title: Development of a novel interferon-α2b gene construct with a repetitive hypoxia-inducible factor binding site and its suppressive effects on human renal cell carcinoma cell lines in vitro.
abstract: BACKGROUND
  Despite the advent of targeted therapies, interferon-alpha (IFN-α) remains a therapeutic option for advanced renal cell carcinoma (RCC), especially in Japan, with a treatment response rate of 15-20 %. To improve the efficacy of IFN-α-based therapies, we evaluated a novel treatment strategy for RCC using an IFN-α2b gene construct with a repetitive hypoxia-inducible factor binding site.
  
  
  METHODS
  We constructed an expression plasmid designated 5HREp-IFN-α2b containing the coding region of the IFN-α2b gene. Five copies of the hypoxia-response element (HRE) sequences were inserted upstream of the IFN-α2b gene, and the construct was transfected into human RCC cell lines ACHN, 786-O and KU19-20. The concentrations of IFN-α2b in the conditioned media were measured by enzyme-linked immunosorbent assay. Cell viabilities were determined by MTS assays.
  
  
  RESULTS
  Construct-induced IFN-α secretion was confirmed in all three cell lines. IFN-α production was significantly enhanced by the hypoxia-mimicking agent deferoxamine mesylate in cell lines expressing the wild-type von Hippel-Lindau (VHL) gene (KU19-20 and ACHN) compared with cells expressing the mutant VHL gene (786-O). The construct exerted significant suppressive effects on the viabilities of all RCC cell lines.
  
  
  CONCLUSIONS
  This is the first study to report on the construction of a cytokine gene with a repetitive hypoxia-inducible factor binding site and its application in the suppression of human cancer cells. Gene therapy using this IFN-α2b gene construct with HREs may represent a novel treatment modality for advanced RCC.
authors.affiliation: Department of Urology, Saitama Cancer Center, 818 Komuro, Ina, Kita-Adachi-gun, Saitama, 362-0806, Japan, na-fukui@cancer-c.pref.saitama.jp.
authors.forename: Naotaka
authors.initials: N
authors.lastname: Fukui
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kageyama
authors.forename: Yotsuo
authors.initials: Y
authors.lastname: Higashi
authors.forename: Kazunori
authors.initials: K
authors.lastname: Kihara
authors.forename: Shinae
authors.initials: S
authors.lastname: Kizaka-Kondoh
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hiraoka
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Shinojima
authors.forename: Kenjiro
authors.initials: K
authors.lastname: Suzuki
authors.forename: Mototsugu
authors.initials: M
authors.lastname: Oya
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D001665:Binding Sites; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D020218:Response Elements; D014162:Transfection; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics / Q000378:metabolism; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D017077:Culture Media, Conditioned; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C497446:VHL protein, human