Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors
| Autor: | Yelena Mironchik, Balaji Krishnamachary, Santosh Kumar Bharti, Zaver M. Bhujwalla, Samata Kakkad, Pierre Danhier |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Male
Cancer Research Blotting Western Green Fluorescent Proteins Transplantation Heterologous Luc luciferase Mice Nude Biology Response Elements lcsh:RC254-282 Time-Lapse Imaging Article Green fluorescent protein 03 medical and health sciences RFP red fluorescent protein 0302 clinical medicine CHX cycloheximide In vivo Cell Line Tumor ODD oxygen-dependent degradation domain Bioluminescence imaging Animals Humans Luciferase Cycloheximide Hypoxia Luciferases 030304 developmental biology Protein Synthesis Inhibitors 0303 health sciences Prostatic Neoplasms Anatomy Transfection lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens Hypoxia-Inducible Factor 1 alpha Subunit EGFP enhanced green fluorescent protein Cell Hypoxia Cell biology Transplantation Gene Expression Regulation Neoplastic Oxygen HIF hypoxia-inducible factor Luminescent Proteins Hypoxia-inducible factors Microscopy Fluorescence HRE hypoxia-response element 030220 oncology & carcinogenesis BLI bioluminescence imaging Ex vivo |
| Zdroj: | Neoplasia (New York, N.Y.) Neoplasia: An International Journal for Oncology Research, Vol 17, Iss 12, Pp 871-881 (2015) |
| ISSN: | 1476-5586 |
| DOI: | 10.1016/j.neo.2015.11.007 |
| Popis: | Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies. |
| Databáze: | OpenAIRE |
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