Characterization of a Folate Transporter in Hela Cells with a Low Ph Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier

6256 Vol. 10, 6256–6264, September 15, 2004 Clinical Cancer Research

Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier

Yanhua Wang, Rongbao Zhao, and acid and PT523, agents for which they have very different I. David Goldman structural specificities. Departments of Medicine and Molecular Pharmacology and Cancer Research Center, Albert Einstein College of Medicine, Bronx, New York INTRODUCTION The major route for the membrane transport of folates and antifolates in mammalian cells is the reduced folate carrier ABSTRACT (RFC) characterized largely in human and murine leukemia cell Studies were undertaken to characterize a low pH lines (1). RFC has a neutral pH optimum in these cells; however, transport activity in a reduced folate carrier (RFC)-null there have been reports of process(es) with low pH optima that HeLa-derived cell line (R5). This transport activity has a transport folates in solid tumors and cell lines of normal tissue ϳ 20-fold higher affinity for pemetrexed (PMX; Kt, 45 origin (2–8). Recently, studies from this laboratory demon- nmol/L) than methotrexate (MTX; K , ϳ1 ␮mol/L) with t strated the ubiquitous nature of low pH folate transport activity comparable V values. The K values for folic acid, max i in a large number of human solid tumor cell lines from a variety ZD9331, and ZD1694 were ϳ 400–600 nmol/L, and the K i of tissue origins (9). In the majority of these cell lines, transport values for PT523, PT632, and trimetrexate were >50 at low pH was equal to or exceeded transport at physiologic pH. ␮mol/L. The transporter is stereospecific and has a 7-fold In only one cell line, the HepG2 hepatoma, was the low pH higher affinity for the 6S isomer than the 6R isomer of 5-formyltetrahydrofolate but a 4-fold higher affinity for the activity negligible. The low pH activity in HeLa cells is greater 6R isomer than the 6S isomer of dideazatetrahydrofolic acid. than transport at physiologic pH [(methotrexate (MTX) influx at ϫ Properties of RFC-independent transport were compared pH 5.5 is 2 greater than that at pH 7.4]. In a derivative HeLa with transport mediated by RFC at low pH using HepG2 cell line, R5, obtained under MTX selective pressure, RFC was cells, with minimal constitutive low pH transport activity, deleted from the genome, but transport at low pH was not reduced (9). Hence, in R5 cells, the low pH transporter is transfected to high levels of RFC. MTX influx Kt was comparable at pH 7.4 and pH 5.5 (1.7 versus 3.8 ␮mol/L), but entirely independent of RFC. This low pH transport activity in R5 cells was shown to contribute to the inhibitory activity of Vmax was decreased 4.5-fold. There was no difference in the ϳ ␮ ϳ MTX when cells were grown at a low pH (9). Kt for PMX ( 1.2 mol/L) or the Ki for folic acid ( 130 ␮mol/L) or PT523 (ϳ 0.2 ␮mol/L) at pH 7.4 and pH 5.5. The mechanism(s) underlying transport activities with low MTX influx in R5 and HepG2 transfectants at pH 5.5 was pH optima have not yet been clarified, although in intestinal trans-stimulated in cells loaded with 5-formyltetrahydrofo- cells there is some evidence to suggest that this may be related ,late, inhibited by Cl؊ (HepG2-B > R5), Na؉ independent, to RFC expression (10–14). The availability of the R5 cell line and uninhibited by energy depletion. Hence, RFC-independ- in which transport at low pH is entirely RFC independent, ent low pH transport activity in HeLa R5 cells is consistent provided an opportunity to characterize this process (one focus with a carrier-mediated process with high affinity for PMX. of this report). An additional issue addressed is the extent to Potential alterations in protonation of RFC or the folate which RFC-mediated transport may be altered at low pH due to molecule as a function of pH do not result in changes in changes in the protonation of the transporter or the folate/ affinity constants for antifolates. Whereas both activities at antifolate substrate molecule. These changes could obfuscate the low pH have similarities, they can be distinguished by folic properties of a RFC-independent low pH activity in cells in which both transport processes are expressed. To clarify this issue, HepG2 cells with low constitutive low pH activity (9) were transfected with RFC to achieve very high levels of carrier Received 4/5/04; accepted 4/22/04. expression so that RFC-mediated transport activity at low pH Grant support: National Institutes of Health grant CA-82621 and a was sufficient to allow its characterization. These properties grant from the Eli Lilly Company. were then compared with RFC-independent transport at low pH The costs of publication of this article were defrayed in part by the in HeLa R5 cells. Finally, these studies use and compare the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to transport properties of two antifolates: MTX, a classical inhib- indicate this fact. itor of dihydrofolate reductase; and pemetrexed (PMX), a novel Requests for reprints: I. David Goldman, Albert Einstein College of new-generation antifolate, which, in its polyglutamyl forms, is a Medicine Cancer Center, Chanin 2, 1300 Morris Park Avenue, Bronx, NY potent inhibitor of thymidylate synthase with lesser inhibitory 10461. Phone: 718-430-2302; Fax: 718-430-8550; E-mail: igoldman@ aecom.yu.edu. potential at the level of glycinamide ribonucleotide formyltrans- ©2004 American Association for Cancer Research. ferase (15).

MATERIALS AND METHODS added, and cells were equilibrated at this temperature for 20 Chemicals. Radiolabeled reagents included [3Ј,5Ј7- minutes. The buffer was then aspirated, and uptake was initiated 3H]MTX from Amersham (Piscataway, NJ) and [3H]PMX along by the addition of 0.5 mL of fresh buffer containing the radio- with nonlabeled PMX, 6R- and 6S-dideazatetrahydrofolic acid active antifolate and other reagents. For determination of trans- (DDATHF), and LY309887 (6R-2Ј,5Ј-thienyl-5,10-dideazatet- stimulation, cells were incubated with 100 ␮mol/L racemic rahydrofolatic acid) provided by the Eli Lilly Co (Indianapolis, 5-CHO-THF in the appropriate buffer at 37°C for 25 minutes. IN). All tritiated reagents were purified before use and main- The cells were then washed twice with ice-cold drug-free MBS tained at high purity by high-performance liquid chromatogra- buffer at pH 5.5, and then MBS at 37°C and pH 5.5 containing phy (16). Nonlabeled folic acid was obtained from Sigma (St. radioactive antifolate was added. Uptake was terminated by the Louis, MO). ZD1694 and ZD9331 were provided by AstraZen- addition of 5 mL of ice-cold HBS, and then cells were washed eca (Wilmington, DE). Trimetrexate was provided by Dr. David three times and lysed by the addition of 0.5 mL of 0.2N NaOH. Fry (Pfizer, New York, NY), aminopterin was provided by Dr. Radioactivity was assessed on 0.4 mL of cell lysate to which Barton Kamen (New Jersey Cancer Institute, New Brunswich, fluor was added. Protein concentration was determined using the NJ), and PT523 and PT632 were provided by Dr. Andre BCA Protein Assay kit (Pierce, Rockford, IL). Cell antifolate is Rosowsky (Dana-Farber Cancer Institute, Boston, MA). Nonla- expressed as nanomoles per gram of protein. beled MTX was provided by the National Cancer Institute Influx kinetics for [3H]MTX and [3H]PMX were deter- (Bethesda, MD). The stereoisomers of 5-formyltetrahydrofolate mined with by a time-course of uptake over 2 minutes, an

(5-CHO-THF) were obtained from Schircks Laboratories (Jona, interval over which initial uptake rates were sustained. Ki values Switzerland). All other reagents were obtained in the highest for nonlabeled folates and antifolates were determined based on purity available from commercial sources. inhibition of 0.5 ␮mol/L [3H]MTX influx with concentrations of Cell Culture. Cells were maintained in RPMI 1640 con- these agents adjusted to produce ϳ50% inhibition of MTX taining 2.3 ␮mol/L folic acid, supplemented with 10% fetal calf transport. serum (Hyclone, Logan, UT), 2 mmol/L glutamine, 20 ␮mol/L Transfections. The HepG2-B cell line was obtained by 2-mercaptoethanol, 100 units/mL penicillin, and 100 ␮g/mL transfection of HepG2 cells with human RFC cDNA that in- streptomycin. The R5 cell line, with a genomic RFC deletion, cluded a hemagglutinin epitope at the COOH terminus (a gift was maintained in the absence of MTX (9). A HepG2 cell line from Dr. Larry Matherly; ref. 20). Transfection was carried out expressing high levels of RFC (HepG2-B) was obtained by using LipofectAMINE Plus (Invitrogen, Carlsbad, CA) accord- transfection of human RFC cDNA, as described below, and ing to the manufacturer’s protocol after HepG2 cells were maintained in 600 ␮g/mL G418. These cells were regrown from grown in 6-well plates to 50% to 75% confluence. After 2 days frozen stock every 1–2 months due to the progressive loss of of growth, the cells were trypsinized, diluted 10-fold, and then RFC expression under growth conditions. Mycoplasma adherent incubated in RPMI 1640 containing G418 (600 ␮g/mL). After 2 to cells produce transport activity with very high affinity for weeks, surviving clones were picked up and replated, and RFC PMX and other folates (17, 18); this is especially prominent in activity was assessed by determination of [3H]MTX influx. mesothelioma cells, but a low level of activity can also be HepG2-B cells are the RFC-transfected clone. HepG2-D cells detected in HeLa and HepG2 cells contaminated with this or- are the vector-transfected control clone. ganism.1 Because of this, cell cultures were monitored regularly RNA Isolation and Northern Blots. Total RNA was with a Mycoplasma detection kit (American Type Culture Col- isolated from cells with Trizol reagent (Invitrogen) according to lection, Manassas, VA) and were shown to be free of this the manufacturer’s protocol. Total RNA (25 ␮g) was resolved microorganism. by electrophoresis on 1.2% agarose gels containing formalde- Transport Studies. Cells were grown in monolayer cul- hyde. RNA was transferred to Nytran N-membranes (Schleicher ture adherent to the bottom of glass scintillation vials, in which & Schuell, Keene, NH) and fixed with a Stratalinker UV-cross transport measurements were made as described previously linker (Stratagene, La Jolla, CA). The blot was first probed with (19). Experiments at pH 5.0 to pH 6.5 were performed in MBS human RFC cDNA and then stripped and reprobed with ␤-actin (20 mmol/L 4-morpholinepropanesulfonic acid, 140 mmol/L cDNA as the loading control.

NaCl, 5 mmol/L KCl, 2 mmol/L MgCl2, and 5 mmol/L glucose) and at pH 7.0 to pH 8.0 in HBS (20 mmol/L HEPES, 140 RESULTS mmol/L NaCl, 5 mmol/L KCl, 2 mmol/L MgCl2, and 5 mmol/L glucose). NaCl-free sucrose buffer was used at pH 7.4 (20 Influx Kinetics and Structural Specificity of the Low mmol/L HBS) or pH 5.5 (20 mmol/L MBS) with 225 mmol/L pH Transporter in Reduced Folate Carrier-Null HeLa R5 ϩ Cells. Influx of MTX at pH 5.5 in RFC-null R5 cells is sucrose, and pH was adjusted with Mg(OH)2.Na -free MBS choline chloride buffer (20 mmol/L 4-morpholinepropanesulfo- mediated by a RFC-independent process. Initial studies demon- 3 3 nic acid,140 mmol/L choline chloride, 5 mmol/L KCl, 2 mmol/L strated that initial uptake rates for [ H]MTX and [ H]PMX were sustained for at least 2 minutes. This was the interval used for MgCl2, and 5 mmol/L glucose) was used at pH 5.5. Before uptake determinations, cells were washed in ice- the determination of influx kinetics. As indicated in the top Ϯ cold transport buffer, and then 1 mL of the buffer at 37°C was panel of Fig. 1, MTX influx was saturable with a Kt of 1.03 ␮ Ϯ 0.11 mol/L and a Vmax of 1.37 0.16 nmol/g protein/min. PMX had an ϳ20-fold higher affinity for this process, with an Ϯ Ϯ influx Kt of 44.9 5.0 nmol/L but a comparable Vmax of 1.09 1 Unpublished observations. 0.14 nmol/g protein/min (Fig. 1, bottom panel). The Kt for MTX

with and only ϳ2-fold higher than the direct measurement of the 3 [ H]PMX influx Kt described above. Influx Kinetics and Structural Specificity in HepG2 Cells Transfected with Reduced Folate Carrier. HepG2 cells, with very low constitutive low pH activity and only modest RFC activity at physiologic pH (9), were transfected with human RFC to achieve high-level carrier expression. The Northern analysis on one transfectant, the HepG2-B clone, is indicated in the top panel of Fig. 3 and demonstrates the high level of RFC message. RFC mRNA in wild-type HepG2 cells was very low, and detection required a long interval of exposure to the X-ray film. Consistent with the high level of carrier expression was the marked increase in initial uptake rates for MTX in the HepG2-B transfectant (Fig. 3, middle panel), which exceeded that of the vector-transfected control cells by a factor of ϳ65 at pH 7.4 (7.82 Ϯ 0.91 versus 0.12 Ϯ 0.05 nmol/g

Fig. 1 MTX (top panel) and PMX (bottom panel) influx kinetics at pH 5.5 in R5 cells that lack RFC activity. Influx was monitored over 2 minutes, an interval during which initial uptake rates were sustained. The data are the average of three independent experiments Ϯ SE.

Fig. 2 The inhibitory effects of folic acid or PT523 on MTX influx in influx mediated by RFC in wild-type HeLa cells at pH 7.4 was R5 cells at pH 5.5. The concentration of [3H]MTX was 0.5 ␮mol/L. The ␮ ϳ2 ␮mol/L (data not shown). concentration of PT523 and folic acid was 20 mol/L. The data are the average of three independent experiments Ϯ SE. Influx of 0.5 ␮mol/L [3H]MTX in R5 cells at pH 5.5 was markedly suppressed by 20 ␮mol/L folic acid but unaffected by 20 ␮mol/L PT523 (Fig. 2). At these concentration ratios, folic acid does not inhibit MTX influx mediated by RFC at pH 7.4, Table 1 The inhibitory effects of folates and antifolates on influx of ͓3 ͔ and PT523 markedly suppresses MTX influx mediated by RFC H MTX into R5 cells at pH 5.5

at pH 7.4 (see Fig. 5). Hence, these agents are good discrimi- Substrate Ki (nmol/L) nators of RFC-mediated transport at neutral pH versus RFC- Pemetrexed 91.0 Ϯ 10.0 independent transport at low pH (see next section). 6S-5-CHO-THF 131.8 Ϯ 7.5 Ϯ Table 1 indicates the inhibition constants for a variety of 6R-5-CHO-THF 873 32 6R-DDATHF 112.9 Ϯ 9.1 folates and antifolates in R5 cells determined at pH 5.5, based 6S-DDATHF 550 Ϯ 120 3 on their inhibition of [ H]MTX influx. The highest affinities LY309887 288 Ϯ 58 were observed for PMX, the natural isomer (6S) of 5-CHO- ZD9331 437 Ϯ 82 THF, and the 6R isomer of DDATHF. Transport was stereospe- ZD1694 531 Ϯ 84 Ϯ cific, with a K of 132 versus 873 nmol/L for the 6S versus 6R Folic acid 622 111 i Aminopterin 1,307 Ϯ 212 isomer of 5-CHO-THF, respectively, and a Ki of 113 versus 550 Edatrexate 1,685 Ϯ 383 nmol/L for the 6R versus 6S isomer of DDATHF, respectively. TMQ Ͼ50,000 The K for LY309887 was 285 nmol/L. This transporter had PT523 Ͼ50,000 i Ͼ somewhat lower affinities for ZD9331, ZD1694, and folic acid PT632 50,000

(Ki values of 437, 531, and 622 nmol/L, respectively). Affinities NOTE. Inhibition constants were determined based on the degree of suppression of ͓3H͔MTX influx (extracellular level of 0.5 ␮mol/L) for aminopterin and edatrexate were lower, with Ki values of ϳ ␮ produced by a concentration of folate/antifolate that produced inhibition 1.3 and 1.7 mol/L). The affinities for PT523, PT532, and in the range of 50% and assuming Michaelis-Menten kinetics. The data Ͼ ␮ TMR were very low ( 50 mol/L). The Ki for PMX, based on are the average of three independent experiments Ϯ SE. 3 inhibition of [ H]MTX influx in these studies, was comparable Abbreviations: 5-CH3-THF, 5-methyltetrahydrofolate.

when the pH was decreased to 5.5 (Fig. 4, bottom panel). Hence, the affinity of RFC for MTX was essentially constant over this

pH range. On the other hand, the Vmax decreased by a factor of 4.5 (59.1 Ϯ 7.2 to 12.8 Ϯ 1.6 nmol/g protein/min) with this fall

in pH. The influx Ki for folic acid at pH 7.4 and pH 5.5 was 142 ␮ and 125 mol/L, respectively, and the influx Ki for PT523 at pH 7.4 and pH 5.5 was 0.24 and 0.19 ␮mol/L, respectively (the average of two experiments). Consistent with these inhibition constants, as indicated in the top panel of Fig. 5, influx of 0.5 ␮mol/L [3H]MTX was essentially unaffected by 20 ␮mol/L folic acid at either pH 7.4 (7.16 Ϯ 0.06 versus 7.08 Ϯ 0.27 nmol/g protein/min) or pH 5.5 (0.86 Ϯ 0.15 versus 0.80 Ϯ 0.13 nmol/g protein/min) but was markedly suppressed (Fig. 5, bottom panel) by equimolar PT523 at pH 5.5 (2.15 Ϯ 0.67 versus 0.18 Ϯ 0.09 nmol/g protein/min) and pH 7.4 (8.85 Ϯ 0.91 versus 0.46 Ϯ 0.13 nmol/g protein/min). There was a small ordinate intercept at pH 5.5 in this series of experiments that was depressed by folic acid without a change in initial uptake rate (Fig. 5, top panel). This likely reflects a very low level of MTX

bound to the cell surface. The Ki for PMX was 1.10 and 1.28 ␮mol/L, respectively, at pH 7.4 and pH 5.5 (the average of two experiments). Trans-Stimulation. One important characteristic of carrier-mediated processes is trans-stimulation: augmentation of

Fig. 3 Properties of MTX transport in HepG2 cells transfected to high levels of RFC. Top panel, Northern blot of RFC expression in vector- transfected control HEPG2 cells (HepG2-D) and a clone transfected with human RFC cDNA (HepG2-B). Middle panel,[3H]MTX (0.5 ␮mol/L) influx in vector-transfected control cells (HepG2-D) and RFC- transfected cells (HepG2-B) at pH 7.4 and pH 5.5. Bottom panel,apH profile of [3H]MTX influx in HepG2-B and vector-transfected control HepG2-D cells. The data are the average of three independent experiments Ϯ SE.

protein/min) and by a factor of ϳ8 at pH 5.5 (1.51 Ϯ 0.05 versus 0.18 Ϯ 0.02 nmol/g protein/min). The bottom panel of Fig. 3 is the pH profile for MTX influx in wild-type and HepG2-B cells over a pH range of 5.0 to 8.0. It can be seen that although the pH optimum is ϳ7.4 to 8.0 in HepG2-B cells, there is still a prominent RFC-mediated transport component at pH 5.5 in the transfected cells that allowed for measurement of RFC-mediated transport characteristics at this pH. Influx kinetics for MTX in HepG2-B cells mediated by Fig. 4 MTX influx kinetics in HepG2-B cells at pH 7.4 (top panel) and RFC at pH 7.4 is indicated in the top panel of Fig. 4. The Kt was 3 Ϯ ␮ pH 5.5 (bottom panel). [ H]MTX influx was determined with multiple 1.70 0.21 mol/L, a level comparable with RFC-mediated measurements over a 2-minute interval, during which initial uptake rates transport 2 ␮mol/L at pH 7.4 in HeLa cells (data not shown). were sustained. The data are the average of three independent experi- Ϯ ␮ Ϯ The influx Kt was only slightly increased to 3.77 0.12 mol/L ments SE.

HepG2-B cells at pH 5.5 was much more sensitive to the presence of chloride in the buffer (Fig. 7, bottom panel). When sodium chloride was removed, influx of MTX increased by a factor of 7.5 (from 1.09 Ϯ 0.11 to 8.18 Ϯ 1.20 nmol/g protein/ min). In neither case was influx altered at all by the substitution of sodium with choline, consistent with the lack of sodium dependence by either process, confirming that the influx stimulation in the absence of sodium chloride is related solely to the absence of chloride. Energy Dependence. Influx mediated by RFC at physiologic pH is energy independent; the process is unchanged or increased under conditions of energy deprivation (28). As indicated in the top panel of Fig. 8, there was no change in influx in R5 cells in the absence of glucose with or without the addition of 10 mmol/L azide at pH 5.5. In HepG2-B cells at pH 5.5 (Fig. 8, bottom panel) and at pH 7.4 (data not shown), influx was stimulated by a factor of 1.50 Ϯ 0.30 in the presence of azide in glucose-free buffer, but it was unchanged in the absence of glucose.

Fig. 5 The inhibitory effects of PT523 and folic acid on MTX influx mediated by RFC in HepG2-B cells at pH 7.4 and pH 5.5. The concentration of folic acid (top panel) and PT523 (bottom panel) was 20 ␮mol/L; the concentration of [3H]MTX was 0.5 ␮mol/L. The data are the average of three independent experiments Ϯ SE. In the bottom panel, Œ and ‚ indicate pH 7.4 and 5.5, respectively.

the unidirectional flux of a substrate into cells by the opposite flux of the same or another substrate out of cells via the same carrier (21, 22). This has been demonstrated at physiologic pH for RFC-mediated transport (23). This was also the case for MTX influx in RFC-null R5 cells loaded with 5-CHO-THF at pH 5.5 (from 0.87 Ϯ 0.15 to 1.19 Ϯ 0.17 nmol/g protein/min, a 45% increase; Fig. 6, top panel). Comparable trans-stimulation (45%) was also demonstrated in the HepG2-B transfectant loaded with 5-CHO-THF not only at pH 7.4 (data not shown) but also at pH 5.5 (a 43% increase from 0.71 Ϯ 0.10 to 1.03 Ϯ 0.25 nmol/g protein/min; Fig. 6, bottom panel). This is strong evidence that the RFC-independent, low pH transport activity in R5 cells is a carrier-mediated process and that trans-stimulation mediated by RFC remains intact at low pH. Anion Sensitivity. RFC is an anion exchanger and is inhibited at physiologic pH by a variety of structurally unrelated inorganic and organic anions (24–26). Likewise, when chloride Fig. 6 Trans-Stimulation of MTX influx in R5 and HepG2-B cells at is removed from the transport buffer, influx mediated by RFC is pH 5.5. Cells were incubated with 100 ␮mol/L 5-CHO-THF for 25 enhanced (24, 25, 27). As indicated in the top panel of Fig. 7, minutes. The cells were then washed with ice-cold MBS buffer at pH 5.5, buffer was removed, and uptake of 0.5 ␮mol/L [3H]MTX was influx mediated by the low pH transporter in R5 cells was anion initiated with the addition of radiotracer at 37°C at pH 5.5. Top panel, sensitive. When sodium chloride was removed, influx was stim- R5 cells. Bottom panel, HepG2-B cells. The data are the average of three ulated by a factor of 2.7. Influx of MTX mediated by RFC in independent experiments Ϯ SE.

ϳ ␮ MTX of 1 mol/L at pH 5.5, a Kt that is only slightly less than that reported for RFC at physiologic pH in these and other human cell lines (1). However, whereas RFC has a 2-fold higher affinity for PMX (30, 31), the low pH transporter has an affinity for PMX (ϳ45 nmol/L) that is ϳ20-fold greater than that for MTX at this pH. This transporter has affinities for the natural (6S) isomer of 5-CHO-THF and the 6R isomer of the glycinamide ribonucleotide formyltransferase inhibitor DDATHF comparable with that of PMX. Affinities for ZD1694 and ϳ ZD9331 were somewhat lower (Ki, 500 nmol/L) and slightly less than their affinities for RFC. On the other hand, the affinity ϳ of folic acid for the low pH route at pH 5.5 in R5 cells (Ki, 620 nmol/L) was 2 orders of magnitude greater than the affinity of folic acid for RFC. The transporter is stereospecific, with a 6-fold higher affinity for the natural isomer of 5-CHO-THF as compared with the 6R form and a 5-fold higher affinity for the 6R isomer as compared with the 6S isomer of DDATHF. The low pH transporter has a very low affinity for PT523, PT632, and trimetrexate agents that either lack or have a modified glutamate moiety, although the affinity for ZD9331 is orders of

Fig. 7 The impact of inorganic ions on influx of MTX in R5 and HepG2-B cells at pH 5.5. In Naϩ-free buffer, choline chloride was substituted for NaϩClϪ. In NaCl-free buffer, sucrose was substituted for both ions. Top panel, R5 cells; bottom panel, HepG2-B cells. The extracellular [3H]MTX concentration was 0.5 ␮mol/L. The data are the average of three independent experiments Ϯ SE.

DISCUSSION RFC is highly expressed in normal human tissues and tumors, and this transporter in human and murine leukemia cells has a neutral pH optimum (1). However, recent studies from this laboratory indicate that in human solid tumors, in general, there is very prominent transport activity at low pH that is usually equal to or often exceeds transport mediated by RFC at physiologic pH (9). The mechanism(s) underlying this low pH transport activity has not been clarified, although there is some evidence suggesting that in cells of intestinal origin this transport activity is related, at least in part and in some way, to RFC (11–14). This study addresses the mechanism of folate transport at low pH using a HeLa cell line with prominent low pH activity in which RFC was deleted from the genome and therefore cannot contribute at all, qualitatively or quantitatively, to the transport characteristics observed. The current studies indicate that the low pH transporter in R5 cells has the properties of a Fig. 8 The impact of energy metabolism on influx of MTX in R5 or carrier-mediated process similar to what has been observed for HepG2-B cells at pH 5.5. For depletion of glucose, cells were incubated members of the superfamily of facilitative carriers (29). This in the absence of this substrate for 20 minutes. Azide was used at a transporter shares many of the characteristics of RFC, but there concentration of 10 mmol/L; cells were pretreated with this agent 15 minutes before transport determinations. Top panel, R5 cells; bottom are some important distinguishing features beyond the differ- panel, HepG2-B cells. The extracellular [3H] MTX concentration was ence in pH optima. 0.5 ␮mol/L. The data are the average of three independent experi- The low pH transport carrier in R5 cells has an affinity for ments Ϯ SE.

magnitude greater. The high affinity of PT523 for RFC at low this organism. Second, there are features of the structural spec-

and high pH and its low affinity for the low pH transporter, ificities that are quite different. For instance, the Ki values for along with the high affinity of folic acid for the low pH trans- MTX, ZD1694, and ZD9331 observed in Mycoplasma-contam- porter and its low affinity for RFC, make these agents good inated mesothelioma cells of ϳ100 ␮mol/L are more than 3 discriminators of these processes. Hence, in cells in which a orders of magnitude higher than those observed at low pH in R5 high level of RFC activity is present along with the low pH cells (17). transport activity, the absence of inhibition by PT523 but potent In a previous study (9), residual MTX transport activity at inhibition by folic acid at low pH would be consistent with a physiologic pH was noted in R5 cells that contributed to the RFC-independent transport process as described in these studies. preservation of MTX activity despite the complete loss of RFC. The observation that influx of MTX is trans-stimulated at This RFC-independent transport activity at pH 7.4 also had a ϳ ␮ low pH in R5 cells that have been loaded with 5-CHO-THF is relatively higher affinity for PMX (Ki 12 mol/L) than for ϳ ␮ the most definitive evidence that this RFC-independent trans- MTX, ZD1694, and folic acid (Ki 90–100 mol/L) and a very ϳ ␮ porter in R5 cells is a carrier-mediated process. These studies low affinity (Ki 250 mol/L) for PT523 (37). R5 cells were, in also demonstrate using the RFC-transfected HepG2-B model fact, collaterally sensitive to PMX when grown in medium in that the trans-stimulation phenomenon for RFC is present at which 5-CHO-THF was the folate growth source. This was both physiologic and low pH. The data indicate that the low pH attributed to the partial preservation of transport along with the transport system in R5 cells is anion sensitive, albeit to a lesser marked contraction of folate cofactor pools under these condi- extent than that observed for RFC. This suggests that in addition tions (9, 37, 38). The mechanism underlying this transport to a proton symport that appears to be a basis for the uphill activity at neutral pH was not clarified, but it is possible that this transport of folates into cells and membrane vesicles of intesti- may represent residual activity of the low pH route within the nal origin at low pH, an anion-exchange component may also physiologic range with pH-dependent alterations in binding play a role in transport mediated by the low pH transporter (10, constants for folates and antifolates. This is currently under 32). For instance, the hydroxyl gradient that occurs across the further study. cell membrane under acidic extracellular conditions would also These studies with HepG2 cells represent the first analysis favor uphill transport into cells. For RFC, uphill transport ap- of RFC-mediated transport properties at low and neutral pH in pears to be related to an exchange with intracellular organic the same cell line in general and in a human solid tumor cell line phosphates that are asymmetrically present at high concentra- in particular. Due to the prevalence and prominence of low pH tions within cells. This type of exchange was recently confirmed folate transport activity in human solid tumors, these studies by the demonstration that both thiamine pyrophosphate and were possible because of the unique lack of this activity in monophosphate are good substrates for RFC, and when cells are HepG2 cells. The data indicate clearly that the absolute and loaded with MTX, efflux of thiamine anions is inhibited, result- relative affinities of RFC for its substrates are minimally af- ing in their enhanced intracellular accumulation (33, 34). Also, fected by pH with very similar properties over a 2-order differ- as observed for RFC (28), the unidirection flux of folates into ence in proton concentration. The reduction in pH did, however, cells via the low pH transporter was not impaired by energy produce a substantial decrease in the maximum transport veloc- depletion. The many similar features between the low pH trans- ity that may be due to an alteration in mobility of the carrier porter in R5 cells and transport mediated by RFC suggest that alone or complexed to folate substrates. Hence, the unique these carriers may share structural as well as functional simi- characteristics of transport at low pH, when the low pH carrier larities and raise the possibility that the low pH route may and RFC activities are present in the same cell, cannot be represent another member of the SLC19 family of membrane attributed to pH-dependent alterations in the protonation of RFC

carriers. However, beyond RFC (SLC19A1), the other two or the folate molecule. This is consistent with the pKa values of members of this family (SLC19A2 and SLC19A3) are thiamine the glutamate carboxyl and amino groups of the folate mole- carriers that do not transport folates (35, 36), and a genome- cules that would not result is an appreciable change in dissoci- wide search does not suggest that there are other members of ation and charge over the range of pH values evaluated in this this transport family. study (39). These observations also appear to exclude alterations It is of interest that the high affinity of the low pH trans- in protonation of RFC or folate substrates as a basis for transport porter in R5 cells for PMX (ϳ45 nmol/L) is only somewhat less activity at low pH in intestine or cell lines of intestinal origin, than what was observed for PMX transport in human mesothe- which has been attributed to RFC (11, 13). lioma cell lines (ϳ30 nmol/L; ref. 17). Subsequent studies have PMX is a new-generation antifolate that, in its polyglu- ␮ clarified that this transport activity in mesothelioma was related tamyl forms, is a potent (Ki, 1.3 mol/L) inhibitor of thymidy- to the presence of Mycoplasma adherent to these cells and likely late synthase with lesser inhibitory activity (Ki, 65 nmol/L) at represents transport into this organism, although the possibility the level of glycinamide ribonucleotide formyltransferase (15). that Mycoplasma might have induced the expression of a novel The drug has activity in mesothelioma and was recently ap- transporter in these human cells was not excluded (18). Whereas proved by the Food and Drug Administration for the treatment Mycoplasma contamination of HeLa cells and HepG2 cells can of this disease in the United States; it is also active in the produce this high-affinity PMX transport, this is of much lower treatment of patients with a variety of other solid tumors (40, magnitude than that observed in mesothelioma cells.1 However, 41).If the low pH transport activity prevalent in other human the low pH activity reported here is unrelated to this process. solid tumor cell lines has a similar high affinity for PMX as First, cells are regularly assessed for Mycoplasma with a highly determined in HeLa cells, this could have important pharmaco- sensitive polymerase chain reaction-based assay and are free of logical consequences in view of the low pH present in the

interstitium of solid tumors (42–44). This advantage for PMX 17. Wang Y, Zhao R, Chattopadhyay S, Goldman ID. A novel folate would be most important long after infusion of the drug (2–3 transport activity in human mesothelioma cell lines with high affinity days), when blood levels fall within the range of the affinity and specificity for the new-generation antifolate, pemetrexed. Cancer Res 2002;62:6434–7. constant of the low pH transporter, and transport via this mech- 18. Wang Y, Zhao R, Chattopadhyay S, Goldman ID. Correction. anism would tend to sustain uptake and continued formation of Cancer Res 2003;63:7004. active polyglutamyl derivatives to maintain suppression of PMX 19. Sharif KA, Goldman ID. Rapid determination of membrane trans- target enzymes. port parameters in adherent cells. BioTechniques 2000;28:926–8, 930–2. 20. Wong SC, Zhang L, Proefke SA, Matherly LH. Effects of the loss ACKNOWLEDGMENTS of capacity for N-glycosylation on the transport activity and cellular We thank Marie Hanscom for expert technical assistance. localization of the human reduced folate carrier. Biochim Biophys Acta 1998;1375:6–12. 21. Bowen D, Diasio RB, Goldman ID. Distinguishing between REFERENCES membrane transport and intracellular metabolism of fluorodeoxyuri- 1. Matherly LH, Goldman DI. Membrane transport of folates. Vitam dine in Ehrlich ascites tumor cells by application of kinetic and high Horm 2003;66:403–56. performance liquid chromatographic techniques. J Biol Chem 1979; 2. Sierra EE, Brigle KE, Spinella MJ, Goldman ID. pH dependence of 254:5333–9. methotrexate transport by the reduced folate carrier and the folate 22. Lowe AG, Walmsley AR. The kinetics of glucose transport in receptor in L1210 leukemia cells. Further evidence for a third route human red blood cells. Biochim Biophys Acta 1986;857:146–54. mediated at low pH. 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Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2004 American Association for Cancer Research. Characterization of a Folate Transporter in HeLa Cells with a Low pH Optimum and High Affinity for Pemetrexed Distinct from the Reduced Folate Carrier

Yanhua Wang, Rongbao Zhao and I. David Goldman

Clin Cancer Res 2004;10:6256-6264.

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