Chronic Imatinib Mesylate Exposure Leads to Reduced Intracellular Drug Accumulation in K562 Cells Measured by High-Performance Liquid Chromatography.

Imatinib mesylate is a selective tyrosine kinase inhibitor that is effective in the treatment of Philadelphia-positive chronic and acute leukemia. Unfortunately disease recurrence due to drug resistance is still the challenge to obtain cure. To investigate the possible mechanisms of imatinib resistance, we established a BCR/ABL⁺ cell line with resistance to imatinib (K562-R) by culturing a wild-type K562 cell line (K562-W) in gradually increased concentrations of imatinib over a period of 6 months, which can survive and was maintained in vitro at 3.0 umol/L of imatinib. K562-W and K562-R cells were cultured to logarithmic phase without imatinib, then treated with imatinib at 3.0 umol/L for 2 hours with or without cyclosporin A (2 ug/ml). The cells were washed thoroughly and incubated for further 0, 15, 30 and 60 minutes. Then cell suspensions containing 2×10⁶ cells was measured for imatinib intracellular concentrations by High-Performance Liquid Chromatography (HPLC) after repeated freezing and thawing these cells. The imatinib concentration of K562-W cell is 0.31 umol/L, which is stable even after incubation for 1 hour. The imatinib concentration of K562-R is decreased significantly, being 0.21, 0.08, 0.06 and less than 0.01 umol/L at different incubation times mentioned above. The high level of cyclosporin A showed no effects on imatinib concentrations of both K562-W and K562-R cells. Flow cytometry showed there was no MDR expression of K562-R, whereas there was a small population (about 20%) of K562-W cells with dim expression of MDR. Compared to K562-W cells, the copies of BCR/ABL fusion genes determined by the real-time PCR showed that there were no amplifications of the fusion genes in K562-R cells and BCR/ABL cDNA sequencing showed no mutations of the imatinib ATP binding sites. The western blot assay showed that the expression of heat shock protein 90(Hsp90) was increased in K562-R cells. These results indicate that the intracellular eliminations of imatinib may be one of possible mechanisms for K562-R cells and Hsp90 as a critical molecular chaperone might be involved in the process.