title: Detailed assessment of gene activation levels by multiple hypoxia-responsive elements under various hypoxic conditions.
abstract: OBJECTIVE
  HIF-1/HRE pathway is a promising target for the imaging and the treatment of intractable malignancy (HIF-1; hypoxia-inducible factor 1, HRE; hypoxia-responsive element). The purposes of our study are: (1) to assess the gene activation levels resulting from various numbers of HREs under various hypoxic conditions, (2) to evaluate the bidirectional activity of multiple HREs, and (3) to confirm whether multiple HREs can induce gene expression in vivo.
  
  
  METHODS
  Human colon carcinoma HCT116 cells were transiently transfected by the constructs containing a firefly luciferase reporter gene and various numbers (2, 4, 6, 8, 10, and 12) of HREs (nHRE+, nHRE-). The relative luciferase activities were measured under various durations of hypoxia (6, 12, 18, and 24 h), O2 concentrations (1, 2, 4, 8, and 16 %), and various concentrations of deferoxamine mesylate (20, 40, 80, 160, and 320 µg/mL growth medium). The bidirectional gene activation levels by HREs were examined in the constructs (dual-luc-nHREs) containing firefly and Renilla luciferase reporter genes at each side of nHREs. Finally, to test whether the construct containing 12HRE and the NIS reporter gene (12HRE-NIS) can induce gene expression in vivo, SPECT imaging was performed in a mouse xenograft model.
  
  
  RESULTS
  (1) gene activation levels by HREs tended to increase with increasing HRE copy number, but a saturation effect was observed in constructs with more than 6 or 8 copies of an HRE, (2) gene activation levels by HREs increased remarkably during 6-12 h of hypoxia, but not beyond 12 h, (3) gene activation levels by HREs decreased with increasing O2 concentrations, but could be detected even under mild hypoxia at 16 % O2, (4) the bidirectionally proportional activity of the HRE was confirmed regardless of the hypoxic severity, and (5) NIS expression driven by 12 tandem copies of an HRE in response to hypoxia could be visualized on in vivo SPECT imaging.
  
  
  CONCLUSIONS
  The results of this study will help in the understanding and assessment of the activity of multiple HREs under hypoxia and become the basis for hypoxia-targeted imaging and therapy in the future.
authors.affiliation: Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa Inage-ku, Chiba, 263-8555, Japan.
authors.forename: Yasuto
authors.initials: Y
authors.lastname: Takeuchi
authors.forename: Masayuki
authors.initials: M
authors.lastname: Inubushi
authors.forename: Yong-Nan
authors.initials: YN
authors.lastname: Jin
authors.forename: Chika
authors.initials: C
authors.lastname: Murai
authors.forename: Atsushi B
authors.initials: AB
authors.lastname: Tsuji
authors.forename: Hironobu
authors.initials: H
authors.lastname: Hata
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Kitagawa
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Saga
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D015179:Colorectal Neoplasms / Q000000981:diagnostic imaging / Q000235:genetics / Q000378:metabolism; D003676:Deferoxamine / Q000494:pharmacology; D048888:Fireflies; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D045325:HCT116 Cells; D006801:Humans; D000860:Hypoxia / Q000000981:diagnostic imaging / Q000235:genetics* / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics* / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008297:Male; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D011859:Radiography; D048790:Renilla; D015899:Tomography, Emission-Computed, Single-Photon; D014162:Transfection
chemical_list: D051793:Hypoxia-Inducible Factor 1; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; D003676:Deferoxamine