Construction of artificial promoters sensitively responsive to sonication in vitro.
OBJECTIVE
To develop artificial promoters that are activated in response to sonication and to determine these properties in vitro.


METHODS
The binding sites of four transcription factors (nuclear factor-kappa B, activating protein-1, nuclear factor-Y, and CArG element binding factor A) that are activated by oxidative stress were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected HeLa cells from human cervical cancer with a plasmid vector containing such a gene cassette were exposed to sonication, and enhancement of luciferase expression was assessed by dual luciferase assay.


RESULTS
Of 62 promoters constructed, two promoters, designated clone 31 and clone 62 promoters, showed a more than tenfold enhancement 6 h after sonication with 1-MHz ultrasound at 1.0 W/cm(2) for 60 s. These promoters were activated in a dose-dependent manner with the intensity and duration of sonication. The activation was attenuated by addition of dimethyl sulfoxide, an antioxidant, suggesting that oxidative stress was involved. The clone 31 promoter responded to each of two serial sonications. When sonicated 24 h after the first sonication, the peak of promoter enhancement was higher than that after the first sonication.


CONCLUSIONS
A promoter sensitively responsive to sonication was constructed using the above method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.
Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Akihiko
A
Watanabe
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Satoshi
S
Kakutani
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan. ogawa@med.u-toyama.ac.jp.
Ryohei
R
Ogawa
Institute of Biomedical Science, Kansai Medical University, Moriguchi, Japan.
Sung-Il
SI
Lee
Second Department of Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Toru
T
Yoshida
Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Akihiro
A
Morii
School of Allied Health Sciences, Kitasato University, Kanagawa, Japan.
Go
G
Kagiya
Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka, Japan.
Loreto B
LB
Feril
Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Hideki
H
Fuse
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Takashi
T
Kondo
oxidative stress; promoter; transcription