Tsujimura T, Furitsu T, Morimoto M, Kanayama Y, Nomura S, Matsuzawa Y, Kitamura Y, Kanakura Y
Department of Pathology, Osaka University Medical School, Japan.
Int Arch Allergy Immunol. 1995 Apr;106(4):377-85. doi: 10.1159/000236870.
The c-kit protooncogene encodes a receptor tyrosine kinase that mediates signals required for differentiation, proliferation and survival of mast cells. We have already shown the constitutive activation of c-kit receptor tyrosine kinase (KIT) in a human mast cell leukemia line (HMC-1) and a murine mastocytoma cell line (P-815). We here examined whether such constitutive activation of KIT occurred in the rat tumor mast cell line RBL-2H3 as well, which is frequently used as a tool for studying functions of mast cells. In RBL-2H3 cells, KIT was constitutively phosphorylated on tyrosine and activated in the absence of autocrine production of its ligand, stem cell factor (SCF). Sequencing analysis revealed that one of c-kit genes of RBL-2H3 cells had a point mutation, resulting in amino acid substitution of Tyr for Asp in codon 817. When rat wild-type c-kit cDNA and mutant-type c-kit cDNA encoding KITTyr817 were transfected into cells of a human embryonic kidney cell line (293T), only mutant form KITTyr817 was constitutively phosphorylated on tyrosine and activated in the absence of SCF. Since mutations at the same Asp codon constitutively activated KIT in all the human HMC-1, murine P-815, and rat RBL-2H3 cell lines, and since the incorporation of antisense oligonucleotides of c-kit messenger RNA significantly suppressed the proliferation of RBL-2H3 cells, the activating mutations in the Asp codon of the c-kit gene appeared to be involved in neoplastic growth of mast cells.
c-kit原癌基因编码一种受体酪氨酸激酶，该激酶介导肥大细胞分化、增殖和存活所需的信号。我们已经在人肥大细胞白血病细胞系（HMC-1）和鼠肥大细胞瘤细胞系（P-815）中证实了c-kit受体酪氨酸激酶（KIT）的组成性激活。我们在此研究了这种KIT的组成性激活是否也发生在大鼠肿瘤肥大细胞系RBL-2H3中，该细胞系经常被用作研究肥大细胞功能的工具。在RBL-2H3细胞中，KIT在酪氨酸上被组成性磷酸化并在其配体干细胞因子（SCF）自分泌产生缺失的情况下被激活。测序分析显示，RBL-2H3细胞的一个c-kit基因有一个点突变，导致密码子817处的天冬氨酸被酪氨酸氨基酸取代。当将大鼠野生型c-kit cDNA和编码KITTyr817的突变型c-kit cDNA转染到人胚肾细胞系（293T）的细胞中时，只有突变形式的KITTyr817在酪氨酸上被组成性磷酸化并在SCF缺失的情况下被激活。由于在所有的人HMC-1、鼠P-815和大鼠RBL-2H3细胞系中，相同天冬氨酸密码子处的突变都能组成性激活KIT，并且由于c-kit信使RNA反义寡核苷酸的掺入显著抑制了RBL-2H3细胞的增殖，c-kit基因天冬氨酸密码子处的激活突变似乎与肥大细胞的肿瘤生长有关。
