Hsieh Ya-Ju, Hwu Luen, Ke Chien-Chih, Huang Ai-Lin, Chen Fu-Du, Wu Shyh-Jong, Chen Sharon Chia-Ju, Zhao Yong-Hua, Liu Ren-Shyan
Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan.
Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, Taipei, Taiwan.
Mol Imaging Biol. 2015 Dec;17(6):802-10. doi: 10.1007/s11307-015-0843-7.
Promoters developed for radiogene therapy always show non-negligible transcriptional activities, even when cells are not irradiated. This study developed a tightly radiation-controlled molecular switch based on radiation responsive element (CArG) repeats for in vivo molecular imaging using the Cre/loxP system.
Different numbers of CArG repeats were cloned as a basal promoter directly, and its pre- and postirradiation transcriptional activities were analyzed by luciferase assay. Nine CArG repeats (E9) were chosen for use as a radiation-controlled molecular switch for the Cre/loxP system, and the feasibility of the switch in vitro and in vivo was demonstrated by luciferase assay and bioluminescence imaging, respectively.
The E9 promoter, which exhibits extremely low transcriptional activity, showed a 1.8-fold enhancement after irradiation with a clinical dose of 2 Gy. Both in vitro and in vivo results indicated that E9 is relatively inert but sufficient to trigger the Cre/loxP system. The luciferase activity of stable H1299/pSTOP-FLuc cells transfected with pE9-NLSCre and exposed to 2-Gy radiation can reach 44 % of that of the same cells transfected with pCMV-NLSCre and not subjected to irradiation. By contrast, no appreciable difference was observed in reporter gene expression in both H1299/pSTOPFluc cells and tumors transfected with pE4Pcmv-NLSCre before and after irradiation, because the strong basal transcriptional activity of the CMV promoter, which acts as a copartner of E4, masked the response of E4 to radiation.
Our results provide detailed insight into CArG elements as a radiation-controlled molecular switch that can facilitate the development of radiogene therapy.
用于放射基因治疗的启动子即使在细胞未受照射时也总是表现出不可忽视的转录活性。本研究基于辐射响应元件（CArG）重复序列开发了一种严格受辐射控制的分子开关，用于使用Cre/loxP系统进行体内分子成像。
将不同数量的CArG重复序列直接克隆为基础启动子，并通过荧光素酶测定分析其照射前后的转录活性。选择九个CArG重复序列（E9）用作Cre/loxP系统的辐射控制分子开关，并分别通过荧光素酶测定和生物发光成像证明该开关在体外和体内的可行性。
表现出极低转录活性的E9启动子在临床剂量2 Gy照射后显示出1.8倍的增强。体外和体内结果均表明，E9相对惰性，但足以触发Cre/loxP系统。用pE9-NLSCre转染并暴露于2 Gy辐射的稳定H1299/pSTOP-FLuc细胞的荧光素酶活性可达到用pCMV-NLSCre转染且未接受照射的相同细胞的44%。相比之下，在用pE4Pcmv-NLSCre转染的H1299/pSTOPFluc细胞和肿瘤中，照射前后报告基因表达未观察到明显差异，因为作为E4共同伙伴的CMV启动子的强基础转录活性掩盖了E4对辐射的反应。
我们的结果为CArG元件作为一种辐射控制分子开关提供了详细的见解，这有助于放射基因治疗的发展。
