2006 Fiscal Year Final Research Report Summary
Analysis of MIL-AF4 leukeinogeriesis for development of molecular target therapy.
| Project/Area Number |
17591014
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | Nippon Medical School |
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Principal Investigator |
DAN Kazuo Nippon Medical School, Graduate School of Medicine, Professor, 大学院・医学研究科, 教授 (90142538)
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| Co-Investigator(Kenkyū-buntansha) |
INOKACKI Koiti Nippon Medical School, Faculty of Medicine, Professor, 医学部, 教授 (10203267)
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| Project Period (FY) |
2005 – 2006
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| Keywords | t(4;11)(q21;q23) / MLL / AF4 / Flt3 / ITD / D835V / 2nd hit |
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| 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.
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