From the Department of Biology, The
Technion, Haifa 32000, Israel and the Departments of ¶ Medical
Oncology and
Rheumatology, Vrije Universiteit Medical
Center, 1081 HV Amsterdam, The Netherlands
Received for publication, September 9, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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We studied the molecular basis of
the up to 46-fold increased accumulation of folates and methotrexate
(MTX) in human leukemia CEM-7A cells established by gradual deprivation
of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and
68-fold decreased LCV and folic acid growth requirements and
23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A
cells displayed a 74-86-fold increase in the reduced folate carrier
(RFC)-mediated influx of LCV and MTX, RFC overexpression per
se cannot induce a prominently increased folate/MTX accumulation
because RFC functions as a nonconcentrative anion exchanger. We
therefore explored the possibility that folate efflux activity mediated
by members of the multidrug resistance protein (MRP) family was
impaired in CEM-7A cells. Parental CEM cells expressed substantial
levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast
cancer resistance protein were undetectable. In contrast, CEM-7A cells
lost 95% of MRP1 levels while retaining parental expression of MRP4
and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the
[3H]folic acid efflux rate constant, which was identical
to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with
parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter
the calcein fluorescence in CEM-7A cells, which markedly lost MRP1
expression. Replenishment of LCV in the growth medium of CEM-7A cells
resulted in resumption of normal MRP1 expression. These results
establish for the first time that MRP1 is the primary folate efflux
route in CEM leukemia cells and that the loss of folate efflux
activity is an efficient means of markedly augmenting cellular
folate pools. These findings suggest a functional role for MRP1 in the
maintenance of cellular folate homeostasis.
Folate cofactors serve as one-carbon donors in the de
novo biosynthesis of purines and thymidylate (1). As such, normal and neoplastic dividing cells have an absolute folate requirement for
DNA replication (1). Disruption of folate biosynthesis with folic acid
antagonists (i.e. antifolates) is the pharmacological basis
for the antitumor activity of methotrexate
(MTX)1 and various
antifolates (2). Because mammalian cells are devoid of folate
biosynthesis, they rely on folate vitamin uptake from exogenous
sources. Membrane transport of folates and MTX is mediated by several
systems (3, 4): (a) the reduced folate carrier (RFC) is the major
uptake route that functions as a bi-directional anion exchanger (5, 6)
taking up folates through an antiport exchange mechanism with
intracellular organic phosphates (7); (b) folate receptors mediate the
unidirectional uptake of folate cofactors into mammalian cells via an
endocytotic process (8); and (c) an apparently independent transport
system with optimal folate uptake activity at low pH (9-11).
Apart from RFC, efflux of folates and MTX (12, 13) is mediated by
multidrug resistance proteins (MRP) MRP1-4 (14-19), which belong to
the ATP-binding cassette superfamily (20, 21). Members of the MRP
family, currently comprising nine genes (i.e. MRP1-9), function as ATP-driven efflux transporters of various natural product
anions and acidic charged drug conjugates (14, 15). Mammalian cells
transfected with MRP1-4 accumulate decreased levels of MTX and
consequently display resistance to this drug, particularly upon short
term drug exposure (16-19). Membrane vesicles isolated from MRP1- and
MRP2-transfected cells exhibit ATP-dependent transport of
MTX (16). Detailed kinetic analysis of folic acid, leucovorin (LCV;
5-formyl-tetrahydrofolate) and MTX transport into MRP1- and MRP3-rich
membrane vesicles reveals Km values in the low
millimolar range (22). Hence, the free intracellular level of folates
and antifolates including MTX is determined by the net activities of
these influx (i.e. RFC) and efflux (RFC and MRP) transport pathways.
CEM-7A is a human leukemia CCRF-CEM subline previously established by
gradual deprivation of LCV from the growth medium (23), resulting in
RFC gene amplification (24) and carrier overexpression (23, 24).
Consequently, CEM-7A cells displayed a marked increase in the influx of
MTX and LCV accompanied by a comparable increase in the steady-state
transmembrane gradient of MTX (23). Surprisingly, however, there was no
increase in the efflux rate constant for MTX (23). This is in contrast
with previous studies (25) demonstrating that upon transfection of RFC
cDNA into murine leukemia cells, there is a marked increase
(5-10-fold) in the bi-directional fluxes (i.e. influx and
efflux) of MTX with only a small increase in the transmembrane
gradient. This discrepancy raised the possibility that the lack of
increase in the MTX efflux rate in CEM-7A cells may be due to a second
alteration, such as a decrease in MRP-mediated folate efflux activity.
To explore this further, studies were undertaken to characterize the
bi-directional fluxes and net transport of folic acid in CEM-7A cells.
Folic acid has a very low affinity for the RFC (26). Thus, folic acid
efflux should be largely mediated by RFC-independent pathways such as
MRPs (14-19). Accordingly, alterations in folic acid efflux (27)
should primarily reflect changes in MRP expression and efflux activity.
We report here that CEM-7A cells exhibit a high influx and
transmembrane gradient for folic acid, LCV, and MTX and that this is
accompanied by a 5-fold decrease in the folic acid efflux rate
constant. This markedly defective folate export appears to be due to a
95% loss of MRP1 expression, thereby resulting in high transmembrane
folate gradients. These results establish for the first time that MRP1
is the primary folate efflux transporter in CEM leukemia cells and that
the loss of this major folate efflux route is an efficient means of
markedly augmenting cellular folate pools. These findings suggest a
functional role for MRP1 in folate homeostasis in mammalian cells.
Chemicals--
Folic acid, LCV (calcium salt), MTX, probenecid,
and N-hydroxysuccinimide were obtained from Sigma.
Trimetrexate (TMQ) glucoronate was a gift from
Warner-Lamber/Parke-Davis (Ann Arbor, MI), edatrexate was kindly
provided by Dr. J. H. Schornagel (Netherlands Cancer Institute,
Amsterdam, NL), and GW1843U89
((S)-2[5-[[(1,2-dihydro-3-methyl-1-oxobenzo[f]quinazolin-9-yl)methyl]-1-oxo-2-isoindolinyl]-glutaric acid) was provided by Dr. G. K. Smith (Glaxo-Wellcome Research Laboratories). [3',5',7',9-3H]folic acid (25 Ci/mmol),
[3H]MTX (15 Ci/mmol), and [3H]leucovorin
(50 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA) and
purified prior to use as previously described (28). Calcein AM
was from Molecular Probes (Eugene, OR), and MK571 was from Cayman
Chemicals (Ann Arbor, MI).
Tissue Culture--
Parental CCRF-CEM leukemia cells were grown
in RPMI 1640 medium (Invitrogen) containing 2.3 µM folic
acid and 10% fetal calf serum (Invitrogen) supplemented with 2 mM glutamine and 100 µg/ml penicillin/streptomycin.
CEM-7A cells, originally isolated by gradual deprivation of LCV from
the growth medium (23), were cultured in folic acid-free RPMI 1640 medium (Biological Industries, Beth-Haemek, Israel) supplemented with
10% dialyzed fetal calf serum (Invitrogen), antibiotics, and 0.25 nM leucovorin as the sole folate source. CEM/MTX (29) and
CEM/GW70 cells (30) were originally obtained by stepwise selection in
gradually increasing concentrations of MTX and GW1843, respectively.
CEM/MTX (26) and CEM/GW70 (30) displayed defective MTX transport
because of mutations in the RFC gene. CEM/MTX and CEM/GW70 were grown in RPMI 1640 medium containing 2.3 µM folic acid and 10%
fetal calf serum as well as 1 µM MTX and 70 nM GW1843, respectively. These cell lines were further
selected by gradual folic acid deprivation, thereby establishing the
sublines CEM/MTX-LF (26) and CEM/GW70-LF (30). These cell lines were
cultured in folate-free RPMI medium containing 10% dialyzed fetal calf
serum, 2 mM glutamine, antibiotics as well as 2 and 5 nM folic acid, respectively, as the sole folate source. The
human ovarian carcinoma cell line, 2008, and its sublines stably
transduced with MRP1, 2, 3, and 5 cDNAs were kindly provided by
Prof. P. Borst and Dr. M. Kool (The Netherlands Cancer Institute, Amsterdam, The Netherlands), whereas HEK293 cells were used as a
control for MRP4 overexpression. These cell lines were cultured in RPMI
1640 medium containing 2.3 µM folic acid, 10% fetal calf serum, 2 mM glutamine, and antibiotics.
Folic Acid and Leucovorin Growth Requirement--
Exponentially
growing CEM and CEM-7A cells were washed three times with
phosphate-buffered saline and transferred to folic acid-free RPMI 1640 medium (Biological Industries) supplemented with 10% dialyzed fetal
calf serum (Invitrogen). Following 4-6 days of growth in folic
acid-deficient medium, cellular growth was arrested. The cells were
then seeded (3 × 104/well) in medium (0.15 ml)
containing increasing concentrations of either folic acid or LCV
ranging from 1 pM to 3 µM in 96-well tissue
culture plates. Following 3 days of incubation at 37 °C, viable cell
numbers were determined by hemocytometer count using Trypan blue
exclusion. EC50 is defined as the folic acid
concentration necessary to produce 50% of maximal cell growth.
Antifolate Growth Inhibition--
Parental CEM cells and their
CEM-7A subline from the mid-logarithmic phase were seeded in 96-well
plates (3 × 104/well) in growth medium (0.15 ml/well)
containing various concentrations of the antifolates MTX or edatrexate.
After 3 days of incubation at 37 °C, cell numbers were determined by
hemocytometer count using Trypan blue exclusion. The 50% inhibitory
concentration (IC50) is defined as the drug concentration
at which cell growth is inhibited by 50% relative to untreated controls.
Transport of Radiolabeled Folates and MTX--
Cells (2 × 107 CEM and 3 × 106 CEM-7A cells) at the
mid-log phase of growth were washed three times in an HBS
transport buffer containing 20 mM Hepes, 140 mM
NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM D-glucose, pH 7.4, with NaOH (27). The cells
were then incubated at 37 °C for 1-30 min in HBS (1-ml suspensions)
containing 2 µM [3H]MTX,
[3H]LCV, or [3H]folic acid. TMQ was
included (5 µM) to block [3H]folic acid and
dihydrofolic acid reduction by dihydrofolate reductase (27). Transport
controls contained a 500-fold excess of unlabeled MTX (1 mM). Transport was stopped by the addition of 10 ml of
ice-cold HBS. Then the cell suspension was centrifuged at 500 × g for 5 min at 4 °C, and the cell pellet was washed twice with 10 ml of ice-cold transport buffer. The final cell pellet was
suspended in 0.2 ml of water, and the radioactivity was determined on
an Ultima Gold (Packard) liquid scintillation spectrometer.
[3H]Folic Acid Efflux--
The cells in
exponential growth were harvested by centrifugation (750 × g for 5 min), washed twice with HBS, and adjusted to a
density of 5 × 106/ml (CEM-7A cells) or 2 × 107 cells/ml (CEM, CEM/MTX-LF, and CEM/GW70-LF cells) in 10 ml HBS (pH 7.4 at 37 °C) and then transferred to 50-ml capped tubes
and placed in a shaking 37 °C water bath. Ten min before the
addition of [3H]folic acid, TMQ was added at 5 µM and was present during the entire period of loading
and efflux. Following a 20-min loading period, the cells were
centrifuged (750 × g for 5 min), the supernatant was
aspirated, and the cell pellet was resuspended in 11 ml of prewarmed
(37 °C) HBS and placed in a shaking water bath at 37 °C. Then
1-ml samples were drawn and transferred to a centrifuge tube containing
10 ml ice-cold HBS. The cells were centrifuged, and the cell pellet was
processed for radioactivity. To distinguish between the RFC-mediated
and MRP-mediated folic acid efflux routes, the irreversible RFC
inhibitor N-hydroxysuccinimide ester of MTX (NHS-MTX, 7.5 µM) (31) and probenecid (1 mM), which
inhibits MRP1 activity (32), were added to the transport buffer.
Because the influx capacities among the leukemia cell lines differed to a large extent, CEM/MTX-LF, CEM/GW70-LF, CEM-7A, and parental CEM cells
were loaded to comparable levels with the following [3H]folic acid concentrations: 2, 2.5, 2.5, and 10 µM, respectively. Folic acid efflux kinetic data were
expressed as the log of the ratio of exchangeable intracellular folate
at a given transport time to the initial exchangeable intracellular
level (27).
Western Blot Analysis of MRPs and RFC Expression--
To examine
the expression of RFC and the various MRPs in parental CEM cells and
its various sublines (2 × 107 cells), total cellular
proteins were extracted in a buffer (250 µl) containing 50 mM Tris, pH 7.5, 50 mM Flow Cytometric Analysis of Calcein AM
Staining--
Exponentially growing CEM and CEM-7A cells were adjusted
to a density of 106/ml and incubated in growth medium
containing 3 nM calcein AM (Molecular Probes, Eugene, OR),
a chromophore that in its intracellular anionic form (i.e.
calcein), is exported also by MRP1 (33). Following 20 min of incubation
at 37 °C, the cells were harvested by centrifugation, washed once
with phosphate-buffered saline, and analyzed for fluorescence
intensity per cell by a FACSCalibur flow cytometer (Becton
Dickenson). Excitation was at 488 nm, and emission was at 525 nm.
The autofluorescence intensities of unstained parental CEM and CEM-7A
cells were recorded and subtracted from those of calcein AM-stained cells.
Folate Growth Requirement and Folate/Antifolate
Accumulation--
Folic acid and LCV growth requirements in parental
CCRF-CEM cells were compared with those of CEM-7A cells adapted to grow in a medium containing 0.25 nM LCV as the sole folate
source (23). The growth requirements of CEM-7A cells for folic acid and
LCV were markedly decreased when compared with parental CEM cells. The
LCV and folic acid concentrations necessary to produce 50% maximal
growth (EC50) of CEM-7A cells were 10- and 68-fold lower than those obtained with parental CEM cells (Table
I). Furthermore, relative to parental CEM
cells, CEM 7-A cells were 23- and 25-fold more sensitive to the
dihydrofolate reductase inhibitors MTX and edatrexate (Table I).
Because these results were consistent with an increased capacity of
CEM-7A cells to accumulate folates and antifolates, the transport of
[3H]LCV (Fig.
1A), [3H]MTX
(Fig. 1B), and [3H]Folic acid (Fig.
1C) was determined in parental CEM and CEM-7A cells. The
accumulation of 2 µM tritiated folates and MTX was markedly increased in CEM-7A cells following 20 min of uptake (Fig. 1);
CEM-7A cells accumulated 490 ± 50 pmol LCV, 510 ± 130 pmol
of MTX, and 30 pmol of folic acid/107 cells, as compared
with 19 ± 4 pmol of LCV, 16 ± 3 pmol of MTX, and 2 pmol of
folic acid/107 cells, respectively, in parental CEM cells
(Table II). Based on the level of MTX
tightly bound to dihydrofolate reductase (determined by efflux
studies), the net MTX and folic acid pools in CEM-7A cells were 46- and
15-fold higher than in parental CEM cells, respectively (Table II).
Consistently, LCV accumulation in CEM 7A cells was 26-fold higher than
that in parental CEM cells (Table II). This marked accumulation of
folates and MTX was not associated with increased activities of
folate-dependent enzymes including dihydrofolate reductase,
thymidylate synthase, and folylpoly- [3H]Folic Acid Efflux--
The markedly increased
levels of folate and MTX accumulation in CEM-7A cells cannot result
from RFC overexpression per se because the latter functions
as an anion exchanger (5, 6) generating very little concentrative
gradients (25). Rather, the high level accumulation of folates and MTX
was suggestive of the loss of some RFC-independent folate efflux
function as previously shown in pyrimethamine-resistant Chinese hamster
ovary cells (27). Hence, folic acid efflux activity in CEM and CEM-7A cells was determined. Whereas LCV and MTX are good RFC transport substrates (Km = 1.4 and 5 µM,
respectively) (23), folic acid is a very poor transport substrate
(Km = 175 µM) (26). To minimize the
RFC-mediated component of efflux and thereby maximize the ATP-driven
folate efflux route(s), [3H]folic acid rather than
[3H]MTX was used in all efflux studies.
[3H]Folic acid efflux was rapid in parental CEM cells
with an average T1/2 of 3 ± 1 min (Fig.
2A), whereas the
T1/2 in CEM-7A was 5-fold higher (15 ± 4 min)
(Fig. 2B). This was consistent with a 5-fold decrease in the
folic acid efflux rate constant in CEM-7A cells relative to parental
CEM cells (Table III). To distinguish
between the relative contributions of RFC and MRPs to the overall
efflux of [3H]folic acid, the
N-hydroxysuccinimide ester of MTX (NHS-MTX, 7.5 µM), and probenecid (1 mM), were used as
transport inhibitors of RFC (31) and MRP1 (32), respectively.
Probenecid induced a 5-fold fall in the folic acid efflux rate constant
in CEM cells (Table III). Remarkably, the folic acid efflux rate
constants obtained under probenecid inhibition in CEM cells and that of
untreated CEM-7A cells were very low and identical (0.05 min Expression of Various MRPs--
Because probenecid has been shown
to inhibit 98% of folic acid efflux mediated by recombinant MRP1 and
MRP3 in purified membrane vesicles from Sf9 insect cells (22),
the expression of various MRPs and other members of the ATP-binding
cassette superfamily was determined in CEM and CEM-7A cells. Parental
CEM cells expressed substantial levels of MRP1 (Fig.
3) and MRP4 (Fig. 3B) and had low levels of MRP5 (Fig. 3B), whereas MRP2, MRP3, BCRP, and
the multidrug resistance transporter Pgp were undetectable (Fig.
3B). In contrast, CEM-7A cells lost 95% of parental MRP1
expression (Fig. 3) but had no decrease in the levels of MRP4 and MRP5
(Fig. 3B). Consistent with the marked increase in LCV and
MTX influx (74-86-fold), CEM-7A cells had a ~30-fold RFC protein
overexpression, as revealed by the reprobing of the MRP Western blot
with anti-hRFC antibodies (Fig. 3A). Flow Cytometric Analysis of Calcein AM Accumulation--
Calcein
AM, a membrane-permeable chromophoric ester of calcein, is rapidly
converted by intracellular esterases to its impermeable anionic form,
calcein. The latter was shown to be a good efflux substrate of MRP1
(33). Consistent with the loss of MRP efflux function, CEM-7A cells
accumulated 2-fold more calcein (Fig. 4). Furthermore, inhibition of
calcein efflux by the amphipathic anion, probenecid (1 mM),
resulted in a 2-fold increase in the intracellular calcein fluorescence
in CEM cells with no change in the fluorescence of probenecid-treated
CEM-7A cells (Fig. 4B). Similarly, treatment of CEM cells
with 20 µM MK571, a specific inhibitor of MRP1 efflux activity (34, 35), resulted in a 60% increase in the calcein fluorescence (Fig. 4B). In contrast, MK571 failed to alter
the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression (Fig. 4B). This further indicates that CEM-7A
cells were deficient in an MRP1-mediated anion efflux function.
[3H]Folic Acid Efflux and MRP1 Expression in CEM
Sublines Harboring RFC Mutations--
To further establish the role of
MRP1 in folic acid efflux, we also studied the sublines CEM/MTX-LF (26)
and CEM/GW70-LF (30) harboring RFC mutations. Following the acquisition
of RFC mutations, these sublines were subjected to a gradual
deprivation of folic acid, resulting in growth at 2-5 nM
folic acid in the medium. Hence, the RFC-mediated component of folic
acid efflux in these cells should be minimal, thereby rendering the
MRP-mediated efflux of folates more amenable for quantitation. Fig.
5 shows that whereas parental CEM cells
displayed a rapid [3H]folic acid efflux
(T1/2 = 3 ± 1), CEM/MTX-LF and CEM/GW70-LF
cells grown in low folate medium containing only 2 and 5 nM
folic acid, respectively, had substantially increased efflux
T1/2 values of 10 ± 2 and 6 ± 1, respectively (Table IV). These translated
into 3- and 2-fold decreased folic acid efflux rates in CEM/MTX-LF
(k = 0.07 min In an initial report (23), CCRF-CEM-7A leukemia cells were shown
to have a markedly augmented concentrative transport of MTX relative to
their parental CCRF-CEM cells. This was associated with RFC gene
amplification (24) and carrier overexpression (23, 24). Because RFC
functions as a bi-directional anion exchanger (5, 6), one would expect
that the markedly augmented influx of MTX in CEM-7A cells should be
accompanied with a symmetrical increase in RFC-mediated efflux of MTX.
However, the highly concentrative transport of folates and MTX in
CEM-7A cells was correlated with a marked increase in folate and MTX
influx but, surprisingly, without any change in efflux (23). We
therefore undertook the present study to further explore the molecular
basis of this unexpected phenomenon. The present data confirm a high
level accumulation of MTX and, in addition, of LCV and folic acid in
CEM-7A cells under conditions in which the reduction and
polyglutamylation of folic acid were suppressed. To explore the role of
alterations in efflux kinetics as the basis for this difference, folic
acid was utilized to exploit its poor affinity for RFC (26), making it
a much better indicator than MTX of changes in MRP-mediated folate
exporter activity.
The following findings are consistent with a marked loss of folate
exporter function in CEM-7A cells: (a) CEM-7A cells had a 5-fold fall
in the folic acid efflux rate constant relative to parental CEM cells;
(b) incubation of parental CEM cells with probenecid, an organic anion
transport inhibitor that also blocks MRP1 efflux activity (32), blocked
78% of folic acid efflux and yielded a folic acid efflux rate constant
(k = 0.05 min Overexpression of murine RFC following cDNA transfection into MTX
transport null mouse leukemia cells resulted in a marked increase in
both MTX influx (10-fold) and efflux (5-fold) (25). This consequently
brought about only a small (2-fold) increase in the concentrative
transport of MTX (25). Hence, the major impact of the increase in RFC
expression was a rapid cycling of the carrier with a much lesser change
in the steady-state MTX level achieved. Because these cells were
obtained by transfection without folate deprivation or antifolate
selective pressure, there was no apparent requirement or stimulus for
secondary alterations in other transport routes (i.e. such
as decreased MRP1 expression) to sustain growth. On the other hand,
during the establishment of CEM-7A cells, the selection of parental CEM
cells was based upon a gradual LCV deprivation. Because the critical
element regulated by the transport processes is the free cellular
folate level achieved, there was an apparent requirement for a
secondary change in efflux with loss of MRP1 expression aimed at
augmenting the intracellular folate pool to a level sufficient to
sustain DNA replication.
In the present paper we provide the first evidence that MRP1 may play a
functional role in the maintenance of cellular folate homeostasis. This
is based on the fact that CEM leukemia cells have, on the one hand, the
ability to down-regulate MRP1 expression and folate efflux activity
under conditions of folate deprivation. On the other hand, upon medium
repletion with normal folate levels (2.3 µM folic acid),
CEM variants (adapted to grow in nM folic acid or LCV
concentrations), resume MRP1 expression. Thus, CEM cells can respond to
extracellular folate status by decreasing MRP1 expression upon folate
deprivation and resume normal MRP1 levels upon repletion of normal
folate concentrations in the growth medium. This mechanism of
adaptation becomes clear when analyzing the cellular folate pools in
CEM-7A, CEM/MTX-LF, and CEM/GW70-LF cells as compared with their
parental CEM cells. Whereas parental cells contain an average cellular
folate pool of 65 pmol/mg protein (23, 26, 36), CEM-7A (26), CEM/MTX-LF
(26), and CEM/GW70-LF cells (30) possess miniscule folate pools of 1.2, 1.1, and 7.7 pmol/mg protein, respectively. Because these dramatically
shrunken folate pools represent the lower folate limit sustaining DNA
replication and cell proliferation (36), the cells must acquire
mechanisms to ensure the maintenance of this minimal cellular folate
pools. Indeed, like CEM-7A, CEM/MTX-LF and CEM/GW70-LF cells had a
simultaneous RFC overexpression (15-30-fold) because of gene
amplification (26, 30) and 3-fold decreased MRP1 expression and folate
efflux activity. Furthermore, replenishment of 2.3 µM
folic acid to CEM/GW70-LF (30) and CEM-MTX-LF cells (26) resulted in
total folate pools of 303 and 500 pmol/mg protein, respectively.
Consistently, CEM-7A cells accumulated 352 pmol of LCV/mg of protein
upon 20 min of exposure to 2 µM LCV (Fig. 1A
and Table II). Thus, replenishment of folates to CEM/GW70-LF, CEM-7A,
and CEM/MTX-LF resulted in 39-, 293-, and 456-fold expansions in
the cellular folate pools when compared with the cellular folate
content obtained with these cells under low folate growth. These folate
pools represented 5-8-fold increases when compared with the total
folate content in parental CEM cells grown under the same conditions.
This dramatic expansion in the cellular folate pools may explain the
resumption of MRP1 expression in these CEM variants upon folate
replenishment. However, the exact molecular mechanism by which
mammalian cells up- or down-regulate MRP1 expression and folate efflux
must await further characterization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol,
0.5% Triton X-100, and the protease inhibitors aprotinin (60 µg/ml), leupeptin (5 µg/ml), phenylmethylsulfonyl fluoride (10 µg/ml), and
EGTA (1 mM). Following 1 h of incubation on ice, the
extract was centrifuged at 15,000 × g for 30 min at
4 °C, and the supernatant containing the fraction of
detergent-soluble proteins was collected. The proteins (10-50 µg)
were resolved by electrophoresis on 7.5% (for MRP) or 10% (for RFC)
polyacrylamide gels containing SDS and electroblotted onto a Nytran
membrane (Schleicher & Schuell). The blots were blocked for 1 h at
room temperature in Tris-buffered saline (150 mM NaCl,
0.5% Tween 20, 10 mM Tris, pH 8.0) containing 1% skim
milk. The blots were then reacted with the following anti-human MRP
monoclonal antibodies (kindly provided by Prof. R. J. Scheper, VU Medical Center, Amsterdam, The Netherlands) at a 1:500
dilution: rat anti-MRP1 (MRP-r1) and mouse anti-MRP2, anti-MRP3,
anti-MRP4 (kindly provided by Dr. G. D. Kruh), anti-MRP5,
anti-BCRP, and anti-Pgp. To determine RFC expression, the blots were
reacted with a polyclonal antiserum (1:700) prepared in mice against a C-terminal hRFC peptide.2 The
blots were then rinsed in the same buffer for 10 min at room temperature and reacted with horseradish peroxidase-conjugated goat
anti-mouse or anti-rat IgG (1:40,000 dilution; Jackson Immunoresearch Labs, West Grove, PA) for 1 h at room temperature. Following three 10-min washes in Tris-buffered saline at room temperature, enhanced chemiluminescence detection was assessed according to the
manufacturer's instructions (Biological Industries). The protein
content was determined using the Bio-Rad protein assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-glutamate synthetase in CEM-7A
cells (23).
Folate growth requirement and antifolate growth inhibition of parental
CEM and CEM-7A cells
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Fig. 1.
Time course of folate and MTX uptake in CEM
and CEM-7A cells. Parental CEM cells (squares) and
their CEM-7A subline (circles) were incubated in the
presence of 2 µM of [3H]LCV (A),
[3H]MTX (B), or [3H]Folic acid
(C). For the transport of [3H]folic acid, 5 µM TMQ was present in the transport buffer to block folic
acid reduction to tetrahydrofolate (27). Following various times of
incubation at 37 °C, the transport of the radiolabeled folate or MTX
was determined as detailed under "Materials and Methods."
Folic acid, leucovorin, and MTX initial uptake rates and accumulation
levels in parental CEM and CEM-7A cells
1; see Table III). Furthermore, incubation of CEM
cells with both 7.5 µM NHS-MTX and 1 mM
probenecid resulted in negligible additional inhibition of folic acid
efflux beyond the effect achieved by probenecid alone in both cell
lines (Table III). These results suggested that the major folic acid
efflux route present in parental CEM cells was RFC-independent,
inhibitable with probenecid, and largely lost in CEM-7A cells.
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Fig. 2.
Time course of [3H]folic acid
efflux in parental CEM cells and their CEM-7A subline. After 30 min of loading (to the same cellular level) of CEM (A) and
CEM-7A cells (B) with 10 and 2.5 µM
[3H]folic acid, respectively, in a transport buffer
containing 5 µM TMQ at 37 °C, the cells were rapidly
centrifuged and resuspended in TMQ-containing buffer lacking folic
acid. Then efflux of [3H]folic acid was followed for up
to 40 min in the absence of inhibitors (solid squares), in
the presence of 1 mM probenecid (open
triangles), 7.5 µM NHS-MTX (open
squares), or both probenecid and NHS-MTX (solid
circles). To determine the folic acid efflux rate constants, the
log value of the percentage of initial free folic acid obtained at each
time point was plotted as a function of time as previously described
(27).
Folic acid efflux rates in CEM and CEM-7A cells in the absence or
presence of RFC and MRP transport inhibitors
-Actin expression
confirmed that equal amounts of proteins were being analyzed in the
various gels (Fig. 3B).
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Fig. 3.
Western blot analysis of RFC and MRP
expression in CEM and CEM-7A cells. Exponentially growing CEM and
CEM-7A cells were harvested by centrifugation and lysed in a buffer
containing 0.5% Triton X-100 (36). Then detergent-soluble proteins
(10-50 µg) were resolved by electrophoresis on 7.5% (for MRP) or
10% (for RFC) polyacrylamide gels containing SDS and electroblotted
onto Nytran membranes. The blots were then reacted with polyclonal
antibodies to hRFC (A, lower panel) or monoclonal
antibodies to MRP1 (A and B, upper
panel), MRP2, MRP3, MRP4, MRP5, Pgp, and BCRP (B). To
confirm the uniformity of protein loading the blots were also reacted
with monoclonal antibodies to -actin (B, lowest
panel). A, lane 1, parental CEM cells;
lane 2, CEM-7A cells grown in 0.25 nM LCV (LF);
lane 3, CEM-7A cells grown in 5 nM LCV for 1 month (LF
HF). The molecular masses (kDa) of MRP1 and RFC are given
on the right. B, lane 1, parental CEM
cells; lane 2, CEM-7A cells grown at 0.25 nM
LCV; lane 3, Various marker cell lines with overexpression
of MRP1, MRP2, MRP3, MRP4, MRP5, BCRP, and Pgp, respectively (starting
from the top of the panel). Reprobing with antibodies to
-actin was performed to confirm that equal amounts of proteins were
being analyzed (bottom).
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Fig. 4.
Flow cytometric analysis of calcein
accumulation in CEM and CEM-7A cells. A, exponentially
growing CEM (dark line) and CEM-7A cells (gray
line) were incubated for 20 min in growth medium at
37 °C in the absence (dashed line), or presence of 3 nM calcein AM (dark and gray lines), washed, and
analyzed by flow cytometry for mean fluorescence per cell.
B, CEM and CEM-7A cells were preincubated for 10 min at
37 °C in the absence (dark symbol), or presence of the
calcein efflux inhibitors MK571 (20 µM; light gray
shading) or probenecid (1 mM; dark gray
shading), after which the cells were incubated for an additional
20 min in the presence of 3 nM calcein AM + MK571 and
probenecid. Then cells were washed and analyzed for mean fluorescence
per cell.
1) and CEM/GW70-LF cells
(k = 0.12 min
1), respectively. Because
these results were suggestive of decreased MRP1-dependent
folate efflux activity, MRP1 expression was determined in CEM/MTX-LF
and CEM/GW70-LF cells (Fig. 6). Relative
to parental CEM cells, MRP1 expression was decreased by 3-fold in
CEM/MTX-LF cells (Fig. 6A). Remarkably, CEM/MTX-LF cells
grown for 1 month in normal folic acid concentration (i.e.
2.3 µM; cells termed CEM/MTX-LF
HF) resumed
parental MRP1 expression (Fig. 6A). Similarly, CEM/GW70-LF
cells selected to grow in medium containing 5 nM folic acid
also expressed 3-fold less MRP1 compared with parental CEM and CEM/MTX
cells growing in 2.3 µM folic acid (Fig. 6B).
CEM/GW70-LF cells grown for 1 month in normal folate concentration
(i.e. 2.3 µM; cells termed CEM/GW70-LF
HF)
resumed parental MRP1 expression (Fig. 6B). Consistently,
CEM-7A cells replenished with 5 nM LCV in the growth medium
for 1 month (termed CEM-7A LF
HF) resumed parental MRP1 expression
(Fig. 3A). These results establish that MRP1 plays a
functional role in folate efflux and suggest a function for MRP1 in
cellular folate homeostasis.
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Fig. 5.
Time course of [3H]folic acid
efflux in parental CEM cells and their sublines CEM/MTX-LF and
CEM/GW70-LF harboring RFC mutations. After 30 min of loading of
CEM (solid squares), CEM/MTX-LF (open squares),
and CEM/GW70-LF cells (circles) with 10, 2, and 2.5 µM [3H]folic acid, respectively, in a
transport buffer containing 5 µM TMQ at 37 °C, the
cells were rapidly centrifuged and resuspended in TMQ-containing buffer
lacking folic acid. Then efflux of [3H]folic acid was
followed for up to 20 min. To determine the folic acid efflux rate
constants, the log value of the percentage of initial free folic acid
obtained at each time point was plotted as a function of time as
previously described (27).
Folic acid efflux rates in parental CEM cells and their RFC transport
defective CEM/MTX-LF and CEM/GW-70-LF sublines
View larger version (27K):
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Fig. 6.
Western blot analysis of MRP1 expression in
parental CEM cells and their CEM/MTX-LF and CEM/GW70-LF sublines grown
under low or high folate concentrations. Triton X-100-soluble
proteins (10-50 µg) from parental CEM (A and
B), CEM/MTX-LF (A), and CEM/GW70-LF cells
(B) were resolved by electrophoresis on 7.5% polyacrylamide
gels containing SDS, electroblotted onto Nytran membranes, and reacted
with monoclonal antibodies to MRP1 as detailed in Fig. 3 legend.
LF refers to growth of CEM/MTX-LF and CEM/GW70-LF under low
folate conditions (2 and 5 nM folic acid, respectively),
whereas LF HF refers to cellular growth at the regular
medium concentration of folic acid (2.3 µM). Reprobing
with antibodies to
-actin was performed to confirm that equal
amounts of proteins were being analyzed (bottom
panels).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1) that was identical to
that obtained with untreated CEM-7A cells; (c) despite the 86-fold
overexpression of RFC-dependent MTX influx in CEM-7A cells,
treatment of CEM-7A and parental CEM cells with a combination of
NHS-MTX (an irreversible inhibitor of RFC) and probenecid resulted in
only a marginal additional inhibition of folic acid efflux when
compared with probenecid alone; (d) when compared with parental CEM,
CEM-7A cells accumulated 2-fold more calcein, a chromophoric anionic
substrate also exported by MRP1 (33, 35); furthermore, treatment of
parental CEM cells with probenecid and the MRP1-specific efflux
inhibitor, MK571, resulted in resumption of a calcein fluorescence that
was nearly identical to that obtained with CEM-7A cells; and (e)
finally, determination of MRP protein levels revealed that the markedly
decreased folate efflux in CEM-7A cells was associated with a dramatic
loss of MRP1 expression in CEM 7A cells. These results establish that MRP1 is the predominant folate efflux route in human leukemia CEM
cells. However, one cannot rule out the possibility that alternative routes exist that may also contribute, at least to some extent, to the
energy-driven efflux of folates and antifolates (e.g. MTX) in parental CEM cells. It was recently shown that the human MRP4 functions not only as an ATP-driven exporter of nucleotide analogues (37) but also as a high capacity (Vmax = 0.2-2
nmol/mg/min), low affinity (Km = 0.2-0.6
mM) efflux transporter of MTX, folic acid, and LCV (38).
Thus, because MRP4 is equally expressed at substantial levels in both
parental CEM and CEM-7A cells, one cannot exclude the possibility that
under physiological conditions MRP4 may also contribute to cellular
efflux of folates. Furthermore, it is also possible that some of the
most recently discovered MRPs (but yet insufficiently characterized)
including MRP6-9 may potentially contribute to folate/MTX efflux, at
least to some extent.
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ACKNOWLEDGEMENTS |
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We are indebted to Prof. I. D. Goldman for the critical reading of the manuscript. We thank Prof. R. J. Scheper for providing monoclonal antibodies to the various MRPs, BCRP, and Pgp and Dr. G. D. Kruh for the monoclonal antibody to MRP4. The technical assistance of Yaffa Both is gratefully acknowledged.
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FOOTNOTES |
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* This work was supported by research grants from the Israel Ministry of Health, the Israel Cancer Association, and the Star Foundation (to Y. G. A.) as well as by Dutch Cancer Society Grant NKB VU 2000-2237 (to G. J. and G. J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Biology, The Technion-Israel Institute of Technology, Haifa 32000, Israel. Tel.: 972-4-829-3744; Fax: 972-4-822-5213; E-mail: assaraf@tx.technion.ac.il.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M209186200
2 I. Ifergan, I. Meller, J. Issakov, and Y. G. Assaraf, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: MTX, methotrexate; MRP, multidrug resistance protein; RFC, reduced folate carrier; LCV, Leucovorin (5-formyl-tetrahydrofolate); TMQ, trimetrexate; BCRP, breast cancer resistance protein; Pgp, P-glycoprotein; NHS-MTX, N-hydroxysuccinimide ester of MTX; AM, acetoxymethyl ester; HBS, Hepes-buffered saline.
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