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• W2079892215 abstract "The present study was designed to investigate howtert-butylhydroquinone (tBHQ) prevents hydrogen peroxide-induced apoptosis in IMR-32 cells. tBHQ pretreatment (10 μm) attenuated hydrogen peroxide-induced cell death and reduced the number of TUNEL (terminal deoxynucleotidyltransferase-mediated, dUTP-incorporated nick end labeling)-positive cells. We hypothesize that tBHQ-mediated activation of the antioxidant responsive element is critical for generating this protective response. Addition of LY294002, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K), 30 min prior to tBHQ treatment completely reversed the protective effect of tBHQ. Oligonucleotide microarrays were used to analyze the gene expression profile associated with tBHQ treatment in the absence and presence of LY294002. Ranking analysis using Affymetrix's difference call indicated that the expression of 137 genes changed with tBHQ treatment. Further analysis using the coefficient of variation for -fold change or average difference change reduced the list to 63 increased and 0 decreased genes. Reverse transcription-PCR for selected genes also confirmed the gene expression pattern. Many of these genes function to combat oxidative stress and increase the detoxification potential of the cells. Inhibition of PI3K significantly blocked the enhanced expression of 49 of the 63 genes induced by tBHQ. These data are the first to show a set of programmed cell life genes involved in conferring protection from an oxidative stress-induced apoptosis. The present study was designed to investigate howtert-butylhydroquinone (tBHQ) prevents hydrogen peroxide-induced apoptosis in IMR-32 cells. tBHQ pretreatment (10 μm) attenuated hydrogen peroxide-induced cell death and reduced the number of TUNEL (terminal deoxynucleotidyltransferase-mediated, dUTP-incorporated nick end labeling)-positive cells. We hypothesize that tBHQ-mediated activation of the antioxidant responsive element is critical for generating this protective response. Addition of LY294002, a selective inhibitor of phosphatidylinositol 3-kinase (PI3K), 30 min prior to tBHQ treatment completely reversed the protective effect of tBHQ. Oligonucleotide microarrays were used to analyze the gene expression profile associated with tBHQ treatment in the absence and presence of LY294002. Ranking analysis using Affymetrix's difference call indicated that the expression of 137 genes changed with tBHQ treatment. Further analysis using the coefficient of variation for -fold change or average difference change reduced the list to 63 increased and 0 decreased genes. Reverse transcription-PCR for selected genes also confirmed the gene expression pattern. Many of these genes function to combat oxidative stress and increase the detoxification potential of the cells. Inhibition of PI3K significantly blocked the enhanced expression of 49 of the 63 genes induced by tBHQ. These data are the first to show a set of programmed cell life genes involved in conferring protection from an oxidative stress-induced apoptosis. central nervous system glutathione antioxidant responsive element NAD(P)H:quinone oxidoreductase glutathione transferase M3 heme oxygenase-1 glutathione reductase thioredoxin reductase γ-glutamylcysteine ligase regulatory subunit γ-glutamylcysteine catalytic subunit poly ADP-ribose polymerase phosphatidylinositol 3-kinase extracellular signal-regulated kinase mitogen-activated protein kinase MAPK kinase 4 NF-E2 related factor 2 terminal deoxynucleotidyltransferase-mediated dUTP-incorporated nick end labeling programmed cell death programmed cell life tert-butylhydroquinone 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-terazolium, inner salt fluorescence isothiocyanate apoptotic index reverse transcription coefficient of variation -fold change average difference change neurofilament heavy subunit butyrate response factor 2 endoplasmic reticulum Accumulated evidence strongly suggests that apoptosis contributes to neuronal cell death in a variety of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease (1Mattson M.P. Nat. Rev. Mol. Cell. Biol. 2000; 1: 120-129Crossref PubMed Scopus (1247) Google Scholar). Central to the apoptotic response is a family of aspartate-directed cysteine proteases termed the caspases. Caspases function both dependently and independently in the disruption of the mitochondria and its release of proapoptotic factors that are known to serve as signal transducers and positive effectors in the apoptotic pathway (2Grubb D.R. Ly J.D. Vaillant F. Johnson K.L. Lawen A. Oncogene. 2001; 20: 4085-4094Crossref PubMed Scopus (39) Google Scholar, 3Bratton S.B. Cohen G.M. Trends Pharmacol. Sci. 2001; 22: 306-315Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Activation of the cysteine protease caspase-3 appears to be a key event in the execution of apoptosis in the central nervous system (CNS).1 The CNS is particularly vulnerable to oxidative stress because of a high rate of oxidative metabolism, which results in high rates of strong oxidant formation. In addition, the CNS contains an abundance of polyunsaturated fatty acids that are susceptible to lipid peroxidation.The cellular toxicity of hydrogen peroxide (H2O2) is initiated by oxidative stress resulting in the rapid modification of cytoplasmic constituents, the depletion of intracellular glutathione (GSH) and ATP, a decrease in NAD+ level, an increase in free cytosolic Ca2+, and lipid peroxidation (4Fernandez-Checa J.C. Kaplowitz N. Garcia-Ruiz C. Colell A. Miranda M. Mari M. Ardite E. Morales A. Am. J. Physiol. 1997; 273: G7-G17Crossref PubMed Google Scholar). H2O2 also activates the mitochondria permeability transition pore and the release of cytochrome c (5Yang J.C. Cortopassi G.A. Free. Radic. Biol. Med. 1998; 24: 624-631Crossref PubMed Scopus (198) Google Scholar). In the cytoplasm, cytochrome c, in combination with Apaf-1, activates caspase-9 leading to the activation of caspase-3 and subsequent apoptosis (6Sugano N. Ito K. Murai S. FEBS Lett. 1999; 447: 274-276Crossref PubMed Scopus (28) Google Scholar, 7Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6183) Google Scholar, 8Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2249) Google Scholar). Because oxidative stress is involved in H2O2-induced cell death, modulation of antioxidant defenses, such as the increased concentration of the intracellular GSH, may protect cells from programmed cell death (PCD).Others have shown that treating cells withtert-butylhydroquinone (tBHQ), a strong inducer of phase II detoxification enzymes via activation of the antioxidant responsive element (ARE), can protect cells from oxidative stress (9Murphy T.H. Miyamoto M. Sastre A. Schnaar R.L. Coyle J.T. Neuron. 1989; 2: 1547-1558Abstract Full Text PDF PubMed Scopus (842) Google Scholar, 10Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar, 11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar). Induction of NAD(P)H:quinone oxidoreductase (NQO1) in N18-RE-105 neuronal cells by tBHQ prior to glutamate treatment was correlated with a significant decrease in glutamate toxicity (11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar). Glutamate toxicity in these cells is not due toN-methyl-d-aspartate receptor activation and calcium influx. Rather, it is a result of competitive inhibition of cysteine uptake, depletion of GSH, increased oxidative stress, and apoptosis (9Murphy T.H. Miyamoto M. Sastre A. Schnaar R.L. Coyle J.T. Neuron. 1989; 2: 1547-1558Abstract Full Text PDF PubMed Scopus (842) Google Scholar). Subsequent studies by Murphy and colleagues (10Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar) using H2O2 and dopamine to induce oxidative stress, however, demonstrated that N18-RE-105 cells overexpressing NQO1 were not resistant to cytotoxicity. These data suggest that the protective effect conferred by tBHQ may not simply be due to an increase in one gene but the coordinate up-regulation of many genes. We hypothesize that tBHQ-mediated activation of the ARE is a principal component generating this protective response.Based on these observations and recent work in our laboratory characterizing a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism of ARE activation for tBHQ in IMR-32 human neuroblastoma cells (12Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar, 13Lee J.M. Hanson J.M. Chu W.A. Johnson J.A. J. Biol. Chem. 2001; 276: 20011-20016Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), we were interested in identifying the ARE-driven gene set increased by tBHQ and correlating this information with the antiapoptotic effect of tBHQ (11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar). The present investigation was designed to: 1) determine if tBHQ pretreatment protects human neuroblastoma cells from H2O2-induced apoptosis; 2) evaluate how the inhibition of PI3K activity modulates any protective effect manifested by treatment with tBHQ; and 3) identify the set of genes conferring this protection using oligonucleotide microarray technology.DISCUSSIONIn this study, H2O2 was used to generate oxidative injury and subsequent apoptosis in cultured IMR-32 cells. Our results clearly show that H2O2 induced IMR-32 cell apoptosis through a caspase-3-dependent pathway. The apoptotic process and development of cell injury in H2O2-treated IMR-32 cells was prevented and/or delayed by strengthening the antioxidant capacity of these cells through the transcriptional activation of ARE-driven genes in response to treatment with tBHQ. Oligonucleotide microarray analysis identified the genes that changed with the treatment. The protective effects of tBHQ pretreatment were reversed by inhibition of PI3K and 46 of the 63 genes increased by tBHQ were significantly blocked. Although we have not screened the entire human genome, the genes identified here represent the first set of antiapoptotic genes encoding proteins that can directly or indirectly attenuate the apoptotic process.The fact that H2O2 toxicity was not totally prevented by tBHQ pretreatment is probably due to the mechanism by which H2O2 causes toxicity. It has been reported that H2O2 depletes NADPH and GSH within 10–30 min after administration (18Tretter L. Adam-Vizi V. J. Neurosci. 2000; 20: 8972-8979Crossref PubMed Google Scholar). The genes induced by tBHQ can increase the steady-state concentration of these essential molecules in the cell and delay but not completely prevent their depletion or the eventual cell death induced by H2O2 depending on the time and dose of exposure. Similarly, Murphy and colleagues (11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar, 19Su J.Y. Duffy S. Murphy T.H. Neurosci. Lett. 1999; 273: 109-112Crossref PubMed Scopus (12) Google Scholar) have demonstrated that pretreatment of rodent neuroblastoma cells with compounds that activated the ARE and increased NQO1 partially protects cells from H2O2- and dopamine-induced cytotoxicity. They also demonstrated that stable overexpression of NQO1 in this cell line did not confer resistance to cytotoxicity, suggesting that the regulation of multiple genes is required for protection (10Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar). Microarray analysis revealed a cluster of phase II detoxification enzymes and other genes, which can coordinately combat oxidative stress and prevent apoptosis. Among these genes, HO1, an enzyme that catalyzes the rate-limiting reaction in heme degradation, is involved in a catabolic pathway that leads to the production of bilirubin, a potent antioxidant. NQO1 catalyzes two-electron reduction of quinones and prevents the participation of such compounds in redox cycling and oxidative stress. GCLR catalyzes the rate-limiting reaction in glutathione biosynthesis. GSTM3 conjugates hydrophobic electrophiles and reactive oxygen species with glutathione. GR catalyzes the reduction of oxidized glutathione to reduced GSH and maintains adequate levels of reduced cellular GSH. TR is part of a family of selenium-containing pyridine nucleotide-disulfide oxidoreductase, which utilize NADPH to catalyze the conversion of oxidized thioredoxin into reduced thioredoxin and to reduce the oxidized forms of ascorbate into reduced ascorbate. Hepatic dihydrodiol dehydrogenase and KIAA0119 are aldo-keto reductases and detoxify reactive carbinyl-containing compounds, which are widely distributed in nature and pose a serious threat to living organisms because of their ability to react with cellular macromolecules. Finally, cytosolic NADP+-dependent malic enzyme, an NADPH-producing cytosolic enzyme, catalyzes both oxidative decarboxylation of malate and reductive carboxylation of pyruvate.Other categories of genes include those that enhance the growth and differentiation of neuron (neuronal olfactomedin-related ER localized protein, axin, NF-H), function as chaperone proteins or respond to heat shock (hsp40 homolog, Bip protein), participate in various signaling pathways (Wnt-5a, MAPKK4, phosphotyrosine-independent ligand p62 for the Lck SH2 domain), and modulate transcription (KIAA0132 and ERF-2). All these genes, in addition to the detoxification and antioxidant genes, may contribute to the antiapoptotic effects of tBHQ treatment. The mechanisms by which they contribute are open to speculation and remain to be determined.tBHQ-mediated activation of ARE is critical to generating the protective response against H2O2-induced apoptosis. We and others (13Lee J.M. Hanson J.M. Chu W.A. Johnson J.A. J. Biol. Chem. 2001; 276: 20011-20016Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 17Ramos-Gomez M. Kwak M.K. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3410-3415Crossref PubMed Scopus (978) Google Scholar) have demonstrated that exposure to tBHQ triggers nuclear accumulation of the transcription factor, Nrf2, which binds to the ARE. These data provide evidence for a novel pathway of signal transduction in response to oxidative stress via the ARE reminiscent of the well-characterized AP-1-mediated or NF-κB-mediated pathway. A search for potential Nrf2 binding sites within the 5′-flanking regions of a selected gene set was performed using MatInspector V2.2 based on TRANSFAC 4.0 (available athttp://transfac.gbf.de) to determine whether the expression profile could be predicted by the presence or absence of Nrf2 binding sites. Indeed, the gene containing no Nrf2 binding site(s) such as GCLC was not increased by tBHQ whereas the promoter regions for the up-regulated genes (e.g.NQO1, GCLR, Wnt5a, TR, andmalate NADP oxidoreductase) contained Nrf2 binding site(s). In contrast, AP-1 and NF-κB are distributed abundantly in the 5′-flanking region of all genes, including GCLC. They are not supposed to play an important role in transcriptional activation by tBHQ. Nrf2 binding sites appear to be in direct correlation with up-regulation of these genes.Recent reports indicate that Nrf2 is normally localized in the cytoplasm bound to a chaperone, Keap1. Exposure of cells to inducers/stressors disrupts the Keap1·Nrf2 complex, and Nrf2 migrates to the nucleus where it binds (in heterodimeric forms with other transcription factors) to the ARE and stimulates transcription. Keap1, a 624-amino acid protein, contains 25 cysteine residues, 9 of which are expected to have highly reactive sulfhydryl groups because they are flanked by one or more basic amino acid residues (20Itoh K. Chiba T. Takahashi S. Ishii T. Igarashi K. Katoh Y. Oyake T. Hayashi N. Satoh K. Hatayama I. Yamamoto M. Nabeshima Y. Biochem. Biophys. Res. Commun. 1997; 236: 313-322Crossref PubMed Scopus (3127) Google Scholar, 21Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarash K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2711) Google Scholar, 22Snyder G.H. Cennerazzo M.J. Karalis A.J. Field D. Biochemistry. 1981; 20: 6509-6519Crossref PubMed Scopus (168) Google Scholar). Because many activators of the ARE can react with sulfhydryl groups, the Keap1·Nrf2 complex is a plausible candidate for the cytoplasmic sensor system that recognizes and reacts with inducers of ARE-driven genes (23Dinkova-Kostova A.T. Massiah M.A. Bozak R.E. Kicks R.J. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3404-3409Crossref PubMed Scopus (518) Google Scholar). A recent study describes the identification of Inrf2, a rat homolog of Keap1, as part of a pathway mediating the response to oxidative stress through the ARE (16Dhakshinamoorthy S. Jaiswal A.K. Oncogene. 2001; 20: 3906-3917Crossref PubMed Scopus (252) Google Scholar). Of particular interest, KIAA0132, a human homolog of Keap1, was up-regulated by tBHQ, whereas other Nrf2 binding proteins, such as the small Maf protein family members (MafK and MafF) present on the U95a array, were unchanged. MatInspector also found putative Nrf2 binding sites in the 5′-flanking region of KIAA0132 indicating that transcription of KIAA0132 can be increased by the transcription factor that it sequesters. We suppose that this feedback effect aims to keep in balance the expression of ARE-driven genes, however, little is understood as to the mechanism by which the Keap1·Nrf2 complex functions as a cytoplasmic sensor for oxidative stress.The signal transduction pathway leading to the activation of gene expression via the Nrf2·ARE interaction has recently been shown to require activation of the PI3K pathway in IMR-32 cells (13Lee J.M. Hanson J.M. Chu W.A. Johnson J.A. J. Biol. Chem. 2001; 276: 20011-20016Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). We exploited this PI3K dependence in the present study to address the question of tBHQ-mediated cytoprotection and identify the associated changes in gene expression. Inhibition of PI3K completely blocked the protective effect seen by tBHQ treatment and significantly inhibited the increased expression of 46 out of the 63 genes increased by tBHQ. Although the data strongly support our hypothesis for the existence of a PI3K-Nrf2·ARE pathway leading to protection following treatment with tBHQ, microarray analysis revealed two important phase II detoxification enzymes, GCLR and TR, that were induced by tBHQ but not inhibited by LY294002. This suggests an alternative pathway for GCLR and TR induction that is dependent on the Nrf2·ARE interaction but independent of PI3K. Others have suggested that ERK 1/2 and/or p38 MAPK are positive modulators of the ARE in several cell lines (24Yu R. Lei W. Mandlekar S. Weber M.J. Der C.J. Wu J. Kong A.T. J. Biol. Chem. 1999; 274: 27545-27552Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 25Yu R. Mandlekar S. Lei W. Fahl W.E. Tan T.H. Kong A.T. J. Biol. Chem. 2000; 275: 2322-2327Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 26Yu R. Chen C. Mo Y.Y. Hebbar V. Owuor E.D. Tan T.H. Kong A.T. J. Biol. Chem. 2000; 275: 39907-39913Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 27Wild A.C. Moinova H.R. Mulcahy R.T. J. Biol. Chem. 1999; 274: 33627-33636Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Because inhibition of ERK1/2 or p38 MAPK does not block tBHQ-mediated activation of the ARE or NQO1 induction, this does not appear to be the case in IMR-32 cells (12Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar). These data imply that, in addition to cell-specific mechanisms for ARE activation, a new level of complexity exists for ARE activation within the same cell.In conclusion, oxidative stress can be defined as an imbalance in which free radicals and their products exceed the capacity of cellular antioxidant defense mechanisms. A gain in product formation or a loss in protective mechanisms can disturb this equilibrium, leading to PCD. Numerous publications have demonstrated a correlation between direct supplementation of medium with chemical antioxidants and a decreased apoptotic rate in cell lines (28Qin F. Rounds N.K. Mao W. Kawai K. Liang C. Cardiovasc. Res. 2001; 51: 736-748Crossref PubMed Scopus (62) Google Scholar, 29Lee M.H. Hyun D.H. Jenner P. Halliwell B. J. Neurochem. 2001; 78: 32-41Crossref PubMed Scopus (120) Google Scholar). Alternatively, induction of multiple antioxidant genes also may provide a way to increase an antioxidative potential and protect cells from apoptosis, as demonstrated here. We refer to this process as programmed cell life or PCL. An equilibrium exists such that any increase in the forces that drive PCD, therefore, must be balanced by increasing the forces that drive PCL. The set of ARE-driven survival genes identified here represents the first set of genes shown to modulate PCD and confirms that the PI3K-Nrf2·ARE pathway is crucial for the transcription of these PCL genes. Accumulated evidence strongly suggests that apoptosis contributes to neuronal cell death in a variety of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease (1Mattson M.P. Nat. Rev. Mol. Cell. Biol. 2000; 1: 120-129Crossref PubMed Scopus (1247) Google Scholar). Central to the apoptotic response is a family of aspartate-directed cysteine proteases termed the caspases. Caspases function both dependently and independently in the disruption of the mitochondria and its release of proapoptotic factors that are known to serve as signal transducers and positive effectors in the apoptotic pathway (2Grubb D.R. Ly J.D. Vaillant F. Johnson K.L. Lawen A. Oncogene. 2001; 20: 4085-4094Crossref PubMed Scopus (39) Google Scholar, 3Bratton S.B. Cohen G.M. Trends Pharmacol. Sci. 2001; 22: 306-315Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Activation of the cysteine protease caspase-3 appears to be a key event in the execution of apoptosis in the central nervous system (CNS).1 The CNS is particularly vulnerable to oxidative stress because of a high rate of oxidative metabolism, which results in high rates of strong oxidant formation. In addition, the CNS contains an abundance of polyunsaturated fatty acids that are susceptible to lipid peroxidation. The cellular toxicity of hydrogen peroxide (H2O2) is initiated by oxidative stress resulting in the rapid modification of cytoplasmic constituents, the depletion of intracellular glutathione (GSH) and ATP, a decrease in NAD+ level, an increase in free cytosolic Ca2+, and lipid peroxidation (4Fernandez-Checa J.C. Kaplowitz N. Garcia-Ruiz C. Colell A. Miranda M. Mari M. Ardite E. Morales A. Am. J. Physiol. 1997; 273: G7-G17Crossref PubMed Google Scholar). H2O2 also activates the mitochondria permeability transition pore and the release of cytochrome c (5Yang J.C. Cortopassi G.A. Free. Radic. Biol. Med. 1998; 24: 624-631Crossref PubMed Scopus (198) Google Scholar). In the cytoplasm, cytochrome c, in combination with Apaf-1, activates caspase-9 leading to the activation of caspase-3 and subsequent apoptosis (6Sugano N. Ito K. Murai S. FEBS Lett. 1999; 447: 274-276Crossref PubMed Scopus (28) Google Scholar, 7Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6183) Google Scholar, 8Budihardjo I. Oliver H. Lutter M. Luo X. Wang X. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2249) Google Scholar). Because oxidative stress is involved in H2O2-induced cell death, modulation of antioxidant defenses, such as the increased concentration of the intracellular GSH, may protect cells from programmed cell death (PCD). Others have shown that treating cells withtert-butylhydroquinone (tBHQ), a strong inducer of phase II detoxification enzymes via activation of the antioxidant responsive element (ARE), can protect cells from oxidative stress (9Murphy T.H. Miyamoto M. Sastre A. Schnaar R.L. Coyle J.T. Neuron. 1989; 2: 1547-1558Abstract Full Text PDF PubMed Scopus (842) Google Scholar, 10Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar, 11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar). Induction of NAD(P)H:quinone oxidoreductase (NQO1) in N18-RE-105 neuronal cells by tBHQ prior to glutamate treatment was correlated with a significant decrease in glutamate toxicity (11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar). Glutamate toxicity in these cells is not due toN-methyl-d-aspartate receptor activation and calcium influx. Rather, it is a result of competitive inhibition of cysteine uptake, depletion of GSH, increased oxidative stress, and apoptosis (9Murphy T.H. Miyamoto M. Sastre A. Schnaar R.L. Coyle J.T. Neuron. 1989; 2: 1547-1558Abstract Full Text PDF PubMed Scopus (842) Google Scholar). Subsequent studies by Murphy and colleagues (10Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar) using H2O2 and dopamine to induce oxidative stress, however, demonstrated that N18-RE-105 cells overexpressing NQO1 were not resistant to cytotoxicity. These data suggest that the protective effect conferred by tBHQ may not simply be due to an increase in one gene but the coordinate up-regulation of many genes. We hypothesize that tBHQ-mediated activation of the ARE is a principal component generating this protective response. Based on these observations and recent work in our laboratory characterizing a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism of ARE activation for tBHQ in IMR-32 human neuroblastoma cells (12Lee J.M. Moehlenkamp J.D. Hanson J.M. Johnson A.J. Biochem. Biophys. Res. Commun. 2001; 280: 286-292Crossref PubMed Scopus (116) Google Scholar, 13Lee J.M. Hanson J.M. Chu W.A. Johnson J.A. J. Biol. Chem. 2001; 276: 20011-20016Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), we were interested in identifying the ARE-driven gene set increased by tBHQ and correlating this information with the antiapoptotic effect of tBHQ (11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar). The present investigation was designed to: 1) determine if tBHQ pretreatment protects human neuroblastoma cells from H2O2-induced apoptosis; 2) evaluate how the inhibition of PI3K activity modulates any protective effect manifested by treatment with tBHQ; and 3) identify the set of genes conferring this protection using oligonucleotide microarray technology. DISCUSSIONIn this study, H2O2 was used to generate oxidative injury and subsequent apoptosis in cultured IMR-32 cells. Our results clearly show that H2O2 induced IMR-32 cell apoptosis through a caspase-3-dependent pathway. The apoptotic process and development of cell injury in H2O2-treated IMR-32 cells was prevented and/or delayed by strengthening the antioxidant capacity of these cells through the transcriptional activation of ARE-driven genes in response to treatment with tBHQ. Oligonucleotide microarray analysis identified the genes that changed with the treatment. The protective effects of tBHQ pretreatment were reversed by inhibition of PI3K and 46 of the 63 genes increased by tBHQ were significantly blocked. Although we have not screened the entire human genome, the genes identified here represent the first set of antiapoptotic genes encoding proteins that can directly or indirectly attenuate the apoptotic process.The fact that H2O2 toxicity was not totally prevented by tBHQ pretreatment is probably due to the mechanism by which H2O2 causes toxicity. It has been reported that H2O2 depletes NADPH and GSH within 10–30 min after administration (18Tretter L. Adam-Vizi V. J. Neurosci. 2000; 20: 8972-8979Crossref PubMed Google Scholar). The genes induced by tBHQ can increase the steady-state concentration of these essential molecules in the cell and delay but not completely prevent their depletion or the eventual cell death induced by H2O2 depending on the time and dose of exposure. Similarly, Murphy and colleagues (11Murphy T.H. DeLong M.J. Coyle J.T. J. Neurochem. 1991; 56: 990-995Crossref PubMed Scopus (70) Google Scholar, 19Su J.Y. Duffy S. Murphy T.H. Neurosci. Lett. 1999; 273: 109-112Crossref PubMed Scopus (12) Google Scholar) have demonstrated that pretreatment of rodent neuroblastoma cells with compounds that activated the ARE and increased NQO1 partially protects cells from H2O2- and dopamine-induced cytotoxicity. They also demonstrated that stable overexpression of NQO1 in this cell line did not confer resistance to cytotoxicity, suggesting that the regulation of multiple genes is required for protection (10Duffy S. So A. Murphy T.H. J. Neurochem. 1998; 71: 69-77Crossref PubMed Scopus (121) Google Scholar). Microarray analysis revealed a cluster of phase II detoxification enzymes and other genes, which can coordinately combat oxidative stress and prevent apoptosis. Among these genes, HO1, an enzyme that catalyzes the rate-limiting reaction in heme degradation, is involved in a catabolic pathway that leads to the production of bilirubin, a potent antioxidant. NQO1 catalyzes two-electron reduction of quinones and prevents the particip" @default.
• W2079892215 created "2016-06-24" @default.
• W2079892215 creator A5033759264 @default.
• W2079892215 creator A5040409719 @default.
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• W2079892215 date "2002-01-01" @default.
• W2079892215 modified "2023-10-16" @default.
• W2079892215 title "Microarray Analysis Reveals an Antioxidant Responsive Element-driven Gene Set Involved in Conferring Protection from an Oxidative Stress-induced Apoptosis in IMR-32 Cells" @default.
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