©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Distinguishing between Folate Receptor--mediated Transport and Reduced Folate Carrier-mediated Transport in L1210 Leukemia Cells (*)

(Received for publication, December 29, 1994)

Michael J. Spinella Kevin E. Brigle Esteban E. Sierra I. David Goldman (§)

From the Departments of Medicine and Pharmacology and the Massey Cancer Center, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia 23298

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

L1210 leukemia cells transport reduced folates and methotrexate via a well defined reduced folate carrier system and, in the absence of low folate selective pressure, do not express an alternate endocytotic route mediated by cell surface folate receptors. This laboratory previously described an L1210 leukemia cell line, MTX^rA, with acquired resistance to methotrexate (MTX) due to the loss of mobility of the reduced folate carrier. We now report on the transfection of MTX^rA with a cDNA encoding the murine homolog of the human folate receptor isoform of KB cells to produce MTX^rA-TF1, which constitutively expresses high levels of FR-alpha. MTX^rA-TF1 and L1210 cells were utilized to compare transport of methotrexate mediated by FR-alpha and the reduced folate carrier, respectively. Methotrexate influx in the two lines was similar when the extracellular level was 0.1 µM, but as the methotrexate concentration increased, influx via the reduced folate carrier increased in comparison to influx mediated by FR-alpha. Transport kinetics indicated both a 20-fold lower influx K and V(max) for MTX^rA-TF1 as compared to L1210 cells. The two cell lines exhibited distinct influx properties. Methotrexate influx in MTX^rA-TF1 was markedly inhibited by 50 nM folic acid and metabolic poisons. In L1210 cells, 1.0 µM folic acid did not affect MTX influx, and metabolic poisons either had no effect on or increased methotrexate influx. Removal of extracellular chloride markedly inhibited transport in MTX^rA-TF1 but stimulated influx in L1210 cells. When the pH was decreased to 6.2, methotrexate influx was not altered in MTX^rA-TF1 but was reduced in L1210 cells. Probenecid and sulfobromophthalein inhibit methotrexate influx in both L1210 and MTX^rA-TF1 cell lines; however, inhibition in MTX^rA-TF1 could be accounted for on the basis of inhibition of methotrexate binding to FR-alpha. The data indicate that the reduced folate carrier and FR-alpha function independently and exhibit distinct properties. FR-alpha expressed at sufficient levels can mediate influx of MTX and folates into cells at rates comparable to the reduced folate carrier and hence has pharmacologic and physiologic importance.


INTRODUCTION

In eukaryotic cells, import of a folate vitamin is required to sustain enzymatic pathways leading to the synthesis of thymidine, purines, and amino acids. The folate-dependent enzymes have long been exploited as chemotherapeutic targets. For example, the folate analog methotrexate (MTX) (^1)is a successful chemotherapeutic agent due to its potent inhibition of dihydrofolate reductase (DHFR). Newer antifolates that directly block folate-dependent purine and pyrimidine biosynthesis (reviewed in (1) ) are now in clinical trials. Membrane transport of antifolates such as MTX has been an area of considerable interest because of the role that this process plays as a determinant of the cytotoxicity, selectivity, and resistance to this class of agents(2, 3, 4, 5) .

Folates and antifolates including MTX enter cells via two major transport systems. The reduced folate carrier has received the most attention because properties of antifolate transport mediated by this system correlate well with the pharmacologic profile of these drugs (2, 3, 4) . This system has the characteristics of carrier mediated processes(6, 7, 8) , generates transmembrane gradients(6, 9, 10) , and has a higher affinity for MTX and reduced folates (1-5 µM) than for folic acid (100-200 µM)(9, 11, 12, 13) . A cDNA encoding this carrier, or a component of the carrier system, has recently been cloned, and the sequence of the protein suggests that it is a member of a superfamily of membrane-spanning transporters(14, 15) .

The second transport system mediates transport of folates by glycosylphosphatidylinositol-anchored folate receptors (reviewed in (16, 17, 18) ) via an endocytotic process. Several folate receptor isoforms (designated FR-alpha, FR-beta, and FR-) have been cloned from human and murine tissues in this and other laboratories(19, 20, 21, 22, 23, 24, 25) . The receptors have a very high affinity for folic acid (1 nM) and the reduced folates 5-formyl- and 5-methyltetrahydrofolate (10-40 nM) and a lower affinity for MTX and other 4-amino-antifolates (0.15-1.7 µM) (16, 17, 18) . Folate receptors are highly expressed in some malignant tissues, suggesting a role in meeting cellular folate requirements and as determinants of antifolate cytotoxicity(26, 27, 28) . The physiologic and pharmacologic properties of folate receptor-mediated transport have not been characterized with the same degree of quantitation and depth as for the reduced folate carrier.

In this report we characterize, in detail, MTX transport mediated by FR-alpha, the murine homolog of the isoform expressed in KB cells(21) . To exclude any contribution to transport via the reduced folate carrier, an L1210 leukemia line (MTX^rA) was utilized in which the reduced folate carrier is not functional(29) . This line was transfected with a cDNA encoding murine FR-alpha and a clonal subline, MTX^rA-TF1, was isolated that constitutively overexpressed this receptor. MTX was utilized as the transport substrate because upon entering the cell it binds rapidly to DHFR, permitting very accurate influx determinations over long intervals obviating back-flux components that occur with natural folates(6, 7, 9) .

The results of this study indicate that transport mediated by FR-alpha is fundamentally different from transport mediated by the reduced folate carrier in terms of energy, ion, and pH dependence. FR-alpha-mediated transport of MTX into cells was quite rapid; MTX influx in the MTX^rA-TF1 line was equivalent to influx mediated by the reduced folate carrier in L1210 cells when the extracellular concentration was 0.1 µM. No evidence was found to support a linkage between these two transport processes. Inhibition of FR-alpha-mediated transport by agents that are inhibitors of transport via the reduced folate carrier (i.e. BSP and probenecid) could be attributed to inhibition of MTX binding to the folate receptor. The results of this study are consistent with the concept that folate receptor-mediated transport of folates and antifolates can achieve influx rates comparable to those achieved by the reduced folate carrier and can be a pathway of both physiologic and pharmacologic importance.


MATERIALS AND METHODS

Chemicals

[3`,5`,7-^3H]MTX and [3`,5`,7,9-^3H]folic acid were obtained from Moravek Biochemicals and purified by high performance liquid chromatography prior to use(30) . Restriction enzymes were obtained from New England Biolabs. Sulfobromophthalein (BSP) and probenecid were purchased from Sigma. CI-920 was provided by Dr. David Fry, Warner-Lambert/Parke Davis Co., and racemic [3`,5`,7,9-^3H]5-methyl-tetrahydrofolate was provided by Dr. Barton Kamen, University of Texas Southwestern Medical Center. All other reagents were obtained in the highest purity available from various commercial sources.

Cell Culture

Murine L1210 leukemia cells and sublines were grown in RPMI 1640 medium (containing 2.3 µM folic acid) supplemented with 10% bovine calf serum (HyClone), 2 mM glutamine, 20 µM 2-mercaptoethanol, penicillin (100 units/ml), and streptomycin (100 µg/ml). For some experiments, cells were cultured in folate-free RPMI 1640 medium supplemented as above with 10% dialyzed bovine calf serum and 200 µM glycine, 100 µM adenosine, and 10 µM thymidine (GAT) 2-4 weeks prior to use. Prior to other experiments, MTX^rA-TF1 (see below) was grown in folate-free RPMI 1640 medium supplemented with 0.5 nM folic acid for 2-4 weeks.

Transfection of MTX^rA with murine FR-alpha

The MTX-resistant L1210 leukemia cell line, MTX^rA was previously developed (29) from a single-step selection in 50 nM MTX and cloned in soft agar. MTX^rA-TF1 was developed by transfecting MTX^rA with an expression vector, pKJ-FR-alpha, encoding the full-length murine FR-alpha cDNA previously isolated from an L1210 subline up-regulated for folate receptor expression by growth in limiting 5-formyltetrahydrofolate(19) . The expression vector, pKJ-1, contains the gene for neomycin resistance (neo^r) under control of the constitutive murine phosphoglycerate kinase promoter, and was kindly provided by Dr. M. W. McBurney, the University of Ottawa, Canada(31) . To construct the pKJ-FR-alpha expression vector, the neo^r cassette in pKJ-1 was removed by restriction with PstI and replaced with an NsiI fragment containing the FR-alpha coding region. Constructs were screened by restriction analysis and orientation confirmed by DNA sequence analysis. pKJ-FR-alpha was co-transfected with pKJ-1 into L1210 and MTX^rA cells by electroporation and selected on 750 µg/ml G418 for 3 weeks. The cell line MTX^rA-TF1 was isolated by dilution cloning in complete RPMI 1640 medium in the presence of G418.

Northern Analysis

Total RNA was isolated using RNAzol B (Biotecx) and poly(A) RNA isolated using Quickprep (Pharmacia LKB Biotechnology Inc.). Northern hybridizations were performed as described previously(19) .

Probes

To confirm that the FR-alpha transcript identified in the transfected cell line was derived from the pJK-FR-alpha expression vector, Northern blots were probed with an oligonucleotide which spans the pGK promoter-FR-alpha junction. The DHFR specific probe is the full-length murine cDNA for this enzyme(32) . The probe for the reduced folate carrier (RFC1) was generated by reverse transcription polymerase chain reaction utilizing RNA isolated from murine erythroleukemia cells using primers based on the sequence reported by Dixon et al.(14) .

Binding Assays

Specific binding of [^3H]folic acid to FR-alpha was determined with whole cells at 0 °C as described previously (33) with the following minor modification. The assay mixture contained 1-4 times 10^6 acid-washed cells (0 °C HBS at pH 4.5) and [^3H]folic acid in HBS (20 mM HEPES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl(2), 5 mM glucose, pH 7.4). Following incubation for 10 min at 0 °C, the samples were centrifuged (12,000 times g, 0 °C, 2 min), the supernatant aspirated, and residual fluid removed by a second centrifugation and aspiration step. Cell pellets were processed and radioactivity determined as described previously(34) . At each concentration tested, specific binding was determined as the difference between [^3H]folic acid bound in the absence and presence of 100 µM unlabeled folic acid. Specific binding of folic acid to FR-alpha in L1210 and MTX^rA cell lines was not detected in this assay. Using this same assay, separate experiments were performed to determine the effects of specific agents (CI-920, BSP, or probenecid) on the binding of 0.1 µM [^3H]MTX, 0.1 µM [^3H]5-methyltetrahydrofolate, or 50 nM [^3H]folic acid to MTX^rA-TF1. Ligand and competitor were presented to the cells simultaneously. Under these conditions specific binding of MTX to the reduced folate carrier in L1210 and MTX^rA is not detectable.

Transport Studies

Influx measurements were performed by methods described previously(34) . Briefly, cells were harvested and acid washed to remove endogenous folates bound to FR-alpha at the cell surface. Cells were then washed twice with 0 °C HBS, pH 7.4 (assay buffer, see above) and resuspended in this buffer to 1 times 10^7 cells/ml. The cell suspensions were equilibrated at 37 °C for 10 minutes, uptake initiated by the addition of [^3H]MTX, and samples taken over the indicated intervals. Influx was terminated by injecting 1.0 ml of the cell suspension into 10.0 ml 0 °C acid HBS (pH 4.5). Cells were collected by centrifugation, washed twice with 0 °C HBS (pH 7.4), and processed for determination of intracellular tritium as described previously (34) . Influx intervals were adjusted so that [^3H]MTX uptake did not exceed the DHFR binding capacity assuring that unidirectional uptake conditions were sustained. For experiments at pH 6.2, MBS buffer was used (20 mM MES, 140 mM NaCl, 5 mM KCl, 2 mM MgCl(2), and 5 mM glucose)(35) . In other experiments NaCl was removed from HBS and replaced with HEPES or other salts. All buffers were adjusted to 290 ± 10 mosm/liter as assessed on a Osmette A osmometer (Precision Systems, Inc., Natich, MA).

Growth Studies

For growth studies, 0.2-ml cultures (8.0 times 10^4 cells/ml) in 96-well plates were continuously exposed to the appropriate concentrations of MTX, folic acid, or 5-formyltetrahydrofolate for 72 h, following which cell numbers were determined by hemocytometer count and viability determined by trypan blue exclusion. Cells were cultured for at least 10 generations in RPMI 1640 medium prior to the analyses of MTX growth inhibition or for 15 generations in folate-free RPMI medium containing GAT prior to assessment of folic acid or 5-formyltetrahydrofolate growth requirements.


RESULTS

Development, Binding Characteristics, and Growth Requirements of the MTX^rA-TF1 Transfectant

These studies utilized a derivative L1210 leukemia cell line, MTX^rA, which is resistant to MTX due to a loss of mobility of the reduced folate carrier(29) . In this line, the carrier is present at the cell surface in unchanged number with unchanged affinity for the drug, but translocation of MTX into the cell does not occur(29) . In the present study, MTX^rA was transfected with an expression vector containing the L1210-derived cDNA encoding the murine folate receptor FR-alpha(19) . Following selection on G418, clones were isolated, expanded, and analyzed for constitutive high level expression of FR-alpha. Several clones were identified, and one of these, MTX^rA-TF1, was used for all subsequent studies. As shown in Fig. 1, the FR-alpha transcript was highly expressed in this line but was not detected in either the L1210 or MTX^rA cell lines. In contrast, each cell line expressed similar levels of message for DHFR and the reduced folate carrier transcript (RFC1) recently identified(14, 15) . For the MTX^rA line, this observation is consistent with previous biochemical studies that indicate a present but nonfunctional reduced folate carrier(29) .


Figure 1: Northern analyses of L1210, MTX-resistant L1210 (MTX^rA), and MTX^rA transfected with FR-alpha (MTX^rA-TF1). Upper panel, total RNA (10 µg) from L1210 (lane1) and MTX^rA cells grown in complete RPMI 1640 medium (lane2) and from MTX^rA-TF1 cells (TF1) grown in either complete RPMI 1640 medium (lane3) folate-free RPMI 1640 medium containing 0.5 nM folic acid (lane4) or folate-free RPMI 1640 medium containing GAT (lane5) was hybridized with an oligonucleotide probe specific for transfected FR-alpha. The blot was stripped and hybridized with specific probes for DHFR and actin. Lower panel, poly(A)-selected RNA (5 µg) from MTX^rA, L1210, and MTX^rA-TF1 was hybridized with a probe specific for the reduced folate carrier (RFC1).



The MTX^rA-TF1 transfectant expresses a functional membrane FR-alpha at a level of 8.9 ± 0.6 nmol/g dry weight of cells (or 1.3 ± 0.1 pmol/10^6 cells) as assessed by [^3H]folic acid binding at 0 °C. Based on Scatchard analysis (Fig. 2), the K(b) for [^3H]folic acid binding was 1.9 ± 0.5 nM, in agreement with values for endogenous murine and human FR-alpha(33, 36) . Over the concentration range of 10 nM to 1 µM, no specific binding of folic acid to L1210 or MTX^rA could be detected consistent with the lack of expression of FR-alpha in these lines (Fig. 1).


Figure 2: Specific binding of [^3H]folic acid to L1210, MTX^rA-TF1, and MTX^rA cells. Specific binding was measured at the indicated concentrations of [^3H]folic acid at 0 °C. box, L1210; bullet, MTX^rA-TF1; , MTX^rA. Inset, Scatchard analysis of the binding data for MTX^rA-TF1. The graph is representative of three experiments.



Consistent with the high affinity of FR-alpha for folic acid(33, 36) , expression of FR-alpha in MTX^rA-TF1 resulted in a 400-fold lower folic acid growth requirement compared to L1210 or MTX^rA (Fig. 3, upper panel). The very small increase in the folic acid growth requirement observed for MTX^rA, which lacks a functional reduced folate carrier, compared to L1210 cells demonstrates that the reduced folate carrier is not a major uptake route for folic acid in either cell line. Compared to MTX^rA, both the MTX^rA-TF1 and L1210 cell lines exhibited a 35-fold lower growth requirement for 5-formyltetrahydrofolate, a good substrate for both FR-alpha (33, 36) and the reduced folate carrier (6, 9, 11, 30, 37) (Fig. 3, middle panel). Overexpression of FR-alpha at the level achieved in the MTX^rA-TF1 line appears to compensate completely for the loss of the reduced folate carrier in meeting cellular demands for folate, even at concentrations of 5-formyltetrahydrofolate comparable to physiologic 5-methyltetrahydrofolate levels.


Figure 3: Folic acid (upper panel) and 5-formyltetrahydrofolate (middle panel) growth requirements and inhibition of growth by MTX (lower panel). Cells were plated at 8 times 10^4 cells/ml in the presence of the indicated concentrations of folate or MTX and incubated for 72 h as described under ``Materials and Methods.'' box, L1210; bullet, MTX^rA-TF1; , MTX^rA. Error bars are S.E. of three experiments.



When grown in the presence of 2.3 µM folic acid (RPMI 1640 medium), the high level of MTX resistance in the MTX^rA line is maintained in the MTX^rA-TF1 transfectant (Fig. 3, lower panel). An extracellular folic acid concentration of 10 nM (a level which fully supports growth of MTX^rA-TF1; Fig. 3, upper panel) resulted in only a small increase in the sensitivity of the MTX^rA-TF1 transfectant to MTX (data not shown). These observations are consistent with transport studies (see below) demonstrating that low levels of folic acid abolish MTX uptake mediated by FR-alpha.

MTX influx kinetics in L1210, MTX^rA-TF1, and MTX^rA Cells

When grown in RPMI 1640 medium containing 2.3 µM folic acid, influx of [^3H]MTX in the MTX^rA-TF1 transfectant was negligible compared to L1210 cells (data not shown). However, when folate was first stripped from FR-alpha by pretreating cells with acid HBS (pH 4.5) at 0 °C, influx of 0.1 µM [^3H]MTX in the transfected line was restored to a level identical to that of L1210 cells (Fig. 4). Acid washing is not a requirement for restoration of transport since similar results were obtained in MTX^rA-TF1 cells grown in folate-free RPMI 1640 medium supplemented with either 0.5 nM folic acid or GAT. [^3H]MTX influx in L1210 or MTX^rA was not altered by the acid wash or growth in folate-free medium; transport was always present in L1210 cells and always absent in MTX^rA cells. At an extracellular MTX concentration of 0.1 µM, influx in MTX^rA was less than 1/75 that of L1210 or MTX^rA-TF1 cells.


Figure 4: Influx of [^3H]MTX in L1210, MTX^rA-TF1, and MTX^rA cells. Cells were pretreated with acid saline, washed, resuspended in HEPES-buffered saline and, at time zero, exposed to 0.1 µM [^3H]MTX at 37 °C. box, L1210; bullet, MTX^rA-TF1; , MTX^rA cells. Error bars are S.E. of 5 experiments.



The [^3H]MTX associated with MTX^rA-TF1 cells was within the intracellular compartment and not simply bound to FR-alpha on the cell membrane since an HBS acid wash (pH 4.5) following [^3H]MTX uptake removed only a trivial amount of drug (data not shown).

The ratio of [^3H]MTX influx in MTX^rA-TF1 to L1210 cells decreased as the extracellular MTX concentration was increased (Fig. 5), suggesting that transport in MTX^rA-TF1 cells saturates at a lower MTX concentration with a lower influx V(max) than in L1210 cells. This was indeed the case. As shown in Table 1, the measured influx, K(t), for MTX in the MTX^rA-TF1 transfectant (0.21 µM) is 1/20 that of L1210 cells (4.2 µM), and is consistent with the MTX K(b) for FR-alpha (0.14 µM)(33) . The influx V(max) for MTX in the MTX^rA-TF1 line (0.18 nmol/g dry weight/min) is 1/16 that observed for L1210 cells (2.9 nmol/g dry weight/min). The level of expression (MTX B(max)) of FR-alpha in MTX^rA-TF1 (8.9 nmol/gram dry weight) is 6 times that observed for the reduced folate carrier in L1210 cells (1.5 nmol/g dry weight)(29) . Hence the cycling rate for the reduced folate carrier in L1210 cells is 100 times faster than the cycling rate for FR-alpha in MTX^rA-TF1. (^2)In contrast, influx of MTX in the MTX^rA line was consistently less than 2% (1/75) of influx in L1210 cells at extracellular MTX concentrations ranging from 0.1 to 10 µM (Fig. 5). This is consistent with a similar K(t) (3.9 µM) and greater than 90-fold lower V(max) for MTX influx in MTX^rA in comparison to L1210 cells (Table 1).


Figure 5: The relationship between extracellular [^3H]MTX concentration and the ratio of [^3H]MTX influx in MTX^rA-TF1 or MTX^rA in comparison to L1210 cells. Cells were pretreated with acid saline, washed, and resuspended in HEPES-buffered saline and, at time zero, exposed to the indicated concentrations (0.1 µM to 10 µM) of [^3H]MTX at 37 °C. Data points represent the ratio of influx in MTX^rA-TF1 or MTX^rA in relation to influx in L1210 cells. bullet, MTX^rA-TF1; MTX^rA cells. Error bars are S.E. of two to four experiments.





Effects of Folic Acid, BSP, Probenecid, or CI920 on [^3H]MTX Influx

To further characterize the mechanism of MTX transport in the transfected line, influx was measured in the presence of either folic acid or the reduced folate carrier inhibitors BSP, probenecid, or CI-920. A concentration of 0.1 µM MTX was used because of the equivalent influx in L1210 and MTX^rA-TF1 cells at this level of drug, while MTX influx in MTX^rA cells is negligible ( Fig. 4and Fig. 5). Influx of [^3H]MTX in the MTX^rA-TF1 transfectant is markedly inhibited by folic acid. While the addition of 1.0 µM folic acid abolishes [^3H]MTX influx in this line, it has no effect on influx in L1210 cells or the residual influx in MTX^rA cells (Fig. 6, upper panel). When folic acid is added after [^3H]MTX influx is established, net uptake of [^3H]MTX is abolished virtually instantaneously (data not shown). The IC for folic acid inhibition in the MTX^rA-TF1 transfectant is less than 10 nM (Fig. 6, lower panel). In contrast, the IC for folic acid inhibition of [^3H]MTX influx via the reduced folate carrier in L1210 cells is 200 µM. The effect of folic acid on [^3H]MTX influx in MTX^rA-TF1 must be due to inhibition at the level of FR, since it occurs at concentrations 5-10 times greater than the folic acid K(b) for FR-alpha but 1/20,000 the folic acid K(m) for the reduced folate carrier.


Figure 6: The effects of folic acid on [^3H]MTX influx in L1210, MTX^rA-TF1, and MTX^rA cells. Upper panel, cells were pretreated with acid saline, washed, resuspended in HEPES-buffered saline, and, at time zero, exposed to 0.1 µM [^3H]MTX at 37 °C, in the presence (opensymbols) or absence (closedsymbols) of 1.0 µM unlabeled folic acid. , box = L1210; bullet, circle= MTX^rA-TF1. = MTX^rA cells in the presence or absence of 1.0 µM folic acid. Error bars are S.E. of three to five experiments. Lower panel, effects of folic acid on 0.1 µM [^3H]MTX influx. Data are expressed as influx as a percentage of the level in the absence of folic acid. box, L1210; bullet, MTX^rA-TF1. Each point is the mean of two experiments.



While structurally unrelated to folates, the organic anion BSP is a potent inhibitor of the reduced folate carrier and has been used to discriminate between folate uptake mediated by this and other routes (35, 38, 39, 40) . BSP (150 µM) inhibited [^3H]MTX influx in L1210 and MTX^rA-TF1 by 97% and 73%, respectively. Increasing the BSP concentration to 350 µM completely abolished the residual [^3H]MTX flux in the MTX^rA-TF1 line (Table 2). Probenecid is also an inhibitor of MTX transport by the reduced folate carrier(41, 42, 43, 44) . At a concentration of 1 mM, probenecid markedly inhibits [^3H]MTX influx in L1210 cells but has only a small effect on influx in MTX^rA-TF1. However, at a level of 10 mM, probenecid markedly suppresses [^3H]MTX influx in both cell lines (Table 2). Despite structural dissimilarity, the antibiotic CI-920 also competes with reduced folates for the reduced folate carrier(45) . At a level of 150 µM, CI920 markedly suppresses [^3H]MTX influx in L1210 cells while inhibiting influx in the MTX^rA-TF1 line by less than 10% (Table 2). In the presence or absence of folic acid, BSP, probenecid, or CI-920 inhibition of MTX influx in the MTX^rA line was negligible (less than 2% of control rate in L1210 and MTX^rA-TF1; Fig. 4Fig. 5Fig. 6and data not shown).



As shown in Fig. 7, each of the reduced folate carrier inhibitors also inhibit MTX binding to FR-alpha. BSP and probenecid inhibit MTX binding with IC values of 100 µM and 4 mM, respectively. The IC for CI-920 was not quantitated but is somewhat less than probenecid. In general, for all of the agents tested, there is good correlation between inhibition of [^3H]MTX influx and binding to FR-alpha in MTX^rA-TF1 (Table 2). Hence, the effects of these agents on [^3H]MTX influx can be attributed to inhibition of [^3H]MTX binding to FR-alpha. Folic acid is a highly specific inhibitor of FR-alpha, and CI-920 is the most selective inhibitor of the reduced folate carrier. MTX binding to the reduced folate carrier was below the level of detection in this assay.


Figure 7: Effects of BSP, probenecid, or CI-920 on [^3H]MTX binding to FR-alpha. Specific binding of 0.1 µM [^3H]MTX was measured in MTX^rA-TF1 cells in the presence of the indicated concentrations of BSP, probenecid, or CI-920 at 0 °C. circle, BSP; bullet, probenecid; box, CI-920. The graph is representative of three experiments. There is no detectable specific binding of MTX to L1210 or MTX^rA cells under these conditions.



The major circulating folate, 5-methyltetrahydrofolate, has been utilized extensively for the characterization of folate uptake in MA-104 cells(38, 46, 47) . Since 5-methyltetrahydrofolate has a 20-fold higher affinity for FR-alpha then does MTX(33, 36) , it was anticipated that its transport would be less sensitive to organic anions and this was indeed the case. However, inhibition of the binding and influx of [^3H]5-methyltetrahydrofolate by either probenecid or BSP was comparable (Table 2). Folic acid, an even stronger ligand for the FR-alpha (150-fold lower K(b) compared to MTX) is insensitive to the effects of BSP on binding and transport in MTX^rA-TF1. There is no measurable transport of 50 nM folic acid into L1210 cells under these conditions.

Energy, Temperature, pH, and Ion Dependence of [^3H]MTX Influx-MTX^rA-TF1 and L1210 cells were used to compare various physical, chemical, and metabolic perturbations on [^3H]MTX influx mediated by FR-alpha or the reduced folate carrier (Table 3). The energetics of MTX transport in L1210 cells is unusual(8, 10, 48) . An energy-requiring efflux pump results in enhanced net cellular transport of MTX in the presence of metabolic inhibitors. Metabolic poisons either enhance or have no effect on influx(10, 48) . [^3H]MTX influx in L1210 cells was enhanced by 10 mM azide and unchanged by 0.5 mM dinitrophenol (DNP). In contrast, [^3H]MTX influx in the MTX^rA-TF1 line was abolished by this concentration of azide and markedly inhibited by dinitrophenol (Table 3). Hence MTX influx in MTX^rA-TF1 is highly energy-requiring and ceases rapidly when energy metabolism is blocked.



The two cell lines also had a markedly different pH dependence. [^3H]MTX influx in L1210 cells was reduced by 70% when the pH of the transport buffer was decreased from 7.4 to 6.2. [^3H]MTX influx in the MTX^rA-TF1 transfectant was insensitive to this change in pH. Influx of [^3H]MTX in both lines is temperature-sensitive. However, the Q for MTX^rA-TF1 cells (7.1) is twice that of L1210 cells (3.8).

MTX influx via the reduced folate carrier is sodium-independent, but is highly sensitive to the anionic composition of the extracellular compartment and is inhibited by a variety of inorganic and organic anions(9, 49, 50) . Replacement of NaCl isosmotically with HEPES markedly stimulated influx of [^3H]MTX in L1210 cells. However, under identical conditions [^3H]MTX influx in the MTX^rA-TF1 transfectant was reduced by 97% (Table 3). MTX influx was not sensitive to the intra- or extra-cellular Na concentration in L1210 or MTX^rA-TF1 cells. Hence influx was unchanged by substituting Na with Li and neither cell line showed sensitivity to ouabain (Table 3). The residual MTX influx in MTX^rA was not significantly altered by azide, dinitrophenol, ouabain, or the substitution of NaCl with HEPES (data not shown).


DISCUSSION

Stable expression of murine FR-alpha in a cell line in which the reduced folate carrier is present but immobile provided the opportunity to explore, in detail, the influx characteristics of transport mediated by FR-alpha. MTX was used as the probe for these studies because of its unique qualities, which facilitate influx measurements(6, 7, 9) .

The data provide strong evidence that MTX influx in the MTX^rA-TF1 cell line is mediated exclusively by FR-alpha and demonstrates that the folate receptor can function independently of the reduced folate carrier. A similar conclusion was made by Dixon et al.(51) utilizing a MTX-resistant human breast cancer cell line (MTX^r ZR-75-1)(52, 53) .

This study defines several parameters that distinguish between FR-alpha-mediated influx and influx mediated exclusively by the reduced folate carrier. These two transport processes are fundamentally different in many respects. 1) There is a marked difference in energy dependence. Consistent with previous reports(9, 10, 48) , influx of MTX in L1210 cells is impervious to the immediate effects of energy depletion. However, influx of MTX in MTX^rA-TF1 cells is markedly inhibited by metabolic poisons, indicating a direct coupling of FR-alpha-mediated transport to energy metabolism. 2) MTX influx in L1210 cells is stimulated by the removal of chloride anion, a manifestation of the nonspecific heteroexchange between anions and the reduced folate carrier(8, 9, 49, 54) . However, this perturbation resulted in cessation of MTX influx in MTX^rA-TF1 cells. Although inorganic anions may have generalized effects on receptor-mediated endocytosis(55) , these data are consistent with previously reported chloride enhancement of folate binding to rat kidney FR(56) . Initial studies suggest a decrease in the MTX binding affinity for FR-alpha in the absence of chloride (data not shown). 3) MTX influx in L1210 cells is markedly suppressed when the pH is decreased from 7.4 to 6.2. However, over this pH range, there is no change in MTX transport in MTX^rA-TF1 cells. This is consistent with reports that binding of folates to the folate receptor does not diminish until the pH falls below 5.0(57) . The data indicate that a proposed alternative route for MTX transport, optimized at pH 6.2, cannot account for rapid influx in MTX^rA-TF1(35) . 4) While MTX influx in both cell lines is temperature-dependent, the influx Q in MTX^rA-TF1 is 2-fold greater. 5) Finally, MTX influx in MTX^rA-TF1 was 5 orders of magnitude more sensitive to folic acid than was L1210 cells. Folic acid is a poor substrate for the reduced folate carrier (K(t) >200 µM)(6, 9, 11, 12) , but has a very high affinity for FR-alpha(33, 36) . FR-alpha expression in MTX^rA-TF1 allowed for growth in low levels of folic acid and 5-formyltetrahydrofolate even in the absence of a functional reduced folate carrier, consistent with similar reports by other laboratories(51, 58, 59, 60, 61) . Since FR-alpha has a 20-fold higher binding affinity for 5-formyltetrahydrofolate compared to FR-beta(33, 36) , the specific folate receptor isoform expressed in normal and malignant cells may influence the efficacy and therapeutic index of treatment regimens that utilize this folate. MTX resistance is maintained in MTX^rA-TF1 even in the presence of very low levels (10 nM) of folic acid, a consequence of the potent inhibitory effect of folic acid on MTX influx in this cell line. Thus, the dual effects of low folate on folate receptor bearing cells in vitro (maintaining cell growth and at the same time blocking FR-mediated MTX cytotoxicity) indicates that folate receptor-mediated transport could support the growth of a reduced folate carrier-defective tumor cell without increasing that cells sensitivity to MTX.

Transport mediated by FR-alpha was quite rapid; the rate of influx of 0.1 µM MTX in MTX^rA-TF1 cells and L1210 cells was identical. However, as the extracellular MTX level was increased, influx mediated by the reduced folate carrier dominated (Fig. 5), the result of both a 20-fold lower influx K(t) and V(max) in MTX^rA-TF1 as compared to L1210 cells. When the relative expression of each transporter was considered along with the influx V(max), the cycling rate of the reduced folate carrier in L1210 cells (116 molecules MTX/binding site/h) was calculated to be 2 orders of magnitude greater than that of the FR-alpha-mediated process in MTX^rA-TF1 (1.2 molecules of MTX/binding site/h). The cycling rate for FR-alpha in the present study is comparable to rates reported for FR-alpha expressed in L1210 and MA-104 cells (0.8-1.1 molecules/binding site/h) (39, 46) and is more than adequate to meet the modest folate requirement of cells under physiologic conditions (16) . Hence when expressed to a sufficient level, FR-alpha may be a significant transport route for folates at physiologic concentrations (10-50 nM) and for antifolates at low blood levels. However, FR-alpha becomes a very minor contributor (Fig. 5) to transport at higher (pharmacologic) levels of folates and antifolates, when the reduced folate carrier dominates.

Folate receptor isoforms have distinct binding affinities for folates and antifolates(33, 36) . The rate of transport at a given folate or antifolate concentration will therefore depend not only on the amount of receptor present but on the specific isoform expressed. Recent studies have shown that folate receptors are widely expressed in normal and malignant tissues and that the pattern of expression is isoform-specific with striking overexpression in certain carcinomas (26, 27, 28) . Specificity in transport mediated by different folate receptor isoforms may be an important element in the development of tumor-specific chemotherapeutic regimens.

While it is clear that folate transport in MTX^rA-TF1 cells is dependent upon the initial association of drug with folate receptor, the remaining steps in the transport process are not clear but probably involve endocytosis of the receptor-ligand complex(62) . In MA104 cells, a monkey kidney epithelial cell line, FR-alpha, as well as other glycosylphosphatidylinositolanchored proteins, appears to cluster in non-clathrin-coated invaginations or caveolae(46, 47, 63, 64) . However, there is recent evidence that this process may not be limited to caveolae(65) . It has been suggested that following acidification of the sealed-off caveolar vesicle, freed folate ligand enters the cytoplasm via the reduced folate carrier. This proposal was based on the observation that the process is sensitive to probenecid(66) . However, the present study indicates that the effects of probenecid and BSP on MTX and 5-methyltetrahydrofolate influx in MTX^rA-TF1 can be explained on the basis of inhibition of binding to FR-alpha. These agents, therefore, cannot reliably discriminate between carrier- and folate receptor-mediated influx. Hence, probenecid sensitivity does not imply participation of the reduced folate carrier in folate influx in MTX^rA-TF1 cells. These observations, together with the multiple qualitative differences in transport noted above, support the conclusion that the reduced folate carrier and FR-alpha operate independently.


FOOTNOTES

*
This work was supported by National Cancer Institute Grants CA-39807, CA-09340, and CA-09349. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Dept. of Medicine, Medical College of Virginia, Box 980230 MCV Station, Richmond, VA 23298-0230. Fax: 804-828-8079.

(^1)
The abbreviations used are: MTX, methotrexate; BSP, sulfobromophthalein; DHFR, dihydrofolate reductase; FR, folate receptor; K, affinity constant of binding; HBS, Hepes-buffered saline; MES, 4-morpholineethanesulfonic acid; DNP, dinitrophenol.

(^2)
The maximum cycling rate for each transporter is equal to: (V(max)(nmol of MTX transported/g dry weight/min))/(B(max)(nmol MTX binding site/g dry weight)) times 60 = mol transported/mol binding site/h.


ACKNOWLEDGEMENTS

We thank Mary Leftwich, Cathy Leyco, and Hilda Terry for technical assistance and Richard Moran and Eric Westin for helpful discussion in the preparation of this manuscript.


REFERENCES

  1. Fleming, G. F., and Schilsky, R. L. (1992) Semin. Oncol. 19, 707-719 [Medline] [Order article via Infotrieve]
  2. Goldman, I. D., and Matherly, L. H. (1985) Pharmacol. Ther. 28, 77-102 [CrossRef][Medline] [Order article via Infotrieve]
  3. Sirotnak, F. M. (1987) NCI Monographs 5, 27-35 [Medline] [Order article via Infotrieve]
  4. Ratnam, M., and Freisheim, J. H. (1990) Folic Acid Metabolism in Health and Disease , pp. 91-120, Wiley-Liss, New York
  5. Grant, S. C., Kris, M. G., Young, C. W., and Sirotnak, F. M. (1993) Cancer Invest. 11, 36-45 [Medline] [Order article via Infotrieve]
  6. Goldman, I. D., Lichtenstein, N. S., and Oliverio, V. T. (1968) J. Biol. Chem. 243, 5007-5017 [Abstract/Free Full Text]
  7. Goldman, I. D. (1971) Biochim. Biophys. Acta 233, 624-634 [Medline] [Order article via Infotrieve]
  8. Fry, D. W., Cybulski, R. L., and Goldman, I. D. (1980) Biochim. Biophys. Acta 603, 157-170 [Medline] [Order article via Infotrieve]
  9. Goldman, I. D. (1971) Ann. N. Y. Acad. Sci. 186, 400-422 [Medline] [Order article via Infotrieve]
  10. Goldman, I. D. (1969) J. Biol. Chem. 244, 3779-3785 [Medline] [Order article via Infotrieve]
  11. Henderson, G. B., Grzelakowska-Sztabert, B., Zevely, E. M., and Huennekens, F. M. (1980) Arch. Biochem. Biophys. 202, 144-149 [Medline] [Order article via Infotrieve]
  12. Sirotnak, F. M. (1985) Cancer Res. 45, 3992-4000 [Medline] [Order article via Infotrieve]
  13. White, J. C., Bailey, B. D., and Goldman, I. D. (1978) J. Biol. Chem. 253, 242-245 [Abstract]
  14. Dixon, K. H., Lanpher, B. C., Chiu, J., Kelley, K., and Cowan, K. H. (1994) J. Biol. Chem. 269, 17-20 [Abstract/Free Full Text]
  15. Williams, F. M. R., Murray, R. C., Underhill, T. M., and Flintoff, W. F. (1994) J. Biol. Chem. 269, 5810-5816 [Abstract/Free Full Text]
  16. Antony, A. C. (1992) Blood 79, 2807-2820 [Medline] [Order article via Infotrieve]
  17. Kane, M. A., and Waxman, S. (1989) Lab. Invest. 60, 737-746 [Medline] [Order article via Infotrieve]
  18. Henderson, G. B. (1990) Annu. Rev. Nutr. 10, 319-335 [CrossRef][Medline] [Order article via Infotrieve]
  19. Brigle, K. E., Westin, E. H., Houghton, M. T., and Goldman, I. D. (1991) J. Biol. Chem. 266, 17243-17249 [Abstract/Free Full Text]
  20. Lacey, S. W., Sanders, J. M., Rothberg, K. G., Anderson, R. G. W., and Kamen, B. A. (1989) J. Clin. Invest. 84, 715-720 [Medline] [Order article via Infotrieve]
  21. Sadasivan, E., and Rothenberg, S. P. (1989) J. Biol. Chem. 264, 5806-5811 [Abstract/Free Full Text]
  22. Ratnam, M., Marquardt, H., Duhring, J. L., and Freisheim, J. H. (1989) Biochemistry 28, 8249-8254 [Medline] [Order article via Infotrieve]
  23. Elwood, P. C. (1989) J. Biol. Chem. 264, 14893-14901 [Abstract/Free Full Text]
  24. Coney, L. R., Tomassetti, A., Carayannopoulos, L., Frasca, V., Kamen, B. A., Colnaghi, M. I., and Zurawski, V. R., Jr. (1991) Cancer Res. 51, 6125-6132 [Abstract]
  25. Shen, F., Ross, J. F., Wang, X., and Ratnam, M. (1994) Biochemistry 33, 1209-1215 [Medline] [Order article via Infotrieve]
  26. Ross, J. F., Chaudhuri, P. K., and Ratnam, M. (1994) Cancer 73, 2432-2443 [Medline] [Order article via Infotrieve]
  27. Campbell, I. G., Jones, T. A., Foulkes, W. D., and Trowsdale, J. (1991) Cancer Res. 51, 5329-5338 [Abstract]
  28. Weitman, S. D., Lark, R. H., Coney, L. R., Fort, D. W., Frasca, V., Zurawski, V. R., Jr., and Kamen, B. A. (1992) Cancer Res. 52, 3396-3401 [Abstract]
  29. Schuetz, J. D., Westin, E. H., Matherly, L. H., Pincus, R., Swerdlow, P. S., and Goldman, I. D. (1989) J. Biol. Chem. 264, 16261-16267 [Abstract/Free Full Text]
  30. Matherly, L. H., Barlowe, C. K., Phillips, V. M., and Goldman, I. D. (1987) J. Biol. Chem. 262, 710-717 [Abstract/Free Full Text]
  31. McBurney, M. W., Sutherland, L. C., Adra, C. N., Leclair, B., Rudnicki, M. A., and Jardine, K. (1991) Nucleic Acids Res. 19, 5775-5761
  32. Simonsen, C. C., and Levinson, A. D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2495-2499 [Abstract]
  33. Brigle, K. E., Spinella, M. J., Westin, E. H., and Goldman, I. D. (1994) Biochem. Pharmacol. 47, 337-345 [CrossRef][Medline] [Order article via Infotrieve]
  34. Fry, D. W., and Goldman, I. D. (1982) J. Membr. Biol. 66, 87-95 [Medline] [Order article via Infotrieve]
  35. Henderson, G. B., and Strauss, B. P. (1990) Cancer Res. 50, 1709-1714 [Abstract]
  36. Wang, X., Shen, F., Freisheim, J. H., Gentry, L. E., and Ratnam, M. (1992) Biochem. Pharmacol. 44, 1898-1901 [Medline] [Order article via Infotrieve]
  37. Sirotnak, F. M., Chello, P. L., Moccio, D. M., Kisliuk, R. L., Combepine, G., Gaumont, Y., and Montgomery, J. A. (1979) Biochem. Pharmacol. 28, 2993-2997 [Medline] [Order article via Infotrieve]
  38. Gewirtz, D. A., Randolph, J. K., and Goldman, I. D. (1980) Cancer Res. 40, 1852-1857 [Medline] [Order article via Infotrieve]
  39. Henderson, G. B., Tsuji, J. M., and Kumar, H. P. (1990) J. Membr. Biol. 101, 247-258
  40. Sirotnak, F. M., and O'Leary, D. F. (1991) Cancer Res. 51, 1412-1417 [Abstract]
  41. Gewirtz, D. A., Plotkin, J. H., and Randolph, J. K. (1984) Cancer Res. 44, 3846-3850 [Abstract]
  42. Sirotnak, F. M., Moccio, D. M., and Young, C. W. (1981) Cancer Res. 41, 966-970 [Abstract]
  43. Henderson, G. B., and Zevely, E. M. (1985) Biochem. Pharmacol. 34, 1725-1729 [Medline] [Order article via Infotrieve]
  44. Fry, D. W., Yalowich, J. C., and Goldman, I. D. (1982) Cancer Res. 42, 2532-2536 [Abstract]
  45. Fry, D. W., Besserer, J. A., and Boritzki, T. J. (1984) Cancer Res. 44, 3366-3370 [Abstract]
  46. Kamen, B. A., and Capdevila, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5983-5987 [Abstract]
  47. Kamen, B. A., Wang, M.-T., Streckfuss, A. J., Peryea, X., and Anderson, R. G. W. (1988) J. Biol. Chem. 263, 13602-13609 [Abstract/Free Full Text]
  48. Fry, D. W., White, J. C., and Goldman, I. D. (1980) Cancer Res. 40, 3669-3673 [Medline] [Order article via Infotrieve]
  49. Henderson, G. B., and Zevely, E. M. (1983) Arch. Biochem. Biophys. 221, 438-446 [Medline] [Order article via Infotrieve]
  50. Jennette, J. C., and Goldman, I. D. (1975) J. Lab. Clin. Med. 86, 834-843 [Medline] [Order article via Infotrieve]
  51. Dixon, K. H., Mulligan, T., Chung, K.-N., Elwood, P. C., and Cowan, K. H. (1992) J. Biol. Chem. 267, 24140-24147 [Abstract/Free Full Text]
  52. Cowan, K. H., and Jolivet, J. (1984) J. Biol. Chem. 259, 10793-10800 [Abstract/Free Full Text]
  53. Dixon, K. H., Trepel, J. B., Eng, S. C., and Cowan, K. H. (1991) Cancer Commun. 3, 357-365
  54. Yang, C.-H., Sirotnak, F. M., and Dembo, M. (1984) J. Membr. Biol. 79, 285-292 [Medline] [Order article via Infotrieve]
  55. Bowen, B. J., and Morgan, E. H. (1988) J. Cell. Physiol. 134, 1-12 [Medline] [Order article via Infotrieve]
  56. Selhub, J., and Franklin, W. A. (1984) J. Biol. Chem. 259, 6601-6606 [Abstract/Free Full Text]
  57. Kamen, B. A., and Caston, J. D. (1986) Biochem. Pharmacol. 35, 2323-2329 [Medline] [Order article via Infotrieve]
  58. Brigle, K. E., Seither, R. L., Westin, E. H., and Goldman, I. D. (1994) J. Biol. Chem. 269, 4267-4272 [Abstract/Free Full Text]
  59. Jansen, G., Westerhof, G. R., Kathmann, I., Rademaker, B. C., Rijksen, G., and Schornagel, J. H. (1989) Cancer Res. 49, 2455-2459 [Abstract]
  60. Jansen, G., Schornagel, J. H., Westerhof, G. R., Rijksen, G., Newell, D. R., and Jackman, A. L. (1990) Cancer Res. 50, 7544-7548 [Abstract]
  61. Van der Veer, L. J., Westerhof, G. R., Rijksen, G., Schornagel, J. H., and Jansen, G. (1989) Leuk. Res. 13, 981-987 [Medline] [Order article via Infotrieve]
  62. Goldstein, J. L., Brown, M. S., Anderson, R. G. W., Russell, D. W., and Schneider, W. J. (1985) Annu. Rev. Cell Biol. 1, 1-39 [CrossRef]
  63. Rothberg, K. G., Ying, Y.-S., Kamen, B. A., and Anderson, R. G. W. (1990) J. Cell Biol. 111, 2931-2938 [Abstract]
  64. Anderson, R. G. W., Kamen, B. A., Rothberg, K. G., and Lacey, S. W. (1992) Science 255, 410-411 [Medline] [Order article via Infotrieve]
  65. Mayor, S., Rothberg, K. G., and Maxfield, F. R. (1994) Science 264, 1948-1951 [Medline] [Order article via Infotrieve]
  66. Kamen, B. A., Smith, A. K., and Anderson, R. G. W. (1991) J. Clin. Invest. 87, 1442-1449 [Medline] [Order article via Infotrieve]

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