TY - JOUR T1 - Enhanced 9-(2-Phosphonylmethoxyethyl)adenine Secretion by a Specific, Indomethacin-Sensitive Efflux Pump in a Mutant 9-(2-Phosphonylmethoxyethyl)adenine-Resistant Human Erythroleukemia K562 Cell Line JF - Molecular Pharmacology JO - Mol Pharmacol SP - 907 LP - 917 DO - 10.1124/mol.54.5.907 VL - 54 IS - 5 AU - Sigrid Hatse AU - Erik De Clercq AU - Jan Balzarini Y1 - 1998/11/01 UR - http://molpharm.aspetjournals.org/content/54/5/907.abstract N2 - We have investigated the molecular basis of the 100-fold resistance of mutant human erythroleukemia K562/PMEA-1 cells to the antiproliferative potential of 9-(2-phosphonylmethoxyethyl)adenine (PMEA). Upon exposure to high PMEA concentrations, comparable intracellular PMEA levels were initially observed in mutant K562/PMEA-1 and wild-type K562/0 cells, indicating that PMEA influx was unaltered. However, after 4 hr of exposure to 0.2 μm[3H]bis(pivaloyloxymethyl)-PMEA [bis(POM)-PMEA], the total intracellular level of unphosphorylated and mono- and diphosphorylated PMEA was 2.8-fold lower in K562/PMEA-1 than in K562/0 cells. Increased PMEA secretion from K562/PMEA-1 cells (compared with K562/0 cells) became more pronounced upon prolonged exposure to bis(POM)-PMEA; after 24 hr, K562/PMEA-1 cells showed 65-fold lower total intracellular PMEA levels than K562/0 cells and at 48 hr, >400-fold less total PMEA was detected in K562/PMEA-1 cells. In addition, PMEA phosphorylation was 25- to 50-fold less efficient in K562/PMEA-1 than in K562/0 cells, pointing to an additional defect at the level of the metabolism of PMEA. The PMEA efflux mechanism was shown to be temperature- and azide-dependent, was markedly inhibited by indomethacin, and did not recognize adenine nucleotides or the phosphorylated metabolites of 3′-azido-3′-deoxythymidine. Also, over a 28-hr period, PMEA efflux was not affected by an inhibitor of RNA synthesis (actinomycin D) or protein synthesis (cycloheximide). Our studies revealed that resistance of K562/PMEA-1 cells to PMEA is the combined result of a severely impaired PMEA phosphorylation on the one hand, and an enhanced PMEA secretion by a highly specific, indomethacin-sensitive efflux pump, different from the classical P-glycoprotein- and multidrug resistance protein-mediated resistance mechanisms, on the other hand. ER -