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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Biochemistry & Molecular Pharmacology (A.M., B.G., J.S.), Basic Pharmaceutical Sciences (P.G.), West Virginia University, Morgantown, West Virginia; Pathology & Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia (S.L., X.S.); and Biomedical Sciences, Edward Via Virginia College of Osteopathic Medicine and Biomedical Sciences & Pathobiology, Virginia Polytechnic Institute & State University, Blacksburg, Virginia (J.S.)
Received October 16, 2005; accepted February 22, 2006.
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Abstract |
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; Piekarz and Bates, 2004; Somech et al., 2004). Differentiation agents cause growth arrest and expression of proteins typical of the normal cell phenotype in cancer cells, but these are transient effects (Munster et al., 2001a,b; Wang et al., 2001; Marks et al., 2004). The ability to trigger apoptosis in tumor cells is critical to the antitumor activity of differentiation agents; the mechanisms leading to tumor apoptosis vary with the individual differentiation agent and tissue type (Marks et al., 2000; Roy et al., 2005; Ungerstedt et al., 2005). We investigated the mechanism of the cell death and differentiation in human breast tumor cells exposed to a new antitumor agent, NSC3852 (5-nitroso-8-quinolinol).
A large number of inhibitors of class I/II histone deacetylases are now characterized, and we know that a variety of chemical structures possess histone deacetylase inhibitory activity (Fig. 1). The main classes of histone deacetylase inhibitor(s) (HDI) are 1) short-chain fatty acids, 2) hydroxamic acids, 3) cyclic tetrapeptides with and without amino-epoxy-oxodecanoic acid residues, and 4) benzamides. The hydroxamic acids, represented by suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), are among the most potent HDI. NSC3852 is a less potent HDI than SAHA, but has many similar effects in MCF-7 cells. Both cause growth arrest and accumulation of cells in G0 phase of the cell cycle, as well as morphologic changes, such as formation of cytoplasmic lipid droplets and an enlargement of the cytoplasm (Munster et al., 2001b; Martirosyan et al., 2004). All HDI now appear to bind the zinc ion at the enzyme active site. NSC3852 lacks the hydroxamic acid moiety that is responsible for SAHA binding to Zn2+ in the histone deacetylase active site. NSC3852 harbors a different Zn2+ chelation motif, 8-quinolinol (Peters et al., 1974). We postulated that NSC3852 might exhibit novel actions in MCF-7 cells, because the quinoline ring and the nitroso substituent place NSC3852 in a chemically unique category of HDI. Our studies revealed that NSC3852, SAHA, and TSA stimulated ROS formation in MCF-7 cells and that NSC3852-induced oxidative stress contributed to apoptosis and differentiation in MCF-7 cells.
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Materials and Methods |
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Chemicals. NSC3852 and NSC2039 (8-quinolinol) were kindly provided by Dr. Robert Schultz (Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Bethesda, MD). In this study, we used concentrations of NSC3852 and NSC2039 that inhibited proliferation of MCF-7 cells by 50% in a MTS cell proliferation assay (Martirosyan et al., 2004). TSA was from Upstate Biotechnology (Lake Placid, NY). SAHA and suberic bishydroxamate (SBHA) were gifts from Dr. Q. Zhou (Johns Hopkins University, Baltimore, MD). N-Acetyl-L-cysteine (NAC) and NG-nitro-L-arginine methyl ester were from Sigma-Aldrich (St. Louis, MO). Dihydroethidium and 2',7'-dihydrodichlorofluorescein diacetate were purchased from Molecular Probes (Eugene, OR).
Western Blot Analyses. Cells (2 x 106/60 mm2) were treated 12 h after plating. Cells were harvested by scraping in 100°C lysis buffer (1% SDS and 10 mM Tris, pH 7.4) and heated (100°C, 5 min), and extracts were prepared by centrifugation in a microcentrifuge (4°C, 5 min). Protein concentrations were determined in aliquots of these extracts using the bicinchoninic acid assay (Pierce, Rockford, IL). Dithiothreitol (1 mM) and protease inhibitors were added [phenylmethylsulfonyl fluoride (1 mM), aprotinin (1 µg/ml), and leupeptin (1 µg/ml)] to the extracts. Protein samples (60–70 µg) were electrophoresed on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Invitrogen, Carlsbad, CA). Membrane blocking and incubations with primary and secondary antibodies were performed as described previously (Zhou et al., 2002). Chemiluminescent signals were recorded on X-ray film and quantified using FluorChem (Alpha Innotech, San Leandro, CA) spot densitometry program with automatic background subtraction.
Free Radical Measurements. All ESR measurements were conducted using a Bruker EMX spectrometer (Bruker Instruments Inc., Billerica, MA) and a flat-cell assembly. Hyperfine couplings were measured (to 0.1 G) directly from magnetic field separation using potassium tetraperoxochromate (K3CrO8) and 1,1-diphenyl-2-picrylhydrazyl as reference standards (Janzen and Blackburn, 1968). The relative radical concentration was estimated by multiplying half of the peak height by (
Hpp)2, where Hpp represents peak-to-peak width. The Acquisit program was used for data acquisitions and analyses. MCF-7 cells (2.0 x 106 cells/60-mm2 dish) were harvested 48 h after plating by trypsinization and collected at room temperature by centrifugation (225g, 5 min). Cells were washed once with ice-cold PBS and resuspended in ice-cold PBS at 2 x 106 cells/ml. Radical production was measured in the presence of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO; Aldrich Chemical Co., Milwaukee, WI). DMPO (200 mM) and 1 x 106 cells ± test agents were mixed in 1.0 ml of PBS, incubated at 37°C for 5 min, and then transferred to a flat cell for ESR measurements.
Intracellular Reactive Oxygen Species Detection. Cells were plated (2 x 105/35-mm2 dish) in 3 ml of DMEM/5% FBS culture medium. After 12 h, cells were exposed to solvent control or 10 µM NSC3852. After the treatment, cells were exposed to 5 µM 2',7'-dihydrodichlorofluorescein diacetate or dihydroethidium for 30 min. Cells were washed twice with PBS, and fluorescence was analyzed (10,000 events) using a FACScalibur flow cytometry (Becton Dickinson, San Jose, CA). Alternatively, cells plated onto sterile glass coverslips were fixed in 10% formaldehyde and examined using fluorescent confocal microscopy (Zeiss LSM 510).
Colorimetric Determination of Reduced and Oxidized Glutathione. The GSH and GSSG concentrations in MCF-7 cells (3 x 106cells/60-mm2 dish) were measured enzymatically using the GSH/GSSG-412 kit (Oxis Research, Portland, OR). GSSG and GSH standards were prepared in 5% metaphosphoric acid. Cell samples were prepared in 5% metaphosphoric acid with or without 1-methyl-2-vinyl-pyridium trifluoromethane sulfonate, a GSH-specific scavenger. Ellman's reagent (5',5-dithiobis-2-nitrobenzoic acid) reacts with GSH to form a product with an absorption maximum at 412 nm. GSSG was determined using glutathione reductase to reduce GSSG to GSH followed by reaction with Ellman's reagent.
Comet Analysis. The comet assay is a single-cell gel electrophoresis method for measuring DNA damage. MCF-7 cells (2 x 105/35-mm2 dish) were treated 12 h after plating. Twenty-four hours later, the cells were harvested and resuspended (1.5 x 105 cells/ml) in ice-cold PBS. The PBS cell suspension (50 µl) was mixed with 500 µl of 42°C low-melting point agarose, spread evenly (75 µl) onto a Comet Slide (Trevigen, Gaithersburg, MD), and allowed to harden. The slides were then immersed in ice-cold lysis solution (Trevigen) for 45 min to lyse the cells and then transferred to freshly prepared alkali solution (300 mM NaOH and 1 mM EDTA, pH 8.0) for 45 min at room temperature to denature the DNA. Electrophoresis was performed at 4°C. Slides were air-dried overnight at room temperature and then stained with SYBR Green (Trevigen). Comets were visualized by fluorescence microscopy at 630x magnification with the aid of an antifade solution. Comet images were analyzed using the LAI Automated Comet Assay Analysis System (Loats Associates, Inc., Westminster, MD). DNA damage was quantified in 80 comets per treatment/experiment using the tail moment [Tail moment = [(%DNA) (distance traveled)]].
Detection of Oxidative Damage to DNA. Oxidative damage to DNA was determined using the OxyDNA assay (Biotrin, Dublin, Ireland). MCF-7 cells were plated onto sterile glass coverslips in 35-mm2 tissue culture dishes and treated with NSC3852 12 h later. After 24 h, cells were fixed and permeabilized with 99% methanol. Nonspecific binding sites were blocked using blocking solution (1 h, room temperature). After washing twice, the cells were incubated in the dark with fluorescein isothiocyanate-conjugated antibody (1 h, room temperature) to identify 8-oxoguanine containing DNA. Cell images were captured using confocal microscopy.
Cell Death Enzyme-Linked Immunosorbent Assay. We quantified cytoplasmic nucleosomes using the Cell Death Detection ELISAPLUS kit (Roche Molecular Biochemicals, Indianapolis, IN) in MCF-7 cells plated in triplicate into 96-well plates. The cytoplasmic fractions from attached cells in each well were assayed in duplicate for the presence of nucleosomes according to the directions from the suppliers. The apoptotic response was measured by the nucleosome-enrichment fraction calculated as the ratio of the absorbance (405 nm) of drug-treated cultures/solvent-exposed cultures.
Statistics. Sigma plot software (SPSS Inc., Chicago, IL), version 5.0, and Prism (GraphPad Software, Inc., San Diego, CA), version 3.0, were used for statistical analysis. Statistically significant differences (P < 0.05) were determined using Student's t test or a one-way ANOVA and Tukey's t test (P < 0.01).
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Results |
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).
We used ESR to examine ROS formation in MCF-7 cell suspensions. Figure 2 shows the ESR signals obtained with MCF-7 cell suspensions containing 10 µM NSC3852 or 8 µM NSC2039. In cells exposed to NSC3852, a 1:2:2:1 quartet signal (with aH = aN = 14.9 G, where aH and aN denote hyperfine splitting of the 
-hydrogen and the nitroxyl nitrogen, respectively), indicative of the DMPO-OH adduct, was observed. In MCF-7 cells treated with NSC2039, no signal was produced demonstrating that the nitroso substituent was required for the generation of ROS (Fig. 2, scans A and B). Figure 3A shows the concentration-response relationship between the ESR-peak height in MCF-7 cells exposed to dimethyl sulfoxide solvent alone, 2 µM NSC3852, and 10 µM NSC3852. Statistically significant DMPO-OH peaks were generated at both the 2 and 10 µM NSC3852 concentrations. Figure 3B compares the time course of the signal intensity of the DMPO-OH peak in cells treated with 2 or 10 µM NSC3852. The MCF-7 ROS response to 2 µM NSC3852 decayed more rapidly than response to 10 µM. In cells treated with 10 µM NSC3852, ROS production was stable for as long as we measured the reaction (25 min). No ROS were detected when NSC3852 or NSC2039 was diluted into PBS without MCF-7 cells, suggesting that cellular metabolism is involved in the ROS response to NSC3852.
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To probe the source of the DMPO-OH ESR signal, either superoxide dismutase (SOD) or catalase was added to cell suspensions. Catalase, a scavenger of H2O2, had no effect upon the signal (Fig. 2, scan C). Therefore, the source of the DMPO-OH signal was unlikely to be OH· derived from H2O2. An alternate route for the formation of DMPO-OH is through the trapping of superoxide to form the unstable adduct, DMPO-OOH that quickly decomposes to DMPO-OH. When SOD was added to the cell suspensions, the ESR signal was quenched (Fig. 2, scan D), consistent with data showing that, when SOD clears the 
(superoxide) very quickly, trapping of the unstable DMPO-OOH intermediate is suppressed. These results support our conclusion that the observed ESR spectra were produced by the decomposition of the DMPO-OOH signal trapped from superoxide (Halliwell and Gutteridge, 1999).
The two major cellular sources for production of are flavoproteins in NADPH oxidase complexes and in the mitochondrial electron transport chain (Berridge and Tan, 2000; Jezek and Hlavata, 2005). Diphenyliodonium is a nonselective flavoprotein inhibitor affecting all of the flavin-dependent enzymes. Because DMPO can enter cells, the ESR spectra in Fig. 2 depict both intracellular and extracellular ROS. The ESR spectra showing suppression of the DMPO-OH signal upon the addition of the enzyme SOD to the cell suspension (Fig. 2D) suggested that at least half of the ESR signal was produced extracellularly. The nonphagocytic form of membrane NADPH oxidase, a flavin-dependent enzyme, is the most likely source of extracellular superoxide (Berridge and Tan, 2000).
We also demonstrated intracellular superoxide production in MCF-7 cells in response to 10 µM NSC3852 using the fluorescent probes dihydroethidium and 2'-7'-dihydrodichlorofluorescein diacetate (data not shown). The identity and relative contribution of the enzymes responsible for the intracellular ROS response of MCF-7 cells to NSC3852 are not fully established; however, the complete inhibition of the ESR signals with DPI (Fig. 2F) strongly implicated flavin-dependent enzymes, such as NADPH oxidase, the mitochondrial Complex I, nitric-oxide synthase, aromatase, and other cytochrome P450-dependent enzymes present in breast cancer (Karihtala et al., 2004; Dumitrescu and Cotarla, 2005). Using 1 mM NG-nitro-L-arginine methyl ester to inhibit nitric-oxide synthase in MCF-7 cells, we showed a 29.5% (P < 0.01) reduction in the DMPO-OH ESR signal intensity, indicating that nitric-oxide synthase is important to the mechanism of action of NSC3852.
Augmentation of the NSC3852-generated DMPO-OH ESR signal by rotenone (Fig. 2E) is evidence for a role of mitochondrial Complex I in the intracellular ROS response to NSC3852. When rotenone binds Complex I, electron transport to ubiquinone is interrupted, resulting in superoxide formation (Majander et al., 1994; Jezek and Hlavata, 2005). Alternatively, there is evidence in brain microglia that rotenone can directly stimulate NADPH oxidase activity, thus enhancing the DMPO-OH signal intensity (Zoccarato et al., 2005). Thus, our data support a model in which NSC3852 stimulates ROS production through a variety of cellular-dependent pathways.
Regulation of G1 Proteins by NSC3852 and Reversal by N-Acetyl-L-cysteine. Cell-cycle arrest in G1 is a prerequisite for differentiation of human breast tumor cells in culture. Initial responses to differentiation stimuli include a shift in the expression of cell-cycle regulatory proteins toward their status in early G1 phase of the cell cycle, specifically hypophosphorylated retinoblastoma protein (Rb), and decreases in E2F1 and Myc protein (Jensen et al., 2001; Munster et al., 2001; Wang et al., 2001). Earlier work showed that MCF-7 cells treated with NSC3852 accumulated in G0 after 48 h but that MCF-7 cells exposed to the related analog, NSC2039, did not (Martirosyan et al., 2004). Based upon the differential ability of NSC3852 and NSC2039 to generate ROS and to cause differentiation in MCF-7 cells, we performed Western blot analyses of Rb, E2F1, and Myc.
NSC3852 caused a time-dependent accumulation of hypophosphorylated Rb and loss of phosphorylated Rb between 12 and 48 h. There were no detectable differences in Rb between control and NSC3852-treated cells at 8 h or earlier. Statistically significant decreases in phosphorylated Rb and E2F1 protein levels were seen at 12 and 24 h. After 24 h, Myc protein levels had decreased significantly, and by 48 h, Myc was undetectable in the Western blots. The time-dependent shift in the profile of Rb, E2F1, and Myc expression is consistent with the progression of NSC3852-treated cells into G0 (Martirosyan et al., 2004).
Figure 4 compares the effects of NSC3852 and NSC2039 on the G1 protein profile at 24 h. There were no statistically significant differences in Rb, E2F1, or Myc protein levels between control and NSC2039-treated cells. In NSC3852-treated cells, E2F1 and Myc levels fell by 50% (n = 3, p < 0.05), and hypophosphorylated Rb levels increased significantly. Figure 4 also shows the dependence upon a NAC-sensitive pathway for the NSC3852-mediated shift in Rb, E2F1, and Myc protein expression. Cells pretreated with 5 mM NAC 1 h before the addition of NSC3852 displayed no significant changes in Rb phosphorylation state, E2F1, and Myc. The effect of NAC pretreatment suggested that a redox-sensitive mechanism in G1 phase might be involved in the actions of NSC3852.
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NSC3852 Effect on [GSH]/[GSSG] Ratio in MCF-7 Cells. The major redox couple in mammalian cells is GSH-GSSG, and decreases in the intracellular [GSH]/[GSSG] ratio are a biological indicator of oxidative stress. NSC3852 stimulated levels within 15 min sufficiently to raise intracellular [GSSG] and thereby decrease the [GSH]/[GSSG] ratio. After 1 h, NSC3852 decreased the [GSH]/[GSSG] by 20% compared with control cells (P < 0.05). By 6 h, the [GSH]/[GSSG] ratios in control and treated cells were statistically indistinguishable, suggesting that NSC3852 mediated a transient oxidative shift in the cellular redox potential.
Free Radical Generation in MCF-7 Cells by Histone Deacetylase Inhibitors. NSC3852 is not unique among tumor differentiation agents in eliciting ROS. Three additional HDI (TSA, SAHA, and SBHA) elicited rapid and robust ESR signals indicative of ROS production in MCF-7 cells. The relative peak heights of the ESR signals (mean ± range, n = 2) were measured in parallel aliquots of 1 x 106 MCF-7 cells treated with 200 nM TSA (99 ± 1 mm), 200 nM SAHA (98 mm), 10 µM 3852 (109 ± 3 mm), or 2 µM 3852 (70 ± 4 mm) for 5 min at 37°C. We conclude that the magnitude of the ROS signals produced by these concentrations of HDI is roughly equivalent (Fig. 5). Previous work using a ROS-sensitive fluorescence dye showed that SAHA and another HDI, MS-275, increased ROS production in human leukemia cells (Rosato et al., 2003; Rahmani et al., 2005). This report is the first to demonstrate ROS generation in response to HDI using ESR.
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). We performed comet assays to test for DNA damage by NSC3852 and measured the extent of damage as the increase in the comet tail moment. NSC3852-induced DNA damage was detected after 12 h and was maximal at 24 h. DNA damage was undetectable after 96 h (Fig. 6A), at which time an apoptotic response might have eliminated cells with damaged DNA. Therefore, after treatment with NSC3852, the cells behaved as if they were exposed to an oxidative stress that was either self-limiting or attenuated by compensatory cell-survival pathways.
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Apoptosis occurred in response to NSC3852, and a 1-h pretreatment with NAC blocked this response (Fig. 8). The peak NSC3852 DNA-damage response occurred at 24 h (Fig. 6A) and preceded the peak in NSC3852-induced apoptosis (48 h) (Fig. 8). This temporal relationship fits a model where NSC3852-induced formation of 8-oxoguanine DNA adducts leads to DNA-strand breakage and apoptotic cell death.
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Discussion |
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). The purpose of this work was to understand the mechanistic basis for its pleiotropic actions in human breast cancer cells that are of potential significance in the treatment of cancer. One important finding was the enrichment of Rb in its hypophosphorylated state in NSC3852-treated cells. Hypophosphorylated Rb is the active tumor suppressor state of Rb and is a marker of cells arrested in G1, cell differentiation, and cell senescence (Yen and Varvayanis, 1995; Knudsen et al., 1998; Yen et al., 2004). We also showed that NSC3852 is a redox-active compound that stimulates superoxide production and a transient rise in intracellular redox potential. Our studies demonstrated that ROS production is important to the mechanism of action of NSC3852. ROS generation is dependent upon the interaction of NSC3852 with the cells and occurs both intracellularly and extracellularly. We propose that ROS production has dual actions in MCF-7 cells stimulating apoptosis through a well established mechanism of oxidative DNA damage (Guetens et al., 2002) and promoting cell differentiation through its actions on Rb protein.
The basis for our hypothesis that NSC3852 acts on Rb phosphorylation status by a redox mechanism is the reversal of NSC3852 activity by N-acetyl-L-cysteine pretreatment and the failure of NSC2039, a non-ROS-generating analog, to change Rb status. The temporal series of events following treatment with NSC3852 suggest that a ROS-initiated cascade of events leads to hypophosphorylated Rb, but our data do not exclude a direct action of ROS upon Rb. Other investigators have also concluded that cellular redox status regulates the phosphorylation state of Rb (Dou et al., 1995; Nelson et al., 1997; Yamauchi and Bloom, 1997; Esposito et al., 2000; Kennedy et al., 2002).
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) suggests that protein phosphatase 2A, the enzyme responsible for the dephosphorylation of Rb, responds to cellular redox status. These investigators showed that, as the intracellular redox potential rises in response to oxidative stress, phosphatase 2A activity is stimulated, increasing hypophosphorylated Rb. The idea that Rb acts as a thiol-dependent nanotransducer of intracellular redox state and cell-cycle progression/differentiation status is intriguing and is the foundation of a model of redox control of cell proliferation proposed by Hoffman et al. (2001). However, there is substantial support for the opposing hypothesis that oxidative stress is mitogenic and antioxidants suppress proliferation (Irani et al., 1997; Chueh, 2000; Menon et al., 2003). All thiol residues on proteins are redox-sensitive and theoretically transduce redox signals rapidly and reversibly via changes in protein structure. A broader examination of redox signaling in cell-cycle regulatory pathways is one approach to resolving this paradox (Li and Oberley, 1998; Moran et al., 2001; Cooper et al., 2002; Kern and Kehrer, 2005; Ungerstedt et al., 2005). Our model describing a dual role for ROS in the cell differentiation and apoptotic responses to NSC3852 in MCF-7 cells appears in Fig. 9. We propose that a transient rise in intracellular redox potential establishes conditions permissive for differentiation to take place. Hypophosphorylated Rb will bind E2F and inhibit activation of genes, such as c-myc, that are required for progression through the cell cycle. Reductions in Myc protein reduce E2F transcription and synthesis. After a permissive state for differentiation exists, what additional stimuli activate the differentiation response? NSC3852 inhibits histone deacetylase activity in vitro at the same concentrations used in this study, and we suggest that inhibition of histone deacetylase is a requisite step in the differentiation response to NSC3852. Furthermore, because TSA, SAHA, and SBHA also generate ROS in MCF-7 cells, ROS production coupled with histone deacetylase inhibition might be a general mechanism for inducing apoptosis and differentiation in breast cancer.
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Acknowledgements |
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Footnotes |
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Portions of this work were presented in abstract form: Strobl J, Martirosyan A, Rahim-Bata R, Freeman A, and Clarke C (2004). Experimental breast cancer differentiation agents NSC3852, NSC69603, NSC10010, and NSC305819, in 1st ISC International Conference on Cancer Therapeutics, Molecular Targets, Pharmacology and Clinical Applications; 2004 February 19–24, International Society of Chemotherapy and Società Italiana di Chemioterapia, Florence, Italy.
ABBREVIATIONS: NSC3852, 5-nitroso-8-quinolinol; DMPO, 5, 5-dimethyl-1-pyrroline-N-oxide; DPI, diphenylene iodonium; DTNB, 5',5-dithiobis-2-nitrobenzoic acid; DMEM, Dulbecco's modified Eagle's medium; ESR, electron spin resonance; GSH, glutathione; GSSG, glutathione disulfide; HDI, histone deacetylase inhibitor(s); MS-275, N-(2-aminophenyl)-4-[N-pyridin-3-yl-methoxycarbonyl)aminomethyl]benzamide; MTS, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl) 2-(4-sulfonphenyl)-2H-tetrazolium; PBS, phosphate-buffered saline, pH 7.4; NAC, N-acetyl-L-cysteine; NSC2039, 8-quinolinol; Rb, retinoblastoma protein; ROS, reactive oxygen species; SAHA, suberoylanilide hydroxamic acid; SBHA, suberic bishydroxamate; SOD, superoxide dismutase; TSA, trichostatin A, [R-(E,E)]-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide; FBS, fetal bovine serum.
Address correspondence to: Dr. Jeannine S. Strobl, Edward Via Virginia College of Osteopathic Medicine, 2265 Kraft Drive, Blacksburg, VA 24060. E-mail jstrobl{at}vcom.vt.edu
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