| Research Abstract |
Drug resistance is a major obstacle in chemotherapy of leukemia patients. Since the treatment responsiveness is different among patients, the therapy should be tailored to specific subtypes of diseases such as defined by drug resistance. To elucidate mechanism(s) of methotrexate (MTX)-resistance as a possible reason behind treatment failure in high-dose MTX regimens combined with leucovorin (LV) rescue, we established MTX-resistant human T-cell leukemia cell line CCRF-CEM cells in the presence of excessive leucovorin, and characterized their property. Continuous exposure of the cells to escalating concentrations of MTX up to 20μM in the presence of 1000 nM leucovorin resulted in establishment of three MTX-resistant sublines with a wide disparity of resistance degree over 4 logarithmic range by approximately 40-, 900- and 44000-fold, respectively. Transraembrane transport of MTX in these sublines was diminished to 52%, 35% and 12%, respectively. Intracellular retention of MTX in these s
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ublines was not different from the parent cells. A cell growth study in various concentrations of leucovorin showed that cells with higher resistance to MTX required more leucovorin supply for their optimal growth. In parallel with the resistance levels, there was increase in mRNA expression of dihydrofolate reductase gene and decrease in that of thymidylate synthase gene, but no change in that of reduced folate carrier (RFC1) gene, as assessed by northern blot analysis. Sequencing of RFC1 gene in all of the 3 sublines revealed a point mutation in codon 47 (TCC→TTC) resulting in substitution of Phe for Ser residue, and additional deletion of CTG of codon 112 in the subline with the highest resistance. In summary, MTX exposure to CCRF-CEM cells in the presence of 1000 nM leucovorin resulted in an establishment of heterogeneous cell populations with a wide range of transport-mediated MTX-resistance, which was associated with differential alterations of RFC gene. Combination of gene expression and mutations involved in the molecular mechanisms of MTX-resistance may give targets for detection and evaluation of it. Less
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