A novel gain of function mutant in C-kit gene and its tumorigenesis in nude mice.
OBJECTIVE
To transfect mutant C-kit cDNA at codon 579 into human embryonic kidney cell line to observe its role in the pathogenesis of gastrointestinal stromal tumor (GIST).


METHODS
Eukaryotic expression vectors of pcDNA3-Kit-NW and pcDNA3-Kit-W were constructed. Then pcDNA3-Kit-NW and pcDNA3-Kit-W plasmids were transfected into human embryonic kidney cell line by Lipofectamine. The resistant clone was screened by G418 filtration and identified by sequencing, Western blotting, and immunocytochemical staining. Human embryonic kidney cells were divided into three groups including pcDNA3-Kit-NW, pcDNA3-Kit-W, and vector control groups. Absorbency value with a wavelength of 574 nm was detected by MTT analysis. Mice were injected with three groups of cells. Volume, mass, and histological examinations of the tumors in different groups were measured and compared.


RESULTS
The C-kit gene and mutant C-kit gene were successfully cloned into the eukaryotic expression vector pcDNA3. pcDNA3-Kit-NW and pcDNA3-Kit-W were successfully transfected into human embryonic kidney cell line and showed stable expression in this cell line. Cell proliferating activity had significant differences between pcDNA3-Kit-NW and pcDNA3, pcDNA3-Kit-NW and pcDNA3-Kit-W (P<0.05), respectively. Tumors were only observed in nude mice implanted with cells transfected with pcDNA3-Kit-NW.


CONCLUSIONS
Mutation of C-kit gene increases the proliferation activity of human cells and plays an important role in the malignant transformation of GIST.
Bai
Chen-Guang
CG
Professor of Department of Pathology, Changhai Hospital, Second Military Medical University, Changhai Road, Shanghai 200433, China. madalie@126.com.
Liu
Xiao-Hong
XH
Xie
Qiang
Q
Feng
Fei
F
Ma
Da-Lie
DL
D000818:Animals; D002460:Cell Line; D046152:Gastrointestinal Stromal Tumors / Q000209:etiology / Q000235:genetics* / Q000473:pathology; D015870:Gene Expression; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D014162:Transfection
D019009:Proto-Oncogene Proteins c-kit