(CANCER RESEARCH 51, 5507-5513, October 15. 1991]

Membrane Transport of Natural Folates and Antifolate Compounds in MurineLI 210 Leukemia Cells: Role of Carrier- and Receptor-mediatedTransport Systems1

G. Robbin Westerhof,2 Gerrit Jansen, Nancy van Emmerik, letje Kathmann, Gert Rijksen, Ann L. Jackman,

and Jan H. SchornagelDepartment of Oncology, [C. R. W., G. J., N. v. £.,/. K.], Free University Hospital, P.O. Box 7057, 1007MB, Amsterdam, Department of Internal Medicine, NetherlandsCancer Institute [J. H. S.J, Amsterdam, and Laboratory of Medical Enzymology, Department of Haematology, University Hospital Utrecht, Utrecht [G. R.], TheNetherlands; and Drug Development Section, Institute of Cancer Research, Sutton, Surrey, United Kingdom [A. L. J.]

ABSTRACT

L1210-B73 cells, variants of 1.1210 cells grown in medium containingnanomolar concentrations of folates, express a membrane associatedfolate binding protein (mFBP) in addition to the classical reduced folate/methotrexate carrier (RF/MTX-carrier) present in L1210 cells grown instandard high folate medium (G. Jansen ft al., Cancer Res., 49: 1959-1963, 1989). In this study we used L1210-B73 and 1.121(1cells as amodel system to study the affinity of the RF/MTX-carrier and the mFBPfor the natural folate compounds folie acid and 5-formyltetrahydrofolate(5-CHO-THF), as well as a number of antifolate compounds. Furthermore we studied the contribution of the RF/MTX-carrier and the mFBPin membrane transport of these (anti)folates, and finally we analyzed therole of the mFBP and RF/MTX-carrier in the cytotoxic effects of theantifolates. The antifolates used were either inhibitors of dihydrofolatereducíase,including methotrexate (MTX) and 10-elhyl-lO-deazaaminop-terine (10-EdAM), or two folate-based inhibitors of thymidylate synthase,/V°-propargyl-5,8-dideazafolic acid (CB3717) and 2-deamino-2-methyl-/V°-propargyl-5,8-dideazafolic acid (ICI-198,583).

The affinity of the RF/MTX-carrier for natural and antifolate compounds declined in the order 10-EdAM > ICI-198,583 > 5-CHO-THF> MTX » CB3717 » folie acid. The mFBP exhibited a high bindingaffinity for CB3717 and ICI-198,583 but a poor binding affinity for MTXand 10-EdAM. Binding affinities of the mFBP decreased in the orderCB3717 > folie acid = ICI-198,583 > 5-CHO-THF » MTX = 10-

EdAM.Over 24 h, at 25 nivi, |3H]folic acid uptake in L1210-B73 cells was

found to proceed for more than 98% via the mFBP. Uptake of |'I I|-5-

CHO-THF, at 50 IIMextracellular concentration, occurred via both themFBP (81%) and the RF/MTX-carrier (19%). With respect to antifolates, the mFBP in L1210-B73 cells contributed for less than 30% in theuptake of |'l 1|M 1\ but was the predominant route (92%) in the uptakeof[3H|ICI-198,583.

Results from affinity and membrane transport observations were consistent with growth inhibition studies on L1210-B73 cells demonstratingthat the mFBP played only a minor role in the cytotoxic effects of MTXor 10-EdAM. On the other hand, L1210-B73 cells were significantlymore sensitive to CB3717 (220-fold) and ICI-198,583 (10-fold) thanparental 1,121(1cells. The putative role of the mFBP in the uptake ofCB3717 and ICI-198,583 in L1210-B73 cells was further supported bythe fact that protection from growth inhibition could be achieved by folieacid rather than by 5-CHO-THF. In 1.1210 cells, expressing only theRF/MTX carrier, no protection with folie acid was observed.

These results suggest that multiple transport systems may play a rolein the uptake of natural folates and nonclassical folate analogues withtargets other than dihydrofolate reducíase.The possible clinical relevanceof these observations will be further discussed.

Received 11/6/90; accepted 8/8/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This study was supported by the Dutch Cancer Society (Grant IKA 89-34).

The authors are very grateful to ICI Pharmaceuticals, Alderly Park, Cheshire,United Kingdom, for their generous funding of the synthesis of the [3H]-ICI-

198,583.2To whom requests for reprints should be addressed.

INTRODUCTION

A high affinity/low capacity carrier system is one of theproteins involved in the membrane transport of natural reducedfolate compounds and folate analogues like MTX3 (1-4). ThisRF-carrier system has been widely characterized in a variety ofnormal and especially neoplastic cells (2, 4-10). Recent studies

have suggested that another class of proteins, mFBP, can mediate the uptake folate compounds as well (reviewed in Refs. 4,11, and 12). This was supported by in vitro observations that:(a) folie acid and 5-CHO-THF could promote growth of cellsexpressing mFBP, which is consistent with the high affinity ofthe mFBP for these compounds (13, 14); (b) folate analoguesfor which the mFBP has a high affinity were potent growthinhibitors of these cells (Refs. 15 and 16; present study); (c)following incubation of cells expressing mFBP with natural andantifolate compounds, these compounds could be identifiedintracci Iuhir as polyglutamate forms (Refs. 16-18; presentstudy), which is the result of a cytoplasmic process (19); andfinally (</)anti-mFBP antibodies and chemically activated estersof folie acid/MTX inhibited the uptake of reduced folate compounds in cells expressing mFBP (20, 21).

Recently we have demonstrated the expression of mFBP ina variant of murine L1210 leukemia cells (L1210-B73) whichwere adapted to grow in folate-conditioned medium containingnanomolar levels of folie acid (22). In addition to mFBP,L1210-B73 cells also expressed the same level of the RF-carrier

system, which is present in parental LI210 cells. ParentalLI 210 cells, grown in standard medium containing 2 ¿IMfolieacid, did not express detectable levels of mFBP.

In the present study we have used L1210-B73 cells as a modelsystem to establish the role of the mFBP and/or the RF/MTX-carrier in membrane transport of natural folates and antifolatecompounds on the basis of (a) affinities of the mFBP and theRF/MTX-carrier for these compounds, (b) uptake studies withradiolabeled (anti)folates, and (c) cytotoxicity studies. The results indicate that membrane transport in L1210-B73 cells oftwo folate-based inhibitors of dihydrofolate reducíase, MTXand 10-EdAM, mainly proceeds via the RF/MTX-carrier sincethe mFBP has a poor affinity for these compounds. On theother hand, the mFBP can be an important roule in ihe uplakeof folie acid and 5-CHO-THF as well as of Iwo folate-basedinhibilors of thymidylale synlhase; /V'°-propargyl-5,8-dideaza-

3The abbreviations used are: MTX, methotrexate; CB 3717, iV'°-propargyl-5,8-dideazafolic acid; ICI-198,583; 2-deamino-2-methyl-A"°-propargyl-5.8-didea-zafolic acid; 10-EdAM; 10-ethyl-lO-deazaaminopterin; TMQ, trimetrexate; 5-CHO-THF, 5-formyltetrahydrofolate; 5-CHj-THF, 5-methyltetrahydrofolate;HBSS, HEPES buffered saline solution; HEPES, 4-(hydroxyethyl)-l-piperazine-ethanesulfonic acid; mFBP, membrane-associated folate binding protein; RF/MTX-carrier, reduced folate/methotrexate carrier; TS, thymidylate synthase;DHFR, dihydrofolate reducíase;IC50, the concentration of inhibitor necessary toinhibit [3H]folic acid binding by 50%; DDATHF, 5,10-dideaza-5,6,7,8-tetrahy-

drofolic acid.

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(ANTI)FOLATE TRANSPORT IN LI210 CELLS

folie acid (CB3717) and ICI-198,583 (23-27). The role of themFBP in the uptake of the thymidylate synthase inhibitorscorrelated with the high affinity of the mFBP for thesecompounds.

MATERIALS AND METHODS

Materials. RPM1 1640, with and without folie acid, fetal calf serum(dialyzed and nondialyzed), and dialyzed horse serum were obtainedfrom Gibco, Grand Island, NY. Folie acid and DL-5-CHO-THF werepurchased from Sigma Chemical Co., St. Louis, MO. Digitonin, dinon-ylphthalate, and dibutylphthalate were purchased from Merck, Darmstadt, Germany. MTX was a gift from Pharmachemie, Haarlem, TheNetherlands. 10-EdAM was a gift from Ciba Geigy, Basel, Switzerland.;V'0-Propargyl-5,8-dideazafolic acid (CB3717) and ICI-198,583 wereprovided by ICI-Pharmaceuticals Division, Alderly Park, Macclesfield,Cheshire, United Kingdom. TMQ was obtained from Warner Lambert,Park Davis, Ann Arbor, MI. [3H]FoIic acid (35 Ci/mmol), [3H]MTX(20 Ci/mmol), and L-[3H]-5-CHO-THF (50 Ci/mmol) were purchased

from Moravek Biochemicals, Brea, CA. Radiolabels were repurified asdescribed previously (10, 14, 22). [3H]ICI-198,583 (25.6 Ci/mmol) was

prepared as the diethyl ester in a custom synthesis by Amersham,Amersham, United Kingdom. The compound was hydrolyzed and thenpurified by high performance liquid chromatography as described before(16).

Cell Cultures. Murine leukemic L1210 cells, expressing the RF/MTX-carrier, were grown in RPMI 1640 containing 2 ^M folie acid,supplemented with 5% fetal calf serum, 2 mM L-glutamine, 100 units/ml of both penicillin and streptomycin, and 50 ¿¿M2-mercaptoethanol.L1210-B73 cells, expressing both the RF/MTX-carrier and mFBP,were grown in folate free RPMI 1640 as described previously (22),supplemented with 10% dialyzed fetal calf serum, glutamine, antibiotics, and 2-mercaptoethanol as described above. A 1 nM concentrationof either folie acid or 5-CHO-THF was added as the sole folate source.Cells were kept at 37°Cin a humidified atmosphere containing 5%

C02.Growth Inhibition Assay. LI210 and L1210-B73 cells in the logarith

mic phase of cell growth were plated at an initial density of 7.5 x IO4/ml into the individual wells of a 24-well tissue culture plate. Themedium for both cell lines was folate free RPMI 1640 supplementedwith 10% dialyzed fetal calf serum and either 1 nM 5-CHO-THF, 20nM 5-CHO-THF, or 20 nM folie acid as the sole folate source. Appropriate concentrations of drug were added at the time of plating. After72 h of drug exposure, cell concentration was determined with a SysmexCC-110 microcell counter and cell viability was determined microscopically by trypan blue exclusion.

DHFR Activity Measurement. Specific activity of DHFR was determined according to the method described by Mini et al. (28).

Binding Studies. Binding of radiolabeled (anti)folates was performedin HBSS buffer (107 mM NaCl, 20 mM HEPES, 26.2 m NaHCO3, 5.3mM KC1, 1.9 mM CaCl2,1.0 mM MgCl2, and 7.0 mM D-glucose adjustedto pH 7.4 with NaOH) at 4°C,as described previously (22). In short,L1210-B73 cells (1 x 10'/15 ml HBSS) were incubated with [3H]folicacid, [3H]-5-CHO-THF, [3H]ICI-198,583 or [3H]MTX (all with a spe

cific activity of 0.5 Ci/mmol) in increasing concentrations. After 10min, the cells were collected by centrifugation (5 min, 800 x g) at 4°C.

The supernatant was removed by suction and residual fluid using cottontissues. Pellets were resuspended in water and analyzed for radioactivityin Optifluor scintillation fluid (United Technologies, Packard, Brussels,Belgium) using an Isocap/300 (Searle, Nuclear Chicago) with a counting efficiency for 3H of 51%. Nonspecific binding of label (usually <2%)

was determined by measuring bound radioactivity in the presence of a1000-fold molar excess of unlabeled folie acid. The relative affinity ofmFBP for natural folate and folate analogues was analyzed by a slightlydifferent procedure. L1210-B73 cells (3 x IO6in 1 ml HBSS, pH 7.4)were incubated for 10 min at 4°Cwith 100 pmol [3H]folic acid in the

absence or presence of increasing concentrations of natural folate andantifolate compounds. Cells were then centrifuged and the radioactivityin the cell pellet was determined as described above. Relative affinities

are defined as the inverse molar ratio of compound required to displace50% of [3H]folic acid from mFBP.

Transport Measurements. L1210 and L1210-B73 cells (5 x IO6) in

the logarithmic phase of growth were suspended in 10 ml folate freeRPMI supplemented with 10% dialyzed fetal calf serum and transferredinto 25-cm2 tissue culture flasks. [3H]MTX, [3H]folic acid, [3H]-5-CHO-THF, or [3H]ICI-198,583 (all with a specific activity of 0.5 Ci/mmol)

were added to final concentrations of 100, 25, 50, and 25 nM, respectively. Control experiments contained the same amounts of 3H-labeled

(anti)folate in the presence of 1 ¿JMfolie acid. Incubations were performed at 37°Cin a humidified atmosphere containing 5% CO2. Cells

were harvested after 1, 4, and 24 h and checked for cell concentrationand viability as described above. After centrifugation (800 x g, 5 min,4°C),the supernatant was removed by suction and the residual fluid

was removed with cotton tissues. To distinguish between surface boundand intracellular 3H-labeled (anti)folate in L1210-B73 cells, the cellswere rinsed with 1 ml ice-cold acidic saline buffer (137 mM NaCl, 20mM HEPES, 5.3 mM KC1, 1.0 mM MgCl2, 1.9 mM CaCl2, and 7.0 mMD-glucose, adjusted to pH 3.5 with acetic acid) to remove surface bound

ligand from the mFBP. After centrifugation for 1 min at 13,000 x gthe total cell associated radioactivity was divided in the supernatantfraction (containing cell surface bound ligand) and the pellet fraction(containing intracellular ligand). As a control experiment to establishwhether radioactivity found in the acid resistant fraction is still associated with the membrane or transferred to the cytoplasm, a rapidseparation of cytosol and paniculate fractions was performed by controlled digitonin induced lysis as described by Rijksen a al. (29). Inshort, the procedure is as follows. In an Eppendorf microfuge tube thefollowing gradient was established: lower layer, 100 u\ HBSS, pH 7.4,containing 10% (v/v) glycerol; middle layer, 500 n\ of a 3:2 (v/v) mixtureof dibutylphthalate and dinonylphthalate; and upper layer, 500 n\ cellsuspension containing l O7L1210-B73 cells. The upper layer was mixed

with 50 n\ 2 mM digitonin for 30 s, followed by centrifugation in anEppendorf centrifuge (45 s, 13,000 x g). The lower layer (containingthe paniculate fraction) and the upper layer (containing the cytosolicfraction, as checked by the release of the cytosolic marker enzymelactate dehydrogenase) were collected and analyzed for radioactivity(see "Binding Studies"). From L1210-B73 cells that were incubated for24 h with 3H-labeled (anti)folates at concentrations indicated above,

more than 85% of the radioactivity in the acid resistant fraction wasrecovered in the cytosolic fraction (results not shown).

Analysis of Polyglutamates. L1210-B73 cells were incubated for 24h with concentrations of [3H]MTX, [3H]folic acid, [3H]-5-CHO-THF,or [3H]ICI-198,583 as indicated above. Cells were harvested by centrifugation and washed once with ice-cold HBSS, pH 7.4, followed by anacidic wash at pH 3.5 (see above) to remove surface bound ligand. Thefinal pellet was resuspended in 50 HIMsodium phosphate buffer, pH5.5, and boiled for 5 min. After centrifugation for 1 min at 13,000 x g,the clear supernatant was applied to a DE52 minicolumn to separatemonoglutamate from polyglutamate forms as described by McGuire etal. (30). Monoglutamates were eluted by washing the column with 35ml, 10 mM Tris-HCl, 125 HIMNaCl, and 2.5 mM dithiothreitol, pH7.5. Polyglutamates were eluted with 3 ml, 0.1 N HC1. The conversionof [3H]folic acid to folylpolyglutamate forms could not be analyzed bythe DE52 anion-exchange method since the elution of [3H]folic acid

from this column started at a NaCl concentration (250 mM) at whichfolylpolyglutamates were eluted as well (not shown).

RESULTS

Affinities of RF/MTX-Carrier and mFBP for (Anti)folateCompounds. The relative affinity of the RF/MTX-carrier for a

series of natural and antifolate compounds was studied byinhibition of [3H]MTX and [3H]-5-CHO-THF transport inLI210 cells (Table 1). The RF/MTX-carrier has a high affinityfor 5-CHO-THF, 10-EdAM, MTX, and ICI-198,583, a lowaffinity for CB3717, and a poor affinity for folie acid. Theaffinities of the mFBP in L1210-B73 cells for these compounds

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(ANTI)FOLATE TRANSPORT IN L1210 CELLS

Table 1 Inhibition off3HJ-5-CHO-THFandf'HJMTX transport via the RF/MTX-carrier and displacement off'HJ-folic add binding to the mFBP by folate

derivatives

1C»(MM)RF/MTX-carrier-iLniO)Inhibitor10-EdAM

ICI- 198,5835-CHO-THFMTXCB3717Folie acid[3H)MTX(5

MM)2.1

3.55.7

10.3114890l'H]-5-CHO-THF(2

MM)1.3

2.93.89.0

80S40mFBP*(LI210-B73;

[3H]Folicacid, 0.1»AI)3.33

(0.03)0.145 (0.69)0.53 (0.19)3.33 (0.03)0.063(1.6)0.10 (1.0)

"L1210 cells (30 X lO'/ml in HBSS, pH 7.4) were incubated at 37'C witheither 5 MM[3H]MTX or 2 MM[3H]-5-CHO-THF in the absence or presence of

increasing concentrations of the indicated inhibitors. After 3 min uptake wasterminated by the addition of ice-cold HBSS, pH 7.4. Cells were washed with ice-cold HBSS buffer, resuspended in 0.5 ml water, and analyzed for radioactivity.Actual influx rates under these conditions were 5.7 pmol/min/107 cells for [3H|MTX and 3.6 pmol/min/107 cells for [3H]-5-CHO-THF. Results are the mean of

two experiments done in duplicate and expressed as concentration of inhibitorrequired to inhibit influx of controls by 50% (1C»).

1 L1210-B73 cells (3 x 10' in 1 ml HBSS, pH 7.4) were incubated for 10 minat 4T with 100 pmol [3H]folic acid in the absence or presence of increasing

concentrations of inhibitors. Relative affinities (given in parentheses) are definedas the inverse molar ratio of compound required to displace 50% of (3H)folic acid

from the mFBP. Relative affinity of the mFBP for folie acid is set to 1. Resultsare the means of at least 3 separate experiments (SD < 15%).

were considerably different. Table 1 shows that the affinity ofthe mFBP for the TS inhibitors CB3717 and ICI-198,583 is inthe same range as for folie acid. The relative affinity for 5-CHO-THF is about 5-fold lower than for folie acid, whereasthe affinity for MTX and 10-EdAM is more than 30-fold lower.

Consistent with these results were the cell surface bindingstudies of [3H]folic acid, [3H]ICI-198,583, [3H]-5-CHO-THFand [3H]MTX to L1210-B73 cells. Concentrations at whichhalf-maximal binding was observed were 0.9, 2.0, 4.2, and 30-35 DM,respectively, and the total cell surface binding capacitywas approximately 100 pmol/107 cells (results not shown).

Membrane Transport of (Anti)folates in L1210/L1210-B73Cells. Uptake over a 24-h time period of the antifolates[3H]-MTX and [3H]ICI-198,583 and the natural folates [3H]folie acid and [3H]-5-CHO-THF by L1210 and L1210-B73 cells

is shown in Fig. 1. To discriminate between internalization viathe RF/MTX-carrier or the mFBP in L1210-B73 cells, experiments were carried out in either the absence or presence of 1¿IMfolie acid. Since at this concentration the mFBP is fullysaturated and folie acid is poorly transported by the RF/MTX-carrier (Table 1), uptake of 'H-labeled compounds under this

condition represents the accumulation via the latter transportsystem. The concentrations of 3H]folic acid (25 nivi), [3H]-5-CHO-THF (50 nM), and [3H]ICI-198,583 (25 nM) were chosenas being approximately 10-fold above the concentration forhalf-maximal binding to the mFBP. At the same time these

concentrations were well below the K„for transport via theRF/MTX-carrier. To compromise for these conditions[3H]-MTX was used at a concentration of 100 nM.

Fig. 1 shows that the uptake of [3H]folic acid by L1210-B73

cells is significantly higher than by LI210 cells. Furthermore,in the presence of 1 ^M unlabeled folie acid, uptake of [3H]folic

acid was reduced by more than 95%, which suggests that folieacid is internalized predominantly via the mFBP in L1210-B73cells. Both the mFBP and the RF/MTX-carrier in L1210-B73cells seem to be functional in the uptake of [3H]-5-CHO-THF.The mFBP and RF/MTX-carrier contributed for approximately 80 and 20%, respectively, in the uptake of [3H]-5-CHO-THF by L1210-B73 cells over a 24-h period of incubation.

Uptake of [3H]MTX was only slightly influenced by the pres

ence of l /¿Mfolie acid, which suggests that the RF/MTX-carrier rather than the mFBP plays a major role in the uptakeof [3H]MTX. In contrast, the main route for uptake of [3H]ICI-198,583 in L1210-B73 cells seems to be the mFBP. AlthoughLI210 cells and L1210-B73 cells accumulated significantamounts of [3H]ICI-198,583 in the presence of folie acid, indi

cating uptake via the RF/MTX carrier, more than 85% of[3H]ICI-198,583 uptake in L1210-B73 cells occurred via the

mFBP.After 24 h incubation of L1210-B73 cells with 25 nM [3H]

ICI-198,583, cells were analyzed for the presence of polygluta-mate forms of the internalized drug. Separation of [3H]ICI-

198,583 monoglutamates from polyglutamate forms showedthat 67% of total intracellular [3H]ICI-198,583 had been con

verted into polyglutamate forms. Similarly, after incubatingL1210-B73 cells for 24 h with 50 nM [3H]-5-CHO-THF more

than 85% was present as folylpolyglutamate forms.Growth Inhibition Studies. Table 2 shows the growth inhibi

tory effects of two folate based inhibitors of DHFR (MTX, 10-EdAM) and two folate based inhibitors of TS (CB3717, ICI-198,583) on LI210 and L1210-B73 cells. In order to establishto what extent the RF/MTX-carrier or the mFBP in LI 210-B73 plays a role in membrane transport of these compounds,growth inhibition studies were performed in the presence of 20nM folie acid or 20 nM 5-CHO-THF. In case of transport via

pmol /IO7ceils

100-[3H]-MTX

50-

25-

[3H]-ICI 198,583

JLJ-n100-

50_

25-

[3H]-Folie acid [3H]-5-CHO-THF

i 4 24 4 24

hours

Fig. 1. Uptake of (3H]MTX, [3H]ICI-198,583. ['HJfolic acid, and [3H]-5-CHO-THF by LI210 cells (D) and L12IO-B73 cells over a 1-, 4-, and 24-h time period.Extracellular concentrations of (anti)folate compounds were 100, 25, 25, and 50nM, respectively. Uptake by L1210-B73 cells was determined either in the absence( ) or presence (8) of 1 (/\i folie acid to discriminate between internalizationvia the RF/MTX-carrier or mFBP. Further details are described in "Materialsand Methods." Results are the mean of at least 3 separate experiments (SD <

21%).

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Table 2 Growth inhibition of LI 210 and LI2IO-B73 cells by MTX, 10-EdAM,TMQ. CB37I7 and ICI-198,583 in the absence or presence of 20 nMfolie acid or

5-CHO-THF as putative protective agents'

InhibitorMTX10-EdAMTMQCB3717ICI-198,583Addition1

nM 5-CHO-THF20 nM 5-CHO-THF20 nM folieacid1

nM 5-CHO-THF20 nM 5-CHO-THF20 nM folieacid1

nM 5-CHO-THF20 nM 5-CHO-THF20 nM folieacid1

nM 5-CHO-THF20 nM 5-CHO-THF20 nM folieacid1

nM 5-CHO-THF20 nM 5-CHO-THF20 nM folie acidICML12102.2

±1.04.9 ±1.42.4 ±1.10.98

±0.192.6 ±1.4

0.99 ±0.122.9

±0.919.2± 10.8

1.7±0.6395

±32790 ±60300 ±659.1

±5.023.0+5.2

6.6 ±2.3(nM)*L1210-B733.4

±1.115.2 + 8.610.3±3.62.9

±0.611.3 ±3.14.9 ±2.08.9

±3.8131 ±25

15.6±7.92.8

±0.44.0 ±0.4

28.7 ±4.50.92

±0.425.9 ±1.4

56.8 ±16.9" Growth inhibition was determined after 72 h continuous exposure to the

drugs.* K .¡iis defined as the drug concentration resulting in 50% inhibition of cell

growth compared to controls. Results are the mean of at least 3 separateexperiments ±SD.

the mFBP, high affinity binding of folie acid could provideprotection from growth inhibition via this route. Likewise,addition of 5-CHO-THF, for which the RF/MTX-carrier has a

high affinity, could result in protection from growth inhibitionat the level of this carrier. Table 2 also shows the growthinhibitory effects of TMQ, an inhibitor of DHFR which, unlikeMTX or 10-EdAM, enters the cell via a process distinct from

the RF/MTX carrier (31).L1210-B73 cells, grown at 1 nM 5-CHO-THF, were found

to be less sensitive (1.5- to 3-fold) to MTX, 10-EdAM, and

TMQ compared to parental LI210 cells. This difference mightbe partially explained by a 2.7-fold higher activity of DHFR inL1210-B73 cells compared to L1210 cells (specific activity,

0.32 versus 0.12 /umol dihydrofolate/h/mg protein, respectively). The addition of 20 nM 5-CHO-THF to L1210-B73 cellsresulted in a 4.5-fold protection from growth inhibition byMTX and 3.9-fold protection from growth inhibition by 10-

EdAM. To a lower extent 20 nM folie acid also provided apartial protection against growth inhibition of L1210-B73 cellsby MTX and 10-EdAM. 5-CHO-THF (20 nM), but not folkacid, gave a significant protection (15-fold) against TMQ cy-totoxicity in L1210-B73 cells. This protection factor was 2-fold

higher than for parental LI210 cells. Table 2 demonstrates thatL1210-B73 cells were highly sensitive to CB3717 compared to

parental cells (IC5o, 2.8 versus 395 nM, respectively). In addition,significant protection from growth inhibition of L1210-B73cells by CB3717 was observed by folie acid (10-fold) rather thanby 5-CHO-THF (1.6-fold). Although L1210 cells were foundto be sensitive for ICI-198,583 (IC50 9.1 HM), L1210-B73 cellswere even 10-fold more sensitive for ICI-198,583 (IC50 0.92nM). Similarly as for CB3717, protection from ICI-198,583induced growth inhibition of L1210-B73 cells was more effective with folie acid (65-fold) than with 5-CHO-THF (6.4-fold).

No protection from growth inhibition induced by the listedantifolates is observed when LI210 cells were plated in thepresence of 20 nM folie acid.

DISCUSSION

Although mFBPs have been identified in various normal andneoplastic cells and tissues (4, 11, 12, 14, 32-47), their physiological and/or pharmacological role in vivo is less well established and is a subject of ongoing discussion: (a) it is evidentthat in cells or tissues obtained under physiological conditions,the cellular expression of mFBP may not be as high as has beenobserved in established cell lines maintained in vitro in folateconditioned medium (13, 14, 20, 22, 32, 33, 44); (b) whenmFBPs are present on the cell surface they will likely besaturated with 5-CH3-THF (the predominant circulating reduced folate compound) for which the mFBP has a high bindingaffinity (4, 11, 12, 17). This may hamper the identification ofmFBPs via binding studies with radiolabeled folate compounds;(c) since the mFBP has a high binding affinity for naturalreduced folates and is therefore able to internalize these compounds very efficiently (17, 18, 48), cells may not necessarilyhave to express high levels of mFBP to meet their folaterequirements for cell growth; (d) it requires sensitive affinityprobes to detect very low cellular levels of mFBP (34, 49). Forthese reasons, the number of studies regarding the identificationand functioning of mFBPs in (non)malignant cellular materialobtained in vivo or maintained in vitro in folate conditioned cellculture medium has been limited at this time.

One of the possible physiological roles of the mFBP may bein the renal retention of folates. There is evidence that mFBPon the brush border membrane of kidney proximal tubules playsa decisive role in maintaining the folate status in mammals (50,51). The mFBP has also been reported to mediate the cellularfolate accumulation in human hematopoietic progenitor cells,a process which was found to be directly related to cell proliferation (35, 36).

It has not been established whether mFBPs, as compared tothe RF-carrier, represent a pharmacologically important routefor uptake of antifolate drugs. In the present study we were ableto address this question by using L1210-B73 cells as a modelsystem. L1210-B73, a subline of murine L1210 leukemia cellsadapted to grow at nanomolar concentrations of 5-CHO-THF,expresses the RF-carrier as well as mFBP within one cell (22).The affinities of both proteins for natural folate compounds,folate based inhibitors of DHFR, and folate based inhibitors ofTS were analyzed in Table 1. The mFBP exhibited a low affinityfor MTX and 10-EdAM as compared to folie acid and 5-CHO-THF. In contrast, the mFBP demonstrated a high affinity forthe folate based TS inhibitors which is nearly identical (ICI-198,583) or even higher (CB3717) than for folie acid. On theother hand, the RF-carrier has a high affinity for MTX, 10-EdAM, and ICI-198,583 but a poor affinity for CB3717. Table2 illustrated that the affinities of the RF-carrier and mFBP forthe antifolate compounds correlated with the uptake studies(Fig. 1) and growth inhibitory effects on L1210 and LI 210-B73 cells. In addition, the degree of protection from folie acidand 5-CHO-THF against the growth inhibitory effects of the

folate based inhibitors of DHFR and TS were consistent withthe relative affinities of the mFBP and RF-carrier for thesenatural folate compounds. Fig. 1 also showed that over a periodof 24 h the accumulation of [3H]-5-CHO-THF in L1210-B73cells is higher than for [3H]folic acid, even though the affinityof mFBP for folie acid is higher than for 5-CHO-THF. It isreasonable to assume that this difference is explained by aslower release of [3H]folic acid from the mFBP and/or reflects

a more efficient metabolism and retention of polyglutamate5510

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(ANTl)FOLATE TRANSPORT IN LI2IO CELLS

forms of [3H]-5-CHO-THF. In this respect, higher rates ofentry of 5-CHO-THF and/or folie acid in L1210-B73 cells may

also contribute to interactions with antifolates within the cell.In the case of MTX and 10-EdAM, competition at the level of

polyglutamylation (52, 53) or transformylation (53, 54) mayresult in a better protective effect of 5-CHO-THF on growthinhibition. The protective effect of 20 nM 5-CHO-THF onTMQ growth inhibition cannot be accounted for on the basisof effects on polyglutamylation, transport, or inhibition of theabove enzymes. Rather, the competition of 5-CHO-THF andits metabolites with TMQ for binding to DHFR, to result in areactivation of the enzyme (55-57), may form the basis for adiminished growth inhibitory effect of TMQ on L1210-B73

cells (Table 1).These results with L1210-B73 cells, as well as other studies

reported by our laboratory for human CCRF-CEM cells (16),suggest that nil HI' is unlikely to play an important role in the

membrane transport of folate based DHFR inhibitors. The lowaffinity of the mFBP for MTX/10-EdAM and the fact thatunder physiological conditions 5-CH.i-THF will strongly compete for binding to the mFBP are unfavorable conditions forbinding and transport via the mFBP. On the other hand, therecan be a role for the mFBP in the uptake of a new series offolate analogues with target enzymes other than DHFR. TheTS inhibitors CB3717 and related compounds like ICI-198,583have been designed in light of the development of cellularresistance to MTX due to increased levels of DHFR (23, 24).Another novel antifolate, DDATHF, has been identified as apotent inhibitor of glycinamide ribonucleotide transformylase,one of the folate dependent enzymes in purine biosynthesis denovo (58, 59). These newly synthesized antifolate drugs havebeen tested in preclinical models and in patients (60-64). In

phase I clinical trials with some of these new antifolates somerather unexpected severe side effects have been observed whichmight be related to the high affinity binding and transport viathe mFBP. In a recent study, Grindey et al. (65) have shownthat toxicity of DDATHF was increased 100-fold when micewere placed on a folate free diet for 2 weeks prior to treatmentwith this compound. High doses of folie acid in the drinkingwater completely reversed this toxicity but also the antitumoreffects, whereas low doses of folie acid prevented the dietary-

induced toxicity while maintaining the growth inhibitory effectson the xenograft carcinomas. Since the binding affinity of themFBP for DDATHF, CB37I7, and ICI-198,583 is very highand folie acid (rather than 5-CHO-THF) is able to provideprotection against growth inhibition by these drugs (Table 2;Ref. 66), these results may be compatible with the involvementof mFBP in membrane transport of these type of antifolatecompounds. Further studies must establish whether the presence of mFBP in a variety of normal cells and tissues is animportant factor in toxicity of normal cells to folate basedinhibitors of TS and glycinamide ribonucleotide transformylase. Based on the high affinities of the RF-carrier for ICI-

198,583 (Table 1; Ref. 66) and DDATHF (66, 67), uptake ofthese compounds in tumor cells can also proceed via the thiscarrier system which is expressed in a great number of neoplas-tic cells (2, 4). The nanomolar concentrations of radiolabeledcompounds used in Fig. I were selected to be saturating for themFBP but not for the RF-carrier in L1210-B73 cells. It can beexpected that at higher extracellular concentrations (micromo-lar range) the contribution of the RF-carrier in the uptake of(anti)folate compounds in L1210-B73 cells may proportionallyincrease due to more favorable transport kinetic conditions for

this carrier system, whereas the saturation of mFBP will beunchanged.

The mechanism of (anti)folate uptake via the mFBP is notwell understood. Immunofluorescence studies with anti-mFBPantibodies on MA104 cells (68) indicated that the mFBP-folatecomplex itself may not be internalized via a classical receptor-mediated endocytosis mechanism. It has been hypothesized ( 18,69) that the mFBP serves as a concentrator of folate compoundswhich are then translocated over the membrane via a secondcarrier system (e.g., the RF-carrier). In L1210-B73 cells, whichexpress both mFBP and RF-carrier, we did not find evidencefor coupling the action of the RF-carrier to the mFBP. Bothtransport systems seem to operate independently from eachother. This can be concluded from the fact that, despite thepresence of mFBP in L1210-B73 cells, there is almost nodifference in growth inhibition by MTX for LI210 and LI 210-B73 cells (Table 2). Furthermore, if the RF-carrier in LI210-B73 cells would be coupled to mFBP, it is difficult to explainthe potent growth inhibitory effect of CB3717 on L1210-B73cells (Table 2) when the RF-carrier has only a poor affinity forthis compound (Table 1). Also, the RF-carrier also has a verylow affinity for folie acid (Table 1), which results in a very lowcellular uptake of this compound in LI210 cells but not inL1210-B73 cells, suggesting that uptake of ['H]folic acid pre

dominantly proceeds via the mFBP. Finally, in human CCRF-CEM leukemia cells which express mFBP and have no functional RF-carrier, uptake of folate-based TS inhibitor can proceed solely via mFBP (16). Currently, studies are under way toinvestigate in more detail the molecular events in (anti)folateuptake via mFBP by using a photoaffinity analogue of folieacid (70).

In summary, this study has evaluated the role of the RF-carrier and mFBP in membrane transport of natural folates andfolate analogues that are inhibitors of DHFR and TS. Theresults suggest that the mFBP plays a minor role in the membrane transport of folate based DHFR inhibitors but could bean important transport route for folate based TS inhibitors.The presence of mFBP in various normal tissues and the highaffinity of mFBP for these types of antifolates indicate thatthere may be a pharmacological role for mFBP, e.g., in dealingwith the toxic effects to normal cells by nonclassical antifolates.

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