real-time imaging of hif-1α stabilization and degradation实时成像hif-1α稳定和退化.pdfVIP

Real-Time Imaging of HIF-1a Stabilization and Degradation 1 2 1 2 4 Ekaterina Moroz , Sean Carlin , Katerina Dyomina , Sean Burke , Howard T. Thaler , Ronald Blasberg1,3*, Inna Serganova1 1 Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 2 Department of Medical Physics, Memorial Sloan- Kettering Cancer Center, New York, New York, United States of America, 3 Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America, 4 Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America Abstract HIF-1a 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-1a chimeric reporter systems, HIF-1a/FLuc and HIF-1a(DODDD)/FLuc, to investigate the tightly controlled level of HIF-1a protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1a in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1a/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1a in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1a 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 affe