Resistance of CCRF-CEM cloned sublines to 5-fluorodeoxyuridine associated with enhanced phosphatase activities

Abstract

Resistance of human CCRF-CEM leukemic cells in tissue culture to 5-fluoro-2′-deoxyuridine (FdUrd) has been examined following a single drug exposure (FS sublines). In two FS sublines generated by soft agar cloning of FdUrd sensitive cells in the presence of 10 nM FdUrd, the level of drug resistance was maintained at 22- to 30-fold following 1 month growth in the absence of FdUrd. Characteristic of the FS sublines was a decreased accumulation and retention of free intracellular 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP) averaging 3% of FdUrd sensitive cells, a more rapid rate of disappearance of free FdUMP and FdUMP-bound thymidylate synthase (EC 2.1.1.45, 5,10-methylenetetrahydrofolate: dUMP C-methyltransferase), and enhanced alkaline and acid phosphatase activities. There was no significant difference in the number of nucleoside transport sites per cell among the FS sublines and FdUrd-sensitive cells, indicating that the decreased accumulation of FdUMP in the resistant sublines was not the result of impaired FdUrd transport across the plasma membrane. The more rapid turnover of FdUMP-bound TMP synthase observed in the FS sublines was neither accompanied by a decreased stability of the TMP synthase-FdUMP-5,10-methylenetetrahydrofolate ternary complex, nor an enhanced rate of degradation of FdUrd to the less potent agent, 5-fluorouracil. In addition, the growth rates of the two FS sublines were similar to that of FdUrd sensitive cells in medium containing hypoxanthine, methotrexate, and thymidine, indicating that there was no depletion of thymidine kinase (EC 2.7.1.21, ATP:thymidine-5′-phosphotransferase) in the FS sublines. Therefore, we propose that enhanced activities of acid and alkaline phosphatases, which influence the intracellular accumulation and retention of FdUMP, are important determinants of stable FdUrd resistance in CCRF-CEM cells.