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

Naotaka Fukui, Yukio Kageyama, Yotsuo Higashi, Kazunori Kihara, Shinae Kizaka-Kondoh, Masahiro Hiraoka, Toshiaki Shinojima, Kenjiro Suzuki, Mototsugu Oya

Research output: Contribution to journalArticle

2 Citations (Scopus)

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. Conclusion: 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.

Original languageEnglish
Pages (from-to)497-504
Number of pages8
JournalInternational Journal of Clinical Oncology
Volume19
Issue number3
DOIs
Publication statusPublished - 2014

Fingerprint

interferon alfa-2b
Renal Cell Carcinoma
Interferons
Binding Sites
Cell Line
Interferon-alpha
Genes
Therapeutics
Deferoxamine
Response Elements
Conditioned Culture Medium
Hypoxia
In Vitro Techniques
Genetic Therapy
Cell Survival
Japan
Plasmids
Enzyme-Linked Immunosorbent Assay

Keywords

  • Gene therapy
  • Hypoxia-inducible factor
  • Interferon
  • Renal cell carcinoma
  • von Hippel-Lindau protein

ASJC Scopus subject areas

  • Oncology
  • Surgery
  • Hematology
  • Medicine(all)

Cite this

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. / Fukui, Naotaka; Kageyama, Yukio; Higashi, Yotsuo; Kihara, Kazunori; Kizaka-Kondoh, Shinae; Hiraoka, Masahiro; Shinojima, Toshiaki; Suzuki, Kenjiro; Oya, Mototsugu.

In: International Journal of Clinical Oncology, Vol. 19, No. 3, 2014, p. 497-504.

Research output: Contribution to journalArticle

Fukui, Naotaka ; Kageyama, Yukio ; Higashi, Yotsuo ; Kihara, Kazunori ; Kizaka-Kondoh, Shinae ; Hiraoka, Masahiro ; Shinojima, Toshiaki ; Suzuki, Kenjiro ; Oya, Mototsugu. / 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. In: International Journal of Clinical Oncology. 2014 ; Vol. 19, No. 3. pp. 497-504.
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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. Conclusion: 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.",
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T1 - 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

AU - Fukui, Naotaka

AU - Kageyama, Yukio

AU - Higashi, Yotsuo

AU - Kihara, Kazunori

AU - Kizaka-Kondoh, Shinae

AU - Hiraoka, Masahiro

AU - Shinojima, Toshiaki

AU - Suzuki, Kenjiro

AU - Oya, Mototsugu

PY - 2014

Y1 - 2014

N2 - 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. Conclusion: 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.

AB - 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. Conclusion: 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.

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KW - Hypoxia-inducible factor

KW - Interferon

KW - Renal cell carcinoma

KW - von Hippel-Lindau protein

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