Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore Formation
The oxygen requirement for chromophore formation potentially limits the use of green fluorescent protein as a reporter under hypoxic conditions. In the light of this, the applicability of a hypoxia-responsive enhanced green fluorescent protein (EGFP)-based system to the measurement of tumor hypoxia...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2001-01-01
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| Series: | Neoplasia: An International Journal for Oncology Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1476558601800087 |
| _version_ | 1811286134801563648 |
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| author | Dirk Vordermark Toru Shibata J. Martin Brown |
| author_facet | Dirk Vordermark Toru Shibata J. Martin Brown |
| author_sort | Dirk Vordermark |
| collection | DOAJ |
| description | The oxygen requirement for chromophore formation potentially limits the use of green fluorescent protein as a reporter under hypoxic conditions. In the light of this, the applicability of a hypoxia-responsive enhanced green fluorescent protein (EGFP)-based system to the measurement of tumor hypoxia was tested in human HT 1080 fibrosarcoma cells stably transfected with a destabilized EGFP vector containing the hypoxia-responsive 5HRE-hCMVmp promoter or, as a positive control, the strong constitutive CMV promoter. After various schedules of hypoxia and reoxygenation, EGFP fluorescence of live cells was assessed by flow cytometry, protein levels were analyzed by Western blot. Fluorescence of CMV promoter positive control cells dropped to 38±5% of aerobic levels after 12 hours at <0.02% oxygen, but was unaffected by higher oxygen concentrations. Following 12 hours at <0.02% oxygen, cells transfected with the hypoxiaresponsive vector exhibited maximum fluorescence after 4 hours of subsequent reoxygenation, reaching 68±2% of the levels in CMV promoter controls under aerobic conditions. With such reoxygenation, these cells exhibited a constant increase in fluorescence between 2% and <0.02% oxygen. EGFP chromophore formation is only affected by near-anoxic oxygen concentrations. The correlation of fluorescence and oxygen concentration is restored by a 4-hour reoxygenation period due to oxidation of pre-synthesized EGFP and a delayed increase in EGFP protein synthesis. |
| first_indexed | 2024-04-13T02:54:28Z |
| format | Article |
| id | doaj.art-913b2958bbad494483379bf82acb7918 |
| institution | Directory Open Access Journal |
| issn | 1476-5586 1522-8002 |
| language | English |
| last_indexed | 2024-04-13T02:54:28Z |
| publishDate | 2001-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Neoplasia: An International Journal for Oncology Research |
| spelling | doaj.art-913b2958bbad494483379bf82acb79182022-12-22T03:05:43ZengElsevierNeoplasia: An International Journal for Oncology Research1476-55861522-80022001-01-013652753410.1038/sj.neo.7900192Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore FormationDirk VordermarkToru ShibataJ. Martin BrownThe oxygen requirement for chromophore formation potentially limits the use of green fluorescent protein as a reporter under hypoxic conditions. In the light of this, the applicability of a hypoxia-responsive enhanced green fluorescent protein (EGFP)-based system to the measurement of tumor hypoxia was tested in human HT 1080 fibrosarcoma cells stably transfected with a destabilized EGFP vector containing the hypoxia-responsive 5HRE-hCMVmp promoter or, as a positive control, the strong constitutive CMV promoter. After various schedules of hypoxia and reoxygenation, EGFP fluorescence of live cells was assessed by flow cytometry, protein levels were analyzed by Western blot. Fluorescence of CMV promoter positive control cells dropped to 38±5% of aerobic levels after 12 hours at <0.02% oxygen, but was unaffected by higher oxygen concentrations. Following 12 hours at <0.02% oxygen, cells transfected with the hypoxiaresponsive vector exhibited maximum fluorescence after 4 hours of subsequent reoxygenation, reaching 68±2% of the levels in CMV promoter controls under aerobic conditions. With such reoxygenation, these cells exhibited a constant increase in fluorescence between 2% and <0.02% oxygen. EGFP chromophore formation is only affected by near-anoxic oxygen concentrations. The correlation of fluorescence and oxygen concentration is restored by a 4-hour reoxygenation period due to oxidation of pre-synthesized EGFP and a delayed increase in EGFP protein synthesis.http://www.sciencedirect.com/science/article/pii/S1476558601800087green fluorescent proteinhypoxiatransfectionreoxygenationfibrosarcoma |
| spellingShingle | Dirk Vordermark Toru Shibata J. Martin Brown Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore Formation Neoplasia: An International Journal for Oncology Research green fluorescent protein hypoxia transfection reoxygenation fibrosarcoma |
| title | Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore Formation |
| title_full | Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore Formation |
| title_fullStr | Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore Formation |
| title_full_unstemmed | Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore Formation |
| title_short | Green Fluorescent Protein is a Suitable Reporter of Tumor Hypoxia Despite an Oxygen Requirement for Chromophore Formation |
| title_sort | green fluorescent protein is a suitable reporter of tumor hypoxia despite an oxygen requirement for chromophore formation |
| topic | green fluorescent protein hypoxia transfection reoxygenation fibrosarcoma |
| url | http://www.sciencedirect.com/science/article/pii/S1476558601800087 |
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