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Assessment of genetic changes in the carcinogenesis of squamous cell lung cancer using in situ PCR.

Research Project

Project/Area Number 07457286
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Thoracic surgery
Research InstitutionTohoku University

Principal Investigator

SATO Masami (1996-1997)  Tohoku University, Institute of Development, Aging and Cancer, Instructor, 加齢医学研究所, 助手 (30250830)

斎藤 泰紀 (1995)  東北大学, 加齢医学研究所, 助手 (90153824)

Co-Investigator(Kenkyū-buntansha) 藤村 重文  東北大学, 加齢医学研究所, 教授 (40006078)
佐藤 雅美  東北大学, 加齢医学研究所, 助手 (30250830)
Project Period (FY) 1995 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 1997: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1996: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1995: ¥2,000,000 (Direct Cost: ¥2,000,000)
Keywordsin situ PCR / K-ras / point mutation / PCR / in situ hybridization / direct in situ PCR / indirect in situ PCR / K-RAS / in situ hybridization / 早期肺癌 / 発癌 / 遺伝子異常 / 肺癌 / 癌関連遺伝子
Research Abstract

In situ PCR is a new technique for the localization of low copy number sequences. We investigated to establish a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR.Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and postfixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated : TGT) and NCI-H460 (wild type : GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the seme preparation confirmed desired amplification.We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations. We also applied direct in situ PCR,but this method failed to detect the point mutation. Huge non-specific incorporation of digoxigenin to several length of DNA fragments caused low specificity of this technique.

Report

(4 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • 1995 Annual Research Report
  • Research Products

    (20 results)

All Other

All Publications (20 results)

  • [Publications] Sato, et al.: "Sequential loss of hereozygosity in the progression of squmous cell carcinoma of the lung." British Journal of Cancer. (in press). (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sagawa, et al.: "K-ras point mutation occurs in the early stage of carcinogenesis in lung cancer." British Journal of Cancer. 77(5). 720-723 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sagawa, et al.: "Detection of K-ras point mutation by insitu PCR in cell suspensions." Lung Cancer. 17. 181-195 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, et al.: "PTENI is frequently mutated in primary endometrial carcinomas." Nature Genetics. 17. 143-144 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, et al.: "Sequential loss of heterozygosity in the progression of squamous cell carcinoma of the lung" British Journal of Cancer. (in press). (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sagawa, et al.: "K-ras point mutation occurs in the early stage of carcinogenesis in lung cancer" British Journal of Cancer. 77-5. 720-723 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sagawa, et al.: "Detection of k-ras point mutation by in situ PCR in cell suspensions" Lung Cancer. 17. 181-195 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, et al.: "PTENI is frequently mutated in primary endometrial carcinomas" Nature Genetics. 17. 143-144 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Sato, et al.: "Sequential loss of heterozygosity in the progression of squamous cell carcinoma of the lung." British Journal of Cancer. (in press). (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sagawa, et al.: "K-ras point mutation occursin the early stage of carcinogenesis in lung canser" British Journal of Cancer. 77(5). 720-723 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sagawa, et al.: "Detection of K-ras point mutation by in situ PCR in cell suspensions." Lung Cancer. 17. 181-195 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Sato, et al.: "PTEN1 is frequently mutated in primary endometrial carcinomas." Nature Genetics. 17. 143-144 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] 佐藤雅美: "肺癌細胞株を用いたK-ras点突然変異のin situ PCR法による検出の試み" 肺癌. 36・5. 670 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 佐藤雅美: "早期肺扁平上皮癌の遺伝子異常" 肺癌. 36・5. 498 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 佐藤雅美: "肺癌集検による早期癌スクリーニング" 日本臨床. 54・5. 1410-1414 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 佐藤雅美: "非小細胞肺癌におけるBcl-2蛋白,アポトーシスおよびp53蛋白の免疫組織学的検討" 肺癌. 36・6. 785-790 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 佐川元保: "PCR法によるI期(_PT_1NoMo)肺扁平上皮癌組織中のHPV16,18,33の検出" 胸部外科. 48. 359-362 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 薄田勝男: "気管支異形成上皮・胸部X線無所見肺癌の核山体形成部位(Ag-NOR)の検討" 日胸外会誌. 43. 836-840 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] 遠藤千顕: "PCR-SSCP法による肺扁平上皮癌組織におけるp53遺伝子異常の検出" 日本臨床細胞学会雑誌. 34. 366 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Saito Yasuki: "Development of dysplasin of the hronchys tollowed by cytelogy and bronchescopy" Acta Cytologin. 39. 348 (1995)

    • Related Report
      1995 Annual Research Report

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Published: 1995-04-01   Modified: 2016-04-21  

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