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Real-Time Imaging of HIF-1α Stabilization and Degradation
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 inv...
Autores principales: | , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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Public Library of Science
2009
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2660410/ https://www.ncbi.nlm.nih.gov/pubmed/19347037 http://dx.doi.org/10.1371/journal.pone.0005077 |
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author | Moroz, Ekaterina Carlin, Sean Dyomina, Katerina Burke, Sean Thaler, Howard T. Blasberg, Ronald Serganova, Inna |
author_facet | Moroz, Ekaterina Carlin, Sean Dyomina, Katerina Burke, Sean Thaler, Howard T. Blasberg, Ronald Serganova, Inna |
author_sort | Moroz, Ekaterina |
collection | PubMed |
description | 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, CoCl(2) 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 (t(1/2) ∼4–6 min) was abolished by the hypoxia-mimetic CoCl(2,) MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t(1/2) ∼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. |
format | Text |
id | pubmed-2660410 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-26604102009-04-04 Real-Time Imaging of HIF-1α Stabilization and Degradation Moroz, Ekaterina Carlin, Sean Dyomina, Katerina Burke, Sean Thaler, Howard T. Blasberg, Ronald Serganova, Inna PLoS One Research Article 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, CoCl(2) 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 (t(1/2) ∼4–6 min) was abolished by the hypoxia-mimetic CoCl(2,) MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t(1/2) ∼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. Public Library of Science 2009-04-04 /pmc/articles/PMC2660410/ /pubmed/19347037 http://dx.doi.org/10.1371/journal.pone.0005077 Text en Moroz et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Moroz, Ekaterina Carlin, Sean Dyomina, Katerina Burke, Sean Thaler, Howard T. Blasberg, Ronald Serganova, Inna Real-Time Imaging of HIF-1α Stabilization and Degradation |
title | Real-Time Imaging of HIF-1α Stabilization and Degradation |
title_full | Real-Time Imaging of HIF-1α Stabilization and Degradation |
title_fullStr | Real-Time Imaging of HIF-1α Stabilization and Degradation |
title_full_unstemmed | Real-Time Imaging of HIF-1α Stabilization and Degradation |
title_short | Real-Time Imaging of HIF-1α Stabilization and Degradation |
title_sort | real-time imaging of hif-1α stabilization and degradation |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2660410/ https://www.ncbi.nlm.nih.gov/pubmed/19347037 http://dx.doi.org/10.1371/journal.pone.0005077 |
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