Real-time imaging of HIF-1alpha stabilization and degradation.
HIF-1alpha 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-1alpha chimeric reporter systems, HIF-1alpha/FLuc and HIF-1alpha(DeltaODDD)/FLuc, to investigate the tightly controlled level of HIF-1alpha protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1alpha in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1alpha/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1alpha in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1alpha 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-1alpha/FLuc protein degradation and quantify the half-life of HIF-1alpha fusion proteins. The rapid clearance component (t(1/2) approximately 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 (t(1/2) approximately 200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1alpha/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1alpha.
Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.
Ekaterina
E
Moroz
Sean
S
Carlin
Katerina
K
Dyomina
Sean
S
Burke
Howard T
HT
Thaler
Ronald
R
Blasberg
Inna
I
Serganova
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction
D017931:DNA Primers; C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit