The role of folates in the development of methotrexate resistance in human leukemia cell line K562

Abstract

The effect of reduced and oxidized folates on the development of methotrexate (MTX) resistance has been examined in human leukemia cell line K562 (K562/S). K562/S cells were made resistant to MTX by soft-agar cloning either in RPMI-1640 medium (K562/MTX-PGA) or in folic-acid-free RPMI-1640 medium containing 10 nM leucovorin (K562/MTX-LV). The optimal concentrations of leucovorin for the growth of K562/S, K562/MTX-PGA and K562/MTX-LV cells were 1 nM, 5 nM and 10 nM respectively. K562/MTX-PGA cells were 24-fold resistant to MTX as noted by impaired MTX transport. In contrast, K562/MTX-LV cells were 26-fold resistant to MTX as noted by gene amplification of dihydrofolate reductase. Furthermore cross-resistance to cytosine arabinoside was only demonstrated in K562/MTX-PGA, while the K562/MTX-LV cells showed no significant cross-resistance to cytosine arabinoside. These results suggest that the type and level of folates used during the development of MTX resistance may play a role in the mechanism for MTX resistance. Leukemia cells that are grown in leucovorin might serve as a model for acquired MTX resistance in vivo.

Large diversity in transport‐mediated methotrexate resistance in human leukemia cell line CCRF‐CEM established in a high concentration of leucovorin

“…The IC 50 values (50%-inhibitory concentration for cell growth) of chemotherapeutic agents in CCRF-CEM/S and CCRF-CEM/MTX-LV was determined by MTT assay, as previously described. 2) LV requirement study. Cells that had been maintained in folatefree RPMI-1640 medium with 10% FCS for 3 days were resuspended in the same fresh medium to a density of 1×10 5 cells/ ml, and finally graded concentrations of LV were added.…”

“…The cells were assayed for radioactivity at six time points from 0 to 60 min, as described previously, with minor modifications. 2) For the retention study of MTX, the cells were incubated in the presence of 0.5 µM [ 3 H]MTX for 2 h, then washed twice and incubated at 37°C. The cells were assayed for radioactivity after 1, 6 and 24 h of incubation, as described above.…”

“…RNA was transferred onto Zeta-Probe nylon membranes (Bio-Rad, Hercules, CA) and then hybridized as previously described. 2) Intensities of blots were quantified by scanning, with National Institutes of Health (NIH) Image software, and normalized as the ratio of CCRF-CEM/S mRNA to those of the sublines. Nucleic acid sequencing of RFC coding region.…”

“…6,7) Our earlier study suggested an important role of folate in the development of MTX resistance. 2) In the presence of a physiological level of a reduced form of folate, LV, MTX-resistant K562 cells emerged with DHFR overexpression due to gene amplification. In contrast, in the presence of an excess of oxidized folate, transport-mediated MTX resistance was established.…”

“…The mechanisms of development of resistance to MTX have been explored in cultured cells. [1][2][3][4] Combinations of an elevated level of the target enzyme, dihydrofolate reductase (DHFR) due to gene amplification, impaired membrane transport of the drug, alteration in DHFR resulting in decreased binding of MTX, reduced polyglutamylation of MTX and a decreased level of thymidylate synthase (TS) have been suggested to be major mechanisms of resistance. To overcome these known mechanisms by which cells become resistant to MTX, highdose MTX regimens have been used in combination with leucovorin (LV) for the rescue of normal cells from MTX cytotoxicity.…”