This Article Free Full Text (Free PDF) Free All Versions of this Article: 1087057106289234v1 11/6/678    most recent References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Woldemichael, G. M. Articles by McMahon, J. B. Search for Related Content PubMed PubMed Citation Articles by Woldemichael, G. M. Articles by McMahon, J. B. Social Bookmarking                   What's this?

Development of a Cell-Based Reporter Assay for Screening of Inhibitors of Hypoxia-Inducible Factor 2-Induced Gene Expression

National Cancer Institute, Molecular Targets Development Program, Center for Cancer Research, Frederick, Maryland

James R. Vasselli

National Cancer Institute, Clinical Research Center, Urologic Oncology Branch, Bethesda, Maryland

Roberta S. Gardella

SAIC-Frederick, Inc., Basic Research Program, NCI-Frederick, Frederick, Maryland

Tawnya C. Mckee

National Cancer Institute, Molecular Targets Development Program, Center for Cancer Research, Frederick, Maryland

W. Marston Linehan

National Cancer Institute, Clinical Research Center, Urologic Oncology Branch, Bethesda, Maryland

James B. McMahon

National Cancer Institute, Molecular Targets Development Program, Center for Cancer Research, Frederick, Maryland

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.

Key Words: HIF-2 • cell-based assay • reporter assay • hypoxia response element • VHL disease

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				K. M. Ruocco, E. I. Goncharova, M. R. Young, N. H. Colburn, J. B. McMahon, and C. J. Henrich A High-Throughput Cell-Based Assay to Identify Specific Inhibitors of Transcription Factor AP-1 J Biomol Screen, February 1, 2007; 12(1): 133 - 139. [Abstract] [PDF]