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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）。尽管KIT的酶活性受其配体干细胞因子（SCF）的调节，但在激酶结构域处用varine或酪氨酸取代天冬氨酸-814导致KIT的组成性失活。为了检测突变KIT的转化潜力，将c-kit^<Val814>cDNA导入鼠白细胞介素-3依赖性IC-2肥大细胞系，其是培养的肥大细胞来源的但不表达KIT，以及具有逆转录病毒载体的正常造血祖细胞。表达KIT-2的IC-2细胞<Val814>在悬浮培养中显示出因子非依赖性生长，并在裸鼠中产生肿瘤。在<Val814>不添加外源性生长因子的情况下，从用KIT^感染的骨髓细胞中产生粒细胞/巨噬细胞、肥大细胞集落和混合红细胞/髓细胞集落。KIT^<Val814>-感染的骨髓细胞的移植导致移植小鼠中急性白血病的发展。此外，表达KIT-1的转基因小鼠<Val814>发生急性白血病或恶性淋巴瘤。我们还研究了在FMA 3小鼠肥大细胞瘤细胞系中KIT组成性激活的分子机制。对c-kit的整个编码区进行测序，结果表明，<814><814>FMA 3细胞中不存在瓦尔^或Tyr^突变，并且FMA 3细胞的c-kit cDNA在胞膜结构域携带编码Thr-Gln-Leu-Pro-Tyr-Asp-His的21个碱基对的框内缺失。在导入21 bp缺失的FMA 3型c-kit cDNA的IC-2细胞中，KIT在没有SCF刺激的情况下被组成型激活并二聚化，表达FMA 3型KIT的IC-2细胞在没有IL-3和SCF的悬浮培养中生长，并在裸鼠体内发生白血病。这些结果表明突变KIT在肥大细胞和造血干细胞的肿瘤发生中的直接作用。