Introduction

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Multidrug resistance protein 4 (MRP4/ABCC4) and multidrug resistance protein 5 (MRP5/ABCC5) belong to the MRP family of multispecific drug transporters (reviewed in Hipfner et al., 1999; Borst et al., 2000). This family now consists of nine members,¹ MRP1-9, which are also called ABCC1-6 and 10-12, respectively. Amino acid sequence comparisons show homology between the MRPs and the multidrug transporter P-glycoprotein. According to secondary structure predictions, MRP1-8 contain two nucleotide binding domains and at least 12 transmembrane segments, the so-called P-glycoprotein-like core, that form the essential transporter. They are mainly localized in the plasma membrane and show ATP-dependent transport of a broad range of compounds. Most MRP substrates are organic anions including many drugs conjugated to glutathione, glucuronide, or sulfate. Transport of nonanionic compounds such as vincristine and doxorubicin is also found in some cases, but this requires (cotransport with) glutathione (Zaman et al., 1995; Versantvoort et al., 1995). Because many MRP substrates are drugs used in cancer therapy, the overexpression of MRPs in cancer cells may contribute to resistance of such cells.

Like the other MRPs, MRP4 and MRP5 are organic anion transporters (Schuetz et al., 1999; McAleer et al., 1999; Wijnholds et al., 2000; Chen et al., 2001), but they have the unique ability to transport nucleoside monophosphate analogs (Schuetz et al., 1999; Jedlitschky et al., 2000; Lee et al., 2000; Wijnholds et al., 2000; Chen et al., 2001) such as the antiviral compound 9-(2-phosphonylmethoxyethyl)adenine. The physiological substrates of these transporters may be cAMP and cGMP (Jedlitschky et al., 2000; Chen et al., 2001). Overexpression of MRP5 (Wijnholds et al., 2000) and MRP4 (Chen et al., 2001) in cells results in resistance to 6-mercaptopurine (6MP) and 6-thioguanine (TG). Both 6MP and TG are purine nucleobase analogs with a sulfur at the C-6 position. 6MP was first synthesized in 1952 and is an important drug in the treatment of acute lymphoblastic leukemia, especially in children (Elion et al., 1952; Burchenal et al., 1953). In addition, 6MP is used as an immunosuppressant [for example, in the treatment of Crohn's disease (Elion, 1989; Markowitz et al., 2000)]. The guanine analog TG, synthesized after 6MP, has a type of action similar to that of 6MP (Elion, 1989; LePage, 1963; Lowe et al., 2001). 6MP and TG are prodrugs and their toxicity depends on their intracellular conversion to thionucleoside monophosphates (tNMPs). Thiopurine metabolism is as extensive as the metabolism of normal purines (Zimm et al., 1985; Martin, 1987; Elion, 1989; Keuzenkamp-Jansen et al., 1995; Aubrecht et al., 1997). After uptake, 6MP is converted into thio-IMP (tIMP) by hypoxanthine-guanine phosphoribosyl transferase (HGPRT), with phosphoribosyl pyrophosphate (PRPP) as the phosphoribosyl donor. In a similar way, TG can be converted into thio-GMP (tGMP). tIMP can be converted into tGMP in two steps: first, thioxanthosine monophosphate (tXMP) is formed by IMP dehydrogenase; second, tGMP is formed by GMP synthetase. tGMP can, in turn, be further phosphorylated to tGTP, which can be incorporated into RNA or, after reduction of the ribose moiety, into DNA. Alternatively, thiopurine methyltransferase (TPMT) can methylate 6MP to form the metabolic end product 6-methyl-MP (MeMP), thereby reducing the toxicity of 6MP (Lennard et al., 1987). Conversely, methylation of tIMP yields 6-methyl-tIMP (MetIMP), an inhibitor of de novo purine synthesis (Vogt et al., 1993). Several steps in purine metabolism have been reported to be involved in resistance of acute lymphoblastic leukemia cells to 6MP: a reduced nucleotide formation caused by low HGPRT activity or low PRPP levels (Rosman et al., 1974; Zimm et al., 1986; Vogt et al., 1993); increased dephosphorylation of the tNMPs by alkaline phosphatase or 5'-nucleotidase (Rosman et al., 1974; Zimm et al., 1986; Pieters et al., 1992; Vogt et al., 1993); and increased methylation of 6MP (Relling et al., 1999).

Because both MRP4 and MRP5 are ATP-dependent efflux pumps, a possible explanation for the resistance observed in cells overexpressing these proteins is the increased removal of an essential thiopurine metabolite from the cells. After preloading cells with radiolabeled 6MP under ATP-depleting conditions, Wijnholds et al. (2000) found that cells overexpressing MRP5 showed an increased efflux of the radiolabel. In similar experiments Chen et al. (2001) found a similar increased efflux for cells overexpressing MRP4. Analysis of the efflux medium showed that several compounds were preferentially effluxed by the MRP5-overexpressing cells, one of which coeluted with tIMP. For MRP4, no attempt was made to identify the effluxed metabolites.

The aim of the current study was to generate a comprehensive picture of the intracellular thiopurine metabolism in HEK293 cells under continuous exposure to thiopurines. We analyzed the metabolites excreted from parental HEK293 as well as from cells overexpressing either MRP4 or MRP5 to compare the substrate spectra of MRP4 and MRP5 and exclude the possibility that modified thiopurine metabolism was involved in thiopurine resistance. To this end, we set up a detection system to investigate the metabolism of thiopurines in HEK293 cells and to identify the metabolites excreted. Upon exposure to the thiopurine nucleobases 6MP and TG, or to 6-methyl-mercaptopurine riboside (MeMPrib), we found that tNMPs are exported by both MRP4 and MRP5 with overlapping substrate specificity. However, only MRP5-overexpressing cells transported tXMP, whereas MRP4-overexpressing cells showed the higher rates of transport of tGMP and MetIMP.

	Experimental Procedures

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Chemicals. Poly-D-lysine, 6MP, TG, thioxanthine (TX), 6-thioguanosine (TGrib), 6-mercaptopurine riboside (MPrib), MeMP, 6-methyl thioguanine, MeMPrib, and PRPP were obtained from Sigma (Zwijndrecht, The Netherlands). tIMP, tGMP, and tXMP were synthesized from 6MP, TG, TX, and PRPP using HGPRT from Saccharomyces cerevisiae (Sigma), as described previously (Wijnholds et al., 2000). MetIMP and 6-methylated tGMP were made from tGMP or tIMP (1 mM) by overnight incubation at room temperature in an aqueous solution containing 70 mM methyl bromide (Sigma) and 3.75% ammonia; thereafter, excess ammonia and methyl bromide were evaporated under vacuum, essentially as described previously (Keuzenkamp-Jansen et al., 1995). TXrib was generated from tXMP by 5'-nucleotidase, and thio-GDP, tGTP, thio-IDP, and thio-ITP were synthesized as described previously (Breter and Mertes, 1990).

Cell Lines. HEK293 parental cells and HEK293/5I and HEK293/5E cells transduced with MRP5 cDNA have been described previously (Wijnholds et al., 2000). The subclone HEK293/5GE was isolated at the same time as HEK293/5I and HEK293/5E but has not been described before. HEK293/5I and HEK293/5E have high MRP5 overexpression, whereas HEK293/5GE has a moderate increase in MRP5 relative to the parental cells (see Results). MRP4-overexpressing cells HEK293/4.3 and HEK293/4.63 were made by transducing HEK293 cells with pMSCV-IRES-EGFP virus containing full-length MRP4 cDNA, the cloning of which has been described elsewhere (Adachi et al., 2002). All cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37°C under 5% CO₂/humidified air. The cells were routinely checked for mycoplasma and MRP4 and MRP5 expression levels.

Monoclonal Antibodies. Anti-MRP5 monoclonal antibody (mAb) NKI-12C5 was generated by cloning a fragment of the mouse Mrp5 cDNA, encoding amino acids 1 to 38, into the pMAL-c vector (New England Biolabs, Beverly, MA) to produce a fusion protein consisting of Escherichia coli maltose-binding protein fused to the N terminus of mouse Mrp5. The affinity-purified protein was injected into rats, and hybridoma cells producing MRP5-specific mAbs were generated, essentially as described previously (Harlow and Lane, 1988). NKI-12C5 specifically recognizes the full-length human MRP5 and mouse Mrp5 protein. The MRP4-specific antibody NKI-12C4 was generated in a similar way using a maltose-binding protein fusion protein containing an internal epitope (amino acids 372-432) from human MRP4. A more complete characterization of these mAbs will be presented elsewhere.

Western Blot Analysis. Protein from a total cell lysate was quantified using the Bio-Rad protein assay (Bio-Rad, Hercules, CA), fractionated on a 8% polyacrylamide slab gel and transferred to a nitrocellulose membrane by electroblotting, essentially as described previously (Kool et al., 1997). After blocking for 1 h in phosphate-buffered saline (PBS) containing 1% nonfat dry milk, 1% bovine serum albumin, and 0.05% Tween 20, the membrane was incubated for 1 h at room temperature with the first antibody. As secondary antibody, horseradish peroxidase-labeled goat anti-rat antibody, preabsorbed for both mouse and human IgGs, was used at a dilution of 1:2000 (Santa Cruz Technology, Santa Cruz, CA). Enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) was used for detection.

Immunohistochemistry. To detect MRP4 and MRP5 by immunofluorescence, cells were grown overnight on poly(D-lysine)-coated microscope slides. The next day, the cells were fixed with methanol (30 s, 20°C) and immunostained with NKI-12C4 and NKI-12C5 (undiluted hybridoma culture medium). The first antibody was visualized using a goat anti-rat-Alexa488-conjugated fluorescent antibody (1:500; Molecular Probes, Leiden, the Netherlands). Cells were mounted using Vectashield (Vector Laboratories, Burlingame, CA) containing 40 µg/ml ethidium bromide to visualize nuclear DNA. Slides were analyzed by confocal laser-scanning microscopy (Leica, Heidelberg, Germany), using a 488-nm laser for excitation and a 530-nm band-pass filter for detection of Alexa488 and a 560-nm high-pass filter for detection of ethidium bromide-stained nuclear DNA.

Cytotoxicity. Relative growth of cells in the presence of thiopurines was tested essentially as described before (Wijnholds et al., 2000). In short, cells were plated in triplicate in 96-well plates (1500 cells/well, 100 µl/well) in conditioned medium. The next day, drug was added at the appropriate dilutions in a volume of 25 µl. After an additional 4 days, cell growth was determined using the CyQuant cell proliferation assay kit (Molecular Probes). The fluorescence in each well was determined using a CytoFluor 4000 plate reader (Applied Biosystems, Foster City, CA) and the IC₅₀ value was defined as the concentration of drug that inhibited growth of the cells by 50%. The relative resistance was then determined as the ratio of the IC₅₀ values of the transduced and parental cells.

Transport Experiments. Cells were seeded at a density of 2 × 10⁶ per well in poly(D-lysine)-coated 12-well plates and were grown overnight. The next day, the cells were washed with PBS and then incubated at 37°C with various thiopurines in Hanks' buffered salt solution (Invitrogen). At the time points indicated, both incubation medium and the cells were collected. The incubation medium was centrifuged and the supernatant was used for analysis. For the analysis of the intracellular metabolites, the cells were washed with ice-cold PBS and extracted using 70% methanol/water, and insoluble material was removed by centrifugation. After complete evaporation of the methanol/water phase at 40°C, the residue was solubilized in 200 µl of HPLC elution buffer A (see below) and analyzed by HPLC.

HPLC Analysis. Cell extracts and incubation media were analyzed by reversed-phase ion-pairing HPLC analysis using a Luna-C18 column (284 Luna, 5 µm, 250 × 4.6 mm; Phenomenex, Cheshire, UK) and a two-buffer gradient system to separate the different nucleotides. The following pump conditions were used: 0-5 min, 100% buffer A; 5-25 min, linear to 70% buffer B; 25-28 min, 70% buffer B; 28-30 min, linear to 100% buffer A; 30-45 min, 100% buffer A. The composition of buffer A was 100 mM NaH₂PO₄, 5.9 mM tetrabutylammonium hydrogen sulfate, 0.34 mM EDTA, and 1% (v/v) acetonitrile. Buffer B was the same as buffer A but contained 25% (v/v) acetonitrile. The flow was kept at 1 ml/min. The complete UV-visible absorbance spectrum (200-800 nm) of the effluent was monitored using a Waters 966 photodiode array detector (Waters Chromatograpy B.V., Etten-Leur, the Netherlands). The metabolites were identified by their specific retention times and UV absorbance maxima (UV_max; see Table 2). Thiopurine metabolites were quantified at their UV_max by relating the peak area of the metabolites detected with the peak area measured for a standard amount of either 6MP, TG, TX, or MeMPrib. Intracellular ATP was monitored to ensure that the cells were metabolically active, and the absence of extracellular ATP confirmed that cells had remained intact during the experiment.

	Results

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MRP4 and MRP5 Levels in Transfected HEK293 Cells. The levels of MRP4 and MRP5 in the HEK293 cell lines transduced with MRP4 or MRP5 cDNA, respectively, were quantified using two new mAbs, NKI-12C4 (MRP4) and NKI-12C5 (MRP5), as shown in Fig. 1. Recombinant MRP4 protein migrates as a broad band of approximately 220 to 230 kDa; MRP4-transduced HEK293/4.3 and HEK293/4.63 cells contain similar levels of MRP4 (Fig. 1A). NKI-12C4 recognizes an endogenous cross-reacting protein of ~90 kDa in all HEK293 variants, in addition to a band of ~70 kDa, which increases in intensity with increasing MRP4 expression. The latter may represent an MRP4 breakdown product. MRP5 migrates as a single band of 220 to 230 kDa (Fig. 1B). Overexpression of MRP5 is high in HEK293/5I and HEK293/5E and intermediate in HEK293/5GE cells. Two smaller bands of ~95 and ~60 kDa may represent breakdown products. In contrast to our previous anti-MRP5 antibody (Wijnholds et al., 2000), the new antibody NKI-12C5 clearly detected endogenous expression of MRP5 in the parental HEK293 cells, whereas the NKI-12C4 mAb detects endogenous MRP4 (Fig. 1, A and B). We see no effect on endogenous levels after introduction of exogenous MRP4 or MRP5 (Fig. 1, A and B). Figure 1C shows the location of MRP4 and MRP5 in HEK293 cells with immunofluorescence. Although we detect weak signals for endogenous MRP4 and MRP5 in the parental line, the intensity was too low to determine the subcellular localization (Fig. 1C).

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Western blot analysis and immunofluorescent detection of MRP4 and MRP5. MRP4 and MRP5 expression in the three cell lines used in this study is shown. For the Western blot, 15 µg of total protein was loaded per lane. MRP4 was detected by rat anti-human MRP4 monoclonal antibody NKI-12C4 (A) and MRP5 by rat anti-mouse MRP5 monoclonal antibody NKI-12C5 (B). Detection of MRP4 and MRP5 by immunofluorescence (C) was done on methanol-fixed cells using the indicated antibodies as outlined under Materials and Methods. MRP4- and MRP5-specific staining is shown in green, and the nuclear DNA stained by ethidium bromide is shown in red. *, ~90 kDa band recognized by NKI-12C4 found in all cell lines; thus, it is most probably an endogenous protein. **, ~75 kDa band in A, two bands of ~95 and ~60 kDa in B, of which the intensity increases with increasing MRP4 or MRP5 expression. The shorter film exposures demonstrate the relative levels of the introduced protein in the MRP4- and MRP5-overexpressing cells.

HPLC Analysis. To analyze the thiopurine metabolites formed by the HEK293 cells, we set up a reversed-phase ion-pairing HPLC system in combination with spectral UV-visible detection. This system is stable and allows the separation of the metabolites of both physiological and thiopurine nucleosides within 30 min (Fig. 2). The combination of elution time and UV-visible absorbance spectra (Fig. 2, insets) makes it possible to discriminate between metabolites. Table 2 provides an overview of UV_max and retention times for the metabolites analyzed in this study.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   HPLC analysis of reference compounds. Chromatograms of mixtures of nucleobases, nucleosides and nucleotides separated by reversed-phase ion-pairing HPLC. A, chromatogram showing the separation of guanosine (1), inosine (2), IMP (3), GMP (4), adenosine (5), AMP (6), and ATP (7), detected at 260 nm. B, chromatogram showing the separation of 6MP (8), TX (9), MPrib (10), tIMP (11), TXrib (12), and tXMP (13), detected at 330 nm. *, peaks that were derived from the Hanks' balanced salt solution in which thiopurines were dissolved. UV absorbance is given in arbitrary units. The insets show examples of absorbance spectra of indicated peaks, which were used to identify different metabolites.

6-Mercaptopurine Uptake by HEK293 Cells. In previous experiments with HEK293/5I cells (Wijnholds et al., 2000), cells were loaded with radiolabeled 6MP under ATP-depletion conditions and efflux was followed after restoration of cellular ATP. This discontinuous incubation is somewhat artificial and results in 10-fold lower 6MP uptake than in the presence of normal cellular ATP levels. We therefore switched to a protocol in which the cells are continuously exposed to 6MP under normal conditions. 6MP is lipophilic and is assumed to rapidly equilibrate across the plasma membrane. Accumulation of 6MP is therefore mainly determined by its intracellular ribophosphorylation, a process that is dependent on HGPRT activity and the intracellular PRPP concentration. Uptake of 6MP by HEK293 cells over 4 h increased with increasing 6MP (Fig. 3A). However, increasing the 6MP concentration 5-fold from 2.5 to 12.5 µM yielded only a 2-fold increase in uptake. This suggests that the uptake of 6MP was no longer linear (Fig. 3B), presumably because the cells were unable to maintain the initial rate of conversion of 6MP into tIMP. HEK293, HEK293/5I, and HEK293/4.3 cells took up similar levels of 6MP from the medium (Fig. 3).

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Uptake of 6MP. A, concentration-dependent uptake of 6MP was determined after 4 h for the HEK293 (), HEK293/5I (), and HEK293/MRP4.3 () cells. Initial 6MP concentrations in the incubation medium were 0.5, 2.5, and 12.5 µM. After 4 h of incubation at 37°C, the 6MP concentration in the medium was determined. Uptake was determined by the difference in 6MP concentration in the medium at t = 0 and t = 4 h. B, time-dependent uptake of 2.5 µM 6MP, with symbols as in A. The extracellular 6MP concentration is plotted as picomoles per 106 cells calculated using the volume of incubation medium (1 ml) and number of cells (2 × 106). Data are the mean ± S.E.M. of three independent determinations.

Thiopurine Efflux Kinetics for HEK293 Cells in the Continuous Presence of 6-Mercaptopurine. Five thiopurine metabolites were present in the chromatograms from HPLC analysis of the medium from HEK293, HEK293/4.3, and HEK293/5I cells after incubation with 2.5 µM 6MP. These were identified as MPrib, tIMP, TX, TXrib, and tXMP. Figure 4 shows the excretion of the four major metabolites, MPrib, tIMP, TXrib and tXMP over time. Table 3 gives the excretion rates for the different thiopurines and shows that the excretion of tIMP was greatly increased in cells overexpressing MRP4 or MRP5. The excretion of tXMP was detected only with HEK293/5I cells. Using the other MRP4 and MRP5 overexpressing HEK293 clones (Fig. 1), we obtained similar results (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   6MP metabolites excreted by HEK293 cells. Metabolites excreted from the HEK293 (), HEK293/5I (), and HEK293/4.3 () during continuous incubation with 6MP (initial concentration 2.5 µM). The levels of tIMP (A), tXMP (B), MPrib (C), and TXrib (D) were quantified at their UVmax wavelength, using chromatograms similar to those shown in Fig. 2. The amounts of the various thioinosine and thioxanthosine metabolites were calculated by relating the peak area of the detected metabolites with the peak area obtained with standard amounts of either 6MP or TX. The data are the mean ± S.E.M of three independent determinations. The indicated data points were significantly different (*, p < 0.05) from the parental cells in a Student's t test.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Intracellular 6MP metabolites. Metabolites found in the intracellular fractions from the HEK293 (A), HEK293/4.3 (B), and HEK293/5I (C) during continuous incubation with 6MP (initial concentration, 2.5 µM). Levels of tIMP (), MPrib (), tXMP (), and TXrib () were quantified by relating the peak areas of detected metabolites with the peak area obtained with a 6MP and TX standards. TX levels were below the detection limit (< 1 pmol/106 cells). The data are the mean ± S.E.M. of three independent determinations.

Thiopurine Metabolism and Transport by HEK293 Cells Incubated with 6-Thioguanine. Because we were unable to detect TG metabolites in cells incubated with 6MP, we tested TG itself, a compound to which the transfectants are resistant (Table 1). The HEK293 cells took up TG at about the same rate as 6MP (results not shown). The only metabolites found consistently were tGMP and TGrib. The excretion of tGMP was increased in both MRP4 and MRP5 transfectants; the increase was most pronounced in the MRP4 cells (Fig. 6A and Table 3). The efflux rates were 0.24, 0.77, and 1.22 pmol/10⁶ cells/min for HEK293, HEK293/5I, and HEK293/4.3 cells, respectively. Excretion of TGrib was also substantial and did not differ for the three cell lines (Table 3 and Fig. 6B).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Continuous incubation with TG. Thiopurine metabolites found in the extracellular (left, A and B) and intracellular (right, C and D) fractions from the HEK293 (), HEK293/5I (), and HEK293/4.3 () cells during continuous incubation with TG (initial concentration 2.5 µM). Thioguanine metabolite levels were quantified by relating the peak areas of detected metabolites with the peak area obtained with a TG standard. The data are the mean ± S.E.M. of three independent determinations. The indicated data points were significantly different (*, p < 0.05) from the parental cells in a Student's t test.

Thiopurine Transport from Cells Incubated with 6-Methyl-mercaptopurine Riboside. In patients treated with 6MP, 6-methylated thiopurines are formed by TPMT. 6MP can be methylated to MeMP, which is not a substrate of HGPRT. In this case, methylation leads to reduction of the effective 6MP dose and decreased cytoxicity (Lennard et al., 1987). However, when tIMP is methylated, this may lead to a build up of MetIMP, which is an inhibitor of purine de novo synthesis and thereby contributes to cytotoxicity (Vogt et al., 1993). To examine whether MRP4 or MRP5 also transport MetIMP, we incubated cells with MeMPrib, which is converted to MetIMP by adenosine kinase. Uptake of MeMPrib was rapid and MetIMP was the only metabolite detected by HPLC analysis in the intracellular and extracellular fractions (Fig. 7A). We did not detect any 6-methylated guanosines or di- or triphosphorylated methyl thiopurines. MetIMP accumulated to high concentrations in the cells (Fig. 7B) and was excreted at substantial rates (Table 3). Average MetIMP efflux rates (determined by linear regression) were 1.3, 1.9, and 3.2 pmol/10⁶ cells/min for HEK293, HEK293/5I, and HEK293/4.3 cells, respectively. The relatively high rate of MetIMP secretion by the HEK293/4.3 cells is mirrored by a decreased intracellular concentration at the 4 h time point in Fig. 7B. Recovery of MeMPrib was 77 ± 23, 85 ± 15, and 79 ± 21% (mean ± S.E.M.) for the HEK293, HEK293/4.3, and HEK293/5I cells. It is possible, therefore, that we may have missed MeMPrib metabolites. The analysis of these metabolites is complicated by their relatively low absorption maximum (Table 2), which results in interference by the standard nucleotides present in cell extracts.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Continuous incubation with MeMPrib. Thiopurine metabolites found in the extracellular (A) and intracellular (B) fractions from the HEK293 (squares), HEK293/5I (circles), and HEK293/4.3 (triangles) cells during continuous incubation with MeMPrib (initial concentration, 2.5 µM). Metabolite levels were quantified by relating the peak areas of detected metabolites with the peak area obtained with a standard amount of MeMPrib. The data are the mean ± S.E.M. of three independent determinations. The indicated data points were significantly different (*, p < 0.05) from the parental cells in a Student's t test.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We have investigated the effect of MRP4 or MRP5 overexpression on the metabolism of thiopurines in HEK293 cells. Previous studies showed that both MRP4- and MRP5-overexpressing cells showed low-level resistance against 6MP and TG. After preloading with radiolabeled 6MP under ATP-depleting conditions, increased efflux of radiolabel was seen from the cells (Chen et al., 2001), but the analysis was hampered by low thionucleotide formation and limitations in the detection method (Wijnholds et al., 2000). Here, we circumvented these problems in two ways. First, we exposed the cells to thiopurines continuously without limiting their ATP-generating capacity; second, we adopted a reversed-phase ion-pairing HPLC system incorporating spectral UV-visible detection. The continuous exposure resulted in a more extensive, and physiologically relevant, thiopurine metabolism in the HEK293 cells, and the new detection system allowed us to more accurately identify thiopurine metabolites formed.

Under constant exposure to 6MP, the HEK293 cells exhibited extensive thiopurine metabolism. Uptake was readily saturable, and at the concentration we used routinely (2.5 µM), accumulation slowed rapidly after 1 h, reaching a maximum uptake after 4 h of 30 to 40% of the total 6MP applied. A similar pattern was observed with exposure to thioguanine. The generation of tIMP or tGMP during exposure to 6MP or TG, respectively, was the limiting step in metabolism, suggesting that the concentration of PRPP determined the amount of tNMP formed. In both cases, cells formed thiopurine nucleotides (tNMPs) and nucleosides, which were recovered both intracellularly and in the medium. The formation of all other metabolites followed the generation of the initial tNMP (see also Fig. 8). Overall, the gradual decrease in extracellular thiopurine concentration during the incubation combined with the subsequent intracellular flux through the different metabolites may approximate both clinical dosing regimens and pharmacokinetics.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Simplified scheme showing how tNMP transport by MRP4 and MRP5 might contribute to thiopurine resistance. The tNMPs transported by MRP4 and/or MRP5 are indicated. The blocked arrow indicates inhibition of de novo purine synthesis by MetIMP.

All cells effluxed tNMPs when exposed to 6MP, TG, or MeMPrib, presented schematically in Fig. 8. Cells overexpressing MRP4 or MRP5 effluxed more tIMP, tGMP, and MetIMP than the parental cells; only MRP5-overexpressing cells effluxed tXMP. Because of the complex nature of the in vivo system used here, we cannot draw definitive conclusions on the relative preference of MRP4 and MRP5 for the different tNMP substrates they transport. However, based on rates calculated by simple linear regression, MRP4 seems to prefer MetIMP and tGMP, and MRP5 seems to prefer tIMP and tXMP. The affinity of MRP4 and MRP5 for tNMPs may be relatively low because our rough estimates of the intracellular concentrations of the effluxed metabolites indicate that these may reach millimolar levels. Accurate determination of the affinity of the pumps for the respective tNMPs requires vesicular uptake studies. It is also likely, in view of the substantial efflux from the parental cells, that they contain other pumps able to extrude tNMPs. Because there is a high overexpression of MRP4 or MRP5 in our transfectants, it seems unlikely that the endogenous levels of these pumps alone can account for the efflux we observe from the parental cells. MRP8 and MRP9 are possible candidates (Bera et al., 2001; Tammur et al., 2001; Yabuuchi et al., 2001).

In addition to tNMPs, the HEK293 cells also generated substantial quantities of thiopurine nucleosides upon exposure to 6MP and TG. In fact, most of the 6MP recovered extracellularly was in the form of MPrib or TXrib, and TG was found predominantly as TGrib. This is consistent with the observation that both alkaline phosphatase and 5'-nucleotidase decrease the intracellular tNMP levels (Rosman et al., 1974; Pieters et al., 1992). Once formed, the nucleosides are probably released from the HEK293 cells by equilibrative nucleoside transporters (our unpublished observations). The amount of MPrib released was equivalent for the three cell lines, whereas TGrib was slightly reduced in the HEK293/4.3 cells, presumably because of the increased tGMP efflux by these cells. Remarkably, we observed a consistent increase in the release of TXrib from HEK293 cells overexpressing MRP4. This was unexpected because TXrib is neither a nucleoside monophosphate nor an organic anion. Because thiopurine metabolism is complex and we could not use TX itself as a substrate to confirm transport, we hesitate to draw conclusions on TXrib transport by MRP4. The transport should be tested in an independent experiment (e.g., in vesicle uptake).

The anticancer activity of thiopurines is dependent on their conversion into tGMP and subsequent incorporation of tGTP into RNA and/or DNA. Any mechanism that reduces the intracellular concentration of tGMP may act as a potential resistance mechanism. As mentioned above, 5'-nucleotidase reduces the tNMP pool and has been linked to thiopurine resistance (Rosman et al., 1974; Skladanowski et al., 1996). Furthermore, TPMT methylates 6MP, forming MeMP, which reduces the 6MP concentration and toxicity (Lennard et al., 1987). Indeed, TPMT activity is inversely related to tolerance of thiopurine chemotherapy; toxicity increases in patients with low TPMT activity, whereas those with no detectable TPMT activity experience severe toxicity (Krynetski et al., 1995; Relling et al., 1999). Additionally, tIMP may be methylated, leading to a build up of MetIMP, an inhibitor of purine de novo synthesis (Vogt et al., 1993). Based on our results, it is possible that during thiopurine treatment, MRP4 and MRP5 modulate the intracellular tNMP pool via the efflux of tIMP, tXMP, tGMP, and MetIMP, and thereby contribute to cellular detoxification (Fig. 8).

In the clinic, both 6MP and TG are normally administered orally. The pharmacokinetics are relativity fast, with maximal plasma concentrations found after 2 to 3 h and a plasma half-life of 1 to 2 h (Lowe et al., 2001). MRP5 expression has been found in almost all tissues, including gut and kidneys (Kool et al., 1997; Belinsky et al., 1998), and the protein is localized to the basolateral membrane of the polarized cell line MDCKII (Wijnholds et al., 2000). MRP4 is highly expressed in the kidney (van Aubel et al., 2002) and the prostate (Lee et al., 2000) and, to a lesser extent, in the gut (unpublished observations). We find expression of both MRP4 and MRP5 in lymphoblasts from leukemia patients (unpublished observations). In light of the results presented here, MRP4 and MRP5 transporters might influence thiopurine pharmacokinetics, as well as the relative toxicity of thiopurines in both normal tissues and leukemic cells.

It is clear that MRP4 and MRP5 can transport most of the tNMPs found in patients treated with 6MP or TG. Because thiopurine nucleotide levels have been found to be predictive for treatment outcome (Lilleyman and Lennard, 1994), MRP4 and MRP5 may have a previously unrecognized involvement in thiopurine resistance and pharmacokinetics in patients. However, the potential impact of MRP4 and MRP5 will depend on the activity of other enzymes that modulate the nucleotide pool, including 5'-nucleotidase (as observed in this study) and TPMT. Most important for patients receiving 6MP treatment is the possibility that overexpression of MRP4 and MRP5 leads to a decrease in thiopurine toxicity in leukemic cells.