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Development of novel bioluminescent assay for telomerase and its application to diagnostic of cancer

Research Project

Project/Area Number 22590537
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Laboratory medicine
Research InstitutionShowa University

Principal Investigator

ARAKAWA Hidetoshi  昭和大学, 薬学部, 教授 (70129807)

Project Period (FY) 2010 – 2012
Project Status Completed (Fiscal Year 2012)
Budget Amount *help
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2012: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2011: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2010: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywordsテロメラーゼ / 生物発光 / ピロリン酸 / ルシフェラーゼ / 腫瘍 / テロメア / ガン診断
Research Abstract

Telomeres are specific structures found at the end of chromosomes in eukaryotes. In human chromosomes, telomeres consist of thousands of copies of 6 base repeats (TTAGGG). Although human somatic cells induce cell-death by reduction of telomeric repeats with cell division, cancer cells induce extension of telomeric repeats by telomerase. Telomerase is a ribonucleoprotein that synthesizes and directs the telomeric repeats onto the 3’ end of existing telomeres using its RNA component as a template. Therefore, telomerase participates in malignant transformation or immortalization of a cell, and attracts attention as anticancer drug screening and diagnostic tumor marker. Recently, telomeric repeat amplification protocol (TRAP) is used as universal method of telomerase assay. However, these approaches generally employ acrylamide gel electrophoresis after amplifying telomeric repeat by polymerase chain reaction (PCR); as a result, the TRAP method requires considerable time and skill for us. I … More n this study, for rapid and high sensitive detection of telomerase activity, we developed novel telomerase assay using bioluminescent detection method; that is, pyrophosphates produced by telomerase reaction and PCR are converted ATP by pyruvate phosphate dikinase (PPDK), and ATP is detected by firefly luciferin-luciferase reaction.As a result, the detection limit of pyrophosphate was 1.0×10-15mol/assay. For optimal bioluminescent assay of telomerase activity, we designed the specific primers to the telomeric repeat and selected the efficient Ta q polymerase for PCR. Sequences of the sense and antisense primers for PCR amplification of telomerase reaction product were 5’-AATCCGTCGAGCAGAGTT-3’ and 5’-CTAACCCTAACCCTAACC-3’, respectively. In study of Taq polymerase, efficient PCR amplification could be obtained by use of TITANIUM Taq DNA polymerase. After the telomerase reaction and subsequent PCR, the released pyrophosphate was detected by the proposed bioluminescent assay. As a result, positive cell (500 cells) and inactive cell (prepared by heating at 85℃ for 10 min) could be clearly identified. Then, time course of bioluminescent intensity are examined. The maximum bioluminescence intensity was maintained for about two minutes. The detection limit of cells with telomerase was examined with 500, 250, 100, 50, 10, 5, 1 cell. 1 cell/assay was detectable by telomerase reaction for 30 minutes and PCR consisting of 33 cycles. PCR cycle number also was examined, 25 cycles was detectable. Presently, we are examining simpler and rapid bioluminescent detecting method, and we are developing its applying to clinical chemistry of cancer and to basic research such as regenerative medicine. Less

Report

(4 results)
  • 2012 Annual Research Report   Final Research Report ( PDF )
  • 2011 Annual Research Report
  • 2010 Annual Research Report
  • Research Products

    (13 results)

All 2013 2012 2011

All Journal Article (5 results) (of which Peer Reviewed: 2 results) Presentation (8 results) (of which Invited: 1 results)

  • [Journal Article] Development of novel telomerase assay using PPDK-luciferin-luciferase detection system2013

    • Author(s)
      K. Karasawa, Y. Sano, H. Arakawa
    • Journal Title

      Luminescence

      Volume: - Issue: 1 Pages: 52-57

    • DOI

      10.1002/bio.2501

    • Related Report
      2012 Annual Research Report 2012 Final Research Report
    • Peer Reviewed
  • [Journal Article] Development of bioluminescent enzyme immnoassay for S-Equol using firefly luciferase and its application to the assessment of Equol-priducer status2011

    • Author(s)
      T. Minekawa, A. Kambegawa, K. Shindome, H. Ohkuma, K. Abe, H. Maekawa and H. Arakawa
    • Journal Title

      Chem. Pharm. Bull

      Volume: 59 Pages: 84-87

    • Related Report
      2012 Final Research Report
  • [Journal Article] Bioluminescent assay for nitric oxide utilizing the biological enzyme activity of soluble guanylate cyclase2011

    • Author(s)
      Y. Sano, M. Seki, S. Abe, S. Suzuki and H. Arakawa
    • Journal Title

      Analytical Letters

      Volume: 44 Pages: 2832-2840

    • Related Report
      2012 Final Research Report 2011 Annual Research Report
  • [Journal Article] Practical application of bioluminescence enzyme immunoassay using enhancer for firefly luciferin-luciferase bioluminescence2011

    • Author(s)
      T. Minekawa, H. Ohkuma, K. Abe, H. Maekawa and H. Arakawa
    • Journal Title

      Luminescence

      Volume: 26 Pages: 167-171

    • Related Report
      2012 Final Research Report 2011 Annual Research Report
  • [Journal Article] Development of bioluminescent enzyme immnoassay for S-Equol using firefly luciferase and its application to the assessment of Equol-2011

    • Author(s)
      Takayuki Minekawa
    • Journal Title

      Chem.Pharm.Bull.

      Volume: 59 Pages: 84-87

    • Related Report
      2011 Annual Research Report
    • Peer Reviewed
  • [Presentation]2012

    • Organizer
      日本臨床化学会(招待講演)
    • Place of Presentation
      盛岡
    • Year and Date
      2012-09-06
    • Related Report
      2012 Final Research Report
  • [Presentation]2012

    • Organizer
      第25回バイオメデカル分析科学シンポジウム
    • Place of Presentation
      東京
    • Year and Date
      2012-08-08
    • Related Report
      2012 Final Research Report
  • [Presentation]2012

    • Organizer
      17thInternational Symposium on Bioluminescence and Chemiluminescence
    • Place of Presentation
      カナダ
    • Year and Date
      2012-05-28
    • Related Report
      2012 Final Research Report
  • [Presentation] 新規腫瘍マーカー・テロメラーゼ分析法の開発2012

    • Author(s)
      荒川秀俊
    • Organizer
      第25回バイオメデイカル分析科学シンポジウム
    • Place of Presentation
      東京
    • Related Report
      2012 Annual Research Report
  • [Presentation] 先端分析によるテロメラーゼ測定法2012

    • Author(s)
      荒川秀俊
    • Organizer
      日本臨床化学会
    • Place of Presentation
      盛岡市
    • Related Report
      2012 Annual Research Report
    • Invited
  • [Presentation] Development of novel telomerase assay by bioluminescent detection method2012

    • Author(s)
      Hidetoshi Arakawa
    • Organizer
      17th International Symposium on Bioluminescence and Chemiluminescence
    • Place of Presentation
      カナダ、トロント
    • Related Report
      2012 Annual Research Report
  • [Presentation]2011

    • Organizer
      Molecular diagnostics summit Europe
    • Place of Presentation
      ドイツ
    • Year and Date
      2011-10-12
    • Related Report
      2012 Final Research Report
  • [Presentation] Development of ultra sensitive bioluminescence EIA for biomarkers2011

    • Author(s)
      Hidetoshi Arakawa
    • Organizer
      Molecular Diagnostics Summit Europe
    • Place of Presentation
      ドイツ(ハノーファー)
    • Year and Date
      2011-10-12
    • Related Report
      2011 Annual Research Report

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Published: 2010-08-23   Modified: 2019-07-29  

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