Journal Article

Assessment of Hypoxia Inducible Factor Levels in Cancer Cell Lines upon Hypoxic Induction Using a Novel Reporter Construct

Biechele, Travis, Dosey, Timothy L., Horwitz, Marshall S., Ruohola-Baker, Hannele, Moon, Randall T., Zhou, Wenyu

2011

Overview

Hypoxia Inducible Factor (HIF) signaling pathway is important for tumor cells with limited oxygen supplies, as it is shown to be involved in the process of proliferation and angiogenesis. Given its pivotal role in cancer biology, robust assays for tracking changes in HIF expression are necessary for understanding its regulation in cancer as well as developing therapies that target HIF signaling. Here we report a novel HIF reporter construct containing tandem repeats of minimum HIF binding sites upstream of eYFP coding sequence. We show that the reporter construct has an excellent signal to background ratio and the reporter activity is HIF dependent and directly correlates with HIF protein levels. By utilizing this new construct, we assayed HIF activity levels in different cancer cell lines cultured in various degrees of hypoxia. This analysis reveals a surprising cancer cell line specific variation of HIF activity in the same level of hypoxia. We further show that in two cervical cancer cell lines, ME180 and HeLa, the different HIF activity levels observed correlate with the levels of hsp90, a cofactor that protects HIF against VHL-independent degradation. This novel HIF reporter construct serves as a tool to rapidly define HIF activity levels and therefore the therapeutic capacity of potential HIF repressors in individual cancers.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Angiogenesis / Apoptosis / Benzoquinones - pharmacology / Binding sites / Biochemistry / Biology / Biotechnology / Cancer / Cancer therapies / Cell Hypoxia - drug effects / Cell Hypoxia - genetics / Cell Line, Tumor / Cervical cancer / Cervix / Development and progression / Fibroblasts / Gene expression / Gene Expression Regulation, Neoplastic - drug effects / Genes, Reporter - genetics / Genomes / Health aspects / Heat shock proteins / HSP90 Heat-Shock Proteins - genetics / HSP90 Heat-Shock Proteins - metabolism / Hsp90 protein / Humans / Hypoxia / Hypoxia-Inducible Factor 1, alpha Subunit - genetics / Hypoxia-Inducible Factor 1, alpha Subunit - metabolism / Kinases / Lactams, Macrocyclic - pharmacology / Medicine / Nucleotide sequence / Oxygen / Oxygen - pharmacology / Protein Binding - drug effects / Proteins / Repetitive Sequences, Nucleic Acid - genetics / Repressors / RNA, Messenger - genetics / RNA, Messenger - metabolism / Signal transduction / Signaling / Stem cells / Transcription factors / Transcription, Genetic - drug effects / Tumor cell lines / Tumor cells / Tumorigenesis / Vascular endothelial growth factor / VHL protein