From the Departments of § Medicine and Molecular
Pharmacology and the Albert Einstein College of Medicine Comprehensive
Cancer Center, Bronx, New York 10461 and the Department
of Biology, The Technion, Israel Institute of Technology,
Haifa 32000, Israel
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ABSTRACT |
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Previous studies described a Chinese hamster ovary cell line, PyrR100, resistant to lipid-soluble antifolates due to the loss of an energy-coupled folate exporter resulting in a marked increase in intracellular folate cofactor accumulation. There was, in addition, an unexplained increase in folic acid influx in PyrR100 cells which is shown in this paper to be mediated by a transporter with a low pH optimum. The pH profile for folic acid influx in parental Chinese hamster ovary AA8 cells indicated peak activity at pH 6; this was increased >3-fold in PyrR100 cells. In contrast, methotrexate (MTX) influx in AA8 cells showed two peaks of comparable activities at pH 6 and 7.5; in PyrR100 cells, the component at pH 6 was increased 2-fold. Folic acid was a potent inhibitor of [3H]MTX or [3H]folic acid influx (1 µM) via the low pH route with IC50 values of ~1 µM. Prostaglandin A1 was a potent inhibitor of [3H]MTX influx via the reduced folate carrier 1 at pH 7.5 with only a small inhibitory effect on the low pH route. The addition of 10 µM folic acid to PyrR100 cells resulted in a MTX influx pH profile identical to that of AA8 cells, consistent with suppression of the low pH route. In contrast, addition of 25 µM prostaglandin A1 to PyrR100 cells resulted in a MTX influx pH profile comparable to that of folic acid, consistent with the loss of the reduced folate carrier-mediated component. Inhibition (~70%) of [3H]folic acid influx by ~10 µM unlabeled folic acid at pH 7.5 indicated that the low pH transporter accounts for the majority of folic acid transport at physiological pH.
This study demonstrates the functional importance of a low pH folate transporter that is increased when enhanced folic acid entry into cells is required as an adaptive response to antifolate selective pressure. This may represent a mechanism of resistance to new antifolate inhibitors of folate cofactor-dependent enzymes in which cytotoxic activity is limited by expanded cellular folate pools.
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INTRODUCTION |
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Previous studies from this laboratory described a novel form of resistance to lipid soluble antifolates in a Chinese hamster ovary (CHO)1 cell line, PyrR100 (1), based upon enhanced cellular accumulation of reduced folate cofactors (2). This augmentation of net folate transport was primarily due to the loss of function of an energy-dependent folate exporter. In addition, there was an unexplained increase in folic acid influx in the resistant cells; this paper explores the mechanism by which this occurs.
Folic acid is the folate source in virtually every mammalian cell culture medium. Transport of this folate is therefore critical to maintaining one-carbon dependent biosynthetic processes and the methylation state of nucleic acids. However, the mechanism by which folic acid is transported into cells under usual growth conditions remains a subject of some controversy (3, 4). In contrast, the reduced folate cofactor, 5-CH3THF is the major folate in the blood of man and rodents and there is a considerable understanding of the mechanism of transport for this and other folate cofactors and 4-amino antifolates.
The major route of transport for 5-CH3THF and 5-CHOTHF is the reduced folate carrier, RFC1 (5-8). Folic acid transport by this pathway is very slow because of its very low affinity for RFC1 and can account for only a small component of uptake at the usual concentration of folic acid in most media (3-5). Consistent with this is the observation that cell lines, in which RFC1 function is lost with the acquisition of MTX resistance, have only a small increase in their folic acid growth requirement (9). Folic acid has a very high affinity (Kd = 1 nM) for the folate receptor which mediates entry via an endocytic process (10-12). But expression of folate receptor is minimal, or not detected at all, in most cultured cell lines. Recently, another transport process, with a low pH optimum, has been identified with an affinity for folic acid in the micromolar range, but the contribution of this pathway to folate transport at physiological pH has not been established (13, 14).
This paper provides new insights into the mechanism of folic acid transport in CHO cells at low and physiological pH. Enhanced folic acid influx into lipophilic antifolate-resistant PyrR100 CHO cells is shown to be due, to a large extent, to increased transport via the low pH route. Furthermore, the data supports the conclusion that it is the activity of this process that mediates a major component of folic acid entry into CHO cells at physiological pH. This is the first demonstration of the importance of the low pH route to folic acid delivery into cells under normal growth conditions in vitro.
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MATERIALS AND METHODS |
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Chemicals--
[3',5',7',9'-3H]folic acid,
[3',5',7'-3H]MTX, and
(6S)[3H]5-CH3THF were obtained
from Amersham. Folic acid and (6R,6S)-5-CHOTHF were from Lederle, Carolina, Puerto Rico. Folate and antifolate compounds were purified by high performance liquid chromatography prior
to use (15). Trimetrexate (TMQ) glucuronate was kindly provided by
Warner-Lambert, Ann Arbor, MI. Prostaglandin A1
(PGA1) was purchased from Cayman Chemical Co., Ann Arbor,
MI. Stock solutions (20 mM) of PGA1 were
prepared before use in ethanol and stored at 20 °C; the final
content of ethanol did not exceed 0.25% in all transport experiments,
a level that did not have any effect on folate transport in AA8 or
PyrR100 cells.
Cell Lines and Tissue Cultures-- PyrR100 cells were obtained by multiple step selection of parental CHO AA8 cells in gradually increasing concentrations of the lipid-soluble antifolate pyrimethamine (1). This stepwise selection was initiated at 100 nM pyrimethamine (the 50% lethal dose, LD50) and terminated at 100 µM. The resultant 1000-fold pyrimethamine-resistant cells, termed PyrR100, retained wild-type dihydrofolate reductase (DHFR) gene copy number and mRNA levels, as well as parental levels of DHFR enzyme activity (1). The affinity of DHFR for various antifolates was preserved in PyrR100 cells, along with parental sensitivity to MTX but these cells were cross-resistant to the lipophilic antifolate trimetrexate and piritrexim (1, 16). AA8 cells and their PyrR100 subline were maintained in monolayer or suspension culture conditions in RPMI 1640 medium containing 2.3 µM folic acid (HyClone, Logan, UT), supplemented with 5% dialyzed fetal bovine serum, Gemini (Bio-Laboratories Inc., Calabasas, CA), 1 mM sodium pyruvate, (Mediatech, Herndon, VA), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. The growth medium of PyrR100 monolayers was supplemented with 100 µM pyrimethamine. Prior to transport experiments, cells were grown in suspension culture in drug-free medium.
Folate Transport and pH Profile-- Transport of tritiated folates was measured as described previously (2). Briefly, exponentially growing cells from suspension cultures (1-3-liter spinner flasks, Wheaton) in drug-free growth medium were collected by centrifugation and washed three times with ice-cold unbuffered saline (140 mM NaCl, 5.3 mM KCl, 1.9 mM CaCl2, 1 mM MgCl2, and 7 mM glucose). Transport experiments at pH 5.0-6.5 were performed in MES-buffered saline (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose) (13, 14) and those at pH 7.0-8.0 in Hepes-buffered saline (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 5 mM glucose). Prior to influx determinations, cells were suspended to a density of 4-8 × 107/ml in the desired buffer including 1 mM pyruvic acid and then equilibrated at 37 °C for 20 min. In folic acid uptake experiments 5 µM TMQ was routinely included to ensure complete blockade of DHFR activity (17). TMQ and pyrimethamine at concentrations of 10 and 100 µM, respectively, did not alter folic acid influx in either parental AA8 or PyrR100 cells. Uptake was initiated by addition of the radiolabeled folate, and terminated by injecting 0.5-1-ml portions of the cell suspension into 9 ml of 0 °C Hepes-buffered saline at pH 7.4. Cells were then collected by centrifugation, washed twice with ice-cold Hepes-buffered saline, and processed for determination of cellular radiolabel as described previously (18). All transport studies were performed at 1 µM [3H]folic acid or 1 µM [3H]MTX unless otherwise indicated. Influx was always measured over an interval that assured initial rates for each folate substrate.
[3H]Folic Acid Binding-- Specific binding of [3H]folic acid to membrane folate receptors was determined with viable cells at 0 °C as described previously (9). The assay mixture contained 2-4 × 107 cells, which were acid-washed three times with MES-buffered saline at pH 4.5 to strip off folic acid bound to folate receptors. Following a 10-min incubation with 100 nM [3H]folic acid (high performance liquid chromatography purified immediately prior to use) at 0 °C, cells were sedimented (12,000 × g for 15 s at 0 °C), the supernatant was aspirated, and the residual fluid removed by a second centrifugation and aspiration. Triplicate samples of cell pellets were processed and radioactivity determined as described previously (18). Specific binding was the difference between [3H]folic acid bound in the absence or presence of a 1000-fold excess of unlabeled folic acid.
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RESULTS |
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pH Dependence of Folic Acid and MTX Influx-- As reported previously, folic acid influx is increased by a factor of ~ two in PyrR100 cells at pH 7.4 (2). However, as indicated in Table I, there is only a minimal increase in 5-CH3-THF (10%) and MTX (20%) influx in PyrR100 relative to parental AA8 cells. These observations suggested that the bulk of the change in folic acid influx is mediated by a process distinct from RFC1. Since there is no evidence for folate receptor-mediated transport in these cells (see below), studies were undertaken to determine whether augmented transport via the low pH route might be the mechanism by which folic acid influx is enhanced under these conditions.
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Discriminating between Transport Mediated by the Low pH Route and
RFC1--
Further studies were undertaken to better discriminate
between transport mediated by the low pH pathway and RFC1. Folic acid is a very poor substrate for RFC1 (3-5), but a very good substrate for
the low pH route in murine leukemia cells and hence has properties that
should permit the separation of these processes (13, 14). As
illustrated in Fig. 2A, when
influx of [3H]folic acid (1 µM) was
monitored in PyrR100 cells as a function of increasing
concentrations of unlabeled folic acid at pH 6.0, about 80% of influx
was blocked by 10 µM folic acid. The remaining 20% was
not inhibited by concentrations of folic acid in excess of 100 µM. The IC50 for the folic acid inhibitable
component was ~1 µM. This established that the major component of influx at pH 6.0 was mediated by a process with a high
affinity for folic acid, clearly different from RFC1 which has an
affinity for this folate which is 2 orders of magnitude lower (3-5).
Similarly, at physiological pH (Fig. 2B), the component of
[3H]folic acid influx inhibited by low concentrations of
folic acid (IC50 ~ 2 µM) in
PyrR100 cells was ~70% and the residual influx (~30%)
was not inhibited by folic acid concentrations of up to 200 µM. Hence, the major component of folic acid influx at
physiological pH appears to be mediated by the low pH transporter.
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The pH Dependence of Net MTX and Folic Acid Transport-- To evaluate whether the pH dependence of folate influx is accompanied by changes in net concentrations achieved within the cell, studies evaluated the full time course of uptake and steady-state levels for folic acid and MTX. As indicated in Fig. 6, a clear increase in the steady-state levels of folic acid was observed in both AA8 (Fig. 6A) and PyrR100 cells (Fig. 6B) at pH 6.0 relative to pH 7.5 under conditions in which folic acid reduction was blocked with TMQ. While the percentage increase was greater in AA8 cells, it should be noted that the absolute levels of folic acid accumulation in PyrR100 cells were 10-fold higher. Steady-state levels of MTX accumulation were comparable in AA8 cells (Fig. 6C) with a small increase in PyrR100 cells (Fig. 6D). Hence, enhanced influx of folic acid and MTX is accompanied by steady-state levels that are the same or greater at pH 6.0 than at pH 7.4.
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Determination of Monolayer Culture Medium pH during Cell Growth-- Since the activity of a low pH transporter was augmented in PyrR100 cells during selection with pyrimethamine in the presence of folic acid (2), studies were undertaken to evaluate the extent of acidification of the medium during growth of CHO cell monolayers under similar conditions. It can be seen in Fig. 7 that after plating, the pH of the growth medium falls steadily to a value of 7.0 by day 4. When AA8 and PyrR100 cells begin to reach confluence at days 6 and 7, respectively, the culture medium reaches a pH of 6.8 and 6.7, respectively. In contrast, the pH of the cell-free growth medium, alone, was stable at about pH 7.3.
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DISCUSSION |
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In a prior report (2), resistance to the lipid-soluble antifolate pyrimethamine was associated with a marked decrease in folate growth requirement in a CHO cell line, PyrR100. Resistance was shown to be due primarily to the loss of an energy-dependent efflux pump resulting in marked augmentation of folate accumulation (2). There was a smaller increase (2-fold) in folic acid influx in PyrR100 cells. This paper clarifies the basis for this increase in folic acid influx within the context of the known routes of transport for folate compounds: RFC1, folate receptor-mediated endocytosis, and a process that functions optimally at low pH. The data indicate that the major change in folic acid influx in PyrR100 cells can be attributed to a substantial increase in transport mediated by the low pH route and that the increase in folic acid influx observed at physiological pH is due largely to the residual activity of this process. Beyond this, the findings represent the first demonstration of the functional overexpression of a low pH transporter as the result of suppression of DHFR activity by a lipophilic antifolate. This is apparently one of several stable functional alterations which take place to meet the need for high intracellular folate levels to overcome the pharmacologic activity of this class of antifolates in the PyrR100 cell line (16).
Several lines of evidence exclude the possibility that the overexpressed folate transporter in PyrR100 cells is a mutated RFC1 with increased affinity for folic acid and an optimal activity at pH 6.0. (i) The pH optimum of a single transporter with an altered RFC1 cannot be acidic for one substrate, folic acid, but with an unchanged physiological pH optimum for another cognate substrate such as MTX. Furthermore, as with AA8 cells, PyrR100 cells displayed a single low pH optimum for folic acid transport but two pH optima for MTX transport, consistent with two distinct transport entities. (ii) The low pH folic acid transport activity is increased 3-fold in PyrR100 cells, whereas wild-type MTX transport activity is retained at physiological pH. A single transport entity (i.e. even a mutated RFC1) cannot at the same time be overexpressed for one substrate (folic acid) without a change in its cognate substrate (MTX). (iii) Folic acid blocks [3H]MTX influx via the overexpressed low pH transport route but leaves the RFC-mediated component of transport unchanged at physiological pH at the same level as in parental AA8 cells. IV PGA1 which was found here to be a potent and selective inhibitor of RFC1 in both parental AA8 and PyrR100 cells, did not suppress the low pH folic acid transporter. These observations further support the conclusion that the low pH folate transporter is independent of, and distinct from, RFC1. Finally, (v) structural alterations (i.e. mutations) of RFC1 are most frequently associated with altered transport of MTX resulting in resistance to this antifolate but PyrR100 cells retain wild-type sensitivity to MTX.
As indicated above, the data show that the low pH route makes an important contribution to folic acid transport at physiological pH under usual growth conditions in CHO cells. This clarifies, for this cell line at least, the mechanism of folic acid transport and its relationship to RFC1, an issue that has been quite controversial (3, 4). Furthermore, as the growth medium of monolayer cultures gradually acidifies, RFC1 activity falls and the cells depend increasingly on the low pH route for delivery of folic acid. Folic acid is a favored substrate for this route; in contrast to the very low affinity of RFC1 for folic acid (3-5), the affinity of the low pH transporter for this folate is orders of magnitude higher (13, 14). An important corollary of this difference in influx is the steady-state levels of folic acid achieved at low pH, the parameter of transport that determines the availability of folates for biosynthetic processes (19). Steady-state levels are not only sustained, but are increased by the low pH transporter. Finally, folate influx at low pH is more prominent than at physiological pH in CHO cells as compared with L1210 cells. MTX influx at pH 6.0 is comparable to that at physiological pH in AA8 cells; however, in murine L1210 leukemia cells, MTX influx at pH 6.0 was one-third of that at physiological pH (13, 14). Thus there is high basal activity of the low pH transporter in CHO cells. Taken together, these factors favor stable overexpression of the low pH route as a mechanism by which CHO cells adapt to the requirement for increased folate accumulation.
PyrR100 cells are 1000-fold resistant to pyrimethamine, 37-fold and 28-fold cross-resistant to TMQ and piritrexim, respectively, all of which are lipid-soluble antifolates that inhibit mammalian DHFR (1). PyrR100 cells were shown to have a 3-fold higher folate pool size than AA8 cells (20). This expanded pool of cellular folates in PyrR100 represents a very useful adaptation to DHFR inhibition, particularly by lipid-soluble antifolates that have a relatively low affinity for this enzyme. Hence, increased pools of reduced folates result in the build-up of high levels of dihydrofolate, as antifolate associates with DHFR and tetrahydrofolates are oxidized, which then compete with the antifolate for the small percentage of enzyme sites that are sufficient to sustain tetrahydrofolate synthesis within the cell (21).
In previous studies lipophilic antifolate-resistant clonal variants
exposed to nanomolar concentrations of the lipid-soluble antifolate TMQ
displayed marked resistance to the selecting agent and prominent
cross-resistance to other lipophilic antifolates, but retained
wild-type sensitivity to MTX without quantitative or qualitative
changes in DHFR. This resistance was genetically stable and the
frequency of emergence of resistant clones was enhanced 100-fold when
cells were treated with a mutagen such as -irradiation prior to drug
selection (17, 22). Thus, it is likely that during the early stages of
exposure to low concentrations of lipophilic antifolates such as
trimetrexate or pyrimethamine, stable mutations occur which abolish
folate exporter function, while other mutations may increase the
expression of a low pH folate transporter.
PyrR100 cells also display 31-fold cross-resistance to the
folate-based glycinamide ribonucleotide transformylase inhibitor,
5,10-dideaza-5,6,7,8-tetrahydrofolic acid, and 27-fold cross-resistance
to the lipid-soluble thymidylate synthase inhibitor AG377 data not
shown. High folate cofactor pools would be a basis for resistance to
these agents by competition at the tetrahydrofolate
cofactor-dependent enzyme sites. Alternatively, high folate
pools could inhibit antifolate polyglutamylation at the level of
folylpoly--glutamyl synthetase thereby blocking the formation of the
active polyglutamate congeners. For instance, an antifolate-resistant
CCRF-CEM cell line with high tetrahydrofolate cofactor pools and
wild-type folylpoly-
-glutamyl synthetase activity, has very low
levels of antifolate polyglutamylation (23). This mechanism of
resistance to antifolates that require polyglutamylation for activity
also has features similar to what was recently reported for
5,10-dideaza-5,6,7,8-tetrahydrofolic acid-resistant L1210 cells in
which folate cofactor accumulation was markedly enhanced (24). In this
case, however, augmented cellular folate levels were due to mutations
in RFC1 that resulted in a marked increase in the affinity of carrier
for folic acid with resultant enhanced transport.
Folate transport systems that operate optimally at acidic pH have been
identified in rat and human small intestine (25-27), and in rat kidney
brush-border membrane vesicles (28, 29). The driving force for
intestinal and kidney folate transport in membrane vesicles is the
transmembrane proton gradient and not the acidic extracellular pH alone
(29, 30). Folate uptake mediated by the mammalian low pH intestinal
transporter has been attributed to either a OH/folate
exchange or H+/folate cotransport (30, 31). The
H+ transmembrane gradient is also the driving force for
intestinal and renal PEPT1 and PEPT2 H+-coupled peptide
transporters (32, 33). The basis for enhanced influx and increased
transmembrane gradients for folic acid at low pH in CHO cells is not
clear. This could be due to a proton-driven folate cotransport
energized by a transmembrane H+ gradient. Alternatively,
the low pH folate transporter could have maximal activity at acidic pH
without H+/folate cotransport. The acidic pH optimum of the
low pH transport route cannot be attributed to the protonation state of
the
- and
-carboxyl residues of folates that have
pKa values of 3.1-3.5 and 4.6-4.8, respectively
(34), since protonation would not change significantly over the pH
range of 6.0-8.0 studied. Nor, for the same reason, could the
ionization of the N-1 nitrogen of the pteridine ring
(pKa = 2.4) be relevant to the pH dependence of
transport (34, 35).
In summary, this paper provides evidence, for the first time, of the functional importance of a folic acid transport route distinct from RFC1 and the folate receptors, that has a low pH optimum. This route accounts for the majority of folic acid transport at physiological pH in CHO cells and is concentrative; the low pH mechanism generates higher transmembrane gradients for folic acid than are achieved by RFC1. While RFC1 is thought to be an anion exchanger the mechanism by which the low pH route achieves uphill transport is not clear and is currently under study. The relationship between this transporter and other low pH folate transport processes in kidney and intestine remains to be clarified.
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FOOTNOTES |
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* This work was supported by Grant CA-39807 from the National Cancer Institute.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: Depts. of Medicine and Molecular Pharmacology, The Albert Einstein Comprehensive Cancer Center, Chanin Two, 1300 Morris Park Ave., Bronx, NY 10461. Fax: 718-430-8550; Tel.: 718-430-2302.
1 The abbreviations used are: CHO, Chinese hamster ovary; DHFR, dihydrofolate reductase; RFC1, reduced folate carrier; MTX, methotrexate; TMQ, trimetrexate; 5-CH3THF, 5-methyltetrahydrofolate; 5-CHOTHF, 5-formyltetrahydrofolate; PGA1, prostaglandin A1; PyrR100, pyrimethamine-resistant cell line; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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