Selection and establishment of gefitinib-resistant PC9 cell line and its gene expression profile
10.3781/j.issn.1000-7431.2008.07.003
- Author:
Bo SU
1
Author Information
1. Department of Oncology
- Publication Type:Journal Article
- Keywords:
Cells, cultured;
Drug resistance, neoplasm;
Gefitinib;
Gene expression;
Lung neoplasms
- From:
Tumor
2008;28(7):552-557
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To establish 6 subclones of gefitinib-resistant cells from gefitinib-sensitive lung adenocarcinoma cell line PC9 and study their biological characteristics. Methods: The PC9 cells were induced to mutate by a mutagen MNNG and selected. The subclone of gefitinib-resistant cell was obtained by limited dilution. The sensitivity and IC50 for gefitinib of various cell lines were determined by MTT assay. The different profile of gene expression between the wildtype PC9 cell and the gefitinib-resistant cells were determined by DNA microarray. Fibronectin and collagen IV expressions in various PC9 cell lines were checked by immunohistochemistry. Cell cycle was analyzed by flow cytometry. Results: Two resistant cell lines to gefitinib were obtained, named PC9/ G2 and PC9/G4, respectively, and the IC50 for the gefitinib was 7-10 μmol/L, about 160 to 260 folds higher compared with wildtype PC9 (IC50:0.03-0.05 μmol/L). The doubling time of PC9 cells and PC9/G2 cells were (16.2 ±3.5)h and(36.5 ± 4.8)h, respectively. The gene expression profile had significant difference between wildtype PC9 cells and resistant PC9/G2 cells. Gene function annotation and grouping showed that in PC9/G2 cells fatty acid metabolism- and oxidative phosphorylation-related genes were down-regulated, while glycolysis-genes were up-regulated; the expressions of ribosomal subproteins were down-regulated, and the gene groups located in Golgi Stack, cisternae, clathrin-coated vesicle were up-regulated; the protein-secreting function was enhanced and ubiquitin-proteosome system was activated; DNA repair and helicase genes were also up-regulated. For the genes with protein kinase activity, 30 of them were up-regulated, while 11 of them were down-regulated; 15 genes with phosphoprotein phosphatase activity were up-regulated. For the genes with GTP binding function, 21 of them were up-regulated, while 4 of them were down-regulated; 8 genes related to cytoskeletal movement and cell adhesion were up-regulated; integrin betal subunit, insulin-like receptor and positive regulative factors for NFκB cascade were up-regulated. Conclusion: Six lines of gefitinib-resistant PC9 cells were successfully established. Our results suggested that moderate resistance to gefitinid might be related with extracellular matrix changes and adhesion signaling cascade, while high resistance may be due to activation of instead signaling pathway and the downstream molecules of signaling pathway.
