Akihiko Watanabe1, Satoshi Kakutani2, Ryohei Ogawa3, Sung-Il Lee4, Toru Yoshida5, Akihiro Morii1, Go Kagiya6, Loreto B Feril7, Hideki Fuse1, Takashi Kondo2. 1. Department of Urology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. 2. Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan. 3. 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. 4. Institute of Biomedical Science, Kansai Medical University, Moriguchi, Japan. 5. Second Department of Surgery, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. 6. School of Allied Health Sciences, Kitasato University, Kanagawa, Japan. 7. Department of Anatomy, School of Medicine, Fukuoka University, Fukuoka, Japan.
Abstract
PURPOSE: 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.
PURPOSE: 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 humancervical 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.
Authors: Amir Abdollahi; Sophie Domhan; Juergen W Jenne; Mazin Hallaj; Giorgio Dell'Aqua; Martina Mueckenthaler; Alexandra Richter; Heather Martin; Juergen Debus; Wilhelm Ansorge; Kullervo Hynynen; Peter E Huber Journal: FASEB J Date: 2004-07-01 Impact factor: 5.191