Development of radiation inducible promoter in prostate cancer

开发了一个启动子文库，该文库由随机延长转录因子顺式作用元件构建的DNA片段组成，该转录因子可能通过辐射在前列腺癌中激活，并连接到TATA盒序列。在文库的LNCaP细胞中选择了一个对X射线反应最强的启动子，并通过引入随机突变进行了改进。由此产生的启动子被命名为克隆880-8，在X射线照射(X射线照射)下表现出最高的剂量依赖性活性增强。将880-8克隆控制下的重组逆转录病毒感染LNCaP，经10GyX射线照射12h后，荧光素酶活性增加9.12±0.36倍。将感染细胞接种到裸鼠体内，10GyX射线照射12h后，荧光素酶的表达增强了4.27±1.36倍。当另一个携带FCY：：Fur基因的重组体在克隆880-8下游感染LNCaP时，经X射线照射后，FCY：：Fur的表达增强。与未经X-射线照射的对照相比，5-氟胞嘧啶可提高剂量依赖性的细胞杀伤率。这些结果表明，本研究所采用的方法是构建对刺激反应的启动子的有效方法。这样的启动子可以用于刺激控制的基因治疗。

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.