Construction of X-ray-inducible promoters through cis-acting element elongation and error-prone polymerase chain reaction

摘要

Background A promoter that is activated by ionizing radiation may be a useful tool for cancer therapy since, with such a promoter, the therapeutic gene can be expressed only in cancer tissues by irradiation. An artificially constructed promoter is advantageous as natural promoters may have physiological limitations. However, reasonably designing a promoter is hampered by shortage of information about the relationship between the structure and properties of a promoter DNA. Materials and methods Binding sites of four transcription factors that were activated by radiation were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected cancer cells with such a vector were exposed to X-ray irradiation and enhancement of luciferase expression was assessed. To improve promoter sensitivity, mutations were randomly introduced into a constructed promoter by error-prone polymerase chain reaction (epPCR). Results Of the 11 promoters constructed, the clone 11 promoter (clone 11 + TATA-box) showed a 5-fold enhancement 6 h after the 10 GyX-ray irradiation in HeLa cells. A mutant designated the clone 11-9-37 promoter generated through two steps of epPCR showed a sensitivity 4.8 times higher than the clone 11 promoter to the 10 Gy X-rays, showing 21.6-fold enhancement of luciferase expression. Clone 11 was composed of 16 cis-acting elements, and the clone 11-9-37 promoter carried six point mutations. Conclusion A sensitively responsive promoter to radiation could be constructed using this method, possibly leading to the construction of a promoter of interest that could be applied for clinical use. Copyright (C) 2007 John Wiley & Sons, Ltd.