellular milieu may help to maintain macrophage functions under hyperoxic conditions. The present study investigates whether ethacrynic acid (EA) affects hyperoxia-induced HMGB1 release from macrophages and improves their functions. Macrophage-like RAW 264.7 cells and bone marrow–derived macrophages were exposed to different concentrations of EA for 24 hours in the presence of 95% O2. EA significantly decreased the accumulation of extracellular HMGB1 in cultured media. Importantly, the phagocytic activity and migration capability of macrophages were significantly enhanced in EA-treated cells. Interestingly, hyperoxia-induced NF-κB activation was also inhibited in these cells. To determine whether NF-κB plays a role in hyperoxia-induced HMGB1 release, BAY 11-7082, an inhibitor of NF-κB activation, was used. Similar to EA, BAY 11-7082 significantly inhibited the accumulation of extracellular HMGB1 and improved hyperoxia-compromised macrophage migration and phagocytic activity. Furthermore, 24-hour hyperoxic exposure of macrophages caused hyperacetylation of HMGB1 and its subsequent cytoplasmic translocation and release, which were inhibited by EA and BAY 11-7082. Together, these results suggest that EA enhances hyperoxia-compromised macrophage functions by inhibiting HMGB1 hyperacetylation and its release from macrophages, possibly through attenuation of the NF-κB activation. Therefore, the activation of NF-κB could be one of the underlying mechanisms that mediate hyperoxia-compromised macrophage functions.

The chelatable iron pool (CIP) is a small but chemically significant fraction of total cellular iron. While this dynamic population of iron is limited, it is redox active and capable of generating reactive oxygen species (ROS) that can lead to oxidative stress which is associated with various pathologies. Nitric oxide (•NO), is a free radical signalling molecule that regulates numerous physiological and pathological conditions. We have previously shown that macrophages exposed to endogenously generated or exogenously administered nitric oxide (•NO) results in its interaction with CIP to form dinitrosyliron complexes with thiol containing ligands (DNICs). In this study we assessed the consequences of DNIC formation in cancer cells as •NO is known to be associated with numerous malignancies. Incubation of cancer cells with •NO led to a time and dose dependent increase in formation of DNICs. The formation of DNICs results in the sequestration of the CIP which is a major source of iron for redox reactions and reactive oxygen species (ROS) generation. Therefore, we set out to test the antioxidant effect of •NO by measuring the ability of DNICs to protect cells against oxidative stress. We observed that cancer cells treated with •NO were partially protected against H(2)O(2) mediated cytotoxicity. This correlated to a concomitant decrease in the formation of oxidants when •NO was present during H(2)O(2) treatment. Similar protective effects were achieved by treating cells with iron chelators in the presence of H(2)O(2). Interestingly, •NO decreased the rate of cellular metabolism of H(2)O(2) suggesting that a proportion of H(2)O(2) is consumed via reactions with cellular iron. When the CIP was artificially increased by supplementation of cells with iron, a significant decrease in the cytoprotective effect of •NO was observed. Notably, •NO concentrations, at which cytoprotective and antioxidant effects were observed, correlated with concentration-dependent increases in DNIC formation. Collectively, these results demonstrate that •NO has antioxidant properties by its ability to sequester cellular iron. This could play a significant role in variety of diseases involving ROS mediated toxicity like cancer and neurodegenerative disorders where •NO has been shown to be an important etiologic factor.

Prolonged exposure to hyperoxia results in acute lung injury (ALI), accompanied by a significant elevation in the levels of proinflammatory cytokines and leukocyte infiltration in the lungs. However, the mechanisms underlying hyperoxia-induced proinflammatory ALI remain to be elucidated. In this study, we investigated the role of the proinflammatory cytokine high mobility group box protein 1 (HMGB1) in hyperoxic inflammatory lung injury, using an adult mouse model. The exposure of C57BL/6 mice to ≥99% O2 (hyperoxia) significantly increased the accumulation of HMGB1 in the bronchoalveolar lavage fluids (BALF) prior to the onset of severe inflammatory lung injury. In the airways of hyperoxic mice, HMGB1 was hyperacetylated and existed in various redox forms. Intratracheal administration of recombinant HMGB1 (rHMGB1) caused a significant increase in leukocyte infiltration into the lungs compared to animal treated with a non-specific peptide. Neutralizing anti-HMGB1 antibodies, administrated before hyperoxia significantly attenuated pulmonary edema and inflammatory responses, as indicated by decreased total protein content, wet/dry weight ratio, and numbers of leukocytes in the airways. This protection was also observed when HMGB1 inhibitors were administered after the onset of the hyperoxic exposure. The aliphatic antioxidant, ethyl pyruvate (EP), inhibited HMGB1 secretion from hyperoxic macrophages and attenuated hyperoxic lung injury. Overall, our data suggest that HMGB1 plays a critical role in mediating hyperoxic ALI through the recruitment of leukocytes into the lungs. If these results can be translated to humans, they suggest that HMGB1 inhibitors provide treatment regimens for oxidative inflammatory lung injury in patients receiving hyperoxia through mechanical ventilation., Graphical abstract, Highlights • Exposure to hyperoxia results in accumulation of high levels of airway HMGB1 that precede inflammatory acute lung injury (ALI). • Airway HMGB1 is critical in mediating hyperoxia-induced inflammatory ALI via recruiting leukocytes including neutrophils. • Extracellular HMGB1-accumulated upon prolonged exposure to hyperoxia is hyperacetylated, existing in different redox states. • Small molecule EP, administrated even after the onset of hyperoxic exposure, can mitigate hyperoxia-induced inflammatory ALI by inhibiting HMGB1 release into the extracellular milieu.

Supraphysiological concentrations of oxygen (hyperoxia) can compromise host defense and increase susceptibility to bacterial infections, causing ventilator-associated pneumonia. The phagocytic activity of macrophages is impaired by hyperoxia-induced increases in the levels of reactive oxygen species (ROS) and extracellular high-mobility group box protein B1 (HMGB1). Ascorbic acid (AA), an essential nutrient and antioxidant, has been shown to be beneficial in various animal models of ROS-mediated diseases. The aim of this study was to determine whether AA could attenuate hyperoxia-compromised host defense and improve macrophage functions against bacterial infections. C57BL/6 male mice were exposed to hyperoxia (≥98% O2, 48 h), followed by intratracheal inoculation with Pseudomonas aeruginosa, and simultaneous intraperitoneal administration of AA. AA (50 mg/kg) significantly improved bacterial clearance in the lungs and airways, and significantly reduced HMGB1 accumulation in the airways. The incubation of RAW 264.7 cells (a macrophage-like cell line) with AA (0–1,000 μM) before hyperoxic exposure (95% O2) stabilized the phagocytic activity of macrophages in a concentration-dependent manner. The AA-enhanced macrophage function was associated with significantly decreased production of intracellular ROS and accumulation of extracellular HMGB1. These data suggest that AA supplementation can prevent or attenuate the development of ventilator-associated pneumonia in patients receiving oxygen support.

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide (•NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to •NO. We demonstrate that the interaction of •NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological •NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of •NO. Augmenting the chelatable iron pool abolished •NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between •NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.

It is well established that nitric oxide ((•)NO) reacts with cellular iron and thiols to form dinitrosyliron complexes (DNIC). Little is known, however, regarding their formation and biological fate. Our quantitative measurements reveal that cellular concentrations of DNIC are proportionally the largest of all (•)NO-derived adducts (900 pmol/mg protein, or 45-90 μM). Using murine macrophages (RAW 264.7), we measured the amounts, and kinetics, of DNIC assembly and disappearance from endogenous and exogenous sources of (•)NO in relation to iron and O(2) concentration. Amounts of DNIC were equal to or greater than measured amounts of chelatable iron and depended on the dose and duration of (•)NO exposure. DNIC formation paralleled the upregulation of iNOS and occurred at low physiologic (•)NO concentrations (50-500 nM). Decreasing the O(2) concentration reduced the rate of enzymatic (•)NO synthesis without affecting the amount of DNIC formed. Temporal measurements revealed that DNIC disappeared in an oxygen-independent manner (t(1/2)=80 min) and remained detectable long after the (•)NO source was removed (24 h). These results demonstrate that DNIC will be formed under all cellular settings of (•)NO production and that the contribution of DNIC to the multitude of observed effects of (•)NO must always be considered.

The role of nitric oxide (NO()) as a mediator of cancer phenotype has led researchers to investigate strategies for manipulating in vivo production and exogenous delivery of this molecule for therapeutic gain. Unfortunately, NO() serves multiple functions in cancer physiology. In some instances, NO() or nitric oxide synthase (NOS) levels correlate with tumor suppression and in other cases they are related to tumor progression and metastasis. Understanding this dichotomy has been a great challenge for researchers working in the field of NO() and cancer therapy. Due to the unique chemical and biochemical properties of NO(), it's interactions with cellular targets and the subsequent downstream signaling events can be vastly different based upon tumor heterogeneity and microenvironment. Simple explanations for the vast range of NO-correlated behaviors will continue to produce conflicting information about the relevance of NO() and cancer. Paying considerable attention to the chemical properties of NO() and the methodologies being used will remove many of the discrepancies in the field and allow for in depth understanding of when NO-based chemotherapeutics will have beneficial outcomes.

Altered nitric oxide (•NO) metabolism underlies cancer pathology, but mechanisms explaining many •NO-associated phenotypes remain unclear. We have found that cellular exposure to •NO changes histone posttranslational modifications (PTM) by directly inhibiting the catalytic activity of JmjC-domain containing histone demethylases. Herein, we describe how •NO exposure links modulation of histone PTMs to gene expression changes that promote oncogenesis. Through high-resolution mass spectrometry, we generated an extensive map of •NO-mediated histone PTM changes at 15 critical lysine residues on the core histones H3 and H4. Concomitant microarray analysis demonstrated that exposure to physiologic •NO resulted in the differential expression of over 6,500 genes in breast cancer cells. Measurements of the association of H3K9me2 and H3K9ac across genomic loci revealed that differential distribution of these particular PTMs correlated with changes in the level of expression of numerous oncogenes, consistent with epigenetic code. Our results establish that •NO functions as an epigenetic regulator of gene expression mediated by changes in histone PTMs. Cancer Res; 75(24); 5299–308. ©2015 AACR.

The chemical origins of nitrated tyrosine residues (NT) formed in proteins during a variety of pathophysiological conditions remain controversial. Although numerous studies have concluded that NT is a signature for peroxynitrite (ONOO − ) formation, other works suggest the primary involvement of peroxidases. Because metal homeostasis is often disrupted in conditions bearing NT, the role of metals as catalysts for protein nitration was examined. Cogeneration of nitric oxide (NO) and superoxide (O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} ), from spermine/NO (2.7 μM/min) and xanthine oxidase (1–28 μM O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} /min), respectively, resulted in protein nitration only when these species were produced at approximately equivalent rates. Addition of ferriprotoporphyrin IX (hemin) to this system increased nitration over a broad range of O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} concentrations with respect to NO. Nitration in the presence of superoxide dismutase but not catalase suggested that ONOO − might not be obligatory to this process. Hemin-mediated NT formation required only the presence of NO \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} and H 2 O 2 , which are stable end-products of NO and O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} degradation. Ferrous, ferric, and cupric ions were also effective catalysts, indicating that nitration is mediated by species capable of Fenton-type chemistry. Although ONOO − can nitrate proteins, there are severe spatial and temporal constraints on this reaction. In contrast, accumulation of metals and NO \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} subsequent to NO synthase activity can result in far less discriminate nitration in the presence of an H 2 O 2 source. Metal catalyzed nitration may account for the observed specificity of protein nitration seen under pathological conditions, suggesting a major role for translocated metals and the labilization of heme in NT formation.

3-Nitrotyrosyl adducts in proteins have been detected in a wide range of diseases. The mechanisms by which reactive nitrogen oxide species may impede protein function through nitration were examined by using a unique model system, which exploits a critical tyrosyl residue in the fluorophoric pocket of recombinant green fluorescent protein (GFP). Exposure of purified GFP suspended in phosphate buffer to synthetic peroxynitrite in either 0.5 or 5 μM steps resulted in progressively increased 3-nitrotyrosyl immunoreactivity concomitant with disappearance of intrinsic fluorescence (IC 50 ≈ 20 μM). Fluorescence from an equivalent amount of GFP expressed within intact MCF-7 tumor cells was largely resistant to this bolus treatment (IC 50 > 250 μM). The more physiologically relevant conditions of either peroxynitrite infusion (1 μM/min) or de novo formation by simultaneous, equimolar generation of nitric oxide (NO) and superoxide (e.g., 3-morpholinosydnonimine; NONOates plus xanthine oxidase/hypoxanthine, menadione, or mitomycin C) were examined. Despite robust oxidation of dihydrorhodamine under each of these conditions, fluorescence decrease of both purified and intracellular GFP was not evident regardless of carbon dioxide presence, suggesting that oxidation and nitration are not necessarily coupled. Alternatively, both extra- and intracellular GFP fluorescence was exquisitely sensitive to nitration produced by heme-peroxidase/hydrogen peroxide-catalyzed oxidation of nitrite. Formation of nitrogen dioxide (NO 2 ) during the reaction between NO and the nitroxide 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide indicated that NO 2 can enter cells and alter peptide function through tyrosyl nitration. Taken together, these findings exemplified that heme-peroxidase-catalyzed formation of NO 2 may play a pivotal role in inflammatory and chronic disease settings while calling into question the significance of nitration by peroxynitrite.

The quintessential nitrosating species produced during NO autoxidation is N(2)O(3). Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt(2)NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N(2)O(3) through the intermediacy of NO(2). This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO(2) was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.

Endothelial nitric oxide (nitrogen monoxide) is synthesized at the intravascular/extravascular interface. We previously have reported the intravascular half-life of NO, as a result of consumption by erythrocytes, as approximately 2 ms. We report here studies designed to estimate the lifetime of NO in the parenchymal (extravascular) tissue and describe the implications of these results for the distribution of NO and oxygen concentration gradients away from the blood vessel. The rate of consumption of NO by parenchymal cells (hepatocytes) linearly depends on both NO and O 2 concentration. We estimate that the extravascular half-life of NO will range from 0.09 to > 2 s, depending on O 2 concentration and thus distance from the vessel. Computer modeling reveals that this phenomenon, coupled with reversible NO inhibition of cellular mitochondrial oxygen consumption, substantially extends the zone of adequate tissue cellular oxygenation away from the blood vessel, with an especially dramatic effect during conditions of increased tissue work (oxygen consumption). This represents a second action of NO, in addition to vasodilation, in enhancing tissue cellular respiration and provides a possible physiological function for the known reversible inhibition of mitochondrial respiration by low concentrations of NO.

Methylation of lysine residues on histone tails is an important epigenetic modification that is dynamically regulated through the combined effects of methyltransferases and demethylases. The Jumonji C domain Fe(II) α-ketoglutarate family of proteins performs the majority of histone demethylation. We demonstrate that nitric oxide ((•)NO) directly inhibits the activity of the demethylase KDM3A by forming a nitrosyliron complex in the catalytic pocket. Exposing cells to either chemical or cellular sources of (•)NO resulted in a significant increase in dimethyl Lys-9 on histone 3 (H3K9me2), the preferred substrate for KDM3A. G9a, the primary methyltransferase acting on H3K9me2, was down-regulated in response to (•)NO, and changes in methylation state could not be accounted for by methylation in general. Furthermore, cellular iron sequestration via dinitrosyliron complex formation correlated with increased methylation. The mRNA of several histone demethylases and methyltransferases was also differentially regulated in response to (•)NO. Taken together, these data reveal three novel and distinct mechanisms whereby (•)NO can affect histone methylation as follows: direct inhibition of Jumonji C demethylase activity, reduction in iron cofactor availability, and regulation of expression of methyl-modifying enzymes. This model of (•)NO as an epigenetic modulator provides a novel explanation for nonclassical gene regulation by (•)NO.

The S-nitrosation (also referred to as S-nitrosylation) of cysteine residues is an important post-translational protein modification that regulates protein function and cell signaling. The original research articles and reviews in this Forum cover important concepts in protein S-nitrosation and identify key developments and opportunities for progress in this area. Defining the mechanisms by which S-nitrosothiols (RSNOs) may be formed and decomposed in cells and tissues, the integration of the biological chemistry associated with nitric oxide (NO) and other derivatives such as nitrite, and the development of new methodologies merging proteomics and direct quantitation are all key issues that we believe would require detailed attention. Antioxid. Redox Signal. 17, 934–936.

S-Nitrosothiol (RSNO) formation is one manner by which nitric oxide (•NO) exerts its biological effects. There are several proposed mechanisms of formation of RSNO in vivo: auto-oxidation of •NO, transnitrosation, oxidative nitrosylation, and from dinitrosyliron complexes (DNIC). Both free •NO, generated by •NO donors, and S-nitrosocysteine (CysNO) are widely used to study •NO biology and signaling, including protein S-nitrosation. It is assumed that the cellular effects of both compounds are analogous and indicative of in vivo •NO biology. A quantitative comparison was made of formation of DNIC and RSNO, the major •NO-derived cellular products. In RAW 264.7 cells, both •NO and CysNO were metabolized, leading to rapid intracellular RSNO and DNIC formation. DNIC were the dominant products formed from physiologic •NO concentrations, however, and RSNO were the major product from CysNO treatment. Chelatable iron was necessary for DNIC assembly from either •NO or CysNO, but not for RSNO formation. These profound differences in RSNO and DNIC formation from •NO and CysNO question the use of CysNO as a surrogate for physiologic •NO. Researchers designing experiments intended to elucidate the biological signaling mechanisms of •NO should be aware of these differences and should consider the biological relevance of the use of exogenous CysNO. Antioxid. Redox Signal. 17, 962–968.

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide ((•)NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to (•)NO. We demonstrate that the interaction of (•)NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological (•)NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of (•)NO. Augmenting the chelatable iron pool abolished (•)NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between (•)NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.

This study was designed to examine the relationship between the level of implementation of recommended middle school practices and school climate in Oklahoma middle schools. The Middle School Practices Index was sent to all middle school principals in the state. MSPI scores were used to identify one group of schools with high levels of implementation of recommended middle school practices and another group with low levels of implementation. Faculty from these two groups of schools were surveyed using the NASSP School Climate Survey. An analysis was performed to identify relationships between the implementation of middle school practices and school climate. Perceptions of school climate by teachers were in the high implementation schools were higher on 7 of the 10 school climate indicators. Two of those differences were found to be significant at the .05 level. A correlational analysis found 19 significant relationships between middle school practices and school climate indicators. Based upon this ...

Nitric oxide (NO) has been invoked in nearly every normal and pathological condition associated with human physiology. In tumor biology, nitrogen oxides have both positive and negative affects as they have been implicated in both promoting and preventing cancer. Our work has focused on NO chemistry and how it correlates with cytotoxicity and cancer. Toward this end, we have studied both concentration- and time-dependent NO regulation of specific signaling pathways in response to defined nitrosative stress levels that may occur within the tumor microenvironment. Threshold levels of NO required for activation and stabilization of key proteins involved in carcinogenesis including p53, ERK, Akt, and HIF have been identified. Importantly, threshold NO levels are further influenced by reactive oxygen species (ROS) including superoxide, which can shift or attenuate NO-mediated signaling as observed in both tumor and endothelial cells. Our studies have been extended to determine levels of NO that are critical during angiogenic response through regulation of the anti-angiogenic agent thrombospondin-1 (TSP-1) and pro-angiogenic agent matrix metalloproteinase-9 (MMP-9). The quantification of redox events at the cellular level has revealed potential mechanisms that may either limit or potentiate tumor growth, and helped define the positive and negative function of nitric oxide in cancer.

Nitric oxide (NO) has earned the reputation of being a signaling mediator with many diverse and often opposing biological activities. The diversity in response to this simple diatomic molecule comes from the enormous variety of chemical reactions and biological properties associated with it. In the past few years, the importance of steady-state NO concentrations has emerged as a key determinant of its biological function. Precise cellular responses are differentially regulated by specific NO concentration. We propose five basic distinct concentration levels of NO activity: cGMP-mediated processes ([NO]1-30 nM), Akt phosphorylation ([NO] = 30-100 nM), stabilization of HIF-1alpha ([NO] = 100-300 nM), phosphorylation of p53 ([NO]400 nM), and nitrosative stress (1 microM). In general, lower NO concentrations promote cell survival and proliferation, whereas higher levels favor cell cycle arrest, apoptosis, and senescence. Free radical interactions will also influence NO signaling. One of the consequences of reactive oxygen species generation is to reduce NO concentrations. This antagonizes the signaling of nitric oxide and in some cases results in converting a cell-cycle arrest profile to a cell survival profile. The resulting reactive nitrogen species that are generated from these reactions can also have biological effects and increase oxidative and nitrosative stress responses. A number of factors determine the formation of NO and its concentration, such as diffusion, consumption, and substrate availability, which are referred to as kinetic determinants for molecular target interactions. These are the chemical and biochemical parameters that shape cellular responses to NO. Herein we discuss signal transduction and the chemical biology of NO in terms of the direct and indirect reactions.

Matrix metalloproteinases (MMPs) are of central importance in the proteolytic remodeling of matrix and the generation of biologically active molecules. MMPs are distinguished by a conserved catalytic domain containing a zinc ion, as well as a prodomain that regulates enzyme activation by modulation of a cysteine residue within that domain. Because nitric oxide (NO) and derived reactive nitrogen species target zinc ions and cysteine thiols, we assessed the ability of NO to regulate MMPs. A dose-dependent, biphasic regulatory effect of NO on the activity of MMPs (MMP-9, -1, and -13) secreted from murine macrophages was observed. Low exogenous NO perturbed MMP/tissue inhibitor of metalloproteinase (TIMP)-1 levels by enhancing MMP activity and suppressing the endogenous inhibitor TIMP-1. This was cGMP-dependent, as confirmed by the cGMP analog 8-bromo-cGMP, as well as by the NO–soluble guanylyl cyclase–cGMP signaling inhibitor thrombospondin-1. Exposure of purified latent MMP-9 to exogenous NO demonstrated a concentration-dependent activation and inactivation of the enzyme, which occurred at higher NO flux. These chemical reactions occurred at concentrations similar to that of activated macrophages. Importantly, these results suggest that NO regulation of MMP-9 secreted from macrophages may occur chemically by reactive nitrogen species-mediated protein modification, biologically through soluble guanylyl-cyclase-dependent modulation of the MMP-9/TIMP-1 balance, or proteolytically through regulation of MMP-1 and -13, which can cleave the prodomain of MMP-9. Furthermore, when applied in a wound model, conditioned media exhibiting peak MMP activity increased vascular cell migration that was MMP-9-dependent, suggesting that MMP-9 is a key physiologic mediator of the effects of NO in this model.

Asbestos exposure is strongly associated with the development of malignant mesothelioma, yet the mechanistic basis of this observation has not been resolved. Carcinogenic transformation or tumor progression mediated by asbestos may be related to the generation of free radical species and perturbation of cell signaling and transcription factors. We report here that exposure of human mesothelioma or lung carcinoma cells to nitric oxide (NO) in the presence of crocidolite asbestos resulted in a marked decrease in intracellular nitrosation and diminished NO-induced posttranslational modifications of tumor-associated proteins (hypoxia-inducible factor-1α and p53). Crocidolite rapidly scavenged NO with concomitant conversion to nitrite (NO2−). Crocidolite also catalyzed the nitration of cellular proteins in the presence of NO2− and hydrogen peroxide. Nitrated protein adducts are a prominent feature of asbestos-induced lung injury. These data highlight the ability of asbestos to induce phenotypic cellular changes through two processes: (a) by directly reducing bioactive NO levels and preventing its subsequent interaction with target molecules and (b) by increasing oxidative damage and protein modifications through NO2 production and 3-nitrotyrosine formation. (Cancer Res 2006; 66(24): 11600-4)

Independently, superoxide (O2-) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and O2- were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1alpha, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated O2- during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1alpha, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with O2- were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of O2- by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of O2- with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from O2-.

Nitric oxide (NO) donors have been shown to stimulate and inhibit the proliferation, migration, and differentiation of endothelial cellsin vitroand angiogenesisin vivo. Recently, we have shown distinct thresholds for NO to regulate p53-Ser-15P, phosphorylated extracellular signal-regulated kinase (pERK), and hypoxia inducible factor 1α in tumor cells. Because these signaling pathways also promote the growth and survival of endothelial cells, we examined their roles in angiogenic responses of venous endothelial cells and vascular outgrowth of muscle explants elicited by NO. An additional protein involved in the regulation of angiogenesis is thrombospondin-1 (TSP1), a matricellular glycoprotein known to influence adhesion, migration, and proliferation of endothelial cells. Here we demonstrate a triphasic regulation of TSP1 mediated by a slow and prolonged release of NO that depends on ERK phosphorylation. Under conditions of 5% serum, a 24-h exposure of NO donor (0.1–1,000 μM) mediated a triphasic response in the expression of TSP1 protein: decreasing at 0.1 μM, rebounding at 100 μM, and decreasing again at 1,000 μM. Under the same conditions, we observed a dose-dependent increase in P53 phosphorylation and inverse biphasic responses of pERK and mitogen-activated protein kinase phosphatase-1. Both the growth-stimulating activity of low-dose NO for endothelial cells and suppression of TSP1 expression were ERK-dependent. Conversely, exogenous TSP1 suppressed NO-mediated pERK. These results suggest that dose-dependent positive- and negative-feedback loops exist between NO and TSP1. Limiting TSP1 expression by positive feedback through the ERK mitogen-activated protein kinase pathway may facilitate switching to a proangiogenic state at low doses of NO.

NO produced in tumors can either positively or negatively regulate growth. To examine this dichotomy, effects of NO concentration and duration on the posttranslational regulation of several key proteins were examined in human breast MCF7 cells under aerobic conditions. We found that different concentration thresholds of NO appear to elicit a discrete set of signal transduction pathways. At low steady-state concentrations of NO (100 nM), whereas p53 serine 15 phosphorylation occurred at considerably higher levels (>300 nM). ERK phosphorylation was transient during NO exposure. HIF-1α stabilization paralleled the presence of NO, whereas p53 serine 15 phosphorylation was detected during, and persisted after, NO exposure. The dose-dependent effects of synthetic NO donors were mimicked by activated macrophages cocultured with MCF7 cells at varying ratios. ERK and HIF-1α activation was similar in breast cancer cell lines either mutant (MB231) or null (MB157) in p53. The stabilization of HIF-1α by NO was not observed with increased MCF7 cell density, demonstrating the interrelationship between NO and O 2 consumption. The findings show that concentration and duration of NO exposure are critical determinants in the regulation of tumor-related proteins.

The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine/NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine/NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the O 2 / \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \pagestyle{empty} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document} couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with O 2 is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.

Endogenous formation of nitric oxide (NO) and related nitrogen oxides in the vascular system is critical to regulation of multiple physiological functions. An imbalance in the production or availability of these species can result in progression of disease. Nitrogen oxide research in the cardiovascular system has primarily focused on the effects of NO and higher oxidation products. However, nitroxyl (HNO), the one-electron-reduction product of NO, has recently been shown to have unique and potentially beneficial pharmacological properties. HNO and NO often induce discrete biological responses, providing an interesting redox system. This article discusses the emerging aspects of HNO chemistry and attempts to provide a framework for the distinct effects of NO and HNO in vivo.

The impact of nitric oxide (NO) synthesis on different biological cascades can rapidly change dependent on the rate of NO formation and composition of the surrounding milieu. With this perspective, we used diaminonaphthalene (DAN) and diaminofluorescein (DAF) to examine the nitrosative chemistry derived from NO and superoxide (O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{-}\end{equation*}\end{document} ) simultaneously generated at nanomolar to low micromolar per minute rates by spermine/NO decomposition and xanthine oxidase-catalyzed oxidation of hypoxanthine, respectively. Fluorescent triazole product formation from DAN and DAF increased as the ratio of O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{-}\end{equation*}\end{document} to NO approached equimolar, then decreased precipitously as O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{-}\end{equation*}\end{document} exceeded NO. This pattern was also evident in DAF-loaded MCF-7 carcinoma cells and when stimulated macrophages were used as the NO source. Cyclic voltammetry analysis and inhibition studies by using the N 2 O 3 scavenger azide indicated that DAN- and DAF-triazole could be derived from both oxidative nitrosylation (e.g., DAF radical + NO) and nitrosation (NO + addition). The latter mechanism predominated with higher rates of NO formation relative to O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{-}\end{equation*}\end{document} . The effects of oxymyoglobin, superoxide dismutase, and carbon dioxide were examined as potential modulators of reactant availability for the O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{-}\end{equation*}\end{document} + NO pathway in vivo . The findings suggest that the outcome of NO biosynthesis in a scavenger milieu can be focused by O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{-}\end{equation*}\end{document} toward formation of NO adducts on nucleophilic residues (e.g., amines, thiols, hydroxyl) through convergent mechanisms involving the intermediacy of nitrogen dioxide. These modifications may be favored in microenvironments where the rate of O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{2}^{-}\end{equation*}\end{document} production is temporally and spatially contemporaneous with nitric oxide synthase activity, but not in excess of NO generation.

We demonstrate herein dramatic acceleration of aqueous nitric oxide (NO) reaction with O 2 within the hydrophobic region of either phospholipid or biological membranes or detergent micelles and demonstrate that the presence of a distinct hydrophobic phase is required. Per unit volume, at low amounts of hydrophobic phase, the reaction of NO with O 2 within the membranes is approximately 300 times more rapid than in the surrounding aqueous medium. In tissue, even though the membrane represents only 3% of the total volume, we calculate that 90% of NO reaction with O 2 will occur there. We conclude that biological membranes and other tissue hydrophobic compartments are important sites for disappearance of NO and for formation of NO-derived reactive species and that attenuation of these potentially damaging reactions is an important protective action of lipid-soluble antioxidants such as vitamin E.