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¹ Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana and ² RTP Laboratories, Durham, North Carolina

Requests for reprints: Anne H. Dantzig, Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. Phone: 317-276-5083; Fax: 317-276-6510. E-mail: Dantzig{at}lilly.com

5'-Fluorouracil (5-FU), used in the treatment of colon and breast cancers, is converted intracellularly to 5'-fluoro-2'-deoxyuridine (5-FUdR) by thymidine phosphorylase and is subsequently phosphorylated by thymidine kinase to 5'-fluoro-2'-dUMP (5-FdUMP). This active metabolite, along with the reduced folate cofactor, 5,10-methylenetetrahydrofolate, forms a stable inhibitory complex with thymidylate synthase that blocks cellular growth. The present study shows that the ATP-dependent multidrug resistance protein-5 (MRP5, ABCC5) confers resistance to 5-FU by transporting the monophosphate metabolites. MRP5- and vector-transfected human embryonic kidney (HEK) cells were employed in these studies. In 3-day cytotoxicity assays, MRP5-transfected cells were 9-fold resistant to 5-FU and 6-thioguanine. Studies with inside-out membrane vesicles prepared from transfected cells showed that MRP5 mediates ATP-dependent transport of 5 µmol/L [³H]5-FdUMP, [³H]5-FUMP, [³H]dUMP, and not [³H]5-FUdR, or [³H]5-FU. The ATP-dependent transport of 5-FdUMP showed saturation with increasing concentrations and had a K_m of 1.1 mmol/L and V_max of 439 pmol/min/mg protein. Uptake of 250 µmol/L 5-FdUMP was inhibited by dUMP, cyclic nucleotide, cyclic guanosine 3',5'-monophosphate, amphiphilic anions such as probenecid, MK571, the phosphodiesterase inhibitors, trequinsin, zaprinast, and sildenafil, and by the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid and glybenclamide. Furthermore, the 5-FU drug sensitivity of HEK-MRP5 cells was partially modulated to that of the HEK-vector by the presence of 40 µmol/L 5-nitro-2-(3-phenylpropylamino)-benzoic acid but not by 2 mmol/L probenecid. Thus, MRP5 transports the monophosphorylated metabolite of this nucleoside and when MRP5 is overexpressed in colorectal and breast tumors, it may contribute to 5-FU drug resistance.

Key Words: Multidrug resistance protein • 5-fluorouracil • MRP-5 (ABCC5) • Metabolites

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Received 11/ 1/04; revised 2/ 8/05; accepted 3/ 2/05.