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Studies on Molecular Diagnostic Tests for Drug Resistance in Leukemia Cells: Molecular Mechanisms of Drug Resistance Analyzed through Genomic Instability and Its application to Molecular Diagnostic Tests

Research Project

Project/Area Number 10672186
Research Category

Grant-in-Aid for Scientific Research (C)

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

Principal Investigator

MIYACHI Hayato  Tokai University, School of Medicine, Associate Professor, 医学部, 助教授 (20174196)

Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordsleukemia cells / multidrug resistance / point mutation / genomic instability / dihydrofolate reductase
Research Abstract

Various gene alterations are involved in drug resistance in leukemia cells. In order to understand the mechanism that underlies emergence of cells with such gene alterations in human leukemia, we performed clonal analysis of the gene expression of mutant dihydrofolate reductase (DHFR) and mdr1 in trimetrexate-resistant human leukemia MOLT-3 cells. 70- and 60-fold trimetrexate-resistant sublines were developed in the presence or the absence of exogenous supply of thymidine (MOLT-3/TMQィイD270ィエD2/Th+, MOLT-3/TMQィイD260ィエD2/Th-, respectively). All of the clonal lines from the two sublines expressed the mutated DHFR mRNA, with a base change (T→C) at the second position of codon 31, as well as the wild type mRNA, in accordance with cross-resistance to methotrexate. On the other hand, mdr1 mRNA expression was demonstrated by reverse-transcription polymerase chain reaction only in clonal lines from MOLT-3/TMQィイD270ィエD2/Th+ cells. Polymerase chain reaction-based assay for separate microsatellites, mfd27 and mfd41, demonstrated genomic instability among clonal and subclonal lines of MOLT-3. The clonal analysis of polymorphic microsatellites also suggested that emergence of MDR in trimetrexate-resistant MOLT-3 cells in thymidine was not only heterogeneous but also fluently expanding among clones. Genomic instability may have a role in establishment and clonal evolution of drug resistance in leukemia cells. The cells would be useful as an in vitro model system for studies on mechanism of clinical drug resistance. To assess the status of the mismatch repair system, we analyzed the integrity of the DNA mismatch repair genes, including hMLH1 and hMSH2, using immunohistochemistry and polymerase chain reaction. However, we have so far not identified any alteration of expression or structure of the genes. Molecular bases behind the genomic instability and drug resistance would provide informative markers for molecular diagnostic tests for evaluation of drug resistance.

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (13 results)

All Other

All Publications (13 results)

  • [Publications] Miyachi Hayato, et al.: "Microsatellite instability and clonal heterogeneity of MDR1 messenger RNA expression in trimetrexate-resistant human leukemia MOLT-3 cells developed in thymdine"International Journal of Cancer. 82. 63-69 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Miyachi Hayato, et al: "Altered expression of CD45 isoforms in differentiation of acute leukemia"American Journal of hematology. 62. 159-164 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kobayashi Hiroyuki et al: "Competitive RT-PCR assay for quantification of human multidrug resistance 1(MDR1) gene expression in fresh leukemia cells"Journal of Laboratory and Clinical Medicine. (印刷中). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Takemura Yuzuru, et al: "Variable expression of folylglutamate syn thetuse gene at the level of mRNA transcription in human leukemia cell lines sensitive, or made resistant to various an tipsute drugs"Anti-Cancer Drugs. 10. 677-683 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kobayashi Hiroyuki, et al.: "Molecular characterization of human acute leukemia cell line resistant to ZD9331, a non-polyglutamable thymidylote synthase inhibitors"Cancer Chemotherapy and Pharmacology. 42. 105-110 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Miyachi, H., Lin Ma, Takemura Y., et al.: "Microsatellite instability and clonal heterogeneity of mdrl mRNA expression in trimetrexate-resistant human leukemia MOLT-3 cells developed in thymidine."International Journal of Cancer. 82. 63-69 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Miyachi, H., Tanaka, Y., Gondo, K., et al.: "Altered expression of CD45 isoforms in differentiation of acute myeloid leukemia."American Journal of Hematology. 62. 159-164 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kobayashi H, Takemura Y, Miyachi H, et al.: "Competitive RT-PCR assay for quantification of human multidrug resistance 1 (MDR1) gene expression in fresh leukemic cells."Journal of Laboratory and Clinical Medicine. (in press).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Takemura Y, Kobayashi H, Miyachi H.: "Variable expression of folylpolyglutamate synthetase gene at the level of mRNA transcription in human leukemia cell lines sensitive, or made resistant to various antifolate drugs."Anti-Cancer Drugs. 10. 677-683 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kobayashi H, Takemura Y, Miyachi H.: "Molecular characterization of human acute leukemia cell line resistant to ZD9331, a non-polyglutamatable thymidylate synthase inhibitor."Cancer Chemotherapy and Pharmacology. 42. 105-110 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Miyachi Hayato, et al.: "Microsatellite instability and clonal heterogeneity of MDR1 messenger RNA expression in trimetrexate-resistant human leukemia MOLT-3 cells ****loped in thymidinl"International Journal of Cancer. 82. 63-69 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Miyachi Hayato, et al.: "Altered expression of CD45 isoforms in differentiation of awte leukemia"American Journal of Hematology. 62. 159-164 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Miyachi Hayato,et al.: "Microsatellite instability and clonal heterogeneity of MORI messenger RNA expression in trimetrexace-resistant human leukemia MOLT-3 cells developed in thymidine" International Journal of Cancer. 69 印刷中. (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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