Vordermark D, Shibata T, Brown J M
Department of Radiation Oncology, Division of Radiation and Cancer Biology, Stanford University School of Medicine, Stanford, CA 94305-5152, USA.
Neoplasia. 2001 Nov-Dec;3(6):527-34. doi: 10.1038/sj.neo.7900192.
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, and 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 hypoxia-responsive 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.
发色团形成所需的氧气可能会限制绿色荧光蛋白在缺氧条件下作为报告基因的应用。鉴于此，在稳定转染了含有缺氧反应性5HRE-hCMVmp启动子的不稳定增强绿色荧光蛋白（EGFP）载体的人HT 1080纤维肉瘤细胞中，测试了基于缺氧反应性增强绿色荧光蛋白（EGFP）的系统在测量肿瘤缺氧方面的适用性，或者作为阳性对照，测试了强组成型CMV启动子的适用性。在进行各种缺氧和复氧方案后，通过流式细胞术评估活细胞的EGFP荧光，并通过蛋白质印迹法分析蛋白质水平。CMV启动子阳性对照细胞在氧气浓度<0.02%的情况下12小时后，荧光降至有氧水平的38±5%，但不受较高氧气浓度的影响。在氧气浓度<0.02%的情况下处理12小时后，转染了缺氧反应性载体的细胞在随后复氧4小时后表现出最大荧光，达到有氧条件下CMV启动子对照水平的68±2%。通过这种复氧，这些细胞在氧气浓度为2%至<0.02%之间时荧光持续增加。EGFP发色团的形成仅受近无氧氧气浓度的影响。由于预合成的EGFP氧化和EGFP蛋白质合成的延迟增加，经过4小时的复氧期后，荧光与氧气浓度的相关性得以恢复。
