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Analysis on the mechanisms of growth, differentiation, and survival of megakaryocytic cells

巨核细胞生长、分化和存活机制分析

基本信息

批准号：
16209033
负责人：
KANAKURA Yuzuru
金额：
$ 29.2万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16209033/
关键词：
FLT3 Notch HOXB4 Anamorsin c-myc hematopoietic Stem Cell apoptosis アナモルシン増殖分化造血器腫瘍

项目摘要

1.To explore activation mechanism of FLT3 TKD mutation, we analysed critical tyrosine residues for the constitutive activation and downstream signaling of the mutant by generating a series of single Tyr→Phe substitution mutant of all 22 cytoplasmic tyrosine residues of murine FLT3 TKD-mutant (mFLT3Asp838Val). Tyr845Phe, Tyr892Phe and Tyr922Phe substitutions suppressed the phosphorylation of mFLT3Asp838Val itself, the activation of Erk1/2,STAT3 and STAT5, and the factor-independent cell proliferation and survival. In contrast, these three Tyr→Phe mutations partially suppressed but maintained the ligand-dependent activation and anti-apoptotic activity of wild-type FLT3, suggesting that these tyrosine residues were more critical for the constitutive activation and signaling of mFLT3Asp838Val.2.We examined the expression of cell cycle regulatory molecules during Notch- and HOXB4-induced self-renewal of hematopoietic stem cells, and found that both molecules induce the expression of c-myc. … More In addition, we determined that HOXB4 activated the c-my promoter through the element between -195 and -161 bp. We also proved that the induction of c-myc activity alone was sufficient for enhancing self-renewal of hematopoietic stem cells in the presence of appropriate cytokines using Myc/ERT, which reveals c-myc activity in response to 4-hydroxytamoxifen. These results indicated that Notch and HOXB4 induce self-renewal of hematopoietic stem cells through the induction of c-myc.3.We generated knock out mice for Anamorsin. Anamorsin^<-/-> mice are embryonic lethal due to the failure of definitive hematopoiesis in the fetal liver, Although the number of hematopoietic stem/progenitor cells in the fetal liver did not decrease in these mice, myeloid and particularly erythroid colony formation was severely disrupted. Also, Anamorsin^<-/-> erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the fetal liver of Anamorsin^<-/-> mice. Less

1.为了探索FLT 3 TKD突变的激活机制，我们通过对小鼠FLT 3 TKD突变体（mFLT 3Asp 838 Val）的所有22个胞浆酪氨酸残基产生一系列单一Tyr→Phe取代突变体，分析了突变体组成性激活和下游信号传导的关键酪氨酸残基。Tyr 845 Phe、Tyr 892 Phe和Tyr 922 Phe取代可抑制mFLT 3Asp 838 Val自身的磷酸化，抑制Erk 1/2、STAT 3和STAT 5的激活，抑制非因子依赖性细胞增殖和存活。相比之下，这三个Tyr→Phe突变部分抑制但维持了野生型FLT 3的配体依赖性激活和抗凋亡活性，表明这些酪氨酸残基对mFLT 3Asp 838 Val的组成性激活和信号传导更为关键。2.我们检测了Notch和HOXB 4诱导的造血干细胞自我更新过程中细胞周期调控分子的表达，并发现这两种分子都诱导c-myc的表达。 ...更多信息此外，我们确定HOXB 4通过-195和-161 bp之间的元件激活c-my启动子。我们还证明，单独诱导c-myc活性足以在适当的细胞因子存在下使用Myc/ERT增强造血干细胞的自我更新，这揭示了c-myc活性响应于4-羟基他莫昔芬。这些结果表明，Notch和HOXB 4通过诱导c-myc的表达来诱导造血干细胞的自我更新。Anamorsin-/->小鼠是胚胎致死的，这是由于胎肝中确定性造血的失败。尽管在这些小鼠中胎肝中造血干细胞/祖细胞的数量没有减少，但髓系和特别是红系集落形成被严重破坏。此外，Anamorsin-1 <-/->红系细胞在终末成熟期间引发凋亡。至于Anamorsin介导的细胞存活的机制，微阵列分析揭示了Bcl-xL和Jak 2的表达在Anamorsin^-/->小鼠的胎肝中严重受损。少