The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites.
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 approximately 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 micromol/L [(3)H]5-FdUMP, [(3)H]5-FUMP, [(3)H]dUMP, and not [(3)H]5-FUdR, or [(3)H]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 micromol/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 micromol/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.
Pratt
Susan
S
Cancer Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA.
Shepard
Robert L
RL
Kandasamy
Ramani A
RA
Johnston
Paul A
PA
Perry
William
W
Dantzig
Anne H
AH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D005467:Floxuridine / Q000378:metabolism*; D005472:Fluorouracil / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D013866:Thioguanine / Q000494:pharmacology; D013940:Thymidylate Synthase / Q000378:metabolism
D000255:Adenosine Triphosphate; D000964:Antimetabolites, Antineoplastic; D005467:Floxuridine; D005472:Fluorouracil; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D013866:Thioguanine; D013940:Thymidylate Synthase; C120809:ABCC5 protein, human; C025034:doxifluridine