Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 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

Thai Khuc  1 , Chia-Wen Amy Hsu  1 , Srilatha Sakamuru  1 , Menghang Xia  2
Affiliations

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

Thai Khuc et al. Methods Mol Biol. 2016.

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.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Concentration-response curves of cobalt chloride in HRE-bla normoxia mode assay. Each data point is presented as mean ± SD from duplicates.
Fig. 2
Fig. 2
Concentration-response curves of topotecan in HRE-bla hypoxia mode assay. Each data point is presented as mean ± SD from duplicates.
Fig. 3
Fig. 3
Concentration-response curves of cobalt chloride in HIF-1a-NanoLuc normoxia mode assay. Each data point is presented as mean ± SD from duplicates.
Fig. 4
Fig. 4
Concentration-response curves of topotecan and YC-1 in HIF-1a-NanoLuc hypoxia mode assay. Each data point is presented as mean ± SD from duplicates.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Xia M, Bi K, Huang R, Cho MH, Sakamuru S, Miller SC, Li H, Sun Y, Printen J, Austin CP, Inglese J. Identification of small molecule compounds that inhibit the HIF-1 signaling pathway. Molecular cancer. 2009;8:117. doi: 10.1186/1476-4598-8-117. - DOI - PMC - PubMed
    1. Jiang BH, Semenza GL, Bauer C, Marti HH. Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension. The American journal of physiology. 1996;271(4 Pt 1):C1172–1180. - PubMed
    1. Wang GL, Semenza GL. Purification and characterization of hypoxia-inducible factor 1. The Journal of biological chemistry. 1995;270(3):1230–1237. - PubMed
    1. Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia-inducible factor 1alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(14):7987–7992. - PMC - PubMed
    1. Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. The Journal of biological chemistry. 1997;272(36):22642–22647. - PubMed

MeSH terms

LinkOut - more resources