Real-Time Imaging of HIF-1α Stabilization and Degradation

• Published in: PloS one 2009-04, Vol.4 (4), p.e5077
• Main Authors: Moroz, Ekaterina, Carlin, Sean, Dyomina, Katerina, Burke, Sean, Thaler, Howard T., Blasberg, Ronald, Serganova, Inna
• Format: Article
• Language: English
• Subjects: Angiogenesis; Biodegradation; Bioluminescence; Brain tumors; Cardellina; Cardiovascular disease; Cell Biology; Cell Biology/Gene Expression; Cell culture; Cell lines; Clonal deletion; Degradation; Drug development; Fusion protein; Gene expression; Glioma; Glioma cells; Health physics; Hypoxia; Immunofluorescence; Localization; Microscopy; Neurology; Oncology; Oxygen; Pathways; Prostate cancer; Proteins; Rodents; Stabilization; Tissues; VHL protein; Xenografts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0005077

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, CoCl2 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 (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼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.