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Neoplastic transformation of mast cells through activating mutations of c-kit receptor

通过激活 c-kit 受体突变实现肥大细胞的肿瘤转化

基本信息

批准号：
07670245
负责人：
TSUJIMURA Tohru
金额：
$ 1.66万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

The c-kit proto-oncogene encodes a receptor tyrosine kinase (KIT). Although the enzymatic activity of KIT is regulated by its ligand, stem cell factoe (SCF), the substitution of varine or tyrosine for aspartic acid-814 at the kinase domain lead to constitutive avtivation of KIT.To examine the transforming potential of the mutant KIT,the c-kit^<Val814> cDNA was introduced into the murine interleukin-3-dependent IC-2 mast cell line, which is of cultured mast cell origin but does not express KIT,and normal hematopoietic progenitor cells with retroviral vector. IC-2 cells expressing KIT^<Val814> showed factor-independent growth in suspension culture and produced tumors in nude athymic mice. From bone marrow cells infected with KIT^<Val814>, granulocyte/macrophage, mast-cell colonies, and mixed erythroid/myeloid colonies developed without the addition of exogenous growth factors. Transplantation of KIT^<Val814>-infected bone marrow cells led to development of acute leukemia in transplanted mice. Furthermore, transgenic mice expressing KIT^<Val814> developed acute leukemia or malignant lymphoma. We also investigated the molecular mechanism of constitutive activation of KIT in the FMA3 mouse mastocytoma cell line. Sequencing of the whole coding region of the c-kit showed that the Val^<814> or Tyr^<814> mutation was absent in FMA3 cells and that the c-kit cDNA of FMA3 cells carried an in-frame deletion of 21 base pairs encoding Thr-Gln-Leu-Pro-Tyr-Asp-His at the juxtamembrane domain. In IC-2 cells introduced with the FMA3-type c-kit cDNA with 21 bp deletion, KIT was constitutively activated and dimerized without the stimulation by SCF.IC-2 cells expressing the FMA3-type KIT grew in suspension culture without IL-3 and SCF and became leukemic in nude athymic mice. These results demonstrate a direct role of the mutant KITs in tumorigenesis of mast cells and hematopoietic stem cells.

c-kit原癌基因编码受体酪氨酸激酶（KIT）。虽然KIT的酶活性受其配体干细胞因子（SCF）的调节，但在激酶结构域以缬氨酸或酪氨酸取代天冬氨酸-814会导致KIT的组成性激活。为了检测突变体KIT的转化潜能，我们将c-kit^<Val814> cDNA导入小鼠白细胞介素-3依赖性的IC-2肥大细胞系，该细胞系来源于培养的肥大细胞，但不表达KIT，并通过逆转录病毒载体将其导入正常造血祖细胞。表达KIT^<Val814>的IC-2细胞在悬浮培养中表现出不依赖因子的生长，并在裸胸腺小鼠中产生肿瘤。从KIT^<Val814>感染的骨髓细胞中，在不添加外源性生长因子的情况下，形成了粒细胞/巨噬细胞、肥大细胞集落和红细胞/骨髓混合集落。KIT^<Val814>-感染的骨髓细胞移植可导致移植小鼠急性白血病的发生。此外，表达KIT^< val814>的转基因小鼠发生急性白血病或恶性淋巴瘤。我们还研究了KIT在FMA3小鼠肥大细胞瘤细胞系中组成性激活的分子机制。c-kit全编码区测序结果显示，FMA3细胞中不存在Val^<814>或Tyr^<814>突变，FMA3细胞c-kit cDNA在近膜域编码thr - gln - leu - pro - tir - asp - his的21个碱基对框内缺失。在缺失21 bp的fma3型c-kit cDNA导入的IC-2细胞中，KIT在没有SCF刺激的情况下被组成性激活和二聚化。表达fma3型KIT的IC-2细胞在无IL-3和SCF的悬浮培养中生长，并在裸胸腺小鼠中发生白血病。这些结果表明突变体kit在肥大细胞和造血干细胞的肿瘤发生中起直接作用。