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International Immunology Advance Access published online on July 2, 2007
International Immunology, doi:10.1093/intimm/dxm058
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Involvement of oxidative and nitrosative stress in modulation of gene expression and functional responses by IFN{gamma}

S. Jyothi Prasanna, Banishree Saha and Dipankar Nandi

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

Correspondence to: Correspondence to: D. Nandi; E-mail: nandi{at}biochem.iisc.ernet.in


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 References
 
IFN{gamma} is a potent immunomodulator which plays important roles in host defense. IFN{gamma} modulates transcription of growth-related genes [N-myc downstream regulator 1, growth arrest and DNA damage inducible {gamma} and inhibitor of DNA binding 2 (Id2)], which is followed by increased growth suppression in the mouse hepatoma cell line, H6. Further studies revealed modulation of genes involved in oxidative and nitrosative stress (iNos, gp91phox and Catalase) and increased generation of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs) upon IFN{gamma} treatment. High amounts of ROS and RNI are responsible for IFN{gamma}-mediated reduction in cell growth as this process is blocked, using either diphenylene iodonium (DPI), an inhibitor of flavin-containing NADPH oxidases, or N-methyl L-arginine (LNMA), an inhibitor of nitric oxide synthase. Based on studies with LNMA and DPI, IFN{gamma}-modulated genes can be categorized into two distinct sets: oxidative and nitrosative stress independent (transporter associated with antigen processing 2, Cd80, Lmp10 and Icosl) and oxidative and nitrosative stress dependent (iNos, gp91phox, Catalase and Id2). In addition, DPI or LNMA blocked IFN{gamma}-induced activation of Ras, demonstrating the involvement of oxidative and nitrosative stress. Manumycin A, a farnesyl transferase inhibitor, blocked Ras activation and reduced NADPH oxidase activity and ROS amounts leading to increased cell growth in the presence of IFN{gamma}. Notably, the IFN{gamma}-induced MHC class I levels are not modulated in cells treated with DPI, LNMA or manumycin A. Together, these results delineate the role of high amounts of ROS, RNI and Ras activation in modulating expression of some genes and, thereby, function by IFN{gamma}. The implications of these results during modulation of immune responses by IFN{gamma} are discussed.

Keywords: cytokine, growth suppression, inflammation, redox, transcriptional profiling


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
IFN{gamma} is a type II IFN produced, primarily, by NK cells and T cells which plays important roles during host defense. Ifn{gamma}–/– mice are very sensitive to infection by several pathogens, including Mycobacterium tuberculosis. Also, humans lacking IFN{gamma} or its receptor display a similar phenotype and are susceptible to mycobacterial infections (1). Apart from modulating host immunity, IFN{gamma} causes pleiotropic effects and modulates inflammatory responses, cell growth and survival (2, 3).

IFN{gamma} employs the Janus kinase (Jak)–signal transducers and activators of transcription coactivator (Stat) pathway to signal from the cell-surface receptor to modulate transcriptional activation of several genes. Jak1, a non-receptor protein tyrosine kinase, phosphorylates IFN{gamma} receptors which generate a binding site for Stat1, a transcriptional coactivator (4, 5). After phosphorylation, Stat1 dimers dissociate from the receptor and translocate to the nucleus and bind to a motif known as the gamma-IFN-activated site (Gas): TTNCNNNAA. Gas sequences are present in several primary responsive IFN{gamma}-modulated genes and Stat1 enhances transcriptional activation by recruiting several transcriptional coactivators (6–8). Cellular responses mediated by IFN{gamma} are, primarily, due to modulation of gene expression. Therefore, identification and studying roles of IFN-stimulated genes (ISGs) during immune responses are an active area of investigation (2, 9–13). IFN-modulated genes can be further classified into primary responsive or secondary responsive genes. Primary responsive genes are induced early due to the binding of the Stat1 dimer to Gas elements present in promoters of genes, e.g. Irf1. The secondary responsive genes are induced following the binding of Irf1 to their promoters (2). Although IFN{gamma} signaling has been studied for several years, accumulating evidences clearly demonstrate the roles of multiple pathways that coordinate to generate functional responses (4). Most IFN{gamma}-responsive genes are dependent on Stat1 although some genes are Stat1 independent (14). Breast cancer 1 (BRCA1), the tumor suppressor, together with Stat1 differentially activates a subset of ISGs (7). Also, IFN{gamma} has been shown to activate a subset of genes in an I{kappa}B kinase-dependent manner (15). Further studies are required to fully comprehend IFN{gamma} signaling and the cross-talk that occurs between several pathways.

One of the potent effects of IFN{gamma} is its growth-suppressive effect. It induces the cyclin-dependent kinase (CDK) inhibitor p21waf1/CIP1 and p27Kip1 which interfere with the actions of CDKs and prevents hyperphosphorylation of Rb and entry into the S phase of the cell cycle (16–19). Also, recruitment of BRCA1 by Stat1 results in activation of the CDK inhibitor, p21 waf1, which may be involved in the induction of the growth-suppressive effects of IFN{gamma} (7). Another mechanism may involve reduction in telomerase activity and telomerase reverse transcriptase (RT) (20). The role of IFN{gamma} in inhibiting cellular proliferation may be physiologically important. For example, in a model of experimental autoimmune encephalomyelitis, Ifn{gamma}–/– mice accumulate 10- to 16-fold more activated T cells compared with wild type (21). Also, mice lacking IFN{gamma} develop more tumors compared with wild-type mice (22–24). IFN{gamma} enhances the immunogenicity of tumor cells that are recognized and eliminated by the host defense. This may be due to increased immune responses, e.g. by enhancing expression of MHC class I (MHC-I) antigen-processing pathway or increased NK activity; in fact, basal NK activity is lowered in mice lacking IFN{gamma} (25). Alternately, IFN{gamma} may directly inhibit tumors due to its growth-suppressive effect (26). Despite numerous studies on the growth-suppressive actions of IFN{gamma}, the role of oxidative and nitrosative stress in this process is not well appreciated. We studied IFN{gamma}-induced gene expression and functional responses in the mouse hepatoma cell line, H6, which greatly enhances the expression of genes involved in the MHC-I assembly pathway (27). In this study, the crucial role of oxidative and nitrosative stress in modulating expression of distinct sets of genes involved in some functions mediated by IFN{gamma} is demonstrated.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Cell culture
H6 cells (hepatoma, H-2a) were cultured in RPMI 1640 medium containing 25 mM HEPES (Sigma, St Louis, MO, USA), 5% heat-inactivated FCS (Sigma), 5 µM ß-mercaptoethanol (Sigma), 100 µg ml–1 penicillin, 250 µg ml–1 streptomycin, 50 µg ml–1 gentamycin (HiMedia Laboratories, Mumbai, India) and 2 mM glutamine (Life Technologies, Gaithersburg, MD, USA) at 37°C in the presence of 5% CO2 in an incubator (Sanyo, UK).

Reagents and antibodies
IFN{gamma} (recombinant mouse IFN{gamma} expressed in Escherichia coli) from PeproTech, Israel, was titrated and used at ~40 U ml–1 for all experiments. Culture supernatants from hybridomas secreting mAbs to detect MHC-I: 11-4-1 (TIB 95; anti-Kk,p,q,r) and 34-2-12S (HB-87; anti-Dd, {alpha}3 domain) as previously described (27). FITC-labeled goat anti-mouse secondary antibodies and peroxidase-conjugated goat anti-mouse antibodies were obtained from Jackson Immunoresearch Laboratories, West Grove, PA, USA. N-methyl L-arginine (LNMA), diphenylene iodonium (DPI), horse heart mitochondrial ferricytochrome c, superoxide dismutase, PBS, NADPH, sulfanilamide and naphthylethylenediamine dihydrochloride were obtained from Sigma whereas 2',7'-dichlorofluorescein diacetate (DCFDA), catalase and manumycin A were purchased from Calbiochem, La Jolla, CA, USA.

RNA isolation and RT–PCRs
Total RNA was extracted from 1 x 106 cells using 1 ml of TRI reagent (Sigma) according to the manufacturer's instructions. Two micrograms of total RNA was reverse transcribed using 50 U of MMLV-RT (MBI Fermentas, Ontario, Canada) and oligo dT(12–18mer) primer (Amersham Pharmacia, Piscataway, NJ, USA) in a 25 µl volume. cDNA (1 µl) was used as template for PCR amplification in buffer containing 0.4 µM of each primer, 500 µM of deoxynucleoside triphosphate mix and 0.25 U of Taq polymerase (Bangalore Genei, India). Gene-specific PCR amplification was carried out at specific temperature and cycles using specific sets of primers (Table 1). The amplified PCR products were fractionated on 1.5% agarose gels and visualized by ethidium bromide staining.


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  		Table 1. Sequences of primers used for RT–PCR

 
To quantitate the modulation of IFN{gamma}-induced gene expression, semi-quantitative PCR was performed using 25–40 cycles followed by a final extension at 72°C for 5 min (27). The number of cycles was determined empirically based on amounts of individual PCR products. The band intensities were quantified using Image Gauge software, Science Lab 2003 (Fujifilm, Japan). To calculate the relative abundance of transcripts, the intensity of each band was plotted against the number of cycles and slope values were determined. After normalizing the slopes with Hprt, the relative induction under each treatment condition was compared with uninduced H6 cells. Modulation by IFN{gamma} of different genes was studied using independent cDNAs and the mean ± SE was determined.

Cell-surface marker analysis
The expression of MHC-I was determined by flow cytometry, as described previously (27). H6 cells were incubated at 37°C in RPMI in the presence of 5% FCS, alone or with IFN{gamma} for different time periods. After these treatments, ~5 x 105 cells were centrifuged and incubated with optimal amounts of primary mAbs for 30 min at 4°C. Cells were washed and incubated with appropriate amounts of secondary antibodies for 30 min at 4°C. Subsequently, cells were washed with FACS buffer and fixed with 0.5 ml of 1% PFA at 4°C. Analysis was performed on a Becton Dickinson FACScanTM flow cytometer and CellQuest (Becton Dickinson) software was used for acquisition. WinList (Verity, Topsham, ME, USA) software was used to calculate mean fluorescence intensity and percentage of cells staining positive with primary mAbs, after subtraction with the isotype control.

Measurement of reactive oxygen species levels
The production of peroxides, peroxynitrites and other reactive oxygen species (ROS) was measured using the oxidation-sensitive fluorescent dye, DCFDA. This compound is a cell-permeable non-fluorescent probe which, after entering cells, is deacetylated by intracellular esterases and upon oxidation by excess ROS intermediates is converted to a highly fluorescent compound. Cells were washed with RPMI and were incubated with 3 µM DCFDA for 20 min at 37°C and acquired on the FACScan (28).

NADPH oxidase assay
H6 cells treated under different conditions were washed with PBS and centrifuged for 10 min. The cell pellet was re-suspended in lysis buffer [10 mM Tris, 10 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid (EGTA), 10 mM EDTA, 1 mM Na-orthovanadate and protease inhibitors, 0.2 mM phenylmethylsulfonylfluoride, 0.33 M leupeptin and 4.8 TIU aprotinin]. Cells were sonicated lightly ~6 s twice and the lysate was centrifuged at 4°C at 2400 x g for 10 min and the cell supernatant containing the membrane fractions was collected for the assay. The protein content of the supernatant was detected by Bradford’s reagent. Twenty micrograms of the sample was mixed and incubated at 37°C for 5 min in buffer containing 20 mM K2PO4, pH 7.0, 1 mM EGTA, 7.6 mM MgCl2, 10 µM flavin adenine dinucleotide and 75 µM cytochrome c. The mixture was divided into two cuvettes, one containing superoxide dismutase at a final concentration of 400 U ml–1. The reaction was started by addition of 0.2 mM NADPH to both cuvettes and the absorbance changes were monitored continuously at 550 nm by a UV spectrophotometer (Shimadzu). O2– production was calculated using linear slope values and an extinction coefficient for cytochrome c of 21 mM–1 cm–1. The specific activity was expressed as µM of O2.– per min per mg protein (29, 30).

Catalase activity assay
Cells were harvested, washed three to four times with PBS and centrifuged at 600 x g for 5 min and the pellet was re-suspended in phosphate buffer solution (pH 7.0). The cells were sonicated and the cell lysate was centrifuged at 6000 x g for 20 to 30 min at 4°C. The supernatant was collected and assayed for catalase activity using 20 µg of total protein (31). This assay was performed at 25°C in a 1 ml of the reaction volume containing clear cell lysates, 100 mM phosphate buffer (pH 7) and 10 mM H2O2. The decomposition of H2O2 was monitored by decrease in absorbance at 240 nm. The enzyme activity was expressed as µM H2O2 decrease per min per mg protein.

Ras activation assay
H6 cells were treated under different conditions, washed twice with ice cold PBS and lysed using buffer containing 25 mM HEPES pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 10 µg ml–1 aprotinin, 10 µg ml–1 leupeptin, 25 mM sodium fluoride and 1 mM sodium orthovanadate. The assay was performed according to the manufacturer’s protocol (Upstate Cell Signaling Solutions, USA). Briefly, lysates were centrifuged at 14 000 x g for 5 min at 4°C and the supernatants were collected. Approximately 0.5–1 ml of the cell extract was incubated with 5–10 µg of Raf1-Ras-binding domain (RBD) agarose (glutathione-S-transferase–RBD fusion protein corresponding to the human RBD, residues 1–149 of Raf1, expressed in E. coli, bound to glutathione agarose) that binds to Ras-GTP. The reaction mixture was kept at 4°C for 45 min with constant agitation, the agarose beads were pelleted by centrifugation (10 s, 14 000 x g, 4°C), washed thrice, re-suspended in Laemmli-reducing sample buffer and boiled for 5 min. After centrifugation, the supernatant was loaded 20 µl of the mixture per lane on a 15% polyacrylamide gel. The protein was then transferred to a nitrocellulose membrane and probed with a mAb to Ras (1:2000). Detection was performed using goat anti-mouse-HRP conjugate (Jackson Immunoresearch Laboratories) at 1:5000 dilution. Antigen–antibody complexes were detected using chemiluminescence (ECL detection, Millipore) and visualized on LAS-3000 (Fuji, Japan). Anti-GAPDH (Upstate Cell signaling Solutions) was used at 1:2000 dilution to verify equal protein loading per lane.

Nitrite quantitation
Griess reagent was used to measure nitrite as an indicator of NO, as described previously (32). Cell culture supernatant (50 µl) from the cells, untreated and treated under different conditions, was transferred into 96-well flat-bottom microtiter plates. The amount of NO produced was measured as a function of nitrite detected by adding 100 µl of a mixture of 1% (w/v) sulfanilamide and 0.1% (w/v) naphthylethylenediamine dihydrochloride prepared in 2.5% phosphoric acid, i.e. Griess reagent. Simultaneously, a standard curve for nitrite was performed using 0.2–208 µM NaNO2 and absorbances were read at 550 nm using a microtiter plate reader (Molecular Devices, Downingtown, PA USA). The linear range of detection was 3.25–208 µM.

Trypan blue exclusion assay
Adherent cells were detached using 100 µl of 0.5% trypsin–EDTA (Sigma) per well. An equal volume of 0.4% trypan blue was added and the cells were counted in a hemocytometer.

Cell cycle analysis
Cells (~5 x 104) were washed with PBS without CaCl2 and fixed in 70% cold ethanol with continuous tapping to avoid clumping of cells. Cells were frozen at –20°C for at least 30 min. Cells were then washed thoroughly with PBS without CaCl2 to remove excess ethanol and incubated with 100 µg ml–1 RNaseA for 30 min at room temperature. Cells were stained with 50 µg ml–1 of propidium iodide for at least 30 min at 37°C. Cells were then acquired on a FACScan on linear FL-2 settings. Gates were applied to measure the proportion of G0/G1 (n), G2/M (2n) and hypodiploid populations (33).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Transcriptional responses of well-characterized ISGs upon IFN{gamma} treatment
H6 cells were treated with IFN{gamma} for different time periods and the cell-surface levels of MHC-I, Kk and Dd, were studied. IFN{gamma} increased cell-surface expression of Kk and Dd and maximum induction was observed after 36–42 h (Fig. 1A). Next, the transcriptional profile of well-characterized ISGs was studied (2). For this purpose, the RNA levels of suppressor of cytokine signaling 1 (Socs1) (a Jak inhibitor), transporter associated with antigen processing 2 (Tap2) [a component of the transporter of antigen processing which transports peptides from the cytosol into the endoplasmic reticulum (ER) during MHC-I antigen processing] and Cd80 (a co-stimulatory ligand) were studied by RT–PCR. IFN{gamma} treatment increased Socs1 expression by 6 h whereas the expression of Tap2 and Cd80 was increased by 12 h (Fig. 1B). Thus, H6 represents a cell culture system in which genes belonging to different functional responses/pathways are highly modulated in response to IFN{gamma}.


Figure 1
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  		Fig. 1. IFN{gamma} enhances expression of ISGs and cell-surface expression of MHC-I. (A) Cells were treated with 40 U ml–1 of IFN{gamma} for different time intervals and surface MHC-I levels were analyzed by flow cytometry. The fold differences, after normalizing with untreated cells, are shown and are represented as mean ± SE of three independent experiments. (B) Total RNA from H6 cells, untreated or treated for different time periods with 40 U ml–1 of IFN{gamma}, was reverse transcribed and gene expression was studied by PCR. The mean fold induction of Socs1, Tap2 and Cd80, after 12 h induction with IFN{gamma} was 5.1 ± 0.3, 13.3 ± 0.3 and 8.3 ± 1.0, respectively; n = 3 experiments.

 
IFN{gamma} suppresses cell growth
Further studies involved screening for IFN{gamma}-modulated genes, using glass slides spotted with ~15 264 mouse expressed sequence tags derived from PCR amplification. These were obtained from the Microarray Centre, Clinical Genomics Centre, University Health Network, Toronto, Canada. As it is appropriate to validate microarray data, several genes identified during this screening process were confirmed by RT–PCR, using independent cDNAs. The transcript levels of some genes involved in cell cycle and survival was studied using RT–PCR (Fig. 2A). Growth arrest and DNA damage inducible {gamma} (Gadd45{gamma}) is induced during DNA damage and stress (34). N-myc downstream regulator 1 (Ndr1) is a serine/threonine protein kinase family member implicated in regulation of cell division (35). Inhibitor of DNA binding 2 (Id2) belongs to a family of proteins that are dominant-negative repressors of proteins belonging to the basic helix loop helix and the retinoblastoma families. Id2 inhibits the growth-suppressive activities of CDK inhibitors p16 and p21 (36). IFN{gamma} treatment increased levels of Gadd45{gamma} and Ndr1. Concurrently, IFN{gamma} treatment decreased amounts of Id2. Overall, the transcriptional profile of the selected cell growth-regulated genes suggested that IFN{gamma} was growth suppressive.


Figure 2
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  		Fig. 2. IFN{gamma} suppresses cell growth. (A) Cells were treated with IFN{gamma} for different time periods, RNA was extracted, cDNAs were synthesized and PCR was performed using gene-specific primers. The mean fold modulation of Ndr1, Gadd45{gamma} and Id2 after 12 h of IFN{gamma} induction was 3.1 ± 0.4, 3.1 ± 0.5 and –3.5 ± 0.8, respectively; n = 3 experiments. (B) Viable cell numbers, untreated or treated with IFN{gamma}, were determined at different time intervals by trypan blue exclusion method. The data are represented as mean ± SE of three independent experiments. (C) Cells were treated with IFN{gamma} for different time periods and propidium iodide staining was performed to study the effects on cell cycling. The numbers in each histogram depicts the percentage of hypodiploid, G0/G1 and G2/M populations (top to bottom), respectively.

 
The effect of IFN{gamma} on H6 cell cycling was further studied in a kinetic manner by propidium iodide staining (Fig. 2B). Treatment of H6 cells with IFN{gamma} decreased numbers of cycling cells (G2/M population) at 36 h followed by a greater decrease at 42 h. Notably, the maximal increase in the hypodiploid population was observed at 42 h after IFN{gamma} treatment. Next, the viability of cells by IFN{gamma} treatment was assessed using trypan blue staining (Fig. 2C). During early time points, i.e. until 24 h, no major difference in cell numbers was observed in untreated H6 cells with those treated with IFN{gamma}. However, decreased cell numbers were observed in cells treated with IFN{gamma} at later time points (36–42 h), which demonstrated that IFN{gamma} decreased cell growth.

Transcriptional profiles of genes involved in oxidative and nitrosative stress upon IFN{gamma} treatment
Stress-related signaling pathways and redox imbalances are known to be associated with cell death (37, 38). Therefore, the relative transcriptional levels of genes encoding some antioxidant enzymes were studied. NADPH oxidase consists of cytosolic (p40phox, p47phox and p67phox) and membrane-bound flavocytochrome b558 consisting of a heterodimer of p22phox and gp91phox. Importantly, IFN{gamma} enhances the expression of gp91phox and p67phox and this enzyme plays a major role in killing microbes engulfed during phagocytosis. On activation, the cytosolic components migrate and are assembled in the membrane to form active NADPH oxidase (39, 40). The inducible form of nitric oxide synthase (NOS), also called immune nitric oxide synthase (iNOS), is present in antigen-presenting cells (APCs) and converts L-arginine into L-citrulline and NO. This enzyme is strongly induced by IFN{gamma} and LPS. NO reacts with inorganic molecules, e.g. NO can combine with OFormula to form peroxinitrite (ONOO–), a potent oxidizing agent. Also, it reacts with pyrimidine bases in DNA, heme prosthetic groups (iron of the heme moiety of soluble guanyl cyclase) and proteins, leading to S-nitrosylation of thiol (S-NO) containing amino acids, nitration of tyrosine (O-NO) or disruption of iron sulphide or Zn-finger domains. Thus, NO modulates the activity of key molecules, enzymes and transcription factors (39). Catalase is a key component of the cellular antioxidant defense network. It is localized in the peroxisomes and catalyzes the conversion of H2O2, a harmful oxidizing agent, to water and molecular oxygen. The regulated expression of catalase is critical for ROS homeostasis (41). IFN{gamma} treatment decreased levels of Catalase whereas increased levels of iNos and gp91phox were observed (Fig. 3A). These results suggested that IFN{gamma} could increase ROS and reactive nitrogen intermediate (RNI) amounts and reduced antioxidant defenses. Together, these would gear cells toward oxidative and nitrosative stress.


Figure 3
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  		Fig. 3. IFN{gamma} modulates the expression of genes involved in oxidative stress and enhances cellular ROS and RNI levels. (A) Total RNA from H6 cells, untreated or treated with IFN{gamma} for different time periods, was reverse transcribed and gene expression was studied by PCR. The mean fold modulation after 12 h of IFN{gamma} treatment of iNos, gp91phox and Catalase was 2.4 ± 0.7, 3.1 ± 0.7 and –2.0 ± 0.5, respectively; n = 3 experiments. (B) Supernatants from cells, untreated or treated with IFN{gamma}, were used to detect the presence of nitrite using Griess reagent. (C) Total levels of ROS were detected by staining the cells with DCFDA and analyzing by flow cytometry. The relative fold increase ± SE in ROS amounts was calculated after normalizing with untreated H6 cells as control for different time points and is representative of three experiments. Similarly, NADPH oxidase activity (D) and catalase activity (E) were assayed in cells in the absence or the presence of IFN{gamma} at different time points. The above data are represented as mean ± SE of three independent experiments.

 
The above observations led to quantitation of amounts of ROS and RNI in cells treated with IFN{gamma}. Nitrite amounts were not detected in unstimulated H6 cells. However, upon IFN{gamma} treatment, nitrite amounts increased greatly within 24 h (Fig. 3B). The amounts of ROS were studied using the oxidation-sensitive florescent dye, DCFDA. ROS amounts increased with IFN{gamma} treatment and maximal amounts were observed at 42 h (Fig. 3C). As observed in Fig. 3(D), NADPH oxidase activity greatly increased after 24 h of IFN{gamma} treatment. Most likely, NADPH oxidase is activated by IFN{gamma}, which increased cellular ROS. In keeping with the gene expression of Catalase in the presence of IFN{gamma}, catalase activity was reduced in a time-dependent manner (Fig. 3E).

Inhibitors of NOS and NADPH oxidase rescue IFN{gamma}-mediated cell death
To investigate the functional role of oxidative and nitrosative stress during IFN{gamma}-mediated cell death, experiments were designed to inhibit the production of excess ROS and RNI in IFN{gamma}-treated H6 cells. Pre-treatment of H6 cells with DPI, an inhibitor of flavin-containing NADPH oxidases, rescued the cell viability of IFN{gamma}-treated cells in a dose-dependent manner (Fig. 4A). A similar rescue in cell viability was observed in cells pre-treated with LNMA, an uncleavable form of arginine which is the substrate of NOS and acts as a competitive inhibitor. These studies clearly demonstrated a functional role of excess ROS and RNI during IFN{gamma}-mediated reduced cell growth and survival.


Figure 4
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  		Fig. 4. IFN{gamma} modulates the expression of a subset of genes and suppresses cell growth via oxidative and nitrosative stress. (A) H6 cells were treated with IFN{gamma} alone or were pre-treated with different doses of DPI or LNMA for 1 h followed by IFN{gamma} treatment for 42 h. Viable cell numbers were determined by trypan blue exclusion method. The mean ± SE (n = 4 independent experiments) for H6 cells normalized to 100% and treated with different agents are as follows: H6 + 0.5 µM DPI 93 ± 11%; H6 + 500 µM LNMA 107 ± 6%; H6 + IFN{gamma} 27 ± 7%; H6 + IFN{gamma} + 0.5 µM DPI 80 ± 10% and H6 + IFN{gamma} + 500 µM LNMA 100 ± 14%. (B) Total RNA was isolated after treatment of cells with 0.5 µM DPI or 500 µM LNMA and/or IFN{gamma} for 12 h, reverse transcribed and cDNA was amplified by PCR. (C) Subsequently, the amounts of nitrite, DCFDA fluorescence were determined in cells treated under similar conditions with IFN{gamma} for 42 h. The data are represented as mean ± SE, n = 3 independent experiments.

 
To investigate whether the modulation of gene expression by IFN{gamma} was directly due to IFN{gamma} signaling or due to a secondary response to oxidative and nitrosative stress, the transcriptional profiles were studied in cells induced with IFN{gamma}, in the presence or absence of LNMA and DPI. Tap2 levels were up-regulated by IFN{gamma} and pre-treatment with either LNMA or DPI did not modulate its induction (Fig. 4B). Similar results were observed during IFN{gamma}-mediated transcriptional responses of genes belonging to this set, i.e. IFN{gamma} modulated and oxidative and nitrosative stress independent: Cd80, Icosl and Lmp10. On the other hand, iNos was induced by IFN{gamma}; however, pre-incubation with LNMA or DPI greatly reduced IFN{gamma}-induced iNos. These results demonstrate that induction of iNos by IFN{gamma} required ROS and RNI which act as positive regulators. This pattern was also observed for genes belonging to this set, i.e. IFN{gamma} and oxidative and nitrosative stress dependent: Gadd45{gamma}, gp91phox, Catalase and Id2. Next, the amounts of IFN{gamma}-induced ROS and RNI were determined after pre-treatment with DPI and LNMA. Increased amounts of nitrite and ROS were severely reduced in cells treated with IFN{gamma} and DPI or LNMA (Fig. 4C). Overall, the kinetics of gene expression (12 h) correlated with generation of excess ROS/RNI (24–36 h) followed by decreased cell growth and survival observed 36–42 h after IFN{gamma} treatment.

Expression and functional roles of IFN{gamma}-induced Ras activation
In order to gain further mechanistic insights into the IFN{gamma}-induced growth suppression, the role of Ras was investigated. Ras is a member of the Ras superfamily of monomeric small GTP-binding proteins. Its active form is localized in the inner face of the plasma membrane by the C-terminal-S-farnesylcysteine. Ras activates multiple signaling cascades involved in growth, differentiation, stress response and senescence. It has been shown to modulate cellular amounts of ROS leading to modulation of cell proliferation (42–45). First, the kinetics of Ras activation by IFN{gamma} was studied in this system. H6 cells were treated with IFN{gamma} for different time intervals and Ras-GTP amounts were assessed by western blot analysis. Increase in active Ras was detected as early as 12 h and was sustained until 36 h after IFN{gamma} treatment (Fig. 5A). In order to determine whether activation of Ras is related to the increased ROS and RNI, the levels of Ras-GTP were determined in cells treated with DPI and LNMA along with IFN{gamma}. In addition, the effect of manumycin A, a farnesyl transferase inhibitor, which inhibits the farnesylation of the C-terminal region of Ras protein resulting in the failure of Ras to bind to the plasma membrane (43), alone or along with IFN{gamma} was also studied. Manumycin A was effective as Ras-GTP was not detected in cells treated with this inhibitor and IFN{gamma}. Importantly, Ras-GTP amounts were not observed in cells treated with IFN{gamma} and DPI or LNMA, suggesting that IFN{gamma}-mediated Ras activation is dependent on ROS and RNI (Fig. 5B).


Figure 5
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  		Fig. 5. IFN{gamma} activation of Ras is dependent on oxidative and nitrosative stress. (A) Ras activity was determined at different time intervals after treating H6 cells with 40 U ml–1 of IFN{gamma}. Cell lysates were prepared and Ras-GTP was bound to glutathione-S-transferase–RBD agarose followed by immunoblotting with mAb to Ras. GAPDH was used as a control for the amount of protein loaded. (B) Cells were treated with IFN{gamma} alone or pre-treated along with 2.5 µM manumycin A, 0.5 µM DPI or 500 µM LNMA for 36 h and assayed for the amount of activated Ras. The data are representative of one of the three independent experiments performed.

 
Next, the effect of Ras inhibition on cell survival and functions was studied. To ensure that the efficacies of inhibiting different pathways were compared, experiments were designed to also study the effects of DPI and LNMA. Cells were treated with IFN{gamma} and different doses of manumycin A for 36 h. The optimal amount of manumycin A that rescued cell viability was ~2.5 µM (Fig. 6A); however, the enhancement of cell viability was less compared with DPI and LNMA. These results demonstrated that Ras was playing a role during IFN{gamma}-mediated growth suppression. The transcript levels of IFN{gamma} induced but oxidative and nitrosative stress independent were not modulated by manumycin A (Fig. 6B). Furthermore, pre-treatment with manumycin A, DPI or LNMA did not affect the induction of MHC-I, Dd and Kk, by IFN{gamma} (Fig. 6C), which is consistent with gene expression patterns. These results demonstrate that excess generation of ROS/RNI and Ras activation does not modulate IFN{gamma}-induced MHC-I cell-surface expression.


Figure 6
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  		Fig. 6. Manumycin A, a farnesyl transferase inhibitor, does not modulate IFN{gamma}-induced MHC-I but rescues cell growth. (A) The viability of cells, untreated, treated with IFN{gamma} alone or together with manumycin A for 36 h, was determined by trypan blue exclusion. The viability of cells treated with 0.5 µM DPI and 500 µM LNMA in the presence of IFN{gamma} was 83.3 ± 15% and 110 ± 36%, respectively. (B) Cells were pre-treated with the inhibitors for 1 h and the treated with IFN{gamma} for 12 h, RNA was isolated, reverse transcribed and cDNA was amplified by PCR using gene-specific primers. (C) MHC-I levels were studied by flow cytometry after treating the cells with IFN{gamma} alone or along with the inhibitors. All the data are represented as mean ± SE, n = 3 independent experiments.

 
Next, the effect of manumycin A was studied on the expression of genes modulated by oxidative and nitrosative stress. Although LNMA and DPI reduced IFN{gamma}-induced iNos, no significant modulation was observed with manumycin A (Fig. 7A). However, the expression of IFN{gamma}-induced gp91phox transcript was slightly reduced with manumycin A. IFN{gamma} decreased Catalase and Id2 transcripts and all three inhibitors were equally effective in reversing this effect. To address the functional roles of inhibition of Ras activation by IFN{gamma}, the cellular amounts of IFN{gamma}-induced RNI and ROS were determined. Consistent with gene expression studies, LNMA and DPI reduced IFN{gamma}-induced nitrite production; however, a slight reduction in nitrite levels was observed with manumycin A (Fig. 7B). However, manumycin A greatly reduced IFN{gamma}-induced ROS amounts (Fig. 7C). Most likely, the reduction in IFN{gamma}-induced ROS was due to decreased activation of NADPH activation due to inhibition of Ras activation (Fig. 7D), as previously reported (46). The decrease in catalase activity by IFN{gamma} was reversed by all three inhibitors to a similar extent (Fig. 7D). Together, these experiments clearly demonstrate a differential role of Ras in delinking the generation of IFN{gamma}-induced RNI versus ROS.


Figure 7
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  		Fig. 7. IFN{gamma}-induced ROS, but not RNI, is dependent on Ras. Cells were treated with 2.5 µM manumycin A, 0.5 µM DPI or 500 µM LNMA alone or together with IFN{gamma}. (A) RNA was extracted from the control and treated cells after 12 h of treatment. cDNA was prepared and PCR was performed using gene-specific primers. (B) Nitrite levels were determined from the supernatant of the cells treated under different conditions for 36 h. (C) Similarly, DCFDA fluorescence was measured by flow cytometry. (D) NADPH oxidase activity and (E) catalase activity were measured under the above conditions. The data are represented as mean ± SE, n = 3 independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
The observation that genes involved in growth of H6 cells were modulated by IFN{gamma} led us to investigate the mechanisms involved. Transcriptional profiling of enzymes involved in regulating cellular oxidative and nitrosative stress demonstrated that IFN{gamma} induced expression of iNos and gp91phox but decreased levels of Catalase. Antioxidant defenses are increased by catalase (41) and its down modulation by IFN{gamma} probably rendered H6 more sensitive to oxidative and nitrosative stress. IFN{gamma} increased amounts of nitrite, NADPH oxidase activity and cellular ROS; however, it decreased catalase activity (Fig. 3). Most likely, the molecular sources of IFN{gamma} increased amounts of NO and OFormula are iNOS and activated NADPH oxidase. The expression kinetics of IFN{gamma}-modulated genes agreed well with functional responses of H6 cells to IFN{gamma}. Although IFN{gamma}-mediated cell death occurred at later time points (36–42 h), the molecular signatures were seen early, i.e. by 12 h. IFN{gamma} reduced cell growth and survival was blocked using either DPI or LNMA, demonstrating the crucial functional role of high amounts of ROS and RNI in this process (Fig. 4).

In primary cultured hepatocytes, IFN{gamma} induces high amounts of ROS in an IFN regulatory factor (IRF1)-independent manner; however, generation of high amounts of ROS is insufficient to induce cell death and a combination of oxidative and ER stress is required to induce apoptosis (47). In the H6 cell culture system, high amounts of ROS and RNI together, but not singly, enhanced transcriptional activation of a subset of IFN{gamma}-responsive genes. Reports in the literature have demonstrated that NO is either a negative (48, 49) or a positive (50) regulator of iNos transcriptional activation. Also, inhibition of excess ROS using DPI reduces iNos transcriptional activation upon activation with IL-1 (51) or the combination of LPS and IFN{gamma} (52). Induction of oxidative stress alone is known to enhance iNOS expression at RNA and protein levels in rat liver (53). Finally, NO amounts are lowered in gp91phox–/– cells treated with IFN{gamma} and/or LPS (52, 54, 55). Importantly, blockade of NO using LNMA or microglial cells, which are resident macrophages in the central nervous system, from iNos–/– mice produce lower amounts of ROS upon LPS activation (55). Our data support the role of ROS and RNI as positive activators of iNos transcription. In addition, this study demonstrates that ROS and RNI were required for IFN{gamma}-modulated transcription of several other genes, including gp91phox, Catalase and Id2. Also, macrophages from mice lacking iNos or gp91phox display distinct gene expression profiles compared with wild-type macrophages in response to IFN{gamma} and/or M. tuberculosis (11). Although maximal amounts of ROS and RNI were observed after ~36 h of activation (Fig. 3), the modulation of gene expression by IFN{gamma} was observed after 12 h of activation (Figs. 1–3GoGo). In fact, addition of DPI or LNMA at –1, 0 or up to 6 h after incubation with IFN{gamma} did not reduce their efficacy in rescuing survival; however, addition of these compounds 24 h after IFN{gamma} incubation greatly reduced their efficacy (data not shown). These data strongly suggest that cellular oxidative and nitrosative stress was initiated early (i.e. by 12 h), resulting in modulation of expression of some genes by IFN{gamma}. The modulation of the oxidative and nitrosative stress-dependent set of IFN{gamma}-modulated genes increased levels of ROS and RNI, resulting in decreased cell growth and survival (Fig. 8). Together, this study clearly demonstrates the crucial role of increased oxidative and nitrosative stress in modulating expression of some genes and functions by IFN{gamma}.


Figure 8
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  		Fig. 8. IFN{gamma} modulates gene expression and functions via oxidative and nitrosative-dependent and -independent pathways. The above model demonstrates that expression of some IFN{gamma}-responsive genes and increased expression of MHC-I is independent of oxidative and nitrosative pathway. However, enhanced amounts of ROS and RNI results in activation of Ras-GTPase which, in turn, leads to increased ROS generation via NADPH oxidase. Notably, IFN{gamma}-induced NO is less dependent on Ras activation. Together, the high amounts of IFN{gamma}-induced ROS and RNI suppress cell growth.

 
Previous studies have described the relationship between Ras and induction of ROS and RNI. Ras has been shown to induce ROS which increases (42, 43) or reduces (44, 45) cell proliferation. This discrepancy can be attributed to the type of Ras-induced ROS, i.e. signaling or stress induced. Therefore, Ras induction of signaling ROS results in cell proliferation (42, 43); however, excess generation of ROS induced by Ras activation, probably due to NADPH activation (44, 45), leads to cellular senescence or death. In the H6 system, it appears that the Ras-induced ROS belongs to the latter category. IFN{gamma}-mediated Ras activation was dependent on the early generation of oxidative and nitrosative stress (Fig. 5). Also, high amounts of cellular ROS are known to stabilize activated Ras probably due to reduced degradation (44, 45). Manumycin A marginally reduced IFN{gamma}-induced nitrite amounts; however, it greatly reduced IFN{gamma}-induced NADPH activity and cellular ROS amounts (Fig. 6) and rescued cell viability. Together, the evidence demonstrates that the early generation of ROS and RNI by IFN{gamma}-activated Ras which, in turn, enhanced the activity of NADPH oxidase leading to greater amounts of cellular ROS (Fig. 8). Conflicting reports exist in literature regarding the role Ras plays in modulating iNOS. A dominant-negative mutant of Ras has been shown to inhibit the iNOS expression in astrocytes (56). Also, injection of a Ras inhibitor in mice reduced inflammatory responses and NO amounts (57). On the other hand, the induction of iNos by IFN{gamma} and LPS is independent of Ras activation in rat C6 glioma cells (58). Our study demonstrates that Ras activation does not play a major role during IFN{gamma}-induced nitrite production (Fig. 7). Notably, it was possible to rescue IFN{gamma}-mediated growth suppression in the presence of nitrite, as observed with the effects of manumycin A (Figs 6 and 7). These results suggest that ROS may play the dominant role compared with RNI in mediating growth suppression by IFN{gamma}. However, the efficiency of manumycin A was less compared with DPI or LNMA (Figs 6 and 7); therefore, both RNI and ROS are the key mediators of the IFN{gamma}-mediated growth suppression (Fig. 8).

Bioinformatics analysis of the promoters of some genes modulated by IFN{gamma} revealed the presence of Gas sequences: Socs1, iNos, Id2 and Gadd45{gamma} (data not shown). Gene expression studies were performed 12 h after IFN{gamma} treatment and it is possible that there are multiple regulatory networks operating to modulate IFN{gamma}-induced gene expression. This study clearly documents the existence of at least three categories of IFN{gamma}-responsive genes: the first is the oxidative and nitrosative stress independent (e.g. Tap2, Lmp10, Cd80 and Icosl). The oxidative and nitrosative stress-dependent (iNos, gp91phox, Catalase and Id2) category can be further classified into further subsets, based on studies with maumycin A. The major effect of manumycin A was to reduce NADPH activation and cellular ROS amounts (Fig. 7). Gene expression studies demonstrated that inhibition of Ras did not affect IFN{gamma}-induced iNos; however, DPI or LNMA greatly reduced IFN{gamma}-induced iNos and gp91phox. Interestingly, all three inhibitors, LNMA, DPI or manumcyin A rescued IFN{gamma}-modulated expression of Catalase and Id2. These observations suggest that expression of some IFN{gamma}-modulated genes may depend on critical amounts and/or ratios of RNI and ROS. Therefore, expression of one category of genes, e.g. iNos, may occur in the presence of low amounts of ROS and RNI, together with IFN{gamma} signaling. These genes are modulated by DPI or LNMA but not by manumycin A, in the presence of IFN{gamma}. The other category of genes requires IFN{gamma} in combination with high amounts of RNI and ROS, e.g. Catalase and Id2. Therefore, a slight reduction in the amounts of cellular ROS and RNI may result in loss in IFN{gamma} modulation of gene expression. Further studies are required to directly identify subsets of IFN{gamma}-modulated genes and their differential regulation by various free radicals.

A feedback role for NO in IFN{gamma} signaling has also been proposed as high levels of NO nitrosylate Stat1 leading to lower IFN{gamma} signaling (59). ROS and RNI species are known to be involved in intracellular signaling events (60) and it is possible that blocking the generation of these free radicals reduced IFN{gamma} signaling and enhanced cell growth. There have also been reports of Ras enhancing Stat1 activation (61) or reducing IFN{gamma}-mediated signaling and global gene expression, i.e. by reducing Stat1/2 and IRF1 activation (62) and reducing MHC expression (63). It is unlikely that ROS/RNI or Ras activation are globally reducing IFN{gamma} signaling in this system: first, IFN{gamma}-induced gene expression of several genes were unaffected in the presence of DPI or LNMA, e.g. Tap2, Cd80, Icosl and Lmp10 (Fig. 6). Second, IFN{gamma}-induced cell-surface MHC-I expression was not significantly modulated in the presence of LNMA, DPI or manumycin A (Fig. 6). This study demonstrates that subsets of genes are modulated and some functions are regulated by oxidative and nitrosative stress in combination with IFN{gamma} signaling.

IFN{gamma} is known to increase ROS and RNI amounts which greatly inhibit intracellular replication of microbes. For example, H2O2 synergizes with NO to greatly reduce the number of viable E. coli (64). The individual contributions of ROS and RNI in mediating the host response to different pathogens may vary: iNOS plays the major role during M. tuberculosis infection (11) whereas both iNOS and NADPH oxidase are involved in controlling Salmonella typhimurium infection (65). Mice lacking both iNos and gp91phox are extremely sensitive to infections by commensal microbes and develop massive abscesses of internal organs. This phenotype is not observed in mice lacking either iNos or gp91phox and demonstrates the crucial roles of both ROS and RNI in encoding host resistance (65). The functional roles of oxidative and nitrosative stress-modulated IFN{gamma}-responsive genes may depend on the type of immune response. For example, high amounts of IFN{gamma}-induced ONOO– increase the anti-microbial activity of APCs (66). On the other hand, high levels of ONOO– enhance inflammatory responses, resulting in host tissue damage (55). These results have implications for the role of antioxidants during immune responses. These compounds may lower the production of ROS/RNI and reduce the ability of macrophages to restrict the replication of intracellular pathogens (67, 68). However, the use of antioxidants may be beneficial as they reduce excess inflammation-induced tissue damage (69, 70). Together, these results underscore the importance of appropriate amounts of ROS and RNI generated during the immune response. This balance is crucial as sufficient amounts are required to restrict the replication of pathogens without causing excessive tissue damage in the host. Further studies utilizing this highly IFN{gamma}-inducible cell culture system described may identify novel mediators of oxidative and nitrosative stress. These studies may lead to a better understanding of signaling pathways and key mediators involved in generation of ROS and RNI in response to IFN{gamma}.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Indian Council of Medical Research, Government of India.


   Acknowledgements
 
We thank O. Joy and Harikrishnan, DBT-FACS facility, and Mohan M. Kumar, M. Aiyaz and Princy Francis for technical help. We greatly appreciate suggestions by E. Hermel, T. Ramasarma, P. Sadhale, R. Mugasimangalam, S. Majumdar, K. Balaji, S. Das, P. Ajitkumar, K. Somasundaram and members of the DpN Laboratory on different aspects of this study.


   Abbreviations
 
APC, antigen-presenting cell
BRCA1, breast cancer 1
CDK, cyclin-dependent kinase
DCFDA, 2',7'-dichlorofluorescein diacetate
DPI, diphenylene iodonium
EGTA, ethyleneglycol-bis(aminoethylether)-tetraacetic acid
ER, endoplasmic reticulum
Gadd45{gamma}, growth arrest and DNA damage inducible {gamma}
Gas, gamma-IFN-activated site
Id2, inhibitor of DNA binding 2
iNOS, immune nitric oxide synthase
IRF1, IFN regulatory factor 1
ISG, IFN-stimulated gene
Jak, Janus kinase
LNMA, N-methyl L-arginine
MHC-I, MHC class I molecule
Ndr1, N-myc downstream regulator 1
NO, nitric oxide
ONOO–, peroxinitrite
RBD, Ras-binding domain
RNI, reactive nitrogen intermediate
ROS, reactive oxygen species
RT, reverse transcriptase
Socs1, suppressor of cytokine signaling 1
Stat, signal transducers and activators of transcription coactivator
Tap2, transporter associated with antigen processing 2

   Notes
 
Transmitting editor: D. Wallach

Received 6 July 2006, accepted 18 April 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

  1. Dorman SE, Holland SM. Interferon-gamma and interleukin-12 pathway defects and human disease. Cytokine Growth Factor Rev. (2000) 11:321.[CrossRef][Web of Science][Medline]
  2. Boehm U, Klamp T, Groot M, Howard JC. Cellular responses to interferon-{gamma}. Annu. Rev. Immunol. (1997) 15:749.[CrossRef][Web of Science][Medline]
  3. Schroder K, Hertzog PJ, Ravasi T, Hume DA. Interferon-{gamma}: an overview of signals, mechanisms and function. J. Leukoc. Biol. (2004) 75:163.[Abstract/Free Full Text]
  4. Platanias LC. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat. Rev. Immunol. (2005) 5:375.[CrossRef][Web of Science][Medline]
  5. Wen Z, Zhong Z, Darnell JE Jr. Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell (1995) 82:241.[CrossRef][Web of Science][Medline]
  6. Zhu M, John S, Berg M, Leonard WJ. Functional association of Nmi with Stat5 and Stat1 in IL-2 and IFN{gamma}-mediated signaling. Cell (1999) 96:121.[CrossRef][Web of Science][Medline]
  7. Ouchi T, Lee SW, Ouchi M, Aaronson SA, Horvath CM. Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-{gamma} target genes. Proc. Natl Acad. Sci. USA (2000) 97:5208.[Abstract/Free Full Text]
  8. Hiroi M, Ohmori Y. The transcriptional coactivator CREB-binding protein cooperates with STAT1 and NF-kappa B for synergistic transcriptional activation of the CXC ligand 9/monokine induced by interferon-gamma gene. J. Biol. Chem. (2003) 278:651.[Abstract/Free Full Text]
  9. Der SD, Zhou A, Williams BR, Silverman RH. Identification of genes differentially regulated by interferon {alpha}, ß, or {gamma} using oligonucleotide arrays. Proc. Natl Acad. Sci. USA (1998) 95:15623.[Abstract/Free Full Text]
  10. de Veer MJ, Holko M, Frevel M, et al. Functional classification of interferon-stimulated genes identified using microarrays. J. Leukoc. Biol. (2001) 69:912.[Abstract/Free Full Text]
  11. Ehrt S, Schnappinger D, Bekiranov S, et al. Reprogramming of the macrophage transcriptome in response to interferon-gamma and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase. J. Exp. Med. (2001) 194:1123.[Abstract/Free Full Text]
  12. Patrone L, Damore MA, Lee MB, Malone CS, Wall R. Genes expressed during the IFN gamma induced maturation of pre-B cells. Mol. Immunol. (2002) 38:597.[CrossRef][Web of Science][Medline]
  13. Kota RS, Rutledge JC, Gohil K, Kumar A, Enelow RI, Ramana CV. Regulation of gene expression in RAW 264.7 macrophage cell line by interferon-gamma. Biochem. Biophys. Res. Commun. (2006) 342:1137.[CrossRef][Web of Science][Medline]
  14. Ramana CV, Gil MP, Han Y, Ransohoff RM, Schreiber RD, Stark GR. Stat1-independent regulation of gene expression in response to IFN-{gamma}. Proc. Natl Acad. Sci. USA (2001) 98:6674.[Abstract/Free Full Text]
  15. Sizemore N, Agarwal A, Das K, et al. Inhibitor of {kappa}B kinase is required to activate a subset of interferon {gamma}-stimulated genes. Proc. Natl Acad. Sci. USA (2004) 101:7994.[Abstract/Free Full Text]
  16. Harvat BL, Jetten AM. {gamma}-Interferon induces an irreversible growth arrest in mid-G1 in mammary epithelial cells which correlates with a block in hyperphosphorylation of retinoblastoma. Cell Growth Differ. (1996) 7:289.[Abstract]
  17. Kominsky S, Johnson HM, Bryan G, et al. IFN{gamma} inhibition of cell growth in glioblastomas correlates with increased levels of the cyclin dependent kinase inhibitor p21WAF1/CIP1. Oncogene (1998) 17:2973.[CrossRef][Web of Science][Medline]
  18. Mandal M, Bandyopadhyay D, Goepfert TM, Kumar R. Interferon-induces expression of cyclin dependent kinase-inhibitors p21WAF1 and p27Kip1 that prevent activation of cyclin-dependent kinase by CDK-activating kinase (CAK). Oncogene (1998) 16:217.[CrossRef][Web of Science][Medline]
  19. Hobeika AC, Etienne W, Torres BA, Johnson HM, Subramaniam P. IFN-{gamma} induction of p21(WAF1) is required for cell cycle inhibition and suppression of apoptosis. J. Interferon Cytokine Res. (1999) 19:1351.[CrossRef][Web of Science][Medline]
  20. Lee SH, Kim JW, Lee HW, et al. Interferon regulatory factor-1 (IRF-1) is a mediator for interferon-{gamma} induced attenuation of telomerase activity and human telomerase reverse transcriptase (hTERT) expression. Oncogene (2003) 22:381.[CrossRef][Web of Science][Medline]
  21. Chu C-Q, Wittmer S, Dalton DK. Failure to suppress the expansion of the activated CD4+ T cell population in interferon {gamma}-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. (2000) 192:123.[Abstract/Free Full Text]
  22. Kaplan DH, Shankaran V, Dighe AS, et al. Demonstration of an interferon {gamma}-dependent tumor surveillance system in immunocompetent mice. Proc. Natl Acad. Sci. USA (1998) 95:7556.[Abstract/Free Full Text]
  23. Shankaran V, Ikeda H, Bruce AT, et al. IFN{gamma} and lymphocytes prevent primary tumour development and shape tumor immunogenicity. Nature (2001) 410:1107.[CrossRef][Medline]
  24. Street SE, Trapani JA, MacGregor D, Smyth MJ. Suppression of lymphoma and epithelial malignancies effected by interferon {gamma}. J. Exp. Med. (2002) 196:129.[Abstract/Free Full Text]
  25. Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA. Multiple defects of immune cell function in mice with disrupted interferon-{gamma} genes. Science (1993) 259:1739.[Abstract/Free Full Text]
  26. Dighe AS, Richards E, Old LJ, Schreiber RD. Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN {gamma} receptors. Immunity (1994) 1:447.[CrossRef][Web of Science][Medline]
  27. Prasanna SJ, Nandi D. The MHC-encoded class I molecule, H-2Kk, demonstrates distinct requirements of assembly factors for cell surface expression: roles of TAP, Tapasin and ß2-microglobulin. Mol. Immunol. (2004) 41:1029.[Web of Science][Medline]
  28. Rosenkranz AR, Schmaldienst S, Stuhlmeier KM, Chen W, Knapp W, Zlabinger GJ. A microplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate. J. Immunol. Methods (1992) 156:39.[CrossRef][Web of Science][Medline]
  29. Mayo LA, Curnette JT. Kinetic microplate assay for superoxide production by neutrophils and other phagocytic cells. Methods Enzymol. (1990) 186:567.[Medline]
  30. Palicz A, Foubert TR, Jesaitis AJ, Marodi L, Mc Phail LC. Phosphatidic acid and diacylglycerol directly activates NADPH-oxidase by interacting with enzyme components. J. Biol. Chem. (2001) 276:3090.[Abstract/Free Full Text]
  31. Chuang SM, Wang IC, Hwua YS, Yang JL. Short-term depletion of catalase suppresses cadmium-elicited c-Jun N-terminal kinase activation and apoptosis: role of protein phosphatases. Carcinogenesis (2003) 24:7.[Abstract/Free Full Text]
  32. Malu S, Srinivasan S, Kumar MP, Rajagopal D, John B, Nandi D. IFN-gamma bioassay: development of a sensitive method by measuring nitric oxide production by peritoneal exudate cells from C57BL/6 mice. J. Immunol. Methods (2003) 272:55.[CrossRef][Web of Science][Medline]
  33. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell. Biol. (1975) 66:188.[Abstract/Free Full Text]
  34. Azam N, Vairapandi M, Zhang W, Hoffman B, Liebermann DA. Interaction of CR6 (GADD45{gamma}) with proliferating cell nuclear antigen (PCNA) impedes negative growth control. J. Biol. Chem. (2000) 276:2766.[CrossRef][Web of Science][Medline]
  35. Piquemal D, Joulia D, Balaguer P, Basset A, Marti J, Commes T. Differential expression of the RTP/Drg1/Ndr1 gene product in proliferating and growth arrested cells. Biochim. Biophys. Acta (1999) 1450:364.[Medline]
  36. Lasorella A, Iavarone A, Israel MA. Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins. Mol. Cell. Biol. (1996) 16:2570.[Abstract]
  37. Brune B. The intimate relation between nitric oxide and superoxide in apoptosis and cell survival. Antioxid. Redox Signal. (2005) 7:497.[CrossRef][Web of Science][Medline]
  38. Mukherjee S, Ahmed A, Nandi D. CTLA4-CD80/CD86 interactions on primary mouse CD4+ T cells integrate signal-strength information to modulate activation with Concanavalin A. J. Leukoc. Biol. (2005) 78:144.[Abstract/Free Full Text]
  39. Bogdan C, Rollinghoff M, Diefenbach A. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr. Opin. Immunol. (2000) 12:64.[CrossRef][Web of Science][Medline]
  40. Geiszt M, Leto TL. The Nox family of NADPH oxidases: host defense and beyond. J. Biol. Chem. (2004) 279:51715.[Free Full Text]
  41. Gurgul E, Lortz S, Tiedge M, Jorns A, Lenzen S. Mitochondrial catalase overexpression protects insulin-producing cells against toxicity of reactive oxygen species and proinflammatory cytokines. Diabetes (2004) 53:2271.[Abstract/Free Full Text]
  42. Irani K, Xia Y, Zweier JL, et al. Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science (1997) 275:1649.[Abstract/Free Full Text]
  43. Nagase T, Kawata S, Tamura S, et al. Manumycin and gliotoxin derivative KT7595 block Ras farnesylation and cell growth but do not disturb lamin farnesylation and localization in human tumour cells. Br. J. Cancer (1997) 76:1001.[Web of Science][Medline]
  44. Lee AC, Fenster BE, Ito H, et al. Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. J. Biol. Chem. (1999) 274:7936.[Abstract/Free Full Text]
  45. Svegliati S, Cancello R, Sambo P, et al. Platelet-derived growth factor and reactive oxygen species (ROS) regulate Ras protein levels in primary human fibroblasts via ERK1/2. Amplification of ROS and Ras in systemic sclerosis fibroblasts. J. Biol. Chem. (2005) 280:36474.[Abstract/Free Full Text]
  46. Seru R, Mondola P, Damiano S, et al. HaRas activates the NADPH oxidase complex in human neuroblastoma cells via the extracellular signal-regulated kinase 1/2 pathway. J. Neurochem. (2004) 91:613.[CrossRef][Web of Science][Medline]
  47. Watanabe Y, Suzuki O, Haruyama T, Akaike T. Interferon-{gamma} induces reactive oxygen species and endoplasmic reticulum stress at the hepatic apoptosis. J. Cell. Biochem. (2003) 89:244.[CrossRef][Web of Science][Medline]
  48. Weisz A, Cicatiello L, Esumi H. Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-{gamma}, bacterial lipopolysaccharide and NG-monomethyl-L-arginine. Biochem. J. (1996) 316:209.[Web of Science][Medline]
  49. Chang K, Lee SJ, Cheong I, et al. Nitric oxide suppresses inducible nitric oxide synthase expression by inhibiting post-translational modification of I{kappa}B. Exp. Mol. Med. (2004) 36:311.[Web of Science][Medline]
  50. Lee BS, Kim YM, Kang HS, Kim HM, Pyun KH, Choi I. Octamer binding protein-1 is involved in inhibition of inducible nitric oxide synthase expression by exogenous nitric oxide in murine liver cells. J. Biochem. (2001) 129:77.[Abstract/Free Full Text]
  51. Mendes AF, Carvalho AP, Caramona MM, Lopes MC. Diphenyleneiodonium inhibits NFkappaB activation and iNOS expression induced by IL-1ß: involvement of reactive oxygen species. Mediators Inflamm. (2001) 10:209.[CrossRef][Web of Science][Medline]
  52. Pawate S, Shen Q, Fan F, Bhat NR. Redox regulation of glial inflammatory response to lipopolysaccharide and interferon {gamma}. J. Neurosci. Res. (2004) 77:540.[CrossRef][Web of Science][Medline]
  53. Miralles C, Busquets X, Santos C, et al. Regulation of iNOS expression and glutathione levels in rat liver by oxygen tension. FEBS Lett. (2000) 476:253.[CrossRef][Web of Science][Medline]
  54. Brennan RE, Russell K, Zhang G, Samuel JE. Both inducible nitric oxide synthase and NADPH oxidase contribute to the control of virulent phase I Coxiella burnetii infections. Infect. Immun. (2004) 72:6666.[Abstract/Free Full Text]
  55. Li J, Baud O, Vartanian T, Volpe JJ, Rosenberg PA. Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes. Proc. Natl Acad. Sci. USA (2005) 102:9936.[Abstract/Free Full Text]
  56. Pahan K, Liu X, McKinney MJ, Wood C, Sheikh FG, Raymond JR. Expression of a dominant-negative mutant of p21(ras) inhibits induction of nitric oxide synthase and activation of nuclear factor-kappaB in primary astrocytes. J. Neurochem. (2000) 74:2288.[CrossRef][Web of Science][Medline]
  57. Na HJ, Lee SJ, Kang YC, et al. Inhibition of farnesyltransferase prevents collagen-induced arthritis by down-regulation of inflammatory gene expression through suppression of p21(ras)-dependent NF-kappaB activation. J. Immunol. (2004) 173:1276.[Abstract/Free Full Text]
  58. Nishiya T, Uehara T, Edamatsu H, Kaziro Y, Itoh H, Nomura Y. Activation of Stat1 and subsequent transcription of inducible nitric oxide synthase gene in C6 glioma cells is independent of interferon-gamma-induced MAPK activation that is mediated by p21ras. FEBS Lett. (1997) 408:33.[CrossRef][Web of Science][Medline]
  59. Llovera M, Pearson JD, Moreno C, Riveros-Moreno V. Impaired response to interferon-{gamma} in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide. Br. J. Pharmacol. (2001) 132:419.[CrossRef][Web of Science][Medline]
  60. Kamata H, Hirata H. Redox regulation of cellular signalling. Cell. Signal. (1999) 11:1.[CrossRef][Web of Science][Medline]
  61. Song JH, So EY, Lee CE. Increased serine phosphorylation and activation of STAT1 by oncogenic Ras transfection. Mol. Cells (2002) 13:322.[Web of Science][Medline]
  62. Klampfer L, Huang J, Corner G, et al. Oncogenic Ki-ras inhibits the expression of interferon-responsive genes through inhibition of STAT1 and STAT2 expression. J. Biol. Chem. (2003) 278:46278.[Abstract/Free Full Text]
  63. Seliger B, Harders C, Lohmann S, et al. Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts. Eur. J. Immunol. (1998) 28:122.[CrossRef][Web of Science][Medline]
  64. Pacelli R, Wink DA, Cook JA, et al. Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia coli. J. Exp. Med. (1995) 182:1469.[Abstract/Free Full Text]
  65. Shiloh MU, MacMicking JD, Nicholson S, et al. Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase. Immunity (1999) 10:29.[CrossRef][Web of Science][Medline]
  66. Lindgren H, Stenman L, Tarnvik A, Sjostedt A. The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages. Microbes Infect. (2005) 7:467.[CrossRef][Web of Science][Medline]
  67. Gantt KR, Goldman TL, McCormick ML, et al. Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi. J. Immunol. (2001) 167:893.[Abstract/Free Full Text]
  68. Eriksson S, Chambers BJ, Rhen M. Nitric oxide produced by murine dendritic cells is cytotoxic for intracellular Salmonella enterica sv. typhimurium. Scand. J. Immunol. (2003) 58:493.[CrossRef][Web of Science][Medline]
  69. Leiro J, Alvarez E, Arranz JA, Laguna R, Uriarte E, Orallo F. Effects of cis-resveratrol on inflammatory murine macrophages: antioxidant activity and down-regulation of inflammatory genes. J. Leukoc. Biol. (2004) 75:1156.[Abstract/Free Full Text]
  70. Zhang HJ, Doctrow SR, Xu L, et al. Redox modulation of the liver with chronic antioxidant enzyme mimetic treatment prevents age related oxidative damage associated with environmental stress. FASEB J. (2004) 18:1547.[Abstract/Free Full Text]

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