Transforming effect of PDGFRA gene mutant on the cell function in gastrointestinal stromal tumor.
- Author:
Lei YANG
1
;
Chen-Guang BAI
;
Xiao-Wei HOU
;
Xiao-Hong LIU
;
Da-Lie MA
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Apoptosis; CHO Cells; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Cricetinae; Cricetulus; Gastrointestinal Stromal Tumors; etiology; genetics; pathology; Mice; Mice, Nude; Mutation; Plasmids; Proto-Oncogene Proteins c-kit; metabolism; Receptor, Platelet-Derived Growth Factor alpha; genetics; metabolism; Transfection
- From: Chinese Journal of Oncology 2009;31(7):500-504
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo explore the effect of malignant transformation of the L839P, a new mutation site of the PDGFRA gene, on the pathogenesis of gastrointestinal stromal tumors.
METHODSAll recombinant plasmids were stably transfected into CHO cells by liposomes. Western blotting was used to detect the expression of PDGFRA protein. The cell growth curve was plotted by cell counting. Flow cytometry was used to detect the cell cycle and apoptosis of CHO cell, respectively. The stably transformed cells were inoculated subcutaneously into the back of nude mice and the mice were used to observe the tumorigenesis. Transient transfection of the mutant-type plasmids of PDGFRA gene and the wild-type plasmids of kit gene into the CHO cells was performed. Western blot was used to detect the expression of kit protein and its phosphorylated forms.
RESULTSPDGFRA protein expressed in the negative control, experimental group and positive control, except the empty vector. The growth curve showed that it was accelerated in the experimental group and positive control. The ratios of cells in proliferative phase were 28.4% (blank), 24.5% (negative control), 43.8% (experimental group) and 40.9% (positive control). Their apoptotic indexes were 1.8%, 1.9%, 1.5% and 1.6%, respectively. After three weeks, tumors were observed in the nude mice of experimental group and positive control, inoculated with the stably transformed cells. Moreover, the expression of phosphorylated protein of kit was enhanced after cotransfection of the mutant-type plasmids of PDGFRA and the wild-type plasmid of kit.
CONCLUSIONThe PDGFRA mutant L839P is a gain-of-function mutation and has obviously malignant transforming effect on normal cells, and may activate kit protein accelerating the tumorigenesis. Gastrointestinal stromal tumors;