Increased activity of a novel low pH folate transporter associated with lipophilic antifolate resistance in chinese hamster ovary cells.

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 microM) via the low pH route with IC50 values of approximately 1 microM. 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 microM 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 microM 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 ( approximately 70%) of [3H]folic acid influx by approximately 10 microM 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.

Previous studies from this laboratory described a novel form of resistance to lipid soluble antifolates in a Chinese hamster ovary (CHO) 1 cell line, Pyr R100 (1), based upon enhanced cel-lular 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-CH 3 THF 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-CH 3 THF and 5-CHOTHF is the reduced folate carrier, RFC1 (5)(6)(7)(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)(4)(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 (K d ϭ 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 Pyr R100 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. Cell Lines and Tissue Cultures-Pyr R100 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, LD 50 ) and terminated at 100 M. The resultant 1000-fold pyrimethamine-resistant cells, termed Pyr R100 , 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 Pyr R100 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 Pyr R100 subline were maintained in monolayer or suspension culture conditions in RPMI 1640 medium containing 2.3 M 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 g/ml streptomycin. The growth medium of Pyr R100 monolayers was supplemented with 100 M pyrimethamine. Prior to transport experiments, cells were grown in suspension culture in drugfree medium.

Chemicals
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 CaCl 2 , 1 mM MgCl 2 , 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 MgCl 2 , 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 MgCl 2 , and 5 mM glucose). Prior to influx determinations, cells were suspended to a density of 4 -8 ϫ 10 7 /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 M TMQ was routinely included to ensure complete blockade of DHFR activity (17). TMQ and pyrimethamine at concentrations of 10 and 100 M, respectively, did not alter folic acid influx in either parental AA8 or Pyr R100 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 M [ 3 H]folic acid or 1 M [ 3 H]MTX unless otherwise indicated. Influx was always measured over an interval that assured initial rates for each folate substrate.
[ 3 H]Folic Acid Binding-Specific binding of [ 3 H]folic acid to membrane folate receptors was determined with viable cells at 0°C as described previously (9). The assay mixture contained 2-4 ϫ 10 7 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 [ 3 H]folic acid (high performance liquid chromatography purified immediately prior to use) at 0°C, cells were sedimented (12,000 ϫ 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 [ 3 H]folic acid bound in the absence or presence of a 1000-fold excess of unlabeled folic acid.

RESULTS
pH Dependence of Folic Acid and MTX Influx-As reported previously, folic acid influx is increased by a factor of ϳ two in Pyr R100 cells at pH 7.4 (2). However, as indicated in Table I, there is only a minimal increase in 5-CH 3 -THF (10%) and MTX (20%) influx in Pyr R100 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. Folic acid influx in AA8 cells was optimal at pH 6.0; the pattern was symmetrical, yet broad (Fig. 1A). This profile narrowed in Pyr R100 cells; here the pH optimum was essentially unchanged but maximum influx at pH 6.0 was increased more than 3-fold relative to AA8 cells (Fig. 1A). Further evidence for increased activity of the low pH transporter was the observation that folic acid influx in Pyr R100 cells at pH 6.0 was nearly 4-fold higher than at pH 7.4, while there is only a 2-fold difference in folic acid influx at the two pH levels in parental AA8 cells. The profile of pH dependence for MTX influx was different ( Fig. 1B). There was a gradual increase in influx in AA8 cells as the pH was increased from 5.0 to 8.0 with a small peak at pH 6.0 and maximal activity at physiological pH. In contrast to other mammalian cell lines in which maximal MTX influx was always observed at physiological pH (13,14), in Pyr R100 cells influx at pH 6.0 exceeded that at pH 7.4 and was 2-fold greater than that in AA8 cells at pH 6.0 (Fig. 1B). Hence, transport of folic acid and MTX is mediated by two processes with different pH optima and the activity of the transporter that is optimal at low pH is substantially increased in Pyr R100 cells.
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)(4)(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 [ 3 H]folic acid (1 M) was monitored in Pyr R100 cells as a function of increasing concentrations of unlabeled folic acid at pH 6.0, about 80% of influx was blocked by Յ10 M folic acid. The remaining 20% was not inhibited by concentrations of folic acid in excess of 100 M. The IC 50 for the folic acid inhibitable component was ϳ1 M. 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)(4)(5). Similarly, at physiological pH (Fig. 2B), the component of [ 3 H]folic acid influx inhibited by low concentrations of folic acid (IC 50 ϳ 2 M) in Pyr R100 cells was ϳ70% and the residual influx (ϳ30%) was not inhibited by folic acid concentrations of up to 200 M. Hence, the major component of folic acid influx at physiological pH appears to be mediated by the low pH transporter.
The components of [ 3 H]MTX influx mediated by RFC1 and the low pH transporter were further distinguished by studies in which folic acid inhibition was evaluated over a broad pH range in Pyr R100 and AA8 cells. It can be seen in Fig. 3 that in the presence of 10 M unlabeled folic acid, the pH profiles of [ 3 H]MTX influx in both Pyr R100 (Fig. 3A) and AA8 cells (Fig.  3B) become essentially identical with a single pH optimum at physiological pH. Hence, the contribution of the low pH trans- porter is abolished and the remaining RFC1 component is retained. The small decline in MTX influx at pH 7.4 likely represents the loss of residual activity of the low pH transporter. Another approach was utilized in an attempt to achieve selective blockade of RFC1 activity while minimizing inhibition of the low pH transporter. An agent that appears to have desirable properties in this regard is PGA 1 . As illustrated in Fig. 4A, PGA 1 inhibits MTX influx at pH 7.4 by ϳ90% at concentrations of ϳ50 M, consistent with near complete suppression of transport activity via RFC1 in Pyr R100 cells. This is further illustrated in the pH profile for MTX influx in Pyr R100 cells in Fig. 5. In the presence of 25 M PGA 1 , the RFC1mediated component is lost and the profile of [ 3 H]MTX influx approximates that of [ 3 H]folic acid influx in Pyr R100 cells. In contrast, PGA 1 (100 M) inhibition of folic acid influx at pH 6.0 in Pyr R100 cells did not exceed ϳ25%, consistent with the major portion of folate transport mediated by the low pH route (Fig.  4B). The small inhibitory effect might be related to a small RFC1-mediated component and/or some inhibition of the low pH route by high PGA 1 levels. In AA8 cells, the portion of [ 3 H]folic acid influx inhibited by PGA 1 at pH 6.0 and 7.5 is even smaller (15-20%, data not shown).
A variety of experimental approaches exclude the possibility that influx of folates is mediated by the folate receptor, endocytic process in these cells. (a) Northern blot analysis with poly(A) ϩ mRNA from wild-type and Pyr R100 cells showed no detectable folate receptor transcript in either cell line (data not shown). (b) Using a sensitive, high-affinity [ 3 H]folic acid (high performance liquid chromatography purified immediately prior to use) binding assay in the presence or absence of competing unlabeled folic acid, no folate receptor expression was detected. (c) Binding of [ 3 H]folic acid via the folate receptor is typically accompanied by a "high intercept" when folic acid uptake is analyzed as a function of time. This was not observed in Pyr R100 cells. (d) Monensin, a potent vesicular transport inhibitor which blocks folate receptor-mediated endocytosis, enhanced rather than inhibited influx of folic acid.
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 Pyr R100 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 Pyr R100 cells were 10-fold higher. Steady-state levels of MTX accumulation were comparable in AA8 cells (Fig. 6C) with a small increase in Pyr R100 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.
Determination of Monolayer Culture Medium pH during Cell Growth-Since the activity of a low pH transporter was augmented in Pyr R100 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

FIG. 1. The pH dependence of folic acid (A) and MTX (B) influx in parental AA8 (triangles) and Pyr R100 cells (squares).
Exponentially growing cells in suspension cultures were harvested by centrifugation, washed three times at 0°C and resuspended in buffer at the appropriate pH as detailed under "Materials and Methods." Following 20 min equilibration at the various pH levels, cells were exposed to 1 M steadily to a value of 7.0 by day 4. When AA8 and Pyr R100 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.

DISCUSSION
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, Pyr R100 . 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 Pyr R100 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 receptormediated endocytosis, and a process that functions optimally at low pH. The data indicate that the major change in folic acid influx in Pyr R100 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 Pyr R100 cell line (16).
Several lines of evidence exclude the possibility that the overexpressed folate transporter in Pyr R100 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, Pyr R100 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 Pyr R100 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 pressed 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 PGA 1 which was found here to be a potent and selective inhibitor of RFC1 in both parental AA8 and Pyr R100 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 Pyr R100 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)(4)(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.
Pyr R100 cells are 1000-fold resistant to pyrimethamine, 37fold and 28-fold cross-resistant to TMQ and piritrexim, respectively, all of which are lipid-soluble antifolates that inhibit mammalian DHFR (1). Pyr R100 cells were shown to have a 3-fold higher folate pool size than AA8 cells (20). This expanded pool of cellular folates in Pyr R100 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.
Pyr R100 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 5,10dideaza-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)(26)(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 Ϫ /folate exchange or H ϩ /folate cotransport (30,31). The H ϩ transmembrane gradient is also the driving force for intestinal and renal PEPT1 and PEPT2 H ϩcoupled 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 ϩ gradient. Alternatively, the low pH folate transporter could have maximal activity at acidic pH without H ϩ /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 pK a 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 (pK a ϭ 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.