Construction of Mutant TKGFP for Real-Time Imaging of Temporal Dynamics of HIF-1 Signal Transduction Activity Mediated by Hypoxia and Reoxygenation in Tumors in Living Mice
Construction of Mutant TKGFP for Real-Time Imaging of Temporal Dynamics of HIF-1 Signal Transduction Activity Mediated by Hypoxia and Reoxygenation in Tumors in Living Mice
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DOI：
10.2967/jnumed.108.061234
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发表时间：
2009-12-01
影响因子：
9.3
通讯作者：
Liu, Ren-Shyan
中科院分区：
文献类型：
--
作者：
Hsieh, Chia-Hung;Kuo, Jung-Wen;Liu, Ren-Shyan
单纯疱疹病毒1型胸苷激酶(HSV1-tk)/绿色荧光蛋白(TKGFP)双报告基因和多模式成像方法在翻译分子-遗传成像中监测治疗性基因表达、免疫细胞转运和蛋白质-蛋白质相互作用方面发挥着关键作用。然而，TKGFP的细胞毒性和低时间分辨率限制了其在需要快速更换记者的研究中的应用。本研究旨在构建一种细胞毒性低、时间分辨率高的突变型TKGFP融合报告基因，用于实时监测体内外缺氧/复氧诱导因子-1(HIF-1)信号转导活性的时间动力学和空间异质性。方法：在N端插入核输出信号(NES)序列，在C端融合小鼠鸟氨酸脱羧酶(DMODC)的降解结构域，构建失稳的TKGFP。用Western印迹分析、HSV1-tk酶活性测定和流式细胞仪检测TKGFP在NG4TL4活细胞中的稳定性。通过将NESTKGFP：dMODC连接到一个由8个拷贝的缺氧反应元件组成的启动子上，研究了它作为转录报告的适宜性，其活性依赖于HIF-1。用NESTKGFP：dMODC或TKGFP检测缺氧和复氧诱导的动态转录事件，并通过光学成像和PET进行检测。结果：与TKGFP不同，NESTKGFP：dMODC在放线菌酮存在下不稳定，蛋白质和酶活性半衰期较短。NESTKGFP：dMODC的快速转换以26S蛋白酶体依赖的方式发生。此外，NESTKGFP：dMODC在活细胞中表达上调，细胞毒性低。对低氧反应的TKGFP和NESTKGFP：dMODC表达的研究表明，作为报告基因的NESTKGFP：dMODC在监测缺氧和复氧介导的动态转录事件方面比TKGFP具有更好的时间分辨率；TKGFP的表达水平不适合用于监测目的。结论：在翻译分子遗传学成像中，NESTKGFP：dMODC作为报告基因，与光学成像和PET一起，可以直接监测转录诱导，并容易确定其与其他生化变化的关系。
The herpes simplex virus type 1 thymidine kinase (HSV1-tk)/green fluorescent protein (TKGFP) dual-reporter gene and a multimodality imaging approach play a critical role in monitoring therapeutic gene expression, immune cell trafficking, and protein-protein interactions in translational molecular-genetic imaging. However, the cytotoxicity and low temporal resolution of TKGFP limits its application in studies that require a rapid turnover of the reporter. The purpose of this study was to construct a novel mutant TKGFP fusion reporter gene with low cytotoxicity and high temporal resolution for use in the real-time monitoring of temporal dynamics and spatial heterogeneity of hypoxia-inducible factor 1 (HIF-1) signal transduction activity mediated by hypoxia and reoxygenation in vitro and in vivo. Methods: Destabilized TKGFP was produced by inserting the nuclear export signal (NES) sequence at the N terminus and fusing the degradation domain of mouse ornithine decarboxylase (dMODC) at the C terminus. The stability of TKGFP in living NG4TL4 cells was determined by Western blot analysis, HSV1-tk enzyme activity assay, and flow cytometric analysis. The suitability of NESTKGFP: dMODC as a transcription reporter was investigated by linking it to a promoter consisting of 8 copies of hypoxia-responsive elements, whose activities depend on HIF-1. The dynamic transcriptional events mediated by hypoxia and reoxygenation were monitored by NESTKGFP: dMODC or TKGFP and determined by optical imaging and PET. Results: Unlike TKGFP, NESTKGFP: dMODC was unstable in the presence of cycloheximide and showed a short half-life of protein and enzyme activity. Rapid turnover of NESTKGFP: dMODC occurred in a 26S proteasome-dependent manner. Furthermore, NESTKGFP: dMODC showed an upregulated expression and low cytotoxicity in living cells. Studies of hypoxia-responsive TKGFP and NESTKGFP: dMODC expression showed that NESTKGFP: dMODC as a reporter gene had better temporal resolution than did TKGFP for monitoring the dynamic transcriptional events mediated by hypoxia and reoxygenation; the TKGFP expression level was not optimal for the purpose of monitoring. Conclusion: In translational molecular-genetic imaging, NESTKGFP: dMODC as a reporter gene, together with optical imaging and PET, allows the direct monitoring of transcription induction and easy determination of its association with other biochemical changes.