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Methods Mol Biol. 2016;1473:23-31. doi: 10.1007/978-1-4939-6346-1_3.

Using β-Lactamase and NanoLuc Luciferase Reporter Gene Assays to Identify Inhibitors of the HIF-1 Signaling Pathway.

Author information

1
National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.
2
National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA. mxia@mail.nih.gov.

Abstract

The hypoxia-inducible factor 1 (HIF-1) is a transcriptional factor involved in the regulation of oxygen within cellular environments. In hypoxic tissues or those with inadequate oxygen concentrations, activation of the HIF-1 transcription factor allows for subsequent activation of target gene expression implicated in cell survival. As a result, cells proliferate through formation of new blood vessels and expansion of vascular systems, providing necessary nourishment needed of cells. HIF-1 is also involved in the complex pathophysiology associated with cancer cells. Solid tumors are able to thrive in hypoxic environments by overactivating these target genes in order to grow and metastasize. Therefore, it is of high importance to identify modulators of the HIF-1 signaling pathway for possible development of anticancer drugs and to better understand how environmental chemicals cause cancer. Using a quantitative high-throughput screening (qHTS) approach, we are able to screen large chemical libraries to profile potential small molecule modulators of the HIF-1 signaling pathway in a 1536-well format. This chapter describes two orthogonal cell based assays; one utilizing a β-lactamase reporter gene incorporated into human ME-180 cervical cancer cells, and the other using a NanoLuc luciferase reporter system in human HCT116 colon cancer cells. Cell viability assays for each cell line are also conducted respectively. The data from this screening platform can be used as a gateway to study mode of action (MOA) of selected compounds and drug classes.

KEYWORDS:

Beta-lactamase; Cancer; Drug; FRET; Fluorescence resonance energy transfer; Genome editing; Hypoxia inducible factor 1; Hypoxia response elements; Luciferase; NanoLuc; Quantitative high-throughput screening; Reporter gene; qHTS

PMID:
27518620
PMCID:
PMC5375166
DOI:
10.1007/978-1-4939-6346-1_3
[Indexed for MEDLINE]
Free PMC Article

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