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Gene therapy of melanoma using adenoviras vector

Research Project

Project/Area Number 12670824
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Dermatology
Research InstitutionEhime University

Principal Investigator

MURAKAMI Sinji  Ehime University, University Hospital, Assistant Professor, 医学部・附属病院, 講師 (50175626)

Co-Investigator(Kenkyū-buntansha) HANAKAWA Yasushi  Ehime University, Faculty of Medicine, Instructor, 医学部, 助手 (90284398)
SHIRAKATA Yuji  Ehime University, Faculty of Medicine, Instructor, 医学部, 助手 (50226320)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2001: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2000: ¥1,900,000 (Direct Cost: ¥1,900,000)
Keywordsmelanoma / ASK1 / adenovirus vector / apoptosiss / gene therapy / nude mouse / メラノーマ
Research Abstract

In order to assess whether adenovirus vector is useful for melanoma, we constructed adenovirus vector carrying apoptosis signal regulating kinase 1. We introduced full length of ASK1 cDNA into plasmid, transfected to E. coli and then amplified. Next, we purified the plasmid and introduced into cosmid cassette. We co-transfected both cosmid cassette and DNA-TPC into 293 cells and further cultured. After several days, we obtained adenovirus vector carrying ASK1 DNA. Then, adenovirus vector was amplified in 293 cells, and vector was purified with cesium chloride gradient centrifugation method followed by dialysis, and stored at -70 C. Twelve melanoma cell line were cultured in 10% FCS/DME or RPMI1640 and divided into 2 groups according to the cell growth assessed by growth assay. We infected adenovirus vector into melanoma cell line and observed by phase contrast microscopy. The infected melanoma cells turned into apoptosis at a density of MOI=20. These results indicate that ASK! induces apoptosis of melanoma cells. Next we investigated the efficacy of adenovirus vector for gene therapy of melanoma in vivo condition. One million of melanoma cells were injected into the subcutaneous of dorsal skin of nude mouse. After 4 weeks, the tumor grew about 15 mm in diameter. Adenovirus vector was injected directly into the tumor, and then we measured the size of tumor. The size of ASK1 injected melanoma decreased whereas that of control virus injected melanoma increase. Histopathological analysis showed a lot of apoptotic cells in the ASK1 injected tumor. Taking these results together, we conclude that ASK1 has the potential to reduce the size of melanoma and should be useful tool for melanoma gene therapy.

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (6 results)

All Other

All Publications (6 results)

  • [Publications] Tohyama M, Shirakata Y, Yamasaki K, Sayama K, Hashimoto K: "Differentiated keratinocytes are responsible for TNF-alpha regulated production of macrophage inflammatory protein 3alpha/CCL20, a potent chemokine for Langerhans cells"Journal of Deramatological Science. 27(2). 130-139 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sayama K, Hanakawa Y, Shirakata Y, Yamasaki K, Sawada Y, Sun L, Yamasaki K, Ichijo H, Hashimoto K: "Apoptosis signal regulating kinase 1(ASK1) is an intracellular inducer of keratinocyte differentiation"Journal of Biological Chemistry. 276. 999-1004 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tohyama M., Shirakara Y., Yamasaki K., Sayama K., Hashimoto K.: "Differentiated keratinocytes are responsible for TNF-alpha regulated production of macrophage inflammatory protein 3alpha/CCL20, a potent chemokine for Langerhans cells"Journal of Dermatological Science. 27 (2). 130-139 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Sayama K., Hanakawa Y., Shirakata Y., Yamasaki K., Sawada Y., Sun L., Yamanishi K., Ichijo H., Hashimoto K.: "Apoptosis signal regulating kinase 1 (ASK1) is an intracellular inducer of keratinocyte differentiation"Journal of Biological Chemistry. 276. 999-1004 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Tohyama M, Shirakara Y, Yamasaki K, Sayama K, Hashimoto K: "Differentiated keratinocytes are responsible for TNF-alpha regulated production of macrophage inflammatory protein 3alpha/CCL20, a potent chemokine for Langerhans cells"Journal of Dermatological Science. 27(2). 130-139 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Sayama K, Hanakawa Y, Shirakata Y, Yamasaki K, Sawada Y, Sun L, Yamanishi K, Ichijo H, Hashimoto K: "Apoptosis signal regulating kinase 1 (ASK1) is an intracellular induce of keratinocyte differentiation"Journal of Biological Chemistry. 276. 999-1004 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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