Analysis of the deregulation mechanism on cell growth and differentiation induced by mutant ELT3

突变型ELT3诱导细胞生长和分化的失调机制分析

• 批准号: 12670982
• 负责人: KIYOI Hitoshi
• 金额: $ 2.11万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670982/
• 关键词: FLT3; tandem duplication; point mutation; Tyrosine kinase; leukemia; tandem duplication

Internal tandem duplication (ITD) of the FLT3 gene (FLT3/ITD) is found in approximately 20% of acute myeloid leukemia (AML) cases, and is strongly associated with the leukocytosis and poor prognosis in AML patients. In addition, we newly identified activating mutations of D835 (D835-Mt) within a TK domain of FLT3. This mutation was found in approximately 7% of AML cases. Mutant FLT3-expressing 32D cells proliferated without IL3 or FL, and mutant FLT3 was constitutively activated. In this study, we elucidated the role of the JM domain in the activation of FLT3. Mutant FLT3 with not only ITD but also an elongating or shortening JM domain transformed 32D cells regardless of the tyrosine residues in the JM domain. These mutant FLT3s were constitutively tyrosine phosphorylated and activated signal-transduction molecules such as SHC, MAP kinase and STAT5a. Notably, co-transfection of the truncated FLT3/ITD lacking kinase and C-terminal domains with the Wt-FLT3 into 32D cells resulted in autonomous proliferation. In these cells, truncated FLT3/ITD generated a hetero-complex with Wt-FLT3, and Wt-FLT3 was constitutively tyrosine phosphorylated. These findings indicated that the FLT3 JM domain plays an important role in receptor activation, and that the length-mutated JM domain induces ligandindependent receptor activation but also activates Wt-FLT3 in a trans-manner.Furthermore, 32D cells are known to differentiate into mature neutrophils in response to G-CSF, while FLT3/ITD-expressing did not differentiate into mature neutrophils when treated with G-CSF. However, treatment with PKC modulator and inhibitors for molecular chaperon recovered the ability to differentiate into mature neutrophils by GCSF.

FLT 3基因的内部串联重复（FLT 3/ITD）在大约20%的急性髓细胞白血病（AML）病例中被发现，并且与AML患者的白细胞增多和不良预后密切相关。此外，我们在FLT 3的TK结构域内新鉴定了D835（D835-Mt）的激活突变。这种突变在大约7%的AML病例中发现。突变型FLT 3表达的32 D细胞在没有IL 3或FL的情况下增殖，并且突变型FLT 3被组成性激活。在这项研究中，我们阐明了JM结构域在FLT 3激活中的作用。无论JM结构域中的酪氨酸残基如何，不仅具有ITD而且具有延长或缩短的JM结构域的突变体FLT 3都转化了32 D细胞。这些突变体FLT 3是组成性酪氨酸磷酸化和激活的信号转导分子，如SHC，MAP激酶和STAT 5a。值得注意的是，缺乏激酶和C-末端结构域的截短的FLT 3/ITD与Wt-FLT 3共转染到32 D细胞中导致自主增殖。在这些细胞中，截短的FLT 3/ITD与Wt-FLT 3产生异源复合物，并且Wt-FLT 3被组成性酪氨酸磷酸化。这些发现表明FLT 3 JM结构域在受体激活中起重要作用，并且长度突变的JM结构域诱导配体依赖性受体激活，但也以反式方式激活Wt-FLT 3。此外，已知32 D细胞响应于G-CSF分化为成熟的中性粒细胞，而表达FLT 3/ITD的细胞在用G-CSF处理时不能分化为成熟的中性粒细胞。然而，用PKC调节剂和分子伴侣抑制剂治疗恢复了通过GCSF分化为成熟中性粒细胞的能力。

项目成果

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Yokozawa T: "Prognostic significance of the cell cycle inhibitor p27Kip1 in acute myeloid leukemia"Leukemia. 14. 28-33 (2000)

Yokozawa T：“细胞周期抑制剂 p27Kip1 在急性髓系白血病中的预后意义”白血病。

Koch P.: "Identification of a novel putative Ran-binding protein and its close homologue"Biochem Biophys Res Commun.. 278(1). 241-9 (2000)

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