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Vol. 63, Issue 5, 1094-1103, May 2003
Division of Molecular Biology and Center of Biomedical Genetics, the Netherlands Cancer Institute, Amsterdam, the Netherlands (G.R., P.W., N.Z., M.d.H., L.v.D., P.B.); and Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium (J.B.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs. Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates. To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5. In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form. MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not. Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5. In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively. Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP. Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps. These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
metabolism and flux of nucleosides and nucleoside analogs is a complex
process involving a large array of intracellular metabolizing enzymes
as well as several families of membrane transport proteins. The
transport of nucleoside analogs into and out of cells and their
activation by cellular kinases has received much attention because of
the influence of these processes on the efficacy of nucleoside
analog-based anticancer and antiviral therapies. Although the release
of nucleoside monophosphates from cells is documented, the transport
proteins involved and their potential effect on nucleoside analog
therapies remained unknown until two of the newer members of the
multidrug resistance protein (MRP) family of ATP-dependent efflux
pumps, MRP4 and MRP5, were shown to transport nucleoside monophosphates
(Schuetz et al., 1999
; Lee et al., 2000; Wijnholds et al., 2000; Chen
et al., 2001). Continuous selection of the human T lymphoblast CEM cell
line with the antiviral drug 9-(2-phosphonomethoxyethyl)adenine (PMEA) led to an amplification of the MRP4 gene with a concomitant
decrease in PMEA accumulation (Schuetz et al., 1999). We showed that
MRP5-transfected HEK293 cells were resistant against both
6-mercaptopurine, a nucleobase analog drug used in the treatment of
acute lymphoblastic leukemia (Wijnholds et al., 1999), and PMEA
(Wijnholds et al., 2000). Further analysis indicated that the
resistance to PMEA was caused by the efflux of PMEA itself, and
resistance to 6-mercaptopurine was caused by the increased efflux of
thiopurine nucleoside monophosphates (Wijnholds et al., 2000; Wielinga
et al., 2002). Several groups then reported that MRP4 and MRP5
transport the second messengers cyclic GMP and cyclic AMP (Jedlitschky
et al., 2000; Chen et al., 2001; Lai and Tan, 2002; van Aubel et al.,
2002). Both pumps transported cGMP with high affinity; MRP4 also
exhibited high-affinity cAMP transport (Jedlitschky et al., 2000; Chen
et al., 2001). Most recently, MRP4 was shown to efflux the
monophosphorylated form of the antiviral drug ganciclovir, an antiviral
agent used clinically for the treatment of cytomegalovirus infections
(Adachi et al., 2002).
The ability of MRP4 and MRP5 to transport monophosphorylated
forms of several nucleoside analog drugs could affect both anticancer and antiviral therapies involving these classes of drug. In addition to
the thiopurines, deoxynucleoside analogs such as cytarabine and
cladribine are also used in the treatment of a variety of leukemias,
and gemcitabine is used in the treatment of a number of solid tumors
(Galmarini et al., 2001). Deoxynucleoside analogs are taken up by
nucleoside transporters and, like thiopurines, are converted to the
active triphosphate by a succession of cellular kinases before
incorporation into nucleic acids. Nucleoside analogs are also integral
components of antiviral therapy; for example, the
2',3'-dideoxynucleoside drugs didanosine (2',3'-dideoxyinosine), zalcitabine [2',3'-dideoxycytidine (ddC)], and stavudine
[2',3'-didehydro-3'deoxythymidine (d4T)] are all used clinically to
treat HIV infections (De Clercq, 2001). Although many compounds of this
class of nucleoside analog inhibit viral replication, they are often of
limited use because they are poorly activated to the monophosphate form
by cellular nucleoside kinases (Starnes and Cheng, 1987; Balzarini et
al., 1989; Johnson and Fridland, 1989). The kinase requirement can be
circumvented via direct intracellular delivery of dideoxynucleosides in
their monophosphate forms (Balzarini et al., 1996b, 2000), for instance
by cell-permeable phosphoramidate triester and cyclosaligenyl dideoxynucleotide prodrugs, exemplified by the aryloxyalaninyl phosphoramidate of d4TMP (So324) and
cyclosaligenyl-d4T-5'-monophosphate (cycloSal-d4TMP), respectively (see
Fig. 1). Likewise, acyclic nucleoside
phosphonates are preformed nucleoside monophosphate analogs that do not
require activation by nucleoside kinases.
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Because both MRP4 and MRP5 have previously been shown to transport nucleoside monophosphates, this is a potential mechanism for reduced efficacy of therapies involving nucleoside analogs. We have investigated this possibility by comparing the resistance profile and transport properties of MRP4- or MRP5-overexpressing HEK293 cells exposed to a range of both clinically used and experimental nucleoside analog drugs. We find that although both transporters efflux a number of nucleoside analogs in the HEK293 background, MRP4 mediates substantial resistance to a wider variety of acyclic nucleoside phosphonates than MRP5, whereas only MRP5 transports the monophosphorylated form of the dideoxynucleoside d4T and its prodrugs.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Materials.
[3H]PMEA,
[3H]PMEG, and
[8-3H]bis-POM-PMEA were purchased from Moravek
Biochemicals (Brea, CA), and [3H]estradiol
glucuronide was from PerkinElmer Life Sciences (Boston, MA). PMEA and
bis-POM-PMEA were kindly provided by N. Bischofberger (Gilead Sciences,
Foster City, CA). PMEDAP, cPr-PMEDAP, and
1-[(S)-3-hydroxy-2-(phosphonomethoxy)propyl]cytosine were a kind gift from A. Holy (Prague, Czech Republic, and Gilead Sciences). The monophosphates of gemcitabine,
1-
-D-arabinofuranosylcytosine and ddC, were
synthesized by C. Meier (Hamburg, Germany) and purified by HPLC.
Fludarabine and [3H]fludara were kind gifts
from Dr. J. Gay (Schering, Berlin, Germany). Cladribine was obtained
from Janssen-Cilag (The Netherlands), gemcitabine was from Eli Lilly
(The Netherlands), MK571 was from Biomol (Plymouth Meeting, PA),
cytarabine was from Pharmacia (Woerden, the Netherlands),
5-fluorouracil was from TEVA (Mijdrecht, the Netherlands), and
sildenafil was from Pfizer (the Netherlands). Azidothymidine, d4T, and
abacavir were obtained from the Pharmacy Department of The Netherlands
Cancer Institute. [3H]Alaninyl-dTMP was
synthesized from [3H]So324 by incubation with
pig liver carboxylesterase (Sigma, St. Louis, MO) as described
previously (Balzarini et al., 1996b). The syntheses of So324 and
cycloSal-d4TMP have been described previously (Balzarini et al., 1996a,
1999; McGuigan et al., 1996; Meier et al., 1998). All other compounds
were purchased from Sigma.
Cell Lines.
HEK293/5I and HEK293/5E cells transduced with
MRP5 and the MRP4-overexpressing HEK293/4.3 and HEK293/4.63 cells were
described previously (Wijnholds et al., 2000; Wielinga et al., 2002).
Analyzing serial dilutions of cell lysates on Western blot by
densitometry shows approximately 25 and 75 times more MRP4 in the
HEK293/4.3 and HEK293/4.63 cells, respectively, and 80 times more MRP5
in the HEK293/5I cells (data not shown). The HEK293/MRP4.59 cells were
derived from the same transduction but do not show MRP4 overexpression; HEK293 parental cells and all transfectants were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf
serum and 100 units of penicillin/streptomycin per milliliter
(Invitrogen), at 37°C under 5% CO2 humidified
air. The cells were routinely checked for the absence of mycoplasma
infection and for MRP4 and MRP5 expression levels.
Cytotoxicity.
Relative growth of the cells in the presence
of the different compounds was tested as described previously
(Wijnholds et al., 2000). In short, cells were plated in triplicate in
96-well plates (1500 cells/well); on the following day, drugs were
added at the appropriate dilutions. On day 5, the cell growth in the
wells was determined using CyQuant cell proliferation assay kit
(Molecular Probes), and fluorescence in each well was determined with a
CytoFluor 4000 plate reader (Applied Biosystems, Foster City,
CA). The relative resistance was calculated as the ratio of the
concentration of drug inhibiting growth by 50%
(IC50) in the transfected cell line divided by
that in the parental cell line.
Cellular Transport. Cells (2 × 106 per well) were seeded in poly(D-lysine)-coated six-well plates and grown overnight. For the transport experiments, cells were loaded with 1 µM [3H]bis-POM-PMEA, 0.27 µM [3H]cycloSal-d4TMP, or 2.5 µM [3H]So324 for 2 h at 37°C, under ATP-depletion conditions (DMEM without glucose, supplemented with 10% dialyzed fetal calf serum, 10 mM sodium azide, and 10 mM 2-deoxyglucose), plus compounds at the indicated concentrations for the inhibition studies. After loading, the cells were washed rapidly with ice-cold phosphate-buffered saline, followed by addition of prewarmed DMEM. The time-dependent efflux was measured at 37°C by drawing samples over time. Radioactivity in the samples was determined by liquid scintillation counting. Initial efflux rates were determined by linear regression analysis of the linear component of efflux [0-30 min for efflux from So324-loaded cells, 0-15 min for cycloSAL-d4TMP-loaded cells, and 0-60 min for bis-POM-PMEA-loaded cells].
HPLC.
Cells were loaded and allowed to efflux as described
above, with the exception that after loading and washing, Hanks'
buffered salt solution (Invitrogen) was added for the efflux
determinations. Metabolites effluxed from cells preloaded with
[3H]bis-POM-PMEA,
[3H]cycloSal-d4TMP, or
[3H]So324 were determined as described
previously (Balzarini et al., 1996a, 1998; Hatse et al., 1998).
Preparation of Membrane Vesicles.
For the preparation of
membrane vesicles, HEK293, HEK293/5I, and HEK293/4.63 cells were grown
as described above. Cells were harvested by centrifugation at 3000 rpm
for 5 min. The pellet was resuspended in ice-cold hypotonic buffer (0.5 mM sodium phosphate, 0.1 mM EDTA, pH 7.4) supplemented with protease
inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 µg aprotinin/ml, 5 µg/ml leupeptin, and 10 µM pepstatin) and incubated at 4°C for 90 min. The suspension was centrifuged at 4°C at 100,000g for
40 min, and the pellet was homogenized in ice-cold TS buffer (50 mM
Tris-HCl, 250 mM sucrose, pH 7.4) using a tight-fitting Dounce
homogenizer. After centrifugation at 500g at 4°C for 10 min, the supernatant was centrifuged at 4°C at 100,000g
for 40 min. The pellet was resuspended in TS buffer and passed 25 times
through a 27-gauge needle. The vesicles were dispensed in aliquots,
frozen in liquid nitrogen, and stored at 
80°C until use.
Vesicular Transport Assays.
The uptake of various substrates
into membrane vesicles was studied after the rapid filtration method as
described previously (Zelcer et al., 2001). MRP4-mediated transport of
[3H]estradiol 17-glucuronide was measured
for 10 min and MRP5-mediated transport of
[3H]alaninyl-d4TMP was measured for 15 min. For
inhibition studies, uptake in the absence and presence of inhibitors
was compared.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Resistance to Nucleoside Analogs Mediated by MRP4 and MRP5.
We
have tested the effect of MRP4 and MRP5
expression in HEK293 cells on the cytotoxicity of a range of base,
nucleoside, and nucleotide analogs. The results are presented in Table
1, with some previously published data to
provide a complete overview. In cells overproducing MRP4, we see
substantial resistance for the acyclic nucleoside phosphonates PMEA
[confirming the results of Lee et al. (2000) and Lai and Tan (2002)]
and its precursor bis-POM-PMEA and PMEDAP and its derivative
cPr-PMEDAP. Resistance was proportional to MRP4 levels, because the
HEK293/4.63 cells contain more MRP4 than the HEK293/4.3 cells (Wielinga
et al., 2002). The MRP4 cells also showed a lower level of resistance to another acyclic nucleoside phosphonate, PMEG. In contrast, PMEA and
bis-POM-PMEA were the only phosphonates significantly affected by MRP5
(this study and Wijnholds et al., 2000). MRP4 and MRP5 also mediated
resistance to the purine-based carbocyclic nucleoside analog abacavir
(2- and 1.9-fold, respectively).
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; Wijnholds et al.,
2000; Wielinga et al., 2002) replicated here, we only found low-level
MRP4-mediated resistance against the anticancer nucleoside analog
cladribine (2.2-fold).
Efflux from Cells Loaded with d4TMP Prodrugs.
In HEK293 cells,
we saw no toxicity with the 2',3'-dideoxynucleosides ddC,
2',3'-dideoxyinosine, azidothymidine, d4T, or the d4TMP prodrug So324
at concentrations of up to 1 mM, the highest concentrations possible in
the presence of less than 1% dimethyl sulfoxide (data not shown). This
was not caused by sluggish uptake, because HEK293 cells were found to
express the nucleoside transporters hENT1 and hENT2 and accumulate
2',3'-dideoxycytidine (data not shown). However, it is known that many
of these analogs are poor substrates of nucleoside kinases; as such,
they are slowly activated to the monophosphate form (Starnes and Cheng,
1987; Balzarini et al., 1989; Johnson and Fridland, 1989). To
circumvent the need for a nucleoside kinase, membrane-permeable
aryloxyalaninylphosphoramidate and cyclosaligenyl prodrugs of
nucleoside 5'-monophosphate derivatives have been developed that result
in direct intracellular delivery of free nucleoside monophosphates
(reviewed in Meier, 1998). These drugs, however, might be susceptible
to efflux by MRP4 or MRP5.
). To
determine the influence of MRP4 and MRP5 on intracellular levels of the
active drug, cells were loaded with 2.5 µM
[3H]So324 or 0.27 µM
[3H]cycloSal-d4TMP under ATP-depleting
conditions, after which efflux was determined (Fig.
2). Efflux of radiolabel from
So324-loaded HEK293/5I (MRP5) cells was rapid, with an initial rate
approximately 4-fold higher than in the parental cells, resulting in
efflux of almost 80% of the loaded radiolabel within the first hour
(Fig. 2A). In contrast, efflux from HEK293/4.3 (MRP4) was equivalent to
the parental cells (Fig. 2A). CycloSal-d4TMP-loaded HEK293/5I cells
effluxed radioactive compounds faster than HEK293 cells, especially
during the first 15 min, although both cell lines effluxed almost all
preloaded radiolabel within 2 h (Fig. 2B). The differences in
efflux were not a result of differential loading, because the cell
lines accumulated equivalent amounts during the preloading period.
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) was 1.5-fold greater than from the HEK293 parental cells (Fig. 3A,
), strongly suggesting that d4TMP was transported by MRP5. Some
intracellular [3H]d4TMP was detected in the
HEK293/5I cells (Fig. 3B), but this was 5-fold lower than in the
parental cells, where it was the major metabolite found. These data
support the conclusion that MRP5 mediates d4TMP efflux, but the
extensive efflux of d4TMP from the parental cells suggests that
unidentified endogenous transporters also efflux this metabolite. In
addition, low levels of phosphorylated forms of
[3H]d4TMP, namely d4TDP and d4TTP, were found
only in the parental cells. Similar results were obtained with another
MRP5 transfectant, HEK293/5E (data not shown). An additional
intracellular metabolite accounting for approximately 10% of the
radioactivity was detected in the parental cells, but the nature of
this metabolite could not be identified with the HPLC system employed.
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). Figure 3 shows that alaninyl-d4TMP is
the major metabolite formed in both HEK293 and HEK293/5I cells. However, the alaninyl-d4TMP formed in the HEK293/5I cells is almost entirely effluxed (Fig. 3C), whereas most of the alaninyl-d4TMP formed
in the HEK293 cells accumulates intracellularly (Fig. 3D). The
activated d4T metabolite, d4TMP, formed from alaninyl-d4TMP after
cleavage of the phosphoramidate bond, was not detectable in the efflux
media of either cell line and was found at low intracellular levels in
the HEK293 cells.
Transport of PMEA by MRP4.
We have shown that MRP5 extrudes
PMEA in an unmodified form (Wijnholds et al., 2000), and we have
verified that this is also the case for MRP4. After loading cells with
1 µM [3H]bis-POM-PMEA, both efflux medium and
intracellular fractions from MRP4-transduced and HEK293 parental cells
were analyzed by HPLC (Table 2). PMEA was
the only species detected in the efflux medium. After a 2-h incubation,
HEK293/4.3 and HEK293/4.63 cells had effluxed 2.1- and 2.9-fold more
PMEA than the parental HEK293 cells, whereas the control clone
HEK293/4.59, which does not express the transfected MRP4
(Wielinga et al., 2002), showed efflux similar to that of the parental
cells (Table 2). Intracellular concentrations of PMEA were decreased in
the HEK293/4.3 and HEK293/4.63 cells, whereas intracellular
concentrations of the phosphorylated derivatives were similar for all
cell lines. We conclude that MRP4, like MRP5, extrudes PMEA in
unmodified form.
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Inhibition of MRP4- and MRP5-Mediated PMEA Efflux.
Given the
ability of MRP4 and MRP5 to cause resistance to some drugs, selective
inhibitors that can inhibit the transporters in intact cells would be
useful. We have tested a range of potential inhibitors on the initial
rate of PMEA extrusion from cells preloaded with 1 µM
[3H]bis-POM-PMEA. Figure
4 shows that the initial rate of efflux from HEK293/4.3 (MRP4) and HEK293/5I (MRP5) cells was comparable, and
considerably greater than that from the parental cells. This was not a
consequence of different initial intracellular concentrations, as all
three cell lines were loaded to the same extent (approximately 300 pmol/106 cells). The inset shows the relative
rates of efflux mediated by MRP4 and MRP5 cells after subtraction of
efflux from the parental line. The influence of various inhibitors (at
subtoxic concentrations) on PMEA efflux is shown in Fig.
5, and the
concentrations inhibiting MRP4 and MRP5 by 50%
(IC50 values) are summarized in Table
3. Of the commonly used inhibitors
of organic anion transport, probenecid inhibited PMEA efflux by MRP5 at
much lower concentrations (IC50, 200 µM) than
those needed to inhibit MRP4 (IC50, 2300 µM),
as did sulfinpyrazone to a lesser extent (relative
IC50 values 300 and 420 µM, respectively),
whereas benzbromarone inhibited both transporters equally (Fig. 5A). In
contrast, MK571 readily inhibited MRP4 at 10 µM but was a poor
inhibitor of MRP5. Based on the structural similarity between
nucleotides and nucleosides, we also tested three inhibitors of
nucleoside transport. Dipyridamole and dilazep inhibited MRP4-mediated
PMEA efflux (IC50, 2 and 20 µM, respectively), but MRP5 was inhibited by dipyridamole only at the highest
concentration tested (Fig. 5B). NBMPR (at 100 µM) inhibited MRP4 but
had no effect on MRP5 function (Fig. 5B).
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). Sildenafil and trequinsin both inhibited
PMEA efflux mediated by MRP4 (IC50, 20 and 10 µM, respectively) more effectively than that mediated by MRP5
(IC50, 80 and 30 µM). In fact, only high
concentrations of these compounds inhibited MRP5. Zaprinast, on the
other hand, inhibited both pumps only at very high concentrations
(IC50, 250 µM for both MRP4 and MRP5). The
P-glycoprotein inhibitor PSC833 and the breast cancer resistance
protein-specific inhibitor Ko143 (Allen et al., 2002) had no effect on
PMEA efflux mediated by either pump at concentrations of up to 1 µM
(data not shown).
Vesicular Uptake by MRP4 and MRP5.
Direct measurements
of nucleoside analog drug transport by MRP4 and MRP5 into inside-out
membrane vesicles have proven difficult. van Aubel et al. (2002)
attempted unsuccessfully to show PMEA uptake into MRP4 containing
vesicles. Similarly, using concentrations of up to 100 µM, we were
unable to demonstrate transport of [3H]PMEA,
[3H]PMEG, or
[3H]Fludara into vesicles derived from either
HEK293/5I or HEK293/4.63 cells. Because cells overexpressing MRP5
transported alaninyl-d4TMP (Fig. 3C), we tested this substrate in
vesicular transport. Using purified
[3H]alaninyl-d4TMP, we found ATP-dependent
uptake with vesicles prepared from HEK293/5I, but not from parental
HEK293 cells (Fig. 6A). At a
concentration of 1 µM alaninyl-d4TMP, uptake by HEK293/5I vesicles
reached a maximum of approximately 7 pmol/mg protein after 30 min.
Although we did not have access to sufficient alaninyl-d4TMP to
determine the Km, transport did
increase linearly with increasing substrate concentration (Fig. 6B) but
was not saturated at the maximum concentration tested (100 µM).
Consistent with the lack of efflux from MRP4 transfectants noted above,
inside-out membrane vesicles derived from HEK293/4.63 cells did not
accumulate [3H]alaninyl-d4TMP.
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-glucuronide (E217G) as substrate, a
compound not transported by MRP5 (unpublished observations). We tested
this at the published Km of 30 µM
(Chen et al., 2001) to avoid the possibility that we would measure
transport mediated by endogenous MRP1 in the HEK293 cells. As shown in
Table 4, none of the nucleoside
monophosphate analogs had a substantial effect on MRP4 or MRP5, even at
a concentration of 1 mM. This confirms our results with intact cells
showing that these compounds are, at best, low-affinity substrates of
MRP4 and MRP5. Only with cPr-PMEDAP did we find 40% inhibition of
MRP4. An unexpected finding, in light of a recent report (Jedlitschky et al., 2000), was that high concentrations of cGMP and cAMP were required to inhibit transport mediated by both transporters. The results in Table 4 also confirm the poor inhibition of MRP4 and MRP5 by
1 µM concentrations of the PDE5 inhibitors sildenafil and trequinsin,
the maximal levels remaining soluble in the absence of serum proteins.
Zaprinast inhibited both MRP4 and MRP5 equally and at concentrations
lower than those required in intact cells, possibly because the pumps
are more accessible to zaprinast in in vitro assays. Although the
results of the growth inhibition assays suggest that the anticancer
nucleoside analogs fludarabine, gemcitabine, and cytarabine are not
substrates of MRP4 or MRP5, we tested this directly with the
monophosphorylated forms. Slight inhibition of MRP4 (and no inhibition
of MRP5) was observed at inhibitor concentrations of 1 mM, further
evidence that these compounds are, at best, very low-affinity
substrates of MRP4 and MRP5.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Nucleoside analogs are important components of both anticancer and
antiviral therapies. In general, these analogs must be taken up by
target cells, activated by phosphorylation, and incorporated into
nucleic acids to exert their toxic effects, processes mediated by a
series of cellular and viral proteins. Changes in the expression of
these proteins have been implicated in the development of resistance to
nucleoside analogs (Galmarini et al., 2001). Although several resistance mechanisms have been identified in cell lines, these rarely
correlate with clinical resistance (Galmarini et al., 2001). Recent
reports demonstrating that the related ATP-binding cassette transporters MRP4 and MRP5 are able to transport nucleoside
monophosphates raise the possibility that the extrusion of
monophosphorylated nucleoside analogs from cells might contribute to
resistance. To investigate this, we have characterized the transport of
different classes of nucleoside analog drugs by MRP4 and MRP5.
In previous studies, the resistance to PMEA conferred by MRP4
overexpression was predicted to result from the efflux of PMEA itself,
not phosphorylated metabolites (Schuetz et al., 1999). We confirm this
prediction here. Because MRP4 and MRP5 were previously shown to confer
similar levels of resistance to PMEA (Lee et al., 2000; Wijnholds et
al., 2000), we tested whether this was the case for other antiviral and
anticancer nucleoside analogs. We found that MRP4 mediated high-level
resistance against other acyclic nucleoside phosphonate drugs, whereas
MRP5 did not. The 17-fold resistance to cPr-PMEDAP conferred by MRP4 on
the HEK293 cells represents the most striking phenotype yet reported
for MRP4-overexpressing cells. In contrast, MRP5, but not MRP4,
mediated efflux from cells preloaded with the d4TMP prodrugs
cycloSal-d4TMP and So324, with d4TMP and alaninyl-d4TMP representing
the first substrates of MRP5 that are not measurably transported by
MRP4. Transport of alaninyl-d4TMP, a dideoxyadenosine monophosphate
prodrug, demonstrates that MRP5 recognizes dideoxynucleosides, although
it should be stressed that HEK293 cells have additional unidentified
endogenous transporters capable of effluxing d4TMP. The fact that
alaninyl-d4TMP is transported shows that a free phosphate moiety is not
a prerequisite for transport. Transport of alaninyl-d4TMP increased
linearly as a function of concentration from 1 and 100 µM, suggesting
a low-affinity interaction.
Like antiviral therapies, chemotherapy protocols for many types of
cancer include nucleoside analogs, and resistance to these drugs
represents a clinical problem. Previous studies investigating the
ability of MRP4 or MRP5 to mediate resistance to anticancer nucleosides
have yielded contrasting results. Chen et al. (2001) found no effect of
MRP4 overexpression on the sensitivity of NIH3T3 cells to cladribine,
whereas Davidson et al. (2002) found substantial resistance to
cytarabine, gemcitabine, and cladribine in MRP5-overexpressing HEK293
cells. In contrast, we find low-level MRP4-mediated resistance against
cladribine but no MRP4- or MRP5-mediated resistance against cytarabine,
gemcitabine, or fludarabine. A possible explanation is that up- or
down-regulation of nucleoside metabolizing enzymes in our HEK293 cells
limits the potential for transport by MRP4 or MRP5. On the one hand,
the toxicity of gemcitabine, cytarabine, and cladribine in cell lines
increases proportionally with deoxycytidine kinase activity (Galmarini
et al., 2001), and transfection with mitochondrial deoxyguanosine
kinase has also been shown to increase sensitivity to the cytostatic
activity of these agents (Zhu et al., 1998). On the other hand,
transfection with human cytosolic nucleotidase imparts HEK293 cells
with resistance against cladribine and gemcitabine (Hunsucker et al.,
2001), and we have previously found that thiopurine nucleotides were
rapidly dephosphorylated in HEK293 cells, limiting the toxicity of
thiopurine drugs (Wielinga et al., 2002). All these examples show how
strongly the levels of nucleoside monophosphate analogs in cells are
influenced by the activity of enzymes producing or using these
compounds. It is therefore possible that MRP4 and MRP5 might be more
important determinants of resistance in cells in which higher
concentrations of transported substrates can accumulate than in the
HEK293 cells used here.
Our results suggest that MRP4 or MRP5 is unlikely to make a major
contribution to drug resistance in clinical practice, because both
transporters have a relatively low affinity for their nucleoside monophosphate substrates. This was already indicated by studies with
PMEA and thiopurines in intact cells, which suggested that intracellular substrate concentrations in the millimolar range are
required to get substantial transport by MRP4 and MRP5 (Schuetz et al.,
1999; Wijnholds et al., 2000; Wielinga et al., 2002). These results are
confirmed by the vesicular transport experiments presented here.
Although alaninyl-d4TMP is transported by MRP5 (Fig. 5), we have been
unable to produce sufficient amounts of this compound to determine a
Km. None of the other nucleoside monophosphates available in radioactive form
PMEA, PMEG, or the monophosphate of fludarabinewas detectably taken up in vesicular transport experiments, even though PMEA is transported by MRP4 and MRP5
out of intact cells. The low affinity of these transporters for
nucleoside monophosphate analogs is confirmed by our competition experiments: both PMEA and the monophosphates of gemcitabine, cytarabine, zalcitabine, and fludarabine inhibited MRP4- and
MRP5-mediated transport by at most 20%, even at a concentration of 1 mM. Similarly, substantial inhibition of MRP4-mediated
E2-17G transport was achieved only with 1 mM
cPr-PMEDAP.
Recently, MRP4 and MRP5 were reported to transport cGMP with high
affinity (Jedlitschky et al., 2000; Chen et al., 2001). In our
vesicular transport experiments, however, uptake of cGMP was low and
not consistently increased in MRP4- or MRP5-overproducing cells
relative to untransfected control cells. In agreement with this result,
we found that cGMP was a poor inhibitor of MRP4 and MRP5 activity in
vesicular uptake experiments. Although cGMP slightly inhibited both
pumps at 100 µM, a concentration of 1 mM was required to exceed 50%
inhibition. Similarly, cAMP was a low-affinity inhibitor of both
transporters. In the presence of 850 µM cAMP, MRP4 and MRP5 transport
was inhibited by only 30 and 50%, respectively. The results of these
vesicular transport experiments are in full agreement with our results
with intact HEK293 cells, in which MRP4- and MRP5-mediated cyclic
nucleotide efflux also exhibits characteristics of a low affinity
transport process (P. R. Wielinga, I. van der Heijden, G. Reid, J. Beijnen, J. Wijnholds, and P. Borst, submitted).
It is unclear why we find a low affinity for cyclic nucleotides,
whereas other labs report a high affinity. The two codon differences in
the independently isolated MRP4 (Belinsky et al., 1998; Adachi et al.,
2002; Lai and Tan, 2002) and MRP5 cDNAs (McAleer et al., 1999;
Jedlitschky et al., 2000; Wijnholds et al., 2000) would not be expected
to yield proteins with such dramatically altered substrate
specificities and affinities. It is remarkable, however, that Chen et
al. (2001, 2002) find a high-affinity uptake of radioactive cGMP in
MRP4 vesicles, but very poor inhibition by cold cGMP of the uptake of
other substrates such as E2-17G and
methotrexate. Chen et al. (2001, 2002) do not provide an explanation for this unusual discrepancy, but the results of their competition experiments are in line with our results. For MRP5, we can only speculate that the sodium butyrate treatment employed by Jedlitschky et
al. (2000) to increase MRP5 expression in their transfectants coincidentally may have up-regulated the expression of an endogenous transporter(s) responsible for the high affinity cGMP transport observed in their vesicles, similar to the findings of Crane (2000). Because we were interested in the transport by MRP4 and MRP5 under more
physiological conditions, we did not treat the cells with any gene
expression modulating agents.
Another major discrepancy with published results is our finding
that transport by MRP5 is rather insensitive to zaprinast, sildenafil,
and trequinsin, inhibitors of cGMP phosphodiesterase 5 (PDE5). Whereas
Jedlitschky et al. (2000) reported a
Ki of 267 nM for the MRP5-mediated
vesicular transport of cGMP, we see no effect of 1 µM sildenafil on
PMEA transport. For the extrusion of PMEA from HEK293 cells, we find an
approximate IC50 of 80 µM. The discrepancy is
not a result of the substrate studied, because we saw no effect of 10 µM sildenafil on MRP-mediated efflux of cGMP from HEK293 cells (our
unpublished results). If the inhibition of cGMP efflux by PDE5
inhibitors has physiological significance, as suggested by Jedlitschky
et al. (2000), then it would probably be caused by inhibition of MRP4,
which is more sensitive to these compounds (Table 2 and Fig. 5). MRP4
is also more sensitive than MRP5 to the nucleoside transporter
inhibitors dipyridamole, dilazep and NBMPR (Table 2). Probenecid, and
to a lesser extent sulfinpyrazone, are the only inhibitors found thus
far that inhibit MRP5 more than MRP4.
Our demonstration that MRP4 and MRP5 transport nucleotide(s) (analogs) with low affinity raises the question of whether nucleotide transport is the main physiological function of these MRPs. For MRP4 this is unlikely, because we have recently found that this transporter has a high affinity for some steroid-sulfates and glucuronides (unpublished observations). It seems likely, therefore, that the transport of such compounds is a more important function of MRP4 than transport of nucleotides. For MRP5, a high-affinity substrate remains to be found.
| |
Acknowledgments |
|---|
We thank Ria Van Berwaer for expert technical assistance and Hein te Riele and John Allen for critical reading of the manuscript.
| |
Footnotes |
|---|
Received November 18, 2002; Accepted January 31, 2003
1 Current address: Netherlands Ophthalmic Research Institute, KNAW, Meibergdreef 47, 1105 BA Amsterdam, the Netherlands.
This work was supported by Dutch Cancer Society grants NKI 2001-2473 to P.B and J.W., 1998-1764 to P.B. and a grant of the European Commission (HPAW-CT-2002-90001 and QLRT-2000-30291) to J.B.
G.R. and P.W. contributed equally to this work.
Address correspondence to: Dr. Piet Borst, Division of Molecular Biology and Center of Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail: p.borst{at}nki.nl
| |
Abbreviations |
|---|
MRP, multidrug resistance protein;
bis-POM-PMEA, bis(pivaloyloxymethyl)-9-(2-phosphonomethoxyethyl)adenine;
cGMP, guanosine 3',5'-cyclic monophosphate;
cPr-PMEDAP, cyclopropyl-PMEDAP;
cycloSAL-d4TMP, cyclosaligenyl-d4T-5'-monophosphate;
ddC, 2',3'-dideoxycytidine;
d4T, 2',3'-didehydro-2',3'-dideoxythymidine;
d4TMP, 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate;
DMEM, Dulbecco's modified Eagle's medium;
E217G, estradiol
glucuronide;
HEK, human embryonic kidney;
HPLC, high-performance liquid
chromatography;
MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl]
propionic acid;
PMEA, 9-(2-phosphonomethoxyethyl)adenine;
PMEDAP, 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine;
PMEG, 9-(2-phosphonomethoxyethyl)guanine;
PDE, phosphodiesterase;
So324, aryloxyalaninylphosphoramidate of d4T-5'-monophosphate (d4TMP).
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine.
J Biol Chem
276:
33747-33754recent advances in the design of efficient tools for the delivery of biologically active nucleoside monophosphates.
Synlett
3:
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), HEK293/5I (
) cells into complete medium was then measured and expressed as a
percentage of the total drug accumulated during loading (150 pmol/106 cells for So324 and 0.5 pmol/106 cells
for cycloSal-d4TMP). The values for So324 efflux are the mean of four
determinations ± S.D.; those for cycloSal-d4TMP are the average
of two determinations ± difference from the mean.
, top) and
MRP5-dependent (
, 
, 
) is expressed as a
percentage of these values. Each point and bar represent mean ± S.D. of three determinations.
