Study On The Potentiality Of Malignant Transformation Of The C-kit Oncogene Mutation In Human Gastrointestinal Stromal Tumor

Aims:To analyze the C-kit gene mutation type with PCR and DNA sequencing and try to find out new mutant sites of GIST; to observe the function state of the mutant C-kit gene in GIST, the recombinant eukaryotic expression vector plasmids with mutant C-kit cDNA was constructed and stably transfected into human embryonic kidney cell line(293 cell line), and the effect of the recombinant plasmids to the cell proliferation and cell cycle was detected; to difine the malignant transformation effect of the mutant C-kit by injecting nude mice with the transfectant with mutant c-kit cDNA. Methods1 Genomic DNA of GIST was extracted from 8 paraffin-embedded samples, C-kit gene mutation of GIST was screened by PCR and DNA sequencing technique.2 Wild-type C-kit cDNA was coloned from embryonic brain tissue of human by RT-PCR technique.3 Site-directed mutagenesis of the wild type C-kit cDNA was performed according to the c-kit mutations that we had detected and reported previous by literature (as a positive control).4 The wild-type and mutant C-kit cDNA were cloned to pcDNA3 eukaryotic expression vector.5 These recombinant plasmids were stably transfected into human embryonic kidney cell line. The changes of cell proliferation and cell cycle of the transfectants were detected by MTT clolorimetic assay and flow cytometer, respectively.6 Tumorgenesis of transfecants with mutant C-kit cDNA in nude mice was detected. Result1 C-kit mutation in GIST: C-kit gene mutations were found in three of eight GIST samples.The first was an insertion of 12 bps at Codon 579; the second was a deletion of 6 bp among Codon 557 and 579 which resulted in the loss of codon 557 and 579.557-559;the third was point mutations at condon 559 and 562which caused the substitution of valine to asparagic acid and isoleucine to methionine.2 The wild-type C-kit cDNA was cloned from human embryonic brain tissue by RT-PCR and was proved to have the right sequence in comparison with the data from Genbank by DNA sequencing. The mutant C-kit cDNA was obtained by site-directed mutagenesis of the wild type C-kit cDNA and were proved by DNA sequencing;3 The 3 recombinant plasmids with wild-type and mutant C-kit cDNA was proved to have been constructed successfully by restriction digestion of BamHl and Xhol.4 hi contrast with the transfecants with wild-type C-kit cDNA and empty pcDNA3 vector, the proliferation activity and growth rate of the transfecants with mutant C-kit cDNA are increased significantly;The analysis result of cell cycle showed that more ratio of the transfecants with mutanted C-kit cDNA remains in proliferation phase ( S+G2+M) than the transfecants without mutated C-kit cDNA.5 Transfecants with mutant C-kit cDNA could grow autonomously and generate tumor in vivo in nude mice.but transfecants with wild C-kit cDNA and empty pcDNA3 cannot generate tumor in nude mice.Conclusions1 Three C-kit gene mutation at exonll was detected in GIST:Two of which lie in hot spot area(Codon550-560) according to reports of literature;the remain one lie outside this hot spot area.This suggested that the C-kit gene mutations in Chinese might occur in this hot spot area and that one mutation took place outside the hot spot area suggested the sites of C-kit mutation in GIST are various and the types of C-kit mutation are complex.2 The effect of mutant C-kit cDNA to the the proliferation activity and growth rate of human embryonic kidney cell line was observed .The results showed that in contrast with the transfecants with wild-type C-kit cDNA and empty pcDNA3 vector, the proliferation activity and growth rate of the transfecants with mutantC-kit cDNA are increased significantly and higher ratio of cells remained in proliferation phase . This suggested that the C-kit mutation that we have detected in GIST is gain-of-function mutation, and mutation of c-kit gene could increase proliferation activity of human cells and bring more cells in resting phase into proliferation phase, but the...