ToxSci Advance Access published online on June 5, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp123
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Identification of Chemical Compounds that Induce HIF-1
Activity
1 NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MD, USA 2 Department of Radiation Oncology, University of Michigan, Comprehensive Cancer Center, Ann Arbor, MI, USA 3 Vascular Biology Program, Institute for Cell Engineering, Departments of Pediatrics, Medicine, Oncology, Radiation Oncology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA 4 SwitchGear Genomics, Menlo Park, CA, USA 5 National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
Address correspondence and reprint requests to Menghang Xia, Ph.D., 9800 Medical Center Drive, NIH Chemical Genomics Center, National Institutes of Health, Bethesda, MD 20892-3370, Phone: 301-217-5718, Fax: 301-217-5736, Email: mxia{at}mail.nih.gov
Received January 27, 2009; revision received May 21, 2009; accepted May 29, 2009
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Abstract |
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Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1
and constitutively-expressed HIF-1β. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia response elements (HRE) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based β-lactamase HRE reporter gene assay in a quantitative high throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, while 2 compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1 inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1 activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.
Key Words: cobalt sulfate heptahydrate; 7-diethylamino-4-methylcoumarin; 7,12-dimethylbenz(a)anthracence; HIF-1 inducers; iodochlorohydroxyquinoline; NTP 1408 compound library; o-phenanthroline, qHTS.