Real-time imaging of HIF-1α stabilization and degredation Journal Article
| Authors: | Moroz, E.; Carlin, S.; Dyomina, K.; Burke, S.; Thaler, H. T.; Blasberg, R.; Serganova, I. |
| Article Title: | Real-time imaging of HIF-1α stabilization and degredation |
| Abstract: | HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl<sub>2</sub> treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t<sub>1/2</sub> ∼4-6 min) was abolished by the hypoxia-mimetic CoCl<sub>2</sub>, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t<sub>1/2</sub> ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α. © 2009 Moroz et al. |
| Keywords: | signal transduction; controlled study; human cell; nonhuman; polymerase chain reaction; protein localization; protein analysis; mouse; animal; metabolism; animals; mice; protein degradation; oxygen; cell line; animal experiment; protein stability; wild type; hybrid protein; cell strain hek293; xenograft; glioma cell; nucleotide sequence; protein transport; reporter gene; cytoplasm; base sequence; fluorescence microscopy; microscopy, fluorescence; cell hypoxia; cell nucleus; dna primers; primer dna; hypoxia inducible factor 1alpha; bioluminescence; cell strain 3t3; cobalt chloride; hif1a protein, human; hif1a protein, mouse; immunofluorescence microscopy; hypoxia-inducible factor 1, alpha subunit |
| Journal Title: | PLoS ONE |
| Volume: | 4 |
| Issue: | 4 |
| ISSN: | 1932-6203 |
| Publisher: | Public Library of Science |
| Date Published: | 2009-04-04 |
| Start Page: | e5077 |
| Language: | English |
| DOI: | 10.1371/journal.pone.0005077 |
| PUBMED: | 19347037 |
| PROVIDER: | scopus |
| PMCID: | PMC2660410 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 3" - "Export Date: 30 November 2010" - "Art. No.: e5077" - "Source: Scopus" |
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