![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Biology, the Technion, Haifa, Israel (M.S., L.R., Y.G.A.); Departments of Rheumatology (G.J.) and Pathology (G.L.S.), VU Medical Center, Amsterdam, the Netherlands; and Departments of Medicine and Molecular Pharmacology, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York (I.D.G.)
Received January 2, 2003; accepted April 10, 2003
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). The reduced folate carrier (RFC; SLC19A1) is a
bidirectional facilitative system that generates transmembrane folate
gradients by exchange with organic phosphates concentrated within cells.
Folate receptors mediate the unidirectional transport of folates into
mammalian cells by a GPI-linked, energy-requiring endocytotic mechanism. There
are, in addition, several organic anion facilitative carriers (the SLC21
family) expressed in epithelial cells and some tumors that transport folates
and antifolates (Hagenbuch and Meier,
2003). These processes are opposed by at least five ATP-driven,
unidirectional ABC exporters, including MRP1–4 and the breast cancer
resistance protein (BCRP) that pump folates out of cells
(Borst and Oude Elferink, 2002;
Volk et al., 2002).
Antifolates use these physiological folate transport pathways to enter
normal and malignant cells. RFC, in particular, is an important determinant of
the activity of antifolates, best characterized for methotrexate (MTX).
Impaired MTX transport caused by decreased RFC expression or altered RFC
function (i.e., mutations) has been well documented in acquired resistance in
a variety of antifolate-resistant murine and human tumor cell lines and
tissues (Matherly and Goldman,
2003). Overexpression of MRP1–4 increases MTX transport into
inverted membrane vesicles and suppresses MTX uptake into intact cells,
resulting in acquired MTX resistance and increased folate growth requirement
(Hooijberg et al., 1999;
Kool et al., 1999;
Lee et al., 2000;
Zeng et al., 2001). Thus far,
however, there is no example of selection with an antifolate resulting in the
induction of primary resistance that is caused by MRP overexpression. In one
case, however, mammalian cells selected for resistance to mitoxantrone because
of overexpression of BCRP were cross-resistant to MTX, and this was associated
with decreased drug accumulation (Volk et al.,
2000,
2002).
It is clear that the level of endogenous physiological folates in cells is
an important determinant of antifolate activity. Folate levels in murine
leukemia cells are very sensitive to the extracellular level; because the
extracellular folate concentration is increased from below to above the
physiological range, there is a near-proportional increase in intracellular
folate pools (Zhao et al.,
2001). High intracellular folate levels inhibit the formation of
active polyglutamate derivatives of antifolates that are retained in cells
(Andreassi and Moran, 2002) and
can compete with antifolates at the level of their target enzymes
(Jackson and Harrap, 1973;
White, 1979;
White and Goldman, 1981). The
contraction of folate pools within cells because of mutations in RFC, with
concurrent impaired transport of reduced folates, can compensate for the loss
of antifolate transport activity by enhancing the rate and extent of
antifolate polyglutamation (Zhao et al.,
2000a,b).
On the other hand, RFC mutations that enhance transport of physiological
folates and expand cellular folate levels result in resistance to antifolates
(Tse et al., 1998;
Tse and Moran, 1998).
The impact of cellular folate pools on the activity of antifolates is the
basis for a novel mechanism of resistance observed in a Chinese hamster ovary
(CHO) PyrR100 cell line selected for resistance to the
lipid-soluble antifolate pyrimethamine
(Assaraf and Slotky, 1993).
There was marked impairment of folate efflux with enhanced accumulation of
physiological folates resulting in decreased activity of pyrimethamine and a
variety of other antifolates (Assaraf and
Goldman, 1997; Jansen et al.,
1999). The current study was undertaken to characterize the basis
for the loss of folate exporter function in PyrR100 cells and to
establish how this affected transport of a structurally different organic
anion, cholate, an MRP substrate
(Henderson et al., 1995;
Jedlitschky et al., 1996).
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Tissue Culture. A clonal subline (C11) of CHO AA8 cells was
subjected to stepwise selection with increasing concentrations of
pyrimethamine, resulting in the establishment of PyrR100 cells as
described previously (Assaraf and Slotky,
1993). Parental CHO AA8 cells and their PyrR100 subline
were maintained as monolayer cultures at 37°C in RPMI-1640 medium
containing 5% fetal calf serum (Invitrogen, Carlsbad, CA), 2.3 µM folic
acid, supplemented with 2 mM glutamine, 100 µg/ml penicillin/streptomycin,
and 1 mM pyruvate. Human ovarian carcinoma 2008 cells and various sublines
stably transduced with the human cDNA encoding for MRP1, MRP2, and MRP3, as
well as HEK293 cells transfected with MRP5, were kindly provided by Prof. P.
Borst and Dr. M. Kool (Netherlands Cancer Institute, Amsterdam, The
Netherlands). HEK293/MRP4 and human breast carcinoma MCF-7/MR cells served as
MRP4 and BCRP overexpressing controls, respectively. These human carcinoma
cell lines were maintained in RPMI-1640 medium containing 2.3 µM folic
acid, 10% fetal calf serum, 2 mM glutamine, and antibiotics.
Growth Inhibition Assay. Cells in mid-logarithmic growth were seeded in 24-well plates (8 x 103 and 104 cells/well, respectively) in medium (0.5 ml/well) containing various concentrations of pyrimethamine, MTX, doxorubicin, vincristine, cholic acid, etoposide, 6-mercaptopurine, or thioguanine. After 3 days, cells were detached by trypsinization, and viability was assessed by trypan blue exclusion. The IC50 is defined as the drug concentration at which growth was inhibited by 50% relative to untreated control cells.
[3H]Cholic Acid Uptake. Exponentially growing cells were
harvested by centrifugation (750g for 5 min), washed twice with
HEPES-buffered saline transport buffer containing: 20 mM HEPES at pH 7.4, 140
mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM D-glucose
supplemented with 1 mM pyruvate. Density was adjusted to 107
cells/ml in the same buffer. Transport measurements were performed as
described previously (Assaraf and Goldman,
1997). Briefly, after 20-min incubation of cells at 37°C,
uptake of [3H]cholic acid (7600–8200 dpm/pmol) at a final
concentration of 50 nM was initiated, after which 1-ml samples were drawn at
the indicated times and transferred to centrifuge tubes containing 10 ml of
ice-cold HEPES-buffered saline. Cells were then centrifuged and washed two
additional times, after which the cell pellet was lysed, scintillation fluid
was added, and radioactivity was measured.
[3H]Cholic Acid Efflux. Wild-type AA8 and PyrR100 cells were loaded with [3H]cholic acid for 20 min at 37°C to comparable intracellular levels by incubation with buffer containing extracellular cholic acid concentrations of 150 and 50 nM, respectively. At the indicated times, cells were processed for the determination of intracellular radioactivity as described above.
Western Blot Analysis of MRP Expression. Microsomal proteins were
extracted from 2 x 107 cells in a buffer (150 µl)
containing: 50 mM Tris, pH 7.5, 50 mM 
-mercaptoethanol, 0.5% Triton
X-100, and the protease inhibitors aprotinin (60 µg/ml), leupeptin (5
µg/ml), phenylmethylsulfonyl fluoride (10 µg/ml), EDTA (1 mM), and EGTA
(1 mM). After a 1-h incubation on ice, the extract was centrifuged at
15,000g for 30 min at 4°C, and the supernatant containing the
fraction of detergent-soluble proteins was collected. Proteins (25–100
µg) were resolved by electrophoresis on 7% polyacrylamide gels containing
SDS and electroblotted onto a Protran membrane (Schleicher and Schuell). Blots
were blocked for 1 h at room temperature in TBS buffer (10 mM Tris at pH 8.0,
150 mM NaCl) containing 20% skim milk and then reacted for 1 h at room
temperature with anti-human MRP and BCRP monoclonal antibodies (kindly
provided by Prof. R.J. Scheper, VU Medical Center, Amsterdam, The
Netherlands). These included various monoclonal antibodies
(Maliepaard et al., 2001): rat
anti-human MRP1 (MRPr1 at a 1:1000 dilution, 1-h incubation at room
temperature), MRP5 (M5I-1, 1:750 dilution, 1-h incubation)
(Maliepaard et al., 2001), as
well as mouse anti-human MRP1 (MRPm5, 1:500 dilution, 1-h incubation), MRP2
(M2III-5, 1:50 dilution, overnight incubation at 4°C), MRP3
(M3II-9, 1:500 dilution, 1-h incubation), MRP4 (M4II-8,
1:15 dilution, overnight incubation), 2001), and BCRP (BXP-21, 1:150 dilution,
overnight incubation) in a TBS buffer containing 2% low fat milk and 0.1%
Tween 20. Blots were then washed three times in TBS containing 0.5% Tween 20
for 10 min at room temperature, and then reacted with horseradish
peroxidase-conjugated goat anti-mouse or anti-rat IgG (1:20,000 dilution;
Jackson Immunoresearch Labs, West Grove, PA) for 1 h at room temperature. To
examine whether the anti-human MRP and BCRP monoclonal antibodies
cross-reacted with the hamster transporter proteins, liver and kidney
microsomal proteins were extracted from fresh Syrian golden hamster
(Mesocricetus auratus) tissues as described previously
(Assaraf and Borgnia, 1994).
Na+-K+-ATPase (
subunit) served as an internal
control and was detected overnight at 4°C with an affinity-purified rabbit
polyclonal antiserum (anti-KETYY) at a 1:3000 dilution (kindly provided by
Prof. S. J. D. Karlish, The Weizmann Institute of Science, Rehovot, Israel),
followed by incubation with a second goat anti-rabbit IgG (1:6000). After
three washes (10 min each) in TBS at room temperature, enhanced
chemiluminescence detection was performed according to the manufacturer's
instructions (Biological Industries, Beth Haemek, Israel). Protein content was
determined using the Bio-Rad protein assay.
Flow Cytometric Analysis of Calcein AM Staining. Exponentially
growing cells (106 cells/60-mm Petri dish) were incubated in growth
medium (5 ml) containing 20 mM HEPES, pH 7.4, and 3 to 300 nM calcein AM, a
chromophore that, in its intracellular anionic form (i.e., calcein), is an
MRP1 (Olson et al., 2001) but
not an MRP5 substrate (McAleer et al.,
1999). After a 20-min incubation at 37°C, cells were harvested
by centrifugation, washed once with phosphate-buffered saline, and analyzed
for fluorescence intensity on a FACSCalibur flow cytometer (BD Biosciences,
San Jose, CA). Excitation and emission were at 488 and 525 nm, respectively.
Autofluorescence intensities of unstained AA8, PyrR100, and
PyrR100/MRP5 cells were recorded and subtracted from those of
calcein AM-stained cells.
Stable Transfections with hMRP1 and hMRP5 Expression Constructs.
Exponentially growing PyrR100 cells (2 x 107) in a
pyrimethamine-free medium were harvested by centrifugation and stably
transfected by electroporation (1000 µF, 234 V) and X-tremeGene reagent
(Roche Applied Sciences, Indianapolis, IN) with 10 and 2 µg, respectively,
of expression vectors containing the hMRP1 (pJ3
-MRP) and MRP5
(pGEM-MRP5) cDNA (kindly provided by Prof. P. Borst and Dr. M. Kool). After 24
h of growth at 37°C, cells were exposed to 400 to 600 µg/ml active
G-418. Stable transfectants obtained after 2 months of G-418 selection were
used for further analyses.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
,
2001). Cross-resistance to the
lipid-soluble thymidylate synthase inhibitor AG337 was also noted.
Interestingly, these cells displayed 3-fold resistance to thioguanine and
6-mercaptopurine, which inhibit purine synthesis by a mechanism distinct from,
and independent of, folate pathways (Table
2).
|
|
Expression of MRPs in Wild-Type AA8 and PyrR100 Cells.
Previous studies demonstrated a marked loss of folate export activity in
PyrR100 cells, resulting in a substantial expansion in the
intracellular folate pool (Assaraf and
Goldman, 1997; Jansen et al.,
1999). To explore the basis for the loss of folate efflux
function, MRP1 through MRP5 and BCRP expression was examined in the microsomal
fraction of wild-type AA8 and PyrR100 cells
(Fig. 1). Whereas wild-type
cells expressed substantial levels of MRP1, PyrR100 cells had no
MRP1 expression on Western blot analysis performed with two different
monoclonal antibodies (Fig.
1A). Furthermore, whereas wild-type AA8 cells expressed MRP5,
albeit to a lesser extent than MRP1, PyrR100 cells had barely
detectable levels of MRP5 (10% of the level of AA8 cells;
Fig. 1A). BCRP was clearly
recognized by the specific monoclonal antibody used but was not detectable in
either wild-type AA8 or in PyrR100 cells
(Fig. 1B). Expression of MRP2
was negligible in both wild-type and resistant cells with a very strong
hamster liver positive control. The antibodies to MRP3 and MRP4 reacted only
weakly to the hamster liver and kidney controls, respectively, and were not
different from the intensity of the wild-type and PyrR100
bands.
|
[3H]Cholic Acid Efflux and Net Accumulation in AA8 and
PyrR100 Cells; Effects of Prostaglandin A1.
MRP1–4 transport a variety of organic anions, and MRP1–3 are known
to transport bile salts, including cholic acid as well as their conjugates
(Henderson et al., 1995;
Jedlitschky et al., 1996). As
indicated in Fig. 2, net cholic
acid efflux from PyrR100 cells loaded with [3H]cholic
acid was markedly reduced compared with efflux from wild-type AA8 cells
(T1/2 of 7.0 ± 0.2 versus 1.7 ± 0.1 min,
respectively). As indicated in the inset, efflux of the major portion of
intracellular [3H]cholic acid can be characterized by a single
exponential with a rate constant in PyrR100 cells that was 25% of
that of wild-type cells (k = 0.075 ± 0.002 versus 0.306
± 0.008/min, respectively). This reduction in cholic acid efflux was
associated with a 
6-fold higher net accumulation of cholate in
PyrR100 cells relative to wild-type AA8 cells (0.794 ± 0.081
versus 0.136 ± 0.02 nmol/g of dry weight, respectively
(Fig. 3A). Furthermore, net
uptake of cholic acid was increased in wild-type AA8 cells to levels
comparable with those obtained with PyrR100 cells in the presence
of 50 µM prostaglandin A1 (PGA1)
(Fig. 3B), an MRP substrate
(Henderson et al., 1995;
Evers et al., 1997), and a
potent inhibitor of folate and MTX export in CHO cells
(Assaraf et al., 1999).
|
|
Cytotoxicity of Cholic Acid and Various MRP1 Substrates to AA8 and
PyrR100 Cells. Cholic acid at high concentrations is cytotoxic
to mammalian cells. Consistent with the low rates of cholic acid efflux and
high levels of net cholic acid accumulation, the cholate IC50 for
inhibition of the growth of PyrR100 cells was 34% of the
concentration for wild-type cells (187 ± 66 versus 543 ± 55
µM, respectively), as indicated in Table
1. Furthermore, consistent with the loss of MRP1 expression,
PyrR100 cells were up to 3-fold more sensitive to other cytotoxic
MRP1 substrates—etoposide, doxorubicin, and vincristine—than
wild-type AA8 cells (Borst and Oude
Elferink, 2002).
Flow Cytometric Analysis of Calcein AM Uptake in AA8 and
PyrR100 Cells. The anionic chromophore calcein is an MRP1
(Olson et al., 2001) but not
an MRP5 substrate (McAleer et al.,
1999) and is therefore a useful probe for deciphering the impact
of the loss of MRP1 in cells in which expression of both exporters is reduced.
Both cell lines showed a concentration-dependent increase in intracellular
calcein fluorescence, but this was much greater for the PyrR100
cells. The fluorescence ratio of PyrR100 to AA8 reached a peak of
5 at an extracellular calcein AM concentration of 100 nM
(Fig. 4A). Furthermore, 2 mM
probenecid, an MRP inhibitor, markedly increased calcein fluorescence in
wild-type AA8 cells to levels comparable with those obtained with
PyrR100 cells but failed to alter the high level of calcein
fluorescence in PyrR100 cells
(Fig. 4B). Finally, calcein
fluorescence in PyrR100 cells transfected with MRP5
(PyrR100/MRP5) was identical to that of nontransfected
PyrR100 cells (Fig.
4C).
|
The Impact of MRP1 and MRP5 Transfection on the Cytotoxicity of Pyrimethamine and MTX in PyrR100 Cells. The loss of MRP1 expression and the marked decrease in the MRP5 level in PyrR100 cells prompted studies to assess the impact of overexpression of these exporters on the cytotoxicity of pyrimethamine and MTX to PyrR100 cells. PyrR100 cells were transfected with expression constructs harboring MRP1 or MRP5 cDNAs, and stable transfectants (PyrR100/MRP1 and PyrR100/MRP5, respectively) growing in G-418 were isolated. PyrR100/MRP5 cells expressed high levels of MRP5, relative to PyrR100 and wild-type AA8 cells (Fig. 1A); however, the IC50 values for pyrimethamine with PyrR100/MRP5 and PyrR100 cells were not different (129 ± 14 versus 122 ± 9 µM, respectively; Fig. 5 and Table 2). Sensitivity of PyrR100/MRP5 cells to MTX was also not different from that of PyrR100 cells (Table 2). Activity of MRP5 in the transfected cells was confirmed by the observation that sensitivity to thioguanine and 6-mercaptopurine was restored. On the other hand, although cells transfected with the MRP1 construct displayed high-level resistance to 600 µg/ml G-418, in five separate transfections in which five to seven independent clones were studied, no expression of MRP1 could be detected (Fig. 1A).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
;
Assaraf and Goldman, 1997;
Jansen et al., 1999). This, in
turn, suppresses the interaction between pyrimethamine and its target enzyme
dihydrofolate reductase, resulting in high-level resistance to this and other
antifolate agents. A variety of observations in the current study indicate
that this marked decrease in folate exporter activity in PyrR100
cells is caused by the loss of MRP1 function:
; Jedlitschky et al.,
1996) was markedly impaired in PyrR100 cells and this
resulted in a marked increase in net cholate accumulation. ;
Evers et al., 1997) and a
potent inhibitor of folate export (Assaraf
et al., 1999) markedly increased net uptake of cholate in
wild-type AA8 cells to levels comparable with that of PyrR100
cells. ). ).
An attempt was made to restore the MRP1 phenotype to that of wild-type CHO
cells. However, multiple transfections with the selection of multiple clones
using an expression vector harboring MRP1 cDNA, by both electroporation and by
the liposomal X-tremeGene reagent, resulted in high-level resistance to G-418
but no detectable expression of MRP1. Because the vector contained both the
neomycin phosphotransferase resistance and the MRP1 genes, the failure of MRP1
expression must have been caused by either deletion of the MRP1 gene or some
other mechanism by which its expression was silenced. Very high MRP1
expression might so deplete cell folates that survival of these clones would
be impossible. In other studies, cellular folate levels were decreased in MRP1
transfectants, but this was not of sufficient magnitude to alter the survival
of these cells (Hooijberg et al.,
2003).
In addition to the decrease in MRP1 expression in PyrR100 cells,
there was a marked decrease in expression of MRP5. This was unexpected because
MRP5 does not seem to transport folates or antifolates. Rather, MRP5
transports 3',5'-cyclic nucleotides, nucleotides, and nucleoside
analogs and confers resistance to 6-mercaptopurine and thioguanine
(Wijnholds et al., 2000).
Furthermore, MTX does not inhibit cGMP transport in inverted erythrocyte
membrane vesicles that are probably mediated, in part, by MRP5
(Sundkvist et al., 2002). Data
in the current article are consistent with these observations and indicate
that the loss of MRP5 does not contribute to the loss of folate export
function in PyrR100 cells. Hence, transfection and high-level
expression of MRP5 in PyrR100 cells had no effect on the level of
resistance to pyrimethamine or MTX. However, sensitivity to thioguanine and
6-mercaptopurine was restored, confirming the activity of the transfected
exporter.
Why MRP5 expression was lost during selection with pyrimethamine remains
unclear. It is possible that this may have occurred because of the stringent
conditions during the establishment of PyrR100 cells; selection was
performed with medium supplemented with only 5% dialyzed serum
lacking ribonucleosides (Assaraf and
Slotky, 1993). The continuous antifolate-mediated blockade of
purine and thymidylate biosynthesis in the absence of exogenous nucleosides
may have resulted in an adaptive down-regulation of this nucleotide
transporter to preserve intracellular nucleotide pools. The expansion of these
pools in PyrR100 cells under usual growth conditions would probably
result in impaired activation of nucleoside antimetabolites. Introduction of
MRP5 into PyrR100 cells restored wild-type sensitivity to
thiopurines, presumably because of a decrease in ribonucleoside pools.
Additional studies will be required to further substantiate this
formulation.
PyrR100 cells have undergone several adaptive changes that
result in expansion of cellular folate pools: 1) the expression of at least
one major ATP-driven folate efflux pathway, MRP1, was completely lost; 2)
influx of folic acid was enhanced 4-fold because of increased activity of a
low pH transporter (Assaraf et al.,
1998), and 3) folylpolyglutamate synthetase activity, and hence
the rate and extent of folate polyglutamylation, was increased 3- to 4-fold
(Jansen et al., 1999). The
latter results in the formation of congeners that are retained within cells
and allow the build-up of high folate levels, because long-chain polyglutamate
conjugates of folates are not substrates for MRP1-MRP4
(Zeng et al., 2001;
Chen et al., 2002).
Collectively, these alterations contribute to a marked increase in the net
accumulation of folates, resulting in a 100-fold decrease in the folic acid
growth requirement in PyrR100 cells relative to parental AA8 cells
(Jansen et al., 1999).
Like the adaptive change in MRP1 expression that occurred in response to
pyrimethamine-selective pressure, other studies indicate that a similar change
can also occur in response to folate deprivation. When CEM-7A cells were
subjected to gradual leucovorin deprivation, there was overexpression of RFC
as well as loss of MRP1 expression (Jansen
et al., 1990; Assaraf et al.,
2003). Because increased RFC expression can produce
near-symmetrical changes in influx and efflux of folates, with only small
increases in steady-state free levels
(Zhao et al., 1997), and
because endogenous MRP activity always suppresses the free folate and
antifolate monoglutamyl levels in cells
(Assaraf et al., 1999;
Zhao et al., 2000a), it is
likely that the loss of MRP1 export is the most efficient way for cells to
substantially enhance concentrative folate uptake.
Recent studies have established that augmentation of cellular
tetrahydrofolate cofactor pools results in marked suppression of the activity
of antifolates. Intracellular folate pools increase almost in proportion to
the increase in extracellular folate concentration, even over the range of
physiological blood folate levels. As the intracellular folate level is
increased, because of an increase in extracellular folate
(Zhao et al., 2001) or
mutations in RFC that enhance folate transport
(Tse et al., 1998;
Tse and Moran, 1998), there
are substantial decreases in the activities of antifolates that do and do not
form polyglutamate derivatives. There are several mechanisms that underlie the
decreased cytotoxic activity of antifolates that encompass resistance to
antifolates observed in this and earlier studies with the PyrR100
cell line:
;
White, 1979;
Seither et al., 1989). This is
the mechanism of resistance to pyrimethamine and the other lipid-soluble DHFR
inhibitors in PyrR100 cells
(Table 1) and to MTX
(Table 2)
(Zhao et al., 2001). ).
This is a very important factor in the observed resistance to
5,10-diadeazatetrahydrofolate and AG2034
(Tse et al., 1998;
Tse and Moran, 1998; Zhao et
al., 2000b,
2001). ). |
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: RFC, reduced folate carrier; BCRP, breast cancer resistance protein; MRP, multidrug resistance protein; MTX, methotrexate; CHO, Chinese hamster ovary; MK571, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid; HEK, human embryonic kidney; DHFR, dihydrofolate reductase; AG2034, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid; AG337, nolatrexed dihydrochloride.
Address correspondence to: Dr. Yehuda G. Assaraf, Department of Biology, The Technion-Israel Institute of Technology, Haifa 32000, Israel. E-mail: assaraf{at}tx.technion.ac.il
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Assaraf YG, Babani S, and Goldman ID (1998) Increased
activity of a novel low pH folate transporter associated with lipophilic
antifolate resistance in Chinese hamster ovary cells. J Biol
Chem 273:
8106–8111.
Assaraf YG and Borgnia MJ (1994) Probing the interaction of the multidrug-resistance phenotype with the polypeptide ionophore gramicidin D via functional channel formation. Eur J Biochem 222: 813–824.[Medline]
Assaraf YG and Goldman ID (1997) Loss of folic acid
exporter function with markedly augmented folate accumulation in lipophilic
antifolate-resistant mammalian cells. J Biol Chem
272:
17460–17466.
Assaraf YG, Rothem L, Hooijberg JH, Stark M, Ifergan I, Kathmann I,
Dijkmans BA, Peters GJ, and Jansen G (2003) Loss of multidrug
resistance protein 1 expression and folate efflux activity results in a highly
concentrative folate transport in human leukemia cells. J Biol
Chem 278:
6680–6686.
Assaraf YG, Sierra EE, Babani S, and Goldman ID (1999) Inhibitory effects of prostaglandin A1 on membrane transport of folates mediated by both the reduced folate carrier and ATP-driven exporters. Biochem Pharmacol 58: 1321–1327.[CrossRef][Medline]
Assaraf YG and Slotky JI (1993) Characterization of a
lipophilic antifolate resistance provoked by treatment of mammalian cells with
the antiparasitic agent pyrimethamine. J Biol Chem
268:
4556–4566.
Borst P and Oude Elferink RO (2002) Mammalian ABC transporters in health and disease. Annu Rev Biochem 71: 537–592.[CrossRef][Medline]
Chen ZS, Lee K, Walther S, Raftogianis RB, Kuwano M, Zeng H, and
Kruh GD (2002) Analysis of methotrexate and folate transport by
multidrug resistance protein 4 (ABCC4): MRP4 is a component of the
methotrexate efflux system. Cancer Res
62:
3144–3150.
Evers R, Cnubben NH, Wijnholds J, van Deemter L, van Bladeren PJ, and Borst P (1997) Transport of glutathione prostaglandin A conjugates by the multidrug resistance protein 1. FEBS Lett 419: 112–116.[CrossRef][Medline]
Hagenbuch B and Meier PJ (2003) The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609: 1–18.[Medline]
Henderson GB, Hughes TR, and Saxena M (1995) Distinct systems mediate the unidirectional efflux of methotrexate and cholate in human CCRF-CEM cells. Arch Biochem Biophys 316: 77–82.[CrossRef][Medline]
Hooijberg JH, Broxterman HJ, Kool M, Assaraf YG, Peters GJ,
Noordhuis P, Scheper RJ, Borst P, Pinedo HM, and Jansen G (1999)
Antifolate resistance mediated by the multidrug resistance proteins MRP1 and
MRP2. Cancer Res 59:
2532–2535.
Hooijberg JH, Peters GJ, Assaraf YG, Kathmann I, Priest DG, Bunni MA, Veerman AJ, Scheffer GL, Kaspers GJ, and Jansen G (2003) The role of multidrug resistance proteins MRP1, MRP2 and MRP3 in cellular folate homeostasis. Biochem Pharmacol 65: 765–771.[CrossRef][Medline]
Jackson RC and Harrap KR (1973) Studies with a mathematical model of folate metabolism. Arch Biochem Biophys 158: 827–841.[CrossRef][Medline]
Jansen G, Barr H, Kathmann I, Bunni MA, Priest DG, Noordhuis P,
Peters GJ and Assaraf YG (1999) Multiple mechanisms of resistance
to polyglutamatable and lipophilic antifolates in mammalian cells: role of
increased folylpolyglutamylation, expanded folate pools and intralysosomal
drug sequestration. Mol Pharmacol
55:
761–769.
Jansen G, Westerhof GR, Jarmuszewski MJA, Kathmann I, Rijksen G,
and Schornagel JH (1990) Methotrexate transport in variant human
CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier.
Selective effect on carrier-mediated transport of physiological concentrations
of reduced folates. J Biol Chem
265:
18272–18277.
Jedlitschky G, Leier I, Buchholz U, Barnouin K, Kurz G, and Keppler
D (1996) Transport of glutathione, glucuronate and sulfate
conjugates by the MRP gene-encoded conjugate export pump. Cancer
Res 56:
988–994.
Kool M, van der Linden M, de Haas M, Scheffer GL, de Vree JM, Smith
AJ, Jansen G, Peters GJ, Ponne N, Scheper RJ, et al. (1999) MRP3,
an organic anion transporter able to transport anti-cancer drugs.
Proc Natl Acad Sci USA
96:
6914–6919.
Lee K, Klein-Szanto AJ, and Kruh GD (2000) Analysis of
the MRP4 drug resistance profile in transfected NIH3T3 cells. J
Natl Cancer Inst 92:
1934–1940.
Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen MA, Pijnenborg
AC, Schinkel AH, Van de Vijver MJ, Scheper RJ, and Schellens JH
(2001) Subcellular localization and distribution of the breast
cancer resistance protein transporter in normal human tissues.
Cancer Res 61:
3458–3464.
Matherly LH and Goldman ID (2003) Membrane transport of folates, in Vitamins and Hormones (Litwack G ed), Academic Press, San Diego, CA.
McAleer MA, Breen MA, White NL, and Matthews N (1999)
PABC11 (also known as MOAT-C and MRP5), a member of the ABC family of
proteins, has anion transporter activity but does not confer multidrug
resistance when overexpressed in human embryonic kidney 293 cells.
J Biol Chem 274:
23541–23548.
Olson DP, Taylor BJ, and Ivy SP (2001) Detection of MRP functional activity: calcein AM but not BCECF AM as a multidrug resistance-related protein (MRP1) substrate. Cytometry 46: 105–113.[CrossRef][Medline]
Seither RL, Trent DF, Mikullecky DC, Rape TJ, and Goldman ID
(1989) Folate-pool interconversions and inhibition of
biosynthetic processes after exposure of L1210 leukemia cells to antifolates.
J Biol Chem 264:
17016–17023.
Sundkvist E, Jaeger R, and Sager G (2002) Pharmacological characterization of the ATP-dependent low Km guanosine 3',5'-cyclic monophosphate (cGMP) transporter in human erythrocytes. Biochem Pharmacol 63: 945–949.[CrossRef][Medline]
Tse A, Brigle K, Taylor SM, and Moran RG (1998)
Mutations in the reduced folate carrier gene which confer dominant resistance
to 5,10-dideazatetrahydrofolate. J Biol Chem
273:
25953–25960.
Tse A and Moran RG (1998) Cellular folates prevent
polyglutamation of 5,10-dideazatetrahydrofolate. A novel mechanism of
resistance to folate antimetabolites. J Biol Chem
273:
25944–25952.
Volk EL, Farley KM, Wu Y, Li F, Robey RW, and Schneider E
(2002) Overexpression of wild-type breast cancer resistant
protein mediates methotrexate resistance. Cancer Res
62:
5035–5045.
Volk EL, Rohde K, Rhee M, McGuire JJ, Doyle LA, Ross DD, and
Schneider E (2000) Methotrexate cross-resistance in a
mitoxantrone-selected multidrug-resistant MCF7 breast cancer cell line is
attributable to enhanced energy-dependent drug efflux. Cancer
Res 60:
3514–3521.
Webber S, Bartlett CA, Boritzki TJ, Hillard JA, Howland EF, Johnston AL, Kosa M, Margosiak SA, Morse CA, and Shetty BV (1996) AG337, a novel lipophilic thymidylate synthase inhibitor: in vitro and in vivo preclinical studies. Cancer Chemother Pharmacol 37: 509–517.[CrossRef][Medline]
White JC (1979) Reversal of methotrexate binding to
dihydrofolate reductase by dihydrofolate. Studies with pure enzyme and
computer modeling using network thermodynamics. J Biol
Chem 254:
10889–10895.
White JC and Goldman ID (1981) Methotrexate resistance
in an L1210 cell line resulting from increased dihydrofolate reductase,
decreased thymidylate synthetase activity and normal membrane transport.
Computer simulations based on network thermodynamics. J Biol
Chem 256:
5722–5727.
Wijnholds J, Mol CA, van Deemter L, de Haas M, Scheffer GL, Baas F,
Beijnen JH, Scheper RJ, Hatse S, De Clercq E, et al. (2000)
Multidrug-resistance protein 5 is a multispecific organic anion transporter
able to transport nucleotide analogs. Proc Natl Acad Sci
USA 97:
7476–7481.
Zeng H, Chen ZS, Belinsky MG, Rea PA, and Kruh GD
(2001) Transport of methotrexate (MTX) and folates by multidrug
resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX
transport. Cancer Res
61:
7225–7232.
Zhao R, Babani S, Gao F, Liu L, and Goldman ID (2000a)
The mechanism of transport of the multitargeted antifolate, MTA-LY231514 and
its cross resistance pattern in cell with impaired transport of methotrexate.
Clin Cancer Res 6:
3687–3695.
Zhao R, Gao F, Babani S, and Goldman ID (2000b)
Sensitivity to 5,10-dideazatetrahydrofolate is fully conserved in a murine
leukemia cell line highly resistant to methotrexate due to impaired transport
mediated by the reduced folate carrier. Clin Cancer
Res 6:
3304–3311.
Zhao R, Gao F, and Goldman ID (2001) Marked suppression of the activity of some, but not all, antifolate compounds by augmentation of folate cofactor pools within tumor cells. Biochem Pharmacol 61: 857–865.[CrossRef][Medline]
Zhao R, Seither R, Brigle KE, Sharina IG, Wang PJ, and Goldman ID
(1997) Impact of overexpression of the reduced folate carrier
(RFC1), an anion exchanger, on concentrative transport in murine L1210
leukemia cells. J Biol Chem
272:
21207–21212.
This article has been cited by other articles:
|
|
 |
|
  P. Wielinga, J. H. Hooijberg, S. Gunnarsdottir, I. Kathmann, G. Reid, N. Zelcer, K. van der Born, M. de Haas, I. van der Heijden, G. Kaspers, et al. The Human Multidrug Resistance Protein MRP5 Transports Folates and Can Mediate Cellular Resistance against Antifolates Cancer Res., May 15, 2005; 65(10): 4425 - 4430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zhao, S. Chattopadhyay, M. Hanscom, and I. D. Goldman Antifolate Resistance in a HeLa Cell Line Associated With Impaired Transport Independent of the Reduced Folate Carrier Clin. Cancer Res., December 15, 2004; 10(24): 8735 - 8742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Fotoohi, G. Jansen, Y. G. Assaraf, L. Rothem, M. Stark, I. Kathmann, J. Gregorczyk, G. J. Peters, and F. Albertioni Disparate mechanisms of antifolate resistance provoked by methotrexate and its metabolite 7-hydroxymethotrexate in leukemia cells: implications for efficacy of methotrexate therapy Blood, December 15, 2004; 104(13): 4194 - 4201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Ifergan, A. Shafran, G. Jansen, J. H. Hooijberg, G. L. Scheffer, and Y. G. Assaraf Folate Deprivation Results in the Loss of Breast Cancer Resistance Protein (BCRP/ABCG2) Expression: A ROLE FOR BCRP IN CELLULAR FOLATE HOMEOSTASIS J. Biol. Chem., June 11, 2004; 279(24): 25527 - 25534. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
