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


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 Gy X-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.


CONCLUSIONS
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.
Ogawa
Ryohei
R
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama 930-0194, Japan. ogawa@med.u-toyama.ac.jp
Lee
Sung-Il
SI
Kagiya
Go
G
Hirano
Hisao
H
Fukuda
Shigekazu
S
Kondo
Takashi
T
Kodaki
Tsutomu
T
D045744:Cell Line, Tumor; D004742:Enhancer Elements, Genetic; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000528:radiation effects*; D016385:TATA Box; D014157:Transcription Factors / Q000235:genetics
D014157:Transcription Factors