Abstract
Genome-wide screens have proven powerful in associating gene products with certain phenotypes or signal transduction pathways, and thus are valuable tools to ascribe gene function. These genomic screens can be extended to discover genes/proteins that attenuate oxidative stress-induced damage, which is implicated in aging, neurodegenerative disorders, and other diseases. One mechanism by which humans protect themselves from oxidative stress is through an endogenous stress response that leads to transcriptional activation of the antioxidant response element (ARE). The ARE is located in the 59-flanking regions of many phase II detoxification and antioxidant enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and regulates the expression of these genes. Increasing the levels of antioxidant enzymes without causing oxidative stress can potentially counteract degeneration and may be therapeutically useful. On a genomic scale, ARE activators can be identified by screening expression cDNA libraries in a high-throughput amenable reporter gene assay. Further validation of putative hits requires testing of cDNAs for their ability to upregulate the expression of endogenous ARE-regulated genes on the transcript and protein levels, and their ability to protect cells from oxidative insults. General screening procedure and subsequent hit validation are discussed in detail.
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References
van Muiswinkel, F. L., and Kuiperij, H. B. (2005). The Nrf2-ARE signalling pathway: promising drug target to combat oxidative stress in neurodegenerative disorders. Curr. Drug Targets CNS Neurol. Disord. 4, 267–281.
Kensler, T. W., Wakabayashi, N., and Biswal, S. (2007). Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47, 89–116.
Nguyen, T., Sherratt, P. J., and Pickett, C. B. (2003). Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol. 43, 233–260.
Li, J., Lee, J. M., and Johnson, J. A. (2002). Microarray analysis reveals an antioxidant responsive element-driven gene set involved in conferring protection from an oxidative stress-induced apoptosis in IMR-32 cells. J. Biol. Chem. 277, 388–394.
Lee, J. M., Calkins, M. J., Chan, K., Kan, Y. W., and Johnson, J. A. (2003). Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J. Biol. Chem. 278, 12029–12038.
Johnson, D. A., Andrews, G. K., Xu, W., and Johnson, J. A. (2002). Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice. J. Neurochem. 81, 1233–1241.
Liu, Y., Kern, J. T., Walker, J. R., Johnson, J. A., Schultz, P. G., and Luesch, H. (2007). A genomic screen for activators of the antioxidant response element. Proc. Natl. Acad. Sci. USA 104, 5205–5210.
Moehlenkamp, J. D., and Johnson, J. A. (1999). Activation of antioxidant/electrophile-responsive elements in IMR-32 human neuroblastoma cells. Arch. Biochem. Biophys. 363, 98–106.
Hu, Q., Klippel, A., Muslin, A. J., Fantl, W. J., and Williams, L. T. (1995). Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science 268, 100–102.
Lee, J. M., Hanson, J. M., Chu, W. A., and Johnson, J. A. (2001). Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells. J. Biol. Chem. 276, 20011–20016.
SantaCruz, K. S., Yazlovitskaya, E., Collins, J., Johnson, J., and DeCarli, C. (2004). Regional NAD(P)H:quinone oxidoreductase activity in Alzheimer’s disease. Neurobiol. Aging 25, 63–69.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Luesch, H., Liu, Y. (2008). Genome-Wide Overexpression Screen for Activators of Antioxidant Gene Transcription. In: Armstrong, D. (eds) Advanced Protocols in Oxidative Stress I. Methods In Molecular Biology, vol 477. Humana Press. https://doi.org/10.1007/978-1-60327-517-0_26
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DOI: https://doi.org/10.1007/978-1-60327-517-0_26
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