Construction of artificial promoters sensitively responsive to sonication in vitro

  • Akihiko Watanabe
  • Satoshi Kakutani
  • Ryohei Ogawa
  • Sung-il Lee
  • Toru Yoshida
  • Akihiro Morii
  • Go Kagiya
  • Loreto B. FerilJr.
  • Hideki Fuse
  • Takashi Kondo
Original Article



To develop artificial promoters that are activated in response to sonication and to determine these properties in vitro.


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.


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/cm2 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.


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.


promoter transcription oxidative stress 


  1. 1.
    Herzog RW, Cao O, Hagstrom JN, et al. Gene therapy for treatment of inherited haematological disorders. Expert Opin Biol Ther 2006;6:509–522.PubMedCrossRefGoogle Scholar
  2. 2.
    Fiandaca M, Forsayeth J, Bankiewicz K. Current status of gene therapy trials for Parkinson’s disease. Exp Neurol 2008;209:51–57.PubMedCrossRefGoogle Scholar
  3. 3.
    Gaffney MM, Hynes SO, Barry F, et al. Cardiovascular gene therapy: current status and therapeutic potential. Br J Pharmacol 2007;152:175–188.PubMedCrossRefGoogle Scholar
  4. 4.
    Cross D, Burmester JK. Gene therapy for cancer treatment: past, present and future. Clin Med Res 2006;4:218–227.PubMedCrossRefGoogle Scholar
  5. 5.
    Rissanen TT, Rutanen J, Yla-Herttuala S. Gene transfer for therapeutic vascular growth in myocardial and peripheral ischemia. Adv Genet 2004;52:117–164.PubMedCrossRefGoogle Scholar
  6. 6.
    Clackson T. Controlling mammalian gene expression with small molecules. Curr Opin Chem Biol 1997;1:210–218.PubMedCrossRefGoogle Scholar
  7. 7.
    Martinelli R, Simone VD. Short and highly efficient synthetic promoters for melanoma-specific gene expression. FEBS Lett 2005;579:153–156.PubMedCrossRefGoogle Scholar
  8. 8.
    Gehrke S, Jerome V, Muller R. Chimeric transcriptional control units for improved liver-specific transgene expression. Gene 2003;322:137–143.PubMedCrossRefGoogle Scholar
  9. 9.
    Li X, Eastman EM, Schwartz RJ, et al. Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences. Nat Biotechnol 1999;17:241–245.PubMedCrossRefGoogle Scholar
  10. 10.
    Ogawa R, Lee S, Kagiya G, et al. Construction of X-ray-inducible promoters through cis-acting element elongation and error-prone polymerase chain reaction. J Gene Med 2007;10:316–324.CrossRefGoogle Scholar
  11. 11.
    Rebillard X, Soulie M, Chartier-Kastler E, et al. High-intensity focused ultrasound in prostate cancer: a systematic literature review of the French Association of Urology. BJU Int 2008;101:1205–1213.PubMedCrossRefGoogle Scholar
  12. 12.
    Umemura S, Yumita N, Umemura K, et al. Sonodynamically induced effect of rose bengal on isolated sarcoma 180 cells. Cancer Chemother Pharmacol 1999;43:389–393.PubMedCrossRefGoogle Scholar
  13. 13.
    Tachibana K, Feril LB, Jr, Ikeda-Dantsuji Y. Sonodynamic therapy. Ultrasonics 2008;48:253–259.PubMedCrossRefGoogle Scholar
  14. 14.
    Yoshida T, Kondo T, Ogawa R, et al. Combination of doxorubicin and low-intensity ultrasound causes a synergistic enhancement in cell killing and an additive enhancement in apoptosis induction in human lymphoma U937 cells. Cancer Chemother Pharmacol 2007;61:559–567.PubMedCrossRefGoogle Scholar
  15. 15.
    Watanabe A, Otake R, Nozaki T, et al. Effects of microbubbles on ultrasound-mediated gene transfer in human prostate cancer PC3 cells: comparison among Levovist, YM454, and MRX-815H. Cancer Lett 2008;265:107–112.PubMedCrossRefGoogle Scholar
  16. 16.
    Newman CM, Bettinger T. Gene therapy progress and prospects: ultrasound for gene transfer. Gene Ther 2007;14:465–475.PubMedCrossRefGoogle Scholar
  17. 17.
    Abdollahi A, Domhan S, Jenne JW, et al. Apoptosis signals in lymphoblasts induced by focused ultrasound. FASEB J 2004;18:1413–1414.PubMedGoogle Scholar
  18. 18.
    Feril LB Jr, Kondo T, Cui ZG, et al. Apoptosis induced by the sonomechanical effects of low-intensity pulsed ultrasound in a human leukemia cell line. Cancer Lett 2005;221:145–152.PubMedCrossRefGoogle Scholar
  19. 19.
    Kagiya G, Ogawa R, Tabuchi Y, et al. Expression of heme oxygenase-1 due to intracellular reactive oxygen species induced by ultrasound. Ultrason Sonochem 2006;13:388–396.PubMedCrossRefGoogle Scholar
  20. 20.
    Ogawa R, Lee S, Izumi H, et al. Enhancement of artificial promoter activity by ultrasound-induced oxidative stress. Ultrason Sonochem 2009;16:379–386.PubMedCrossRefGoogle Scholar
  21. 21.
    Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2001.Google Scholar
  22. 22.
    Tabuchi Y, Kondo T, Ogawa R, et al. DNA microarray analyses of genes elicited by ultrasound in human U937 cells. Biochem Biophys Res Commun 2002;290:498–503.PubMedCrossRefGoogle Scholar
  23. 23.
    Tabuchi Y, Ando H, Takasaki I, et al. Identification of genes responsive to low-intensity pulsed ultrasound in a human leukemia cell line Molt-4. Cancer Lett 2007;246:149–156.PubMedCrossRefGoogle Scholar
  24. 24.
    Kagiya G, Ogawa R, Hatashita M, et al. Generation of a strong promoter for Escherichia coli from eukaryotic genome DNA. J Biotechnol 2005;115:239–248.PubMedCrossRefGoogle Scholar

Copyright information

© The Japan Society of Ultrasonics in Medicine 2009

Authors and Affiliations

  • Akihiko Watanabe
    • 1
  • Satoshi Kakutani
    • 2
  • Ryohei Ogawa
    • 2
  • Sung-il Lee
    • 3
  • Toru Yoshida
    • 4
  • Akihiro Morii
    • 1
  • Go Kagiya
    • 5
  • Loreto B. FerilJr.
    • 6
  • Hideki Fuse
    • 1
  • Takashi Kondo
    • 2
  1. 1.Department of Urology, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  2. 2.Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  3. 3.Institute of Biomedical ScienceKansai Medical UniversityMoriguchiJapan
  4. 4.Second Department of Surgery, Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan
  5. 5.School of Allied Health SciencesKitasato UniversityKanagawaJapan
  6. 6.Department of Anatomy, School of MedicineFukuoka UniversityFukuokaJapan

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