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FLT3 TKD突变的激活机制，我们通过对鼠FLT3 TKD突变体的所有22个细胞质酪氨酸残基产生一系列单Tyr→Phe取代突变，分析了突变体组成型激活和下游信号转导的关键酪氨酸残基（ mFLT3Asp838Val) Tyr845Phe、Tyr892Phe 和 Tyr922Phe 取代受到抑制mFLT3Asp838Val 本身的磷酸化、Erk1/2、STAT3 和 STAT5 的激活以及因子无关的细胞增殖和存活相反，这三种 Tyr→Phe 突变部分抑制但维持了配体依赖性激活和抗凋亡活性。野生型 FLT3 的突变，表明这些酪氨酸残基对于 mFLT3Asp838Val.2 的组成型激活和信号转导更为关键。我们检查了细胞周期调节分子在Notch-和HOXB4诱导的造血干细胞自我更新过程中的表达，发现这两种分子都诱导c-myc的表达……此外，我们确定HOXB4激活了c-my启动子。通过-195和-161 bp之间的元件我们还证明，在存在适当细胞因子的情况下，单独诱导c-myc活性足以增强造血干细胞的自我更新。 Myc/ERT，揭示了对4-羟基他莫昔芬的c-myc活性。这些表明Notch和HOXB4通过诱导c-myc来诱导造血干细胞的自我更新。3.我们敲除Anamorsin产生的小鼠。阿那莫辛^<-/->小鼠由于胎儿肝脏中确定的造血功能失败而导致胚胎致死，尽管胎儿肝脏中的造血干/祖细胞数量这些小鼠的肝脏并没有减少，骨髓细胞，特别是红细胞集落的形成被严重破坏，而且，Anamorsin^-/->红细胞在终末成熟过程中引发细胞凋亡，微阵列分析显示。表明 Bcl-xL 和 Jak2 在 Anamorsin^<-/-> 小鼠胎儿肝脏中的表达严重受损。