Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors

Science

Volumn 279, Issue 5350, 1998, Pages 577-580

  Hirota, Seiichi ^a   Isozaki, Koji ^a   Moriyama, Yasuhiro ^a   Hashimoto, Koji ^a   Nishida, Toshirou ^a   Ishiguro, Shingo ^a   Kawano, Kiyoshi ^a   Hanada, Masato ^a   Kurata, Akihiko ^a   Takeda, Masashi ^a   Tunio, Ghulam Muhammad ^a   Matsuzawa, Yuji ^a   Kanakura, Yuzuru ^a   Shinomura, Yasuhisa ^a   Kitamura, Yukihiko ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CD34 ANTIGEN; COMPLEMENTARY DNA; STEM CELL FACTOR;

ARTICLE; CELL INTERACTION; ENZYME ACTIVITY; GASTROINTESTINAL TUMOR; GENE MUTATION; GENE SEQUENCE; GENETIC TRANSFECTION; MALIGNANT TRANSFORMATION; MEMBRANE POTENTIAL; PRIORITY JOURNAL;

AMINO ACID SEQUENCE; ANIMALS; ANTIGENS, CD34; CELL LINE; CELL TRANSFORMATION, NEOPLASTIC; DIGESTIVE SYSTEM; DNA, COMPLEMENTARY; ESOPHAGEAL NEOPLASMS; GASTROINTESTINAL NEOPLASMS; HUMANS; INTESTINAL NEOPLASMS; LIGANDS; MICE; MICE, NUDE; MOLECULAR SEQUENCE DATA; MUTATION; PHOSPHORYLATION; PHOSPHOTYROSINE; PROTO-ONCOGENE PROTEINS C-KIT; RECOMBINANT PROTEINS; SEQUENCE DELETION; STEM CELL FACTOR; STOMACH NEOPLASMS; TRANSFECTION;

MURINAE;

EID: 15644363454     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5350.577     Document Type: Article

References (45)

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33. The coding region of the human wild-type c-kit was cloned into Xba I site of expression vector pEF-BOS. The Sna BI-Mro I fragment (nucleotide 1141 to 2282) of the human wild-type c-kit in the expression vector was replaced by the corresponding fragments of the mutant-type cDNA obtained from each GIST. The expression vectors were transfected into the 293T HEK cell line by calcium phosphate precipitation (5).
34. The procedures of cell lysis, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and immunoblotting were done as in (5). Mouse mAb to human KIT (MCA955; Serotec, Oxford, UK) was used for the immunoprecipitation. Immunoblotting was done with mouse mAb to phosphotyrosine (4G10; Upstate Biotechnology, Lake Placid, NY) or rabbit polyclonal anti-human KIT (K963).
35. The immune complex kinase assay was done as in (5). Rabbit polyclonal anti-human KIT (K963) was used for the immunoprecipitation.
36. To generate the murine-type c-kit cDNAs containing the same mutation as the GISTs, we performed site-directed mutagenesis (5). The Nde I-SpI I fragment with the mutation was isolated and inserted into the expression vector pEF-BOS containing murine wild-type c-kit cDNA.
37. A Sea I-cut expression vector pEF-BOS containing the mouse-type mutated c-kit cDNA and the Bam HI-cut expression vector pSV2-neo were cotransfected into the IL-3-dependent Ba/F3 murine lymphoid cell line with the use of GENE PULSER II (Bio-Rad Laboratories). After transfection, the cells expressing neomycin-resistant gene were selected by cultivation in medium containing G418 (0.6 mg/ ml) and rmIL-3 for 4 weeks. Cloning of the cells was done with the limiting-dilution method, and the expression of each mutated c-kit was confirmed by flow cytometry, protein immunoblotting, and RT-PCR with sequencing.
38. To quantitate cell proliferation, we used the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma) rapid colorimetric assay. The procedure was done as in (6).
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45. We thank A. Fukuyama and K. Morihana for preparing histological sections. Supported by grants from the Japanese Ministry of Education, Science, Culture, and Sports.