title: Regulation of gene expression in retrovirus vectors by X-ray and proton beam radiation with artificially constructed promoters.
abstract: BACKGROUND
  We previously obtained an X-ray responsive promoter from 11 promoters that we constructed. In the present study, we aimed to determine the efficiency of our promoter construction method. In addition, the reactivity of the promoter to X-rays in vivo is also investigated.
  
  
  METHODS
  Promoters constructed by linking the TATA box to randomly combined binding sequences of transcription factors activated by radiation were cloned to prepare a promoter library. Combinations of promoters and various genes were stably-transfected into HeLa cells to establish recombinant cell lines, which were then exposed to X-rays or a proton beam to observe gene expression enhancement with or without anti-oxidants. Tumors of luciferase-expressing recombinant cells on mice were exposed to X-rays and promoter activation was evaluated by detecting bioluminescence. As a model for in vitro suicide gene therapy, fcy::fur-expressing recombinant cells were exposed to X-rays before incubation with 5-fluorocytosin. Cell viability was determined with WST-8.
  
  
  RESULTS
  Twenty-five of the 62 promoters in the library enhanced luciferase activity over five-fold, 6 h after receiving 10 Gy of X-ray irradiation, suggesting the effectiveness of our method. Luciferase activity in recombinant cells was enhanced by X-rays and, to a lesser extent, by a proton beam. Anti-oxidants attenuated the enhancement, suggesting the involvement of oxidative stress. Promoters were less reactive to X-rays in tumors on mice. In our suicide gene therapy model, survival of post-irradiated cells decreased dose-dependently with 5-fluorocytosin.
  
  
  CONCLUSIONS
  Our method was efficient in generating radiation responsive promoters. Furthermore, we have successfully shown a potential therapeutic use for one of these promoters.
authors.affiliation: Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan. ogawa@med.u-toyama.ac.jp
authors.forename: Ryohei
authors.initials: R
authors.lastname: Ogawa
authors.forename: Akihiro
authors.initials: A
authors.lastname: Morii
authors.forename: Akihiko
authors.initials: A
authors.lastname: Watanabe
authors.forename: Zheng-Guo
authors.initials: ZG
authors.lastname: Cui
authors.forename: Go
authors.initials: G
authors.lastname: Kagiya
authors.forename: Shigekazu
authors.initials: S
authors.lastname: Fukuda
authors.forename: Kyo
authors.initials: K
authors.lastname: Kume
authors.forename: Takashi
authors.initials: T
authors.lastname: Hasegawa
authors.forename: Masanori
authors.initials: M
authors.lastname: Hatashita
authors.forename: Hironori
authors.initials: H
authors.lastname: Izumi
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ishimoto
authors.forename: Loreto B
authors.initials: LB
authors.lastname: Feril
mesh_terms: D000818:Animals; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects / Q000528:radiation effects; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors / Q000528:radiation effects*; D006367:HeLa Cells; D006801:Humans; D008353:Mannitol / Q000494:pharmacology; D051379:Mice; D009374:Neoplasms, Experimental / Q000378:metabolism; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic / Q000528:radiation effects; D011522:Protons; D012190:Retroviridae; D016385:TATA Box / Q000235:genetics / Q000528:radiation effects; D015533:Transcriptional Activation / Q000235:genetics / Q000528:radiation effects*; D014965:X-Rays
chemical_list: D011522:Protons; D008353:Mannitol; D004121:Dimethyl Sulfoxide