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.
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 microM [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.
Hatse
S
S
Laboratory of Virology and Experimental Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
De Clercq
E
E
Balzarini
J
J
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis; D018528:ATP-Binding Cassette Transporters / Q000032:analysis; D000225:Adenine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology / Q000502:physiology; D000227:Adenine Nucleotides / Q000378:metabolism; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D000963:Antimetabolites / Q000378:metabolism / Q000494:pharmacology; D000998:Antiviral Agents / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D001692:Biological Transport; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005110:Extracellular Space / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007213:Indomethacin / Q000494:pharmacology*; D020014:K562 Cells / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009154:Mutation; D063065:Organophosphonates; D011355:Prodrugs / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013696:Temperature; D015215:Zidovudine / Q000378:metabolism / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000225:Adenine; D000227:Adenine Nucleotides; D000894:Anti-Inflammatory Agents, Non-Steroidal; D000963:Antimetabolites; D000998:Antiviral Agents; D003513:Cycloheximide; D003609:Dactinomycin; D007213:Indomethacin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D063065:Organophosphonates; D011355:Prodrugs; D011500:Protein Synthesis Inhibitors; D015215:Zidovudine; C089823:bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine; C053001:adefovir