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Tumor hypoxia plays a crucial role in tumorigenesis. Under hypoxia, hypoxia-inducible factor 1 alpha (HIF-1 alpha) regulates activation of genes promoting malignant progression. Under normoxia, HIF-1 alpha is hydroxylated on prolines 402 and 564 and is targeted for ubiquitin-mediated degradation by interacting with the von Hippel-Lindau protein complex (pVHL). We have developed a novel method of studying the interaction between HIF-1 alpha and pVHL using the split firefly luciferase complementation-based bioluminescence system in which HIF-1 alpha and pVHL are fused to amino-terminal and carboxy-terminal fragments of the luciferase, respectively. We demonstrate that hydroxylation-dependent interaction between the HIF-1 alpha and pVHL leads to complementation of the two luciferase fragments, resulting in bioluminescence in vitro and in vivo. Complementation-based bioluminescence is diminished when mutant pVHLs with decreased affinity for binding HIF-1 alpha are used. This method represents a new approach for studying interaction of proteins involved in the regulation of protein degradation.

Type

Journal article

Journal

Mol Imaging

Publication Date

05/2008

Volume

7

Pages

139 - 146

Keywords

Animals, Cell Line, Tumor, Diagnostic Imaging, HCT116 Cells, Humans, Hypoxia-Inducible Factor 1, alpha Subunit, Luciferases, Firefly, Luminescent Measurements, Mice, Mice, Nude, Models, Molecular, Mutation, Proline, Recombinant Fusion Proteins, Von Hippel-Lindau Tumor Suppressor Protein, Whole Body Imaging