Constitutive activation of c-kit in FMA3 murine mastocytoma cells caused by deletion of seven amino acids at the juxtamembrane domain

Abstract

A peculiar point mutation results in constitutive activation of c-kit receptor tyrosine kinase (KIT) in three different tumor mast cell lines; ie, the HMC-1, P-815, and RBL-2H3. Because constitutive activation of KIT was also observed in the FMA3 mouse mastocytoma cell line, we investigated the molecular mechanism. Sequencing of the whole coding region of the c-kit showed that the point mutation found in HMC- 1, P-815, and RBL-2H3 cells was absent in FMA3 cells and that the c-kit cDNA of FMA3 cells carried an in-frame deletion of 21 base pairs (bp) encoding Thr-Gln-Leu-Pro-Tyr-Asp-His at codons 573 to 579 at the juxtamembrane domain. The FMA3-type c-kit cDNA with 21 bp deletion was introduced into the IC-2 cell line, which was derived from murine cultured mast cells. IC-2 cells were dependent on interleukin (IL)-3 and did not express KIT on the surface. In IC-2 cells introduced with the FMA3-type c-kit cDNA, KIT was constitutively phosphorylated on tyrosines and activated. Moreover, the FMA3-type KIT was dimerized without the stimulation by stem cell factor (SCF), a ligand for KIT. The spontaneously dimerized FMA3-type KIT without SCF binding was not internalized even after the activation. IC-2 cells expressing the FMA3- type KIT grew in suspension culture without IL-3 and SCF and became leukemic in nude athymic mice. The deletion of seven amino acids at the juxtamembrane domain appeared to be a new activating mutation of KIT that might be involved in neoplastic growth of mast cells.

c‐kit proto‐oncogene exon 8 in‐frame deletion plus insertion mutations in acute myeloid leukaemia

“…c-kit receptor activation due to small deletions, albeit in the juxta-membrane domain, has been previously reported. Tsujimura et al (1996) described a seven amino acid deletion (Thr573-His579) in a murine mast cell line (FMA3), and Hirota et al (1998) reported c-kit activation in human gastrointestinal stromal tumours resulting from deletions located within an 11-amino acid stretch (Lys550-Val560). The role of the fourth immunoglobulin-like domain in activating the transforming potential of the related receptor, c-fms, has been highlighted (van Daalen Wetters et al, 1992).…”

“…Recently, two groups have reported receptor activation resulting from small juxtamembrane domain deletions. Tsujimura et al (1996) reported a seven amino acid deletion (Thr573-His579) in a murine mast cell line (FMA3), and Hirota et al (1998) reported c-kit activation in human gastrointestinal stromal tumours resulting from deletions located within an 11-amino acid stretch (Lys550-Val560).…”

Roles for deregulated receptor tyrosine kinases and their downstream signaling molecules in hematologic malignancies

“…The JM mutations of c-Kit were first identified in mast cell lines: a point mutation (Val560Gly) in a human mast cell leukemia line, HMC-1; the deletion of seven amino acids (ΔThr573-His579) in a murine mastocytoma cell line (FMA3). (12,13) In addition, JM c-Kit mutations were detected in canine mast cell tumors, one of the most popular and aggressive neoplasms in dog. (14) Subsequently, similar JM mutations of c-Kit were found to be present in 50 -80% of cases of gastrointestinal stromal tumors (GISTs).…”

Imatinib inhibits various types of activating mutant kit found in gastrointestinal stromal tumors

“…3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylcetrazolium bromide (MTT) colorimetric assay was carried out according to previously described methods. 29 Briefly, cells were plated in a 96-well plate at a concentration of 5 ϫ 10 4 cells/well and cultured in DMEM supplemented with FBS in the presence of Imatinib at the concentration of 0, 0.0001, 0.001, 0.01, 0.1, 1, 10 or 100 M respectively for 48 hr. Then the cells were further cultured for 4 hr in the presence of MTT (5 mg/ml).…”

“…A tumorigenicity assay with administration of Imatinib in nude mice was carried out according to previously described methods with minor modification. 29,30 The transfectants and original Ba/F3 cells were subcutaneously injected into the posterior flank of nude mice, which had been purchased from Japan SLC Co. Ltd. (Hamamatsu, Shizuoka, Japan). For each type of transfectants, 12 mice were studied.…”