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
- Laboratory of Virology and Experimental Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Abstract
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.
Footnotes
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Send reprint requests to: Dr. Jan Balzarini, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: jan.balzarini{at}rega.kuleuven.ac.be
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These investigations were supported by grants from the Fonds voor Wetenschappelijk Onderzoek−Vlaanderen (G.0104.98), the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek (3–0180-95), the A.S.L.K. Cancer Fund and the Flemish Geconcerteerde Onderzoeksacties (95/5). S.H. is a Research Assistant of the Fonds voor Wetenschappelijk Onderzoek−Vlaanderen.
- Abbreviations:
- PMEA
- 9-(2-phosphonylmethoxyethyl)adenine
- PMEAp
- monophosphorylated form of 9-(2-phosphonylmethoxyethyl)adenine
- PMEApp
- diphosphorylated form of 9-(2-phosphonylmethoxyethyl)adenine
- AZT
- 3′-azido-3′-deoxythymidine
- bis(POM)-PMEA
- bis(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine
- MDR
- multidrug resistance
- mono(POM)-PMEA
- mono(pivaloyloxymethyl)-9-(2-phosphonylmethoxyethyl)adenine
- PMEDAP
- 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine
- PMEG
- 9-(2-phosphonylmethoxyethyl)guanine
- HIV
- human immunodeficiency virus
- HPLC
- high performance liquid chromatography
- PBS
- phosphate-buffered saline
- PBST
- phosphate-buffered saline containing 0.1% Tween-20
- MRP
- multidrug resistance protein
- P-gp
- P-glycoprotein
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- Received April 24, 1998.
- Accepted August 13, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
