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Experimental Therapeutics, Preclinical Pharmacology |
Departments of Medicine and Molecular Pharmacology and the Albert Einstein Comprehensive Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30%) than that for methotrexate (MTX). Unlike MTX, MTA was
rapidly polyglutamated in L1210 cells; hence, a
folylpoly-
-glutamate synthetase-deficient L1210 variant was
used to assess net transport and efflux properties. The MTA
transmembrane gradient for exchangeable drug was 2.5 times greater than
the MTX gradient, attributable primarily to an efflux rate constant
40% that of MTX. No MTA was bound to dihydrofolate reductase.
When grown with folic acid, MTX-resistant L1210 variants with mutations
in the reduced folate carrier demonstrated cross-resistance to MTA,
markedly reduced MTA accumulation, and only a slightly decreased
intracellular folate cofactor pool as compared to L1210 cells. However,
when 5-formyltetrahydrofolate was the growth substrate, these
MTX-resistant cells were less resistant or negligibly resistant to MTA,
accumulated more MTA, and had a lower folate pool as compared to L1210
cells. MTA activity and the intracellular folate pool in L1210 cells
were inversely related. These data indicate that MTA polyglutamation in
L1210 cells is favored by both the generation of high intracellular
drug levels and high MTA affinity for FPGS relative to MTX. Cells
resistant to MTX because of impaired transport may retain appreciable
sensitivity to MTA because of a concurrent reduction in
tetrahydrofolate cofactor transport resulting in cellular folate
depletion, which diminishes endogenous folate suppression of MTA
polyglutamation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7 nM) at least 3 orders of magnitudes
less than that of MTX (1)
. The pharmacological
perturbations produced by MTA are complex and appear to represent a
combination of suppression of both thymidyate and purine synthesis.
Both physiological substrates are required to completely circumvent MTA
cytotoxicity, but in cells with high expression of TS, a source of
purine alone is sufficient to prevent drug activity (1
, 2)
. MTA is a excellent substrate for FPGS, 2 orders of magnitude
better than MTX and 1 order of magnitude better than DDATHF, a property
that favors formation of its active derivatives in cells
(3)
. In clinical studies, this agent appears to have
activity against a variety of tumors, including non-small cell lung
cancer, colorectal carcinoma, and mesothelioma (4, 5, 6, 7, 8)
.
Membrane transport of antifolates is a critical determinant of
activity, and loss of transport is a frequent mechanism of resistance
to MTX (9, 10, 11, 12, 13, 14, 15)
. The mechanism of transport of MTA has not
been established, although the Ki for MTA inhibition
of MTX influx suggests a high affinity for RFC1 (16)
, a
member of the major facilitator superfamily of transporters
(17)
. This agent also has a very high affinity for folate
receptor 
, a GPI-linked endocytotic pathway, that is comparable to
the affinity of the preferred substrate for this route, folic acid
(16)
. This report represents the first detailed assessment
of the transport properties of this agent focusing on unidirectional
fluxes and the level of concentrative transport achieved within the
context of a comparison with MTX. The study also explores
cross-resistance patterns to MTA in several cell lines with primary
resistance to MTX because of impaired transport and demonstrates the
critical role that the folate substrate in the growth medium plays in
transport-related resistance phenomena.
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MATERIALS AND METHODS |
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Cell Lines, Culture Conditions, and Growth Inhibition Assays.
The MTXrA line is an MTX-resistant L1210 murine
leukemia variant obtained under MTX selective pressure with an A130P
mutation in RFC1 that results in loss of carrier mobility
(19)
. L1210-G1a cells were selected in the presence of MTX
with 5-CHO-THF as the folate source and harbors an S46N mutation in
RFC1 (20)
. L51 is a DDATHF-resistant L1210 variant
isolated by chemical mutagenesis in this laboratory with a marked
defect in FPGS activity (21)
. All cells were grown, unless
otherwise noted, in complete RPMI 1640 containing 2.2 µM
folic acid, 5% bovine calf serum (HyClone), 2 mM
glutamine, 20 µM 2-mercaptoethanol, penicillin (100
units/ml), and streptomycin (100 µg/ml) at 37°C in a humidified
atmosphere of 5% CO2. Cells were also grown in
folate-free RPMI (HyClone) containing 5% dialyzed bovine calf serum
(Life Technologies, Inc.), 2 mM glutamine, 20
µM 2-mercaptoethanol, penicillin (100 units/ml), and
streptomycin (100 µg/ml) supplemented with 25 nM
5-CHO-THF. To assess MTA and MTX growth inhibition, cells were grown in
96-well plates (1 x 105 cells/ml) and
exposed continuously to appropriate concentrations of the antifolates.
After 72–96 h, cell numbers were determined by hemocytometer count,
and viability was assessed by trypan blue exclusion.
Measurements of Folate Pools and MTA Accumulation.
Cells (3 x 106) grown in complete
RPMI 1640 were washed twice with folate-free RPMI and resuspended into
the same medium supplemented with either 2 µM
[3
H]folic acid (30 dpm/pmol) or 25
nM [3
H]5-CHO-THF (200 dpm/pmol).
After 1 week of exponential growth, cells were harvested, washed twice
with ice-cold HBS, and processed for intracellular tritium as described
for transport studies. For measurement of MTA accumulation, cells grown
in complete RPMI containing 2.2 µM folic acid or in
folate-free RPMI 1640 supplemented with 25 nM 5-CHO-THF
were incubated with 50 nM [3
H]MTA
(200 dpm/pmol) in the presence of 200 µM glycine, 100
µM adenosine, and 10 µM thymidine to
circumvent the inhibitory effects of this agent. Cells were harvested
after 3 days of exponential growth, washed twice with ice-cold HBS, and
processed for intracellular tritium and polyglutamates (see below).
High-performance Liquid Chromatography Analysis of MTA
Polyglutamates.
Cells exposed to 1 µM
[3
H]MTA for 2 h were washed three times
with 0°C HBS. One portion of the cell pellet was processed for dry
weight and total tritium as described below. Another portion was
processed according to a reported protocol (22)
. Cell
pellets were suspended in 50 mM phosphate buffer at pH 6.0
containing 100 mM 2-mercaptoethanol and boiled for 5 min.
The precipitate was removed by centrifugation, and the supernatant
containing radiolabeled MTA and its metabolites, spiked with unlabeled
MTA and MTA-triglutamate, was separated on a reversed-phase
high-performance liquid chromatography column (Waters Spherisorb, 5
µm ODS2 4.6 x 250 mm) as reported previously with minor
modifications (23)
. Separation of the different
polyglutamate derivatives was achieved by elution with 0.1
M sodium acetate, pH 5.5, for 5 min followed by two linear
gradients of 0–30 and 30–50% acetonitrile in 0.1 M
sodium acetate over 35 and 20 min, respectively, and then 100%
acetonitrile for 10 min. The flow rate was 1 ml/min, and 1-ml fractions
were collected. The level of MTA and its polyglutamate derivatives were
normalized to units of nmol/g of dry wt of cells.
Folic Acid Binding Assay.
Cells (2 x 107) were harvested and
washed once with ice-cold acid buffer (10 mM NaAc, 140
mM NaCl, pH 3.5) and twice with cold PBS. The cells were
incubated in 1 ml of PBS containing 5 nM
[3
H]folic acid (26 Ci/mmol) at 4°C for 30
min, separated by centrifugation, and then washed twice with cold PBS
and finally with the acid buffer (0.5 ml) to extract bound
[3
H]folic acid. Tritium in the supernatant was
assessed by liquid scintillation spectrometry, and the pellet was dried
and weighed. The folic acid binding capacity was expressed in units of
pmol/g of dry weight of cells.
Transport Studies.
Cells were harvested, washed twice with HBS (20 mM
HEPES, 140 mM NaCl, 5 mM KCl, 2 mM
MgCl2, 5 mM glucose, pH 7.4) and
resuspended in HBS to 1.5 x 107 cells/ml.
For measurement of influx at pH 5.5, cells were washed once with
unbuffered saline (140 mM NaCl, 5.3 mM KCl, 1.9
mM CaCl2, 1 mM
MgCl2, 7 mM glucose) and then washed
with and resuspended in 2-(4-morpholino)-ethanesulfonic
acid-buffered saline (20 mM
2-(4-morpholino)-ethanesulfonic acid, 140 mM NaCl, 5
mM KCl, 2 mM MgCl2, 5
mM glucose, pH 5.5). Cell suspensions were incubated at
37°C for 20 min, after which uptake was initiated by the addition of
[3
H]MTA or [3
H]MTX, and
samples were taken at the indicated times. Uptake was terminated by
injection of 1 ml of the cell suspension into 10 ml of ice-cold HBS.
Cells were collected by centrifugation, washed twice with ice-cold HBS,
dried, and digested with 1 N NaOH in an 8-ml vial, and
after fluor was added, radioactivity was assessed in a liquid
scintillation spectrometer (23)
. When appropriate, uptake
intervals were adjusted so that unidirectional conditions were
sustained. For efflux measurements, cells were loaded with tritiated
MTA or MTX, a small portion was taken for measurement of intracellular
antifolate, and remaining cells were separated by centrifugation and
resuspended into a large volume of drug-free buffer. Subsequently,
samples were taken and injected into 10 ml of ice-cold HBS, separated
by centrifugation, washed twice, and then processed for intracellular
tritium as described above.
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RESULTS |
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. This continued net uptake of MTA was
attributed to rapid accumulation of nonexchangeable radiolabel. Hence,
when cells were separated by centrifugation after exposure to MTA for
30 or 120 min and resuspended into MTA-free buffer, a large
nonexchangeable component was detected that increased in parallel (Fig. 1
, – – – –) with the overall accumulation of the drug (——) and
accounted entirely for the increase in net uptake of radiolabel over
this interval. The level of exchangeable MTA did not change during this
time, indicating that this intracellular MTA component was at steady
state with extracellular drug.
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and see below), precluded accurate
assessment of MTA transmembrane gradients and efflux kinetics mediated
by the membrane transport process(es) that govern translocation of this
agent across the cell membrane. To eliminate the complication of
polyglutamation, an L1210 leukemia cell line, L51, was used in which
FPGS was mutated such that enzyme activity was negligible. The L51 cell
line is 23-fold resistant to MTA as compared to L1210 cells upon
continuous exposure to the drug (21)
. MTA influx in L51
cells was comparable to influx in L1210 cells (Fig. 2
, inset). However, unlike
L1210 cells, a MTA steady-state level of 5 nmol/g dry wt was reached
in L51 cells within 30 min (Fig. 2)
.
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. The steady-state levels for these
antifolates were comparable under these conditions despite the fact
that most intracellular MTX is tightly bound to DHFR. To determine
whether MTA was also bound to DHFR, 10 µM TMQ was added
to the transport buffer 15 min prior to the antifolates. This agent is
a very potent inhibitor of DHFR and should block binding of both MTX
and MTA to this enzyme without altering RFC1-mediated transport. It can
be seen that addition of TMQ did not change the initial uptake rates
for either MTA or MTX (Fig. 3
, inset) but produced a 2-fold
decrease in steady-state MTX. In contrast, the MTA steady-state level
was not affected at all by TMQ (Fig. 3)
. When cells were resuspended
into a large volume of drug-free buffer, 80–90% of both antifolates
exited the cells rapidly. The exchangeable MTA level achieved
(1.45 ± 0.02 µM) was 2.5 times greater
that that of MTX (0.61 ± 0.02 µM), with
an MTA chemical gradient across the cell membrane of 1.5:1 based upon a
ratio of intracellular water to dry weight of 3.5 µl/mg in L1210
cells (23)
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. Radioactive MTA and MTA-triglutamate
were identified by comparing their elution times with those of
nonlabeled standard compounds on a reverse phase column. Identification
of MTA triglutamate, eluted at 43 min, and the monoglutamate, eluted at
57 min, permitted assignment of the diglutamate at 49 min. The two
congeners that eluted more rapidly at 36 and 39 min were assigned as
the penta- and tetraglutamates, respectively. In L51 cells, only a
small fraction of total MTA was polyglutamated, as compared to L1210
cells, in which majority of drug was metabolized. The bottom
panel of Fig. 4
compares the levels
of MTA and MTA polyglutamates in the two cell lines per mg of dry
weight. Total drug accumulation after a 2-h exposure to MTA in L51
cells was about 35% that of L1210 cells. Polyglutamates in L51 cells
were about 12% that of L1210 cells, whereas the MTA monoglutamate
level was slightly greater (19%) than that of L1210 cells.
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, increased influx of
MTA as compared to MTX was attributable to a 2-fold higher affinity for
RFC1, an effect opposed by a lesser decrease in the influx
Vmax for this agent. This resulted in
an influx Vmax/Kt
for MTA that was 30% greater than observed for MTX. Efflux kinetics
for these agents are also shown in Table 1
. Here, L51 cells were loaded
with either tritiated MTX or MTA, and the rate constants for the loss
of exchangeable drug were measured. The rate constant for MTA efflux
was 40% that of MTX. Hence, the small increase in MTA influx kinetics
along with the much larger decrease in the efflux rate constant
accounted for the enhanced concentrative transport of this agent
relative to MTX.
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, the
exchangeable intracellular MTA level, when extracellular drug was 0.3
µM, approximated the exchangeable intracellular
MTX level when extracellular MTX was 1 µM,
consistent with the observation that MTA transport is more
concentrative than MTX under these conditions.
PGA1 at 7 µM increased
the steady-state exchangeable MTX level by 90%, but exchangeable
MTA was increased only 30% in L51, a 3-fold difference. When cells
were brought to the steady state at equimolar extracellular drug levels
(1 µM), the addition of 7
µM PGA1 increased the
ratio of steady-state exchangeable MTX to MTA to 1.8 ± 0.1 (based
upon the average of four separate experiments; data not shown). Hence,
MTA appears to be a poorer substrate for the RFC1-independent exporters
than MTX.
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9 and 4% that of L1210 cells, respectively (Table 2)
, as compared to 1.0 and 2.6% for MTX
influx (20)
. Associated with the decreased influx, the MTA
IC50 was increased by factors of 5.8 and 22, and
total MTA accumulation decreased by 74% and 90%, respectively, in
L1210-G1a and MTXrA cells as compared to L1210
cells when folic acid was the folate substrate in the growth medium.
The IC50 for MTX was increased by factors of 12
and 71, respectively, in these cell lines (20)
. Thus, both
RFC1 mutations resulted in impaired MTA transport and drug resistance,
but to a lesser extent than for MTX.
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100 nM, obtained after cells were depleted of endogenous
folates (20)
, MTXrA cells grew with
25 nM 5-CHO-THF at only a slightly reduced rate. MTA influx
under these conditions was decreased by 40% in L1210 cells,
slightly increased in the MTXrA line, and
unchanged in L1210-G1a cells. Most striking was the impact of the
folate source on growth inhibition. Hence, as compared to wild-type
L1210 cells, the MTA IC50 in
MTXrA cells increased by a factor of only 2.4, as
compared to a 22-fold increase when folic acid was the growth source,
and the change in IC50 was about one-half that
observed with folic acid in the L1210-G1a line (Table 2)
. MTX
resistance was also decreased in the MTXrA line,
but to a lesser extent than MTA, and was about the same in L1210-G1a
cells when the folate source was shifted from folic acid to 5-CHO-THF.
Accompanying the decrease in MTA resistance in L1210-G1 and
MTXrA cells was a 2 or 3-fold increase,
respectively, in MTA accumulation when 5-CHO-THF was used as
folate source.
MTA influx in the resistant lines could not be attributed to transport
via folate receptors despite the high affinity of this drug for this
transporter (16)
. Hence, as indicated in Table 3
, folic receptor expression, as assessed
by surface binding, was quite low in all of the cell lines. Folate
receptor expression actually decreased in the L1210-G1 line and was
essentially unchanged in MTXrA cells, as compared
to expression in wild-type L1210 cells. Folate receptor expression was
minimally increased (1.5–2 fold) when cells were grown in 5-CHO-THF
versus folic acid, consistent with the lack of difference in
MTA influx in the L1210-G1a line and a very minimal change in influx in
the MTXrA cells under these conditions
(Table 3)
. There was no significant change in MTA influx measured at pH
5.5 in cells growing in 5-CHO-THF versus folic acid (data
not shown). Thus, the low-pH folate transport system in L1210 cells
(31
, 32)
did not contribute to alterations in MTA
accumulation or activity when MTX-resistant cells were grown in
5-CHO-THF as compared to folic acid.
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; Ref.
33
). However, if 25 nM
[3
H]5-CHO-THF was the folate source, the folate
pool size in L1210-G1a and MTXrA cells was
reduced to 47 and 13% that of L1210 cells, respectively. Thus, the
8-fold drop in the MTA IC50 in
MTXrA cells was associated with a 3-fold increase
in the accumulation of the drug (Table 2)
and an 11-fold decrease in
the natural folate pool (Fig. 6)
when the folate source was changed
from folic acid to 5-CHO-THF.
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, the level of
intracellular folates rose as extracellular 5-CHO-THF was increased,
whereas MTA sensitivity fell in an inverse relationship. Hence, as
extracellular 5-CHO-THF was increased 39-fold from 0.64 to 25
nM and the intracellular folate pool increased 20-fold from
0.35 to 7.5 µM, the MTA IC50
increased by a factor of 13, from 1.3 to 17.3 nM. There was
also an increase in the MTX IC50 but to a lesser
extent, 3.5-fold. These data demonstrate the considerable impact the
intracellular level of THF cofactors play in modulating the cell
growth-inhibitory potency of MTA.
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DISCUSSION |
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Polyglutamylation of MTA not only ensures cellular retention of the drug with the build-up of high levels of inhibitor within cells, but the addition of glutamate moieties markedly increases affinity of MTA for its two target enzymes, TS and GARFT. On the other hand, the affinity of MTA for DHFR is orders of magnitude lower, and is unchanged, when the drug is polyglutamated (1) . Data in this paper indicate that there is essentially no binding of MTA to DHFR when the drug is present in cells at a concentration of 1.5 µM, a level more than 2 orders of magnitude greater than the Ki of MTA for this enzyme. Although some suppression of DHFR might be possible under conditions in which high levels of MTA polyglutamates accumulate within cells, there is considerable evidence to suggest that this is not of pharmacological importance. Hence, in MCF-7 breast and H630 colon human carcinoma cells lines that overexpress TS, the addition of hypoxanthine completely reverses the cytotoxicity of MTX, an effect that would not be possible if MTA depletes THF cofactor pools (2) . Further, when cell are incubated with MTA at a concentration that totally suppresses cell growth, dihydrofolate does not accumulate as occurs if DHFR activity is eliminated, as observed with MTX (34, 35, 36) .
MTA was very rapidly polyglutamated in wild-type L1210 cells under conditions in which there was essentially no polyglutamation of MTX; there was a profound difference in this property between the two antifolates. Enhanced polyglutamation of MTA is attribution both to high FPGS activity (3) for this agent and higher free MTA substrate levels relative to MTX in these cells. Polyglutamation was markedly reduced in an L1210 cell line, L51, in which FPGS activity is negligible but RFC1-mediated transport was unchanged relative to wild-type L1210 cells. This cell line proved to be a very useful model system for studying MTA transport properties that are complicated by rapid metabolism, such as efflux kinetics and steady-state transmembrane gradients. These studies documented the greater concentrative capacity for MTA relative to MTX and excluded the possibility that the higher levels of exchangeable intracellular MTA could be attributable to rapidly reversible binding to DHFR. Nor is it possible that there was appreciable reversible binding of the monoglutamate to TS or GARFT because the affinities of the monoglutamate for these enzymes are lower than that for DHFR (1) .
The free levels of folates and antifolates in L1210 cells are governed by RFC1, a bidirectional anion exchanger, which creates small transmembrane folates gradients and is opposed by ATP-coupled exporters (23 , 37 , 38) , likely to be members of the cMOAT family (39) . Recent studies suggest that efflux of antifolates can be mediated by multidrug resistant proteins (MRP1 and MRP2; Ref. 40 ). Hence, influx of MTA and MTX is mediated by RFC1 alone; efflux is mediated by RFC1 and distinct exporters. In most cases, alterations in concentrative transport of MTX correlate with alterations in the RFC1-mediated entry process without significant change in efflux parameters in MTX-resistant lines (13) .
In exploring the basis for enhanced concentrative transport of MTA
relative to MTX in L1210 cells, the data suggested that this could not
be attributed to differences in transport mediated by RFC1 alone.
Hence, the MTA influx
Vmax/Kt was only
30% greater than that of MTX. On the other hand, the MTA efflux
rate constant was 40% that of MTX (Table 1)
, suggesting a large
difference in transport mediated by the energy-coupled exporters. This
conclusion was supported by the greater augmentation of free MTX levels
relative to MTA by the exporter inhibitor, PGA1,
as would occur if a much lesser fraction of MTA efflux was mediated by
this transporter under physiological conditions. Careful
characterization of MTA versus MTX efflux mediated by
energy-dependent exporters will require further study with an
inside-out membrane vesicle system.
These studies demonstrate clearly that the THF cofactor pool size plays a critical role in modulating the growth-inhibitory effect of MTA. As the intracellular THF cofactor level increased with an increase in the extracellular 5-CHO-THF concentration encompassing, in part, the physiological range, there was a substantial increase in the MTA IC50. This is consistent with studies in mice in which a folate-deficient diet markedly increased MTA activity (41) . Likewise, the activity and toxicity of DDATHF is highly sensitive to the folate status of animals (42) . The level of natural folate pools within cells might modulate MTA activity by at least two mechanisms. First, as indicated as the current study, intracellular folates compete with and inhibit MTA polyglutamation at the level of FPGS. Consistent with this is the observation that the augmentation of the cellular folate pool, as a result of acquired mutations in RFC1 that enhanced carrier affinity for folic acid, markedly suppressed polyglutamation of DDATHF, rendering cells resistant to this agent (43) . High folate pools may also compete with antifolates at the level of their target enzymes. For instance, expansion of folate pools increased the MTX IC50, but to a lesser extent than MTA, probably attributable to an increased build-up of dihydrofolate behind the block at DHFR as cellular THF cofactors are interconverted and then oxidized at TS, resulting in increased competition with MTX for the small fraction of DHFR required to sustain THF synthesis (35 , 36) . A related phenomenon was observed in a cell line selected for high-level resistance to pyrimethamine, a drug that does not form polyglutamate derivatives. In this case, resistance was attributable to loss of exporter activity with markedly increased THF cofactor pools. These cells were cross-resistant to DDATHF and, to a lesser extent, to MTX (44) .
Impaired antifolate transport, which results from mutations in RFC1, leads to cellular "folate deficiency," even when the extracellular 5-CHO-THF concentration is in the physiological range. This is attributable to a concurrent, although not always comparable, decrease in transport of the reduced folates that use the same carrier (20) . The folate pool was not, however, substantially decreased when cells were grown with folic acid as the folate source because folic acid is a poor substrate for RFC1 (45) and enters cells largely by other mechanisms (27 , 33) , one of which is a low pH route (46) . Most commercial media contain folic acid rather than 5-CHO-THF, which resembles the physiological substrate, 5-CH3-THF. With these media, transport-related resistance to MTA, as well as to related agents, will be overestimated because cellular folate pools are minimally perturbed. It should be noted that even when MTXrA and L1210-G1a cells were grown with 5-CHO-THF, they maintained a reduced, but still high-level, resistance to MTX but much lower or negligible resistance to MTA. Thus, tumors resistant to MTX because of deficiencies in transport via RFC1 may still retain appreciable sensitivity to MTA because of compensatory depletion of cellular THF cofactors. It follows then that any loss of transport, even that attributable to low RFC1 expression, as might occur with a change in regulation (47) , would not necessarily be accompanied by comparable cross-resistance to MTA or related agents that require polyglutamation in the cell for activation and/or act at sites downstream to DHFR. Hence, these agents may have specificity and utility for use against tumors with acquired or intrinsic resistance to MTX attributable to impaired membrane transport.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by National Cancer
Institute Grants CA-39807 and CA-82621. 
2 To whom requests for reprints should be
addressed, at Albert Einstein College of Medicine Comprehensive Cancer
Research Center, Chanin 2, 1300 Morris Park Avenue, Bronx, NY 10461.
Phone: (718) 430-2302; Fax: (718) 430-8550; E-mail: igoldman{at}aecom.yu.edu
3 The abbreviations used are:
5-CH3-THF, 5-methyltetrahydrofolate; 5-CHO-THF,
5-formyltetrahydrofolate; DDATHF,
(6R)-5,10-dideazatetrahydrofolate; DHFR, dihydrofolate
reductase; FPGS, folylpoly--glutamate synthetase; HBS,
HEPES-buffered saline; MTA, multitargeted
antifolate
N-(4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo(2,3)pyrimidine-5-yl)ethyl]-benzoyl]-L-glutamic
acid; MTX, methotrexate; PGA1, prostaglandin
A1; RFC1, reduced folate carrier; THF,
tetrahydrofolate; TS, thymidylate synthase.
Received 11/22/99; revised 5/27/00; accepted 5/31/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-glutamyl derivatives of methotrexate and their association with dihydrofolate reductase as assessed by high pressure liquid chromatography in the Ehrlich ascited tumor cell in vitro. J. Biol. Chem., 257: 1890-1896, 1982.This article has been cited by other articles:
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  E. S. Unal, R. Zhao, and I. D. Goldman Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1 Am J Physiol Cell Physiol, July 1, 2009; 297(1): C66 - C74. [Abstract] [Full Text] [PDF]
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 R. Zhao, A. Qiu, E. Tsai, M. Jansen, M. H. Akabas, and I. D. Goldman The Proton-Coupled Folate Transporter: Impact on Pemetrexed Transport and on Antifolates Activities Compared with the Reduced Folate Carrier Mol. Pharmacol., September 1, 2008; 74(3): 854 - 862. [Abstract] [Full Text] [PDF]
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 C. Muller, R. Schibli, E. P. Krenning, and M. de Jong Pemetrexed Improves Tumor Selectivity of 111In-DTPA-Folate in Mice with Folate Receptor-Positive Ovarian Cancer J. Nucl. Med., April 1, 2008; 49(4): 623 - 629. [Abstract] [Full Text] [PDF]
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 A. Llombart-Cussac, M. Martin, N. Harbeck, R. M. Anghel, A. E. Eniu, M. W. Verrill, P. Neven, J. De Greve, A. S. Melemed, R. Clark, et al. A Randomized, Double-Blind, Phase II Study of Two Doses of Pemetrexed as First-Line Chemotherapy for Advanced Breast Cancer Clin. Cancer Res., June 15, 2007; 13(12): 3652 - 3659. [Abstract] [Full Text] [PDF]
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C. H. Takimoto, L. A. Hammond-Thelin, J. E. Latz, L. Forero, M. Beeram, B. Forouzesh, J. de Bono, A. W. Tolcher, A. Patnaik, P. Monroe, et al. Phase I and Pharmacokinetic Study of Pemetrexed with High-Dose Folic Acid Supplementation or Multivitamin Supplementation in Patients with Locally Advanced or Metastatic Cancer Clin. Cancer Res., May 1, 2007; 13(9): 2675 - 2683. [Abstract] [Full Text] [PDF]
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 S. Malempati, H. S. Nicholson, J. M. Reid, S. M. Blaney, A. M. Ingle, M. Krailo, L. C. Stork, A. S. Melemed, R. McGovern, S. Safgren, et al. Phase I Trial and Pharmacokinetic Study of Pemetrexed in Children With Refractory Solid Tumors: The Children's Oncology Group J. Clin. Oncol., April 20, 2007; 25(12): 1505 - 1511. [Abstract] [Full Text] [PDF]
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 S. Chattopadhyay, R. G. Moran, and I. D. Goldman Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications Mol. Cancer Ther., February 1, 2007; 6(2): 404 - 417. [Abstract] [Full Text] [PDF]
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S. Chattopadhyay, R. Zhao, S. A. Krupenko, N. Krupenko, and I. D. Goldman The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line. Mol. Cancer Ther., February 1, 2006; 5(2): 438 - 449. [Abstract] [Full Text] [PDF]
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R. Zhao, S. Zhang, M. Hanscom, S. Chattopadhyay, and I. D. Goldman Loss of Reduced Folate Carrier Function and Folate Depletion Result in Enhanced Pemetrexed Inhibition of Purine Synthesis Clin. Cancer Res., February 1, 2005; 11(3): 1294 - 1301. [Abstract] [Full Text] [PDF]
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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]
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S. Chattopadhyay, Y. Wang, R. Zhao, and I. D. Goldman Lack of Impact of the Loss of Constitutive Folate Receptor {alpha} Expression, Achieved by RNA Interference, on the Activity of the New Generation Antifolate Pemetrexed in HeLa Cells Clin. Cancer Res., December 1, 2004; 10(23): 7986 - 7993. [Abstract] [Full Text] [PDF]
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 T. L. Witt, S. E. Stapels, and L. H. Matherly Restoration of Transport Activity by Co-expression of Human Reduced Folate Carrier Half-molecules in Transport-impaired K562 Cells: LOCALIZATION OF A SUBSTRATE BINDING DOMAIN TO TRANSMEMBRANE DOMAINS 7-12 J. Biol. Chem., November 5, 2004; 279(45): 46755 - 46763. [Abstract] [Full Text] [PDF]
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Y. Wang, R. Zhao, and I. D. Goldman Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier Clin. Cancer Res., September 15, 2004; 10(18): 6256 - 6264. [Abstract] [Full Text] [PDF]
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A. A. Adjei Pemetrexed (ALIMTA), A Novel Multitargeted Antineoplastic Agent Clin. Cancer Res., June 15, 2004; 10(12): 4276S - 4280S. [Abstract] [Full Text] [PDF]
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R. Zhao and I. D. Goldman Enter Alimta(R): A New Generation Antifolate Oncologist, June 1, 2004; 9(3): 242 - 244. [Full Text] [PDF]
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 R. Zhao, M. Hanscom, S. Chattopadhyay, and I. D. Goldman Selective Preservation of Pemetrexed Pharmacological Activity in HeLa Cells Lacking the Reduced Folate Carrier: Association with the Presence of a Secondary Transport Pathway Cancer Res., May 1, 2004; 64(9): 3313 - 3319. [Abstract] [Full Text] [PDF]
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R. Zhao, F. Gao, M. Hanscom, and I. D. Goldman A Prominent Low-pH Methotrexate Transport Activity in Human Solid Tumors: Contribution to the Preservation of Methotrexate Pharmacologic Activity in HeLa Cells Lacking the Reduced Folate Carrier Clin. Cancer Res., January 15, 2004; 10(2): 718 - 727. [Abstract] [Full Text] [PDF]
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M. Stark, L. Rothem, G. Jansen, G. L. Scheffer, I. D. Goldman, and Y. G. Assaraf Antifolate Resistance Associated with Loss of MRP1 Expression and Function in Chinese Hamster Ovary Cells with Markedly Impaired Export of Folate and Cholate Mol. Pharmacol., August 1, 2003; 64(2): 220 - 227. [Abstract] [Full Text] [PDF]
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Y. Wang, R. Zhao, S. Chattopadhyay, and I. D. Goldman A Novel Folate Transport Activity in Human Mesothelioma Cell Lines with High Affinity and Specificity for the New-Generation Antifolate, Pemetrexed Cancer Res., November 15, 2002; 62(22): 6434 - 6437. [Abstract] [Full Text] [PDF]
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I. D. Goldman Membrane Transport of Chemotherapeutics and Drug Resistance: Beyond the ABC Family of Exporters to the Role of Carrier-mediated Processes Clin. Cancer Res., January 1, 2002; 8(1): 4 - 6. [Full Text] [PDF]
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)
or MTX (
), and uptake was monitored over 2 h. A portion of
cells incubated with MTA for 30 min or 2 h was separated by
centrifugation and resuspended in a large volume of MTA-free HBS for 30
and 40 min (
and 
, respectively) to determine
nonexchangeable MTA. 
, exchangeable MTA level. – – – –,
projected increase in nonexchangeable drug. Inset
compares initial uptake rates from the same experiment. Data are
representative of three experiments.
