Development of radiation inducible promoter in prostate cancer

我们开发了一个启动子文库，该文库由随机延长可能在前列腺癌中被辐射激活的转录因子的顺式作用元件构建的DNA片段组成，并连接到TATA盒序列。选择文库中LNCap细胞中对x射线反应性最强的一个启动子，并通过引入随机突变对其进行改进。由此产生的启动子被命名为克隆880-8，在x射线照射（x -辐照）下显示出最高剂量依赖性的活性增强。将克隆880-8为对照的表达荧光素酶基因的重组逆转录病毒感染LNCap，在10 Gy x射线照射12 h后，荧光素酶活性增强9.12±0.36倍。将感染的细胞接种于裸鼠，10 Gy x射线照射12 h后荧光素酶的表达增强4.27±1.36倍。当LNCap下游感染另一个携带fcy::fur基因的重组体至克隆880-8时，x射线照射增强了fcy::fur的表达。与没有x射线照射的对应物相比，它还显示增加了5-氟胞嘧啶的剂量依赖性细胞杀伤率。这些结果表明，本研究采用的方法可以有效地构建对刺激有反应的启动子。这些启动子可用于刺激控制基因治疗。

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.