Development of radiation inducible promoter in prostate cancer

开发了一个启动子文库，该文库由通过随机延长推测在前列腺癌中通过辐射激活的转录因子的顺式作用元件并连接到TATA盒序列而构建的DNA片段组成。选择在文库的LNCap细胞中对X射线具有最强反应性的一个启动子，并通过引入随机突变来改进。所得的启动子被命名为克隆880-8，其在X射线照射（X-照射）下显示出最高的剂量依赖性活性增强。在克隆880-8的控制下表达荧光素酶基因的重组逆转录病毒感染LNCap，LNCap在10戈伊的X射线照射后12小时显示荧光素酶活性增强9.12 ± 0.36倍。当感染细胞接种到裸鼠上时，在10戈伊的X射线照射后12 h，荧光素酶表达增强4.27 ± 1.36倍。当LNCap感染另一个重组携带fcy：：fur基因下游克隆880-8，fcy：：fur表达增强X射线照射。与没有X射线照射的对应物相比，还显示用5-氟胞嘧啶增加剂量依赖性细胞杀伤率。这些结果表明，在本研究中采用的方法是有效的，以构建一个启动子响应刺激。这样的启动子可用于刺激控制的基因疗法。

A promoter library was developed that is composed of DNA fragments constructed by randomly elongating cis-acting elements of transcription factors presumably activated in prostate cancer by radiation, and linking to the TATA box sequence. One promoter with the strongest reactivity to X-ray in LNCap cells of the library was chosen and improved by the introduction of random mutations. A resultant promoter was designated clone 880-8 showing the highest dose-dependent activity enhancement with X-ray irradiation (X-irradiation). A recombinant retrovirus expressing the luciferase gene under the control of clone 880-8 was infected to LNCap that showed 9.12 ± 0.36-fold enhancement of luciferase activity 12 h after X-irradiation at 10 Gy. When the infected cells were inoculated onto nude mice, enhancement of luciferase expression was 4.27 ± 1.36-fold 12 h after X-irradiation at 10 Gy. When LNCap was infected with another recombinant carrying the fcy::fur gene downstream to clone 880-8, fcy::fur expression was enhanced by X-irradiation. It was also shown to increase dose-dependent cell killing ratio with 5-fluorocytosine compared to a counterpart without X-irradiation. These results suggest that the method employed in this study is effective to construct a promoter responsive to stimulation. Such promoters can be utilized for stimulation controlled-gene therapies.