Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
J Gene Med. 2012 May;14(5):316-27. doi: 10.1002/jgm.2625.
We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated.
Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8.
Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin.
Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.
我们之前从构建的 11 个启动子中获得了一个对 X 射线有反应的启动子。在本研究中，我们旨在确定我们的启动子构建方法的效率。此外，还研究了启动子在体内对 X 射线的反应性。
通过将 TATA 盒与辐射激活的转录因子的随机组合结合序列连接，构建启动子文库。将启动子与各种基因的组合稳定转染到 HeLa 细胞中，建立重组细胞系，然后用 X 射线或质子束照射，观察有无抗氧化剂存在时基因表达的增强。用 X 射线照射表达荧光素酶的重组细胞的肿瘤，通过检测生物发光来评估启动子的激活。作为体外自杀基因治疗的模型，用 X 射线照射表达 fcy::fur 的重组细胞，然后用 5-氟胞嘧啶孵育。用 WST-8 测定细胞活力。
文库中的 62 个启动子中有 25 个在接受 10GyX 射线照射 6 小时后，使荧光素酶活性增强了 5 倍以上，表明我们的方法是有效的。X 射线和质子束都能增强重组细胞中的荧光素酶活性，但程度较低。抗氧化剂减弱了增强作用，提示氧化应激的参与。在小鼠肿瘤中，启动子对 X 射线的反应性较低。在我们的自杀基因治疗模型中，照射后的细胞存活率随 5-氟胞嘧啶的剂量依赖性下降。
我们的方法在生成辐射反应性启动子方面是有效的。此外，我们已经成功地展示了其中一个启动子的潜在治疗用途。
