title: Assessment of hypoxia inducible factor levels in cancer cell lines upon hypoxic induction using a novel reporter construct.
abstract: 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.
authors: Department of Biology, University of Washington, Seattle, Washington, United States of America.
authors: Wenyu
authors: W
authors: Zhou
authors: Timothy L
authors: TL
authors: Dosey
authors: Travis
authors: T
authors: Biechele
authors: Randall T
authors: RT
authors: Moon
authors: Marshall S
authors: MS
authors: Horwitz
authors: Hannele
authors: H
authors: Ruohola-Baker
mesh_terms: D016227:Benzoquinones / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics*; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D016227:Benzoquinones; D018841:HSP90 Heat-Shock Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047029:Lactams, Macrocyclic; D012333:RNA, Messenger; C112765:tanespimycin; D010100:Oxygen