MRP3, an organic anion transporter able to transport anti-cancer drugs

Abstract

The human multidrug-resistance protein (MRP) gene family contains at least six members: MRP1, encoding the multidrug-resistance protein; MRP2 or cMOAT, encoding the canalicular multispecific organic anion transporter; and four homologs, called MRP3, MRP4, MRP5, and MRP6. In this report, we characterize MRP3, the closest homolog of MRP1. Cell lines were retrovirally transduced with MRP3 cDNA, and new monoclonal antibodies specific for MRP3 were generated. We show that MRP3 is an organic anion and multidrug transporter, like the GS-X pumps MRP1 and MRP2. In Madin-Darby canine kidney II cells, MRP3 routes to the basolateral membrane and mediates transport of the organic anion S-(2,4-dinitrophenyl-)glutathione toward the basolateral side of the monolayer. In ovarian carcinoma cells (2008), expression of MRP3 results in low-level resistance to the epipodophyllotoxins etoposide and teniposide. In short-term drug exposure experiments, MRP3 also confers high-level resistance to methotrexate. Neither 2008 cells nor Madin-Darby canine kidney II cells overexpressing MRP3 showed an increase in glutathione export or a decrease in the level of intracellular glutathione, in contrast to cells overexpressing MRP1 or MRP2. We discuss the possible function of MRP3 in (hepatic) physiology and its potential contribution to drug resistance of cancer cells.

Figure 1

Immunohistochemical staining of human liver. (A and B) Immunohistochemical detection of MRP3 in the lateral membranes of cholangiocytes (indicated by arrows) and (baso)lateral membranes of hepatocytes surrounding the portal tracts by using monoclonal antibody M₃II-9. B is an enlargement of A. (C) Immunohistochemical detection of cytokeratin 7 in bile-duct epithelium cells. (D) Immunostaining with M₂III-6 against MRP2, showing canalicular staining of the hepatocytes through out the entire liver lobule.

Figure 2

Western blot analysis of MRP3 protein in parental cells and several clones of transduced 2008 and MDCKII cells. Total cell lysates (10 μg/lane for the 2008 cells and 5 μg for the MDCKII cells) were size fractionated in a 7.5% polyacrylamide gel containing 0.5% SDS. The fractionated proteins were transferred to a nitrocellulose membrane, and MRP3 was detected by incubation with monoclonal antibody M₃II-9. (A) 1 = 2008; 2 = 2008/MRP3–4; 3 = 2008/MRP3–8; 4 = MDCKII; 5 = MDCKII/MRP3–17; 6 = MDCKII/MRP3–18; 7 = MDCKII/MRP3–20. (B) Time course in hours of MRP3 expression in 2008/MRP3–8 cells after treatment with tunicamycin (5 μg/ml). Ten μg of total cell lysate was used per lane.

Figure 3

Immunolocalization of MRP3 in 2008 cells and MDCKII monolayers by confocal laser scanning microscopy. MRP3 is detected by indirect immunofluorescence (green signal) with monoclonal antibody M₃II-9. Nucleic acids were detected by counterstaining with propidium iodide (red signal). (A) Parental 2008 cells; (B) 2008/MRP3–8 cells; (C) parental MDCKII cells; (D) MDCKII/MRP3–17 cells; (E) vertical X/Z section of the monolayer shown in D. The arrow in D indicates the position where the X/Z section was made. (Bar = 20 μm.)

Figure 4

Representative growth inhibition curves for cytotoxicity experiments with (A) etoposide, (B) teniposide, (C) podophyllotoxin, and (D) methotrexate by using 2008 cells (♦), 2008/MRP1–4 cells overexpressing MRP1 (■), and 2008/MRP3–4 (▴) and 2008/MRP3–8 cells (X) both overexpressing MRP3. A–C, experiments with continuous (96-h) drug exposure. D, experiments with short-term (4-h) drug exposure. The vertical bars for each data point represent ± SD of three replicate determinations. The IC₅₀ values of 2008, 2008/MRP1–4, 2008/MRP3–4, and 2008/MRP3–8 cells in this particular experiment were for etoposide: 0.51 nM, 10.29 nM, 0.88 nM, and 1.99 nM; for teniposide: 0.074 μM, 1.388 μM, 0.120 μM, and 0.226 μM; for podophyllotoxin: 21.68 nM, 21.85 nM, 20.54 nM, and 21.20 nM; for MTX: 0.027 mM, 2.504 mM, 0.887 mM, and 2.030 mM.

Figure 5

(A) Accumulation and polyglutamylation of [³H]MTX. 2008 and 2008/MRP3–8 cells were incubated 1 μM [³H]MTX for 4 and 24 h. Black bars represent the total intracellular pool of [³H]MTX and short-chain [³H]MTX-polyglutamates (MTX-Glu_2–3). Hatched bars represent the total intracellular pool of long-chain polyglutamates of [³H]MTX (MTX-Glu_4–6). Results presented are the mean ± SD (for total pool of all [³H]MTX) of three to four independent experiments. (B–D) Export of [¹⁴C]DNP-GS from MDCKII and MDCKII/MRP3–17 cells grown in monolayers. Cells were incubated at room temperature with 1-chloro-2,4-dinitro[¹⁴C]benzene (2 μM) in both the apical and basal compartment. Samples were taken at t = 1, 3, 6, 12, and 20 min and extracted with ethyl acetate. The experiments were done in duplicate. Variation between measurements was in most cases within the size of the symbols. ■ = transport to basal compartment; ♦ = transport to apical compartment. (B) Intracellular [¹⁴C]DNP-GS levels at the end of the experiment (t = 20); (C) DNP-GS efflux from MDCKII cells; (D) DNP-GS efflux from MDCKII/MRP3–17 cells.