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Analysis of MIL-AF4 leukeinogeriesis for development of molecular target therapy.

Research Project

Project/Area Number 17591014
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Hematology
Research InstitutionNippon Medical School

Principal Investigator

DAN Kazuo  Nippon Medical School, Graduate School of Medicine, Professor, 大学院・医学研究科, 教授 (90142538)

Co-Investigator(Kenkyū-buntansha) INOKACKI Koiti  Nippon Medical School, Faculty of Medicine, Professor, 医学部, 教授 (10203267)
Project Period (FY) 2005 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2005: ¥2,600,000 (Direct Cost: ¥2,600,000)
Keywordst(4;11)(q21;q23) / MLL / AF4 / Flt3 / ITD / D835V / 2nd hit
Research Abstract

1. We successfully established a cell line expressing MLL-AF4 from proB ALL patients with t(4;11)(q21;q23).
2. We have also succeeded to clone cDNA MLL-AF4 from this cell line, and we got MI length FLT3 cDNA from OM-GENE. After making FLT3-ITD and FLT3D835V mutation (FLT3-mut) by site-directed mutagenesis, we used them to confirm leukemogenetic mechanisms. Murine IL3 dependent call line 32Dc was transduced with lentiviral vector(pCL20c MpFLT3 EFlaDsRedExp). human MLL-AF4 cDNA (pCL20cMp+MLL/AF4sEF1a-GFP) and/or FLT3-ITD or-mut (pCL20c MpFLT3 EFlaDsRedExp). After confirming both mRNA expressions by RT-PCR and protein expressions by Western blot, each clone was isolated by FACSVantageSE.
3. We examined growth profile under IL3 deprivation in each transduced 32Dc cell lines. 32Dc with FLT3-mut, and MLL-AF4 temporally grew and tended to show anti-apoptotic effeet (day 5), but finally did not grow and demonstrated apoptotic cell death (day 10) under IL3 deprivation. By contrast, 32Dc with FLT3-ITD, and MLL-AF4 and FLT3-mut (MLL-FLT3-mut) could permanently grow and tended to show anti-apoptotic effect.
4.We examined their ability to confer clonogenic growth on 32Dc in semisolid media with presence of IL3.Only 32Dc with FLT3-ITD and MLL-FLT3-mut could form coloies in semisolid media without IL3.
5. We examined myeloid differentiation of each transduced 32Dc cell lines in response to granulocyte colony stimulating factor (G-CSF). Stimulation by G-CSF couldn't promote morphologic differentiation of 32Dc with MLL-FLT3-mut, but promoted 32Dc with MLL-AF4 or FLT3-mut to granulocytes.
These results clarify that MLL-AF4 plays an important role in a multi step leukemogenesis. Especially MLL rearrangement plays anti-apoptotic effect and AF4 rearrangement inhibit myeloid differentiation. However FLT3-mut may be necessary and sufficient for secondary genotoxicity on leukemogenesis.

Report

(3 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • Research Products

    (4 results)

All 2007 2006

All Journal Article (4 results)

  • [Journal Article] Pure whith cell aplasia : report of the・・・2007

    • Author(s)
      Tamura H, Okamoto M, et al.
    • Journal Title

      Int J Hematol 85(2)

      Pages: 97-100

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Clinical features of polycythemia vera and essential・・・2006

    • Author(s)
      Dan K, Yamada T, et al.
    • Journal Title

      Int J Hematol 83(5)

      Pages: 439-442

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Diagnostic application of flow cytometric・・・2006

    • Author(s)
      Ogata K, et al.
    • Journal Title

      Blood 108(3)

      Pages: 1037-1044

    • Related Report
      2006 Annual Research Report
  • [Journal Article] Oral administration of imatinib to P230・・・2006

    • Author(s)
      Inami M, Inokuchi K, et al.
    • Journal Title

      Int J Hematol 84(4)

      Pages: 346-353

    • Related Report
      2006 Annual Research Report

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Published: 2005-04-01   Modified: 2016-04-21  

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