title: Development of a cell-based reporter assay for screening of inhibitors of hypoxia-inducible factor 2-induced gene expression.
abstract: Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.
authors.affiliation: National Cancer Institute, Molecular Targets Development Program, Center for Cancer Research, Frederick, Maryland 21702, USA.
authors.forename: Girma M
authors.initials: GM
authors.lastname: Woldemichael
authors.forename: James R
authors.initials: JR
authors.lastname: Vasselli
authors.forename: Roberta S
authors.initials: RS
authors.lastname: Gardella
authors.forename: Tawnya C
authors.initials: TC
authors.lastname: McKee
authors.forename: W Marston
authors.initials: WM
authors.lastname: Linehan
authors.forename: James B
authors.initials: JB
authors.lastname: McMahon
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D014407:Tumor Cells, Cultured; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D000099162:Endothelial PAS Domain-Containing Protein 1