Regulation of gene expression in human prostate cancer cells with artificially constructed promoters that are activated in response to ultrasound stimulation.
We chose promoters responsive to sonication in LNCap cells, a prostate cancer cell line, out of a library composed of DNA fragments constructed by linking the TATA box sequence to randomly combined cis-acting elements of transcription factors activated in response to radiation in prostate cancer cells. When a plasmid containing the luciferase gene under control of a promoter was transfected into LNCap cells and sonicated with 1 MHz ultrasound at 0.5 W/cm(2), 10% DF for 60s, 13 promoters showed more than 10-fold enhancement compared with their counterparts without sonication 12h after sonication. As to their responsiveness to sonication, the best two promoters were then compared to clone 880-8, a derivative from clone 880 that was created by random introduction of point mutations and was shown to have an improved response to X-ray irradiation. We then took clone 880-8 for further analyses since it showed the highest enhancement to sonication, though not statistically significant from the others. Next, we employed a retrovirus vector and stably introduced the luciferase gene under control of clone 880-8 into LNCap cells to establish a cell line. When the cell line was sonicated with 1 MHz ultrasound at 0.5 W/cm(2), 10% DF for 60s, luciferase expression was enhanced up to 14.8-fold 12h after sonication. We then established another cell line by replacing the luciferase gene with the fcy::fur gene, a suicide gene, and when the cell line was sonicated with 1 MHz ultrasound at 0.5 W/cm(2), 10% DF for 60s, expression of the gene was enhanced, showing the maximum expression 12-24h after sonication. When the cells were incubated in medium containing 5-fluorocytosine, cell survival ratio decreased dose dependently with 5-fluorocytosine only after sonication treatment, suggesting this promoter could be utilized for gene expression control with ultrasound.
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. ogawa@med.u-toyama.ac.jp
Ryohei
R
Ogawa
Akihiro
A
Morii
Akihiko
A
Watanabe
Zheng-Guo
ZG
Cui
Go
G
Kagiya
Takashi
T
Kondo
Nobutaka
N
Doi
Loreto B
LB
Feril
D045744:Cell Line, Tumor; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D041722:Genes, Transgenic, Suicide / Q000235:genetics; D015316:Genetic Therapy; D006801:Humans; D008297:Male; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D013010:Sonication; D016385:TATA Box / Q000235:genetics; D014162:Transfection / Q000379:methods*
D004247:DNA