title: Molecular imaging of hypoxia-inducible factor 1 alpha and von Hippel-Lindau interaction in mice.
abstract: 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.
authors.affiliation: Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA 94305-5152, USA.
authors.forename: Clara Y H
authors.initials: CY
authors.lastname: Choi
authors.forename: Denise A
authors.initials: DA
authors.lastname: Chan
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Paulmurugan
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Sutphin
authors.forename: Quynh-Thu
authors.initials: QT
authors.lastname: Le
authors.forename: Albert C
authors.initials: AC
authors.lastname: Koong
authors.forename: Wayne
authors.initials: W
authors.lastname: Zundel
authors.forename: Sanjiv S
authors.initials: SS
authors.lastname: Gambhir
authors.forename: Amato J
authors.initials: AJ
authors.lastname: Giaccia
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003952:Diagnostic Imaging / Q000379:methods*; D045325:HCT116 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics / Q000378:metabolism*; D051598:Whole Body Imaging / Q000379:methods
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011993:Recombinant Fusion Proteins; D011392:Proline; D049409:Luciferases, Firefly; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C497446:VHL protein, human