Objective: To establish an imatinib resistance cell line and to study its resistant principia.
Methods: K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line. MTT assay, RT-PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance.
Results: (1) Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5 micromol/L. K562R cells were maintained in the media containing 5 micromol/L imatinib. (2) Proliferation data showed that cell growth of K562R was not inhibited in 5 micromol/ L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2.0 micromol/L imatinib. (3) The IC50 of K562R was about 7.5 micromol/L which was ten times higher than that of the parental cell. (4) HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27. 8-fold). By flow cytometry, P-gp was not detected on K562R cell, indicating that low intracellular imatinib concentration may not be due to P-gp mediated efflux. (5) Sequence analysis of the 374 bp ABL kinase domain showed no mutation in K562R cell.
Conclusion: An imatinib resistance cell line K562R has been successfully established. Low intracellular imatinib concentration is a key link in the chain of cell resistance.