Establishment And Study Of An Imatinib Resistance Cell Line K562R

objective: To establish an imatinib resistance cell line and to study its resistant principia which is hoped to be conducive to study on imatinib resistance as a new cell model.Methods:1.The induction and purification of imatinib resistance cell line K562R: K562 cells were cultured in gradually increased concentrations of imatinib over a period of several months to generate their resistance line and were purificated through single cell clone.2. The analysis of K562R characteristics:The growth curve was drawn by counting cells and the cytotoxic effects of K562S and K562R treated with different concentration of imatinib were analyzed by MTT assay.3. The preliminary studies of the resistant mechanisms:RT-PCR, flow cytometry, sequence analysis were used to clarify the possible mechanisms of the resistance.Results:1. The establishment of K562R and its characteristics: We established six imatinib resistance cell lines by single cell clone which showed exponential growth in 5.0uM imatinib. Proliferation data showed that cell growth of K562R was not inhibited in 5.0uM imatinib, whereas the parental sensitive cell was significantly inhibited by up to 0.5μM imatinib. The IC50 of K562R was 10.17±0.8510μM which was 15 times higher than that of the parental cell. However, it was testified that there was no statistic difference after comparing the cytotoxic effects of the different cell lines.2. The analysis of the induced resistance mechanisms:By flow cytometry, P-gp was detected on K562R-6 cell line, indicating that the posible resistance mechanisms may be due to P-gp mediated effiux. Sequence analysis of the 863 bp Abl kinase domain showed no mutation in K562R cell.Conclusions:1. In vitro, We establish six resistant cell lines with resistance to 5.0uM Imatinib and 15 fold resistance as compared with that ofK562S.2. The potential mechanisms of resistance of K562R-6 involve amplification of P-gp.3. Sequence analysis of the 863bp ABL kinase domain did not show mutation and it needs a long way to definitude the resistance of imatinib.