KIT of IMC-G4 cells was more apparently phosphorylated under the conditions of both presence and absence of IL-3. also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology. gene, exon 17, gastrointestinal stromal tumor, germline mutation, model mouse, histamine synthesis, mast cell neoplasm, imatinib, nilotinib Introduction The function of the c-gene product, KIT, is essential for the development of five cell lineages such as erythrocytes, melanocytes, germ cells, mast cells and interstitial cells of Cajal (ICCs) [1]. ICCs are considered to be a pacemaker of peristalsis in gastrointestinal tract. Since mutant mice have loss-of-function mutations of the c-gene, they show five phenotypes of anemia, white coat color, infertility, deficiency of mast cells and abnormal gastrointestinal movement due to the impaired development of above-mentioned five cell lineages [1]. On the other hand, gain-of-function mutations of the c-gene are known to be detected in leukemia, malignant melanomas, seminomas, mast cell neoplasms and gastrointestinal stromal tumors (GISTs) at different frequency [1]. Since ICCs and GISTs express both KIT and CD34 in common and since ICCs are the only proper cells in gastrointestinal tract that express KIT, ICCs are considered to be the origin of GISTs [1,2]. Most of the sporadic GISTs have somatic gain-of-function mutations of the c-gene. The mutations are most frequently detected at exon 11 (70-80%), less frequently at exon 9 (approximately 10%) and rarely at exon 8, exon 13 and exon 17 (less than 2% each) [1-4]. Various types of exon 11 mutations are observed, while exon 9, exon 13 and exon 17 mutations usually show the particular types. In addition, several types of germline gain-of-function mutations of the c-gene have been detected in approximately 30 families with multiple GISTs [5-29]. Again, the mutations in the familial GISTs are most frequently detected at exon 11, but exon 8, exon 13 and exon 17 mutations are also reported [5-29]. Development of multiple GISTs with ICC hyperplasia is the essentially observed phenotype in the families [5-29], but some families have mast cell neoplasms [9,14] and/or hyperpigmentation of the digital, perioral and perineal regions [6,8,9,11,14,15,20,26]. Three types of mouse models for familial GISTs with germline gain-of-function mutations of the c-gene have been generated through the knock-in strategy. One has a deletion of codon 558 (valine) at exon 11 (KIT-del-Val558) related to human being familial GIST case with human being KIT-del-Val559 [30]. Another has a substitution mutation Eliglustat tartrate of codon 641 from lysine to glutamic acid at exon 13 (KIT-Lys641Glu) related to human being familial GIST case with human being KIT-Lys642Glu [31], and the other has a substitution of codon 818 from aspartic acid to tyrosine at exon 17 (KIT-Asp818Tyr) related to human being familial GIST case with human being KIT-Asp820Tyr [32]. All types of model mice show development of a cecal GIST with ICC hyperplasia. As mentioned above, mast cell neoplasms and hyperpigmentation at the particular sites are sometimes observed in human being multiple GIST family members, and ectopic pigmentation at the lower esophagus is observed in some of the model mice [30,32]. On the other hand, mast cell figures in the skin of the three model mice are different from each other as compared to respective crazy type mice. Quantity of pores and skin mast cells in the model mice with KIT-del-Val558 raises [30], that with KIT-Lys641Glu decreases [31], and that with KIT-Asp818Tyr is definitely unchanged [32]. In sporadic human being mast cell neoplasms, most of the c-gene mutations are present at exon 17 (KIT-Asp816Val or KIT-Asp816Tyr) [33,34]. In mouse neoplastic mast cell lines, moreover, KIT-Asp814Val or KIT-Asp814Tyr related to human being KIT-Asp816Val or KIT-Asp816Tyr has been reported [35]. In human being mast cell neoplasms, several types of mutations of the c-gene have also been recognized at exon 8, exon 9 or exon 11 [36]. In the present study, we characterized the various types of.Nilotinib also inhibited KIT autophosphorylation in Ba/F3 cells expressing KIT-del-Val558&Val559 in the concentration of 0.1 microM, and did that in BMMCs derived from KIT-Asp818Tyr/KIT-Asp818Tyr mice and IMC-G4 cells and Ba/F3 cells expressing KIT-Asp818Tyr in the concentration of 0.1 microM (B). Inhibitory effect of imatinib and nilotinib about cell proliferation Imatinib almost completely inhibited cell proliferation of Ba/F3 cells expressing KIT-del-Val558&Val559 in the concentration of 0.1 microM. we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) related to human being familial GIST case with human being KIT-Asp820Tyr to clarify the part of the c-gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were Ace utilized for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term tradition were also used. Ultrastructure, histamine material, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We shown that KIT-Asp818Tyr did not impact ultrastructure and proliferation profiles but did histamine material in BMMCs. IMC-G4 cells Eliglustat tartrate experienced an additional novel c-gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell collection to investigate mast cell biology. gene, exon 17, gastrointestinal stromal tumor, germline mutation, model mouse, histamine synthesis, mast cell neoplasm, imatinib, nilotinib Intro The function of the c-gene product, KIT, is essential for the development of five cell lineages such as erythrocytes, melanocytes, germ cells, mast cells and interstitial cells of Cajal (ICCs) [1]. ICCs are considered to be a pacemaker of peristalsis in gastrointestinal tract. Since mutant mice have loss-of-function mutations of the c-gene, they display five phenotypes of anemia, white coating color, infertility, deficiency of mast cells and irregular gastrointestinal movement due to the impaired development of above-mentioned five cell lineages [1]. On the other hand, gain-of-function mutations of the c-gene are known to be recognized in leukemia, malignant melanomas, seminomas, mast cell neoplasms and gastrointestinal stromal tumors (GISTs) at different rate of recurrence [1]. Since ICCs and GISTs communicate both KIT and CD34 in common and since ICCs are the only appropriate cells in gastrointestinal tract that communicate KIT, ICCs are considered to be the origin of GISTs [1,2]. Most of the sporadic GISTs have somatic gain-of-function mutations of the c-gene. The mutations are most frequently recognized at exon 11 (70-80%), less regularly at exon 9 (approximately 10%) and hardly ever at exon 8, exon 13 and exon 17 (less than 2% each) [1-4]. Various types of exon 11 mutations are observed, while exon 9, exon 13 and exon 17 mutations usually show the particular types. In addition, several types of germline gain-of-function mutations of the c-gene have been recognized in approximately Eliglustat tartrate 30 family members with multiple GISTs [5-29]. Again, the mutations in the familial GISTs are most frequently recognized at exon 11, but exon 8, exon 13 and exon 17 mutations will also be reported [5-29]. Development of multiple GISTs with ICC hyperplasia is the essentially observed phenotype in the family members [5-29], but some families possess mast cell neoplasms [9,14] and/or hyperpigmentation of the digital, perioral and perineal areas [6,8,9,11,14,15,20,26]. Three types of mouse models for familial GISTs with germline gain-of-function mutations of the c-gene have been generated through the knock-in strategy. One has a deletion of codon 558 (valine) at exon 11 (KIT-del-Val558) related to human being familial GIST case with human being KIT-del-Val559 [30]. Another has a substitution mutation of codon 641 from lysine to glutamic acid at exon 13 (KIT-Lys641Glu) related to human being familial GIST case with human being KIT-Lys642Glu [31], and the other has a substitution of codon 818 from aspartic acid to tyrosine at exon 17 (KIT-Asp818Tyr) related to human being familial GIST case with human being KIT-Asp820Tyr [32]. All types of model mice show development of a cecal GIST with ICC hyperplasia. As mentioned above, mast cell neoplasms and hyperpigmentation at the particular sites are sometimes observed in human being multiple GIST family members, and ectopic pigmentation at the lower esophagus is observed in some of the model mice [30,32]. On the other hand, mast cell figures in the skin of the three.