Multiple Membrane Transport Systems for the Uptake of Folate-Based Thymidylate Synthase Inhibitors'

(CANCER RESEARCH 50, 7544-7548. December I. I990| Multiple Membrane Transport Systems for the Uptake of Folate-based Thymidylate Synthase Inhibitors'

Gerrit Jansen,2 Jan H. Schornagel, G. Robbin Westerhof, Gert Rijksen, David R. Newell, and Ann L. Jackman

Department of Internal Medicine, Oncology Unii /G. J., J. H. S.. G. K. W.Â¡,and Laboratory of Medical Enzymology, Department of Haematology /G. R.J, University Hospital Utrecht, P. O. Box 85500, 3508 CA Utrecht, The Netherlands; and Drug Development Section, Institute of Cancer Research, Sutton, Surrey, United Kingdom Â¡D.R. N., A. L. J.J

ABSTRACT inhibited MTX uptake by this carrier (5, 15). On the other hand, MTX carrier-defective mutants did not show marked /V10-Propargyl-5,8-dideazafolic acid (CB3717) and 2-desamino-2- methyl-/V"'-propargyl-5,8-dideazafolicacid (ICI-198,583) are potent fol cross-resistance to CB3717 (14-19) suggesting that other trans port route(s) may be utilized. In this study we have investigated ate-based inhibitors of thymidylate synthase. We studied the membrane the membrane transport characteristics of ['H]ICI-198,583 and transport and the growth-inhibitory effects of the two thymidylate syn thase inhibitors on human CCRF-CEM leukemia cells with different CB3717 in human CCRF-CEM leukemia cells and variants transport properties for folie acid, reduced folates, and methotrexate with different folate transport properties. These variants include (MTX). Membrane transport of |'H|ICI-198,583 can proceed via the CEM cells which either lack (20, 21) or overexpress (22) the high affinity/low capacity reduced folate carrier as supported by findings reduced folate/MTX carrier system present in control CCRF- that (a) uptake of |'H]ICI-198,583 was significantly impaired in CEM CEM cells (23-25). In addition, CEM cells were used that do cells which have a transport defect for MTX, (A) variants of CEM cells not have the reduced folate carrier but express a membrane- which overproduce the reduced folate carrier system showed a concomi tant increase in the uptake of |'H|ICI-198,583 as for |'H|MTX, (c) MTX associated FBP (21, 25). The results suggest that membrane inhibited transport of |'II|I( 1 l'),S,58i, and (aminopterin, r>t.-5-formyltetrahydrofolate, r>i.-5-methyltetrahy- reduced folate/MTX carrier. This high sensitivity was related to a high drofolate, and jY-hydroxysuccinimide were purchased from Sigma affinity of the FBP for CB3717 and ICI-198,583 (Ka 2-3 nM), which is Chemical Co., St. Louis, MO. Unlabeled methotrexate was a gift from only 3-fold lower than for folie acid (A,, 1 IIM) but significantly higher Pharmachemie, Haarlem, The Netherlands. 10-Ethyl-10-deazaaminop- than for MTX (A,, 100 nM). Furthermore, after incubation of CEM-FBP cells for 24 h at 10 nM ('HjICI-198,583, the high affinity binding of the terin was a gift from Ciba Geigy, Basel, Switzerland. CB3717 and ICI- FBP for ICI-198,583 allowed a 600-fold concentrative uptake of |-'H]- 198,583 were provided by ICI-Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, United Kingdom. ['HjFolic acid (0.5 Ci/mmol) ICI-198,583 and its conversion to polyglutamate forms. was purchased from Amersham, United Kingdom, and ['HJMTX (10- These results indicate that multiple folate transport systems may be 20 Ci/mmol) was from Moravek Biochemicals, Brea, CA. Radiolabels involved in the uptake of folate-based thymidylate synthase inhibitors. were purified prior to use as described previously (21, 23, 26). ['HjICl- 198,583 (25.6 Ci/mmol) was prepared as the diethyl ester as a custom INTRODUCTION synthesis by Amersham, United Kingdom, and provided in solution at a radiochemical concentration of 1 mCi/ml in ethanohwater (50:50, v/ TS1 (EC 2.1.1.45), one of the key enzymes in folate metabo v). The stock solution was stored at -20Â°C prior to use. The diethyl lism, is a recognized target of cytotoxic antifolates (1-4). Two ester of [5H]ICI-198,583 was hydrolyzed as required as follows. To 2 of these folate-based TS inhibitors, the quinazoline folate ana ml of the ethanolic solution of ['HJIC1-198,583 was added 0.08 ml of logues CB3717 and ICI-198,583, have activity in the preclinical l M NaOH following which the solution was allowed to stand in the (CB3717, ICI-198,583) and clinical (CB3717) settings (5-14). dark at room temperature for 4 h. Acetic acid (0.16 ml, 1 M) was then added and the solution was concentrated by blowing with nitrogen at A question still unanswered is whether membrane transport 30-40Â°C.The concentrated solution of ['HjICI-198,583 was then pu of these compounds proceeds via mechanisms similar to those rified by high performance liquid chromatography. The stationary operative for folate analogues that are inhibitors of DHFR (e.g., phase was a 15 x 0.46-cm Spherisorb C6 column and the ['H]IC1- MTX). A putative role for the high affinity/low capacity re 198,583 was eluted with a linear gradient from 5:95 CH,CN:0.1 M duced folate carrier, which is shared by MTX, has been sup sodium acetate, pH 5, to 15:85 CH_,CN:0. l M sodium acetate, pH 5, ported by observations that CB3717 (and related compounds) over 20 min starting the injection of |'H]ICI-198,583 onto the column. The fractions containing |'H]ICI-198,583 were pooled and reanalyzed Received 1/30/90; accepted 9/5/90. using a separate column, containing the same stationary phase, and the The costs of publication of this article were defrayed in part by the payment above solvent system. The radiochemical purity of |'H]ICI-198,583 of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate (his fact. prepared in this manner was >99.5% and the UV and H-nuclear ' This study was supported by the Dutch Cancer Society (Grant UUKC 89- magnetic resonance spectra were consistent with the structure proposed. 06) and grants from the Cancer Research Campaign and the Medical Research NHS-MTX was synthesized as described previously (21, 23, 27). Council (o D. R. N. and A. L. J. 2To whom requests for reprints should be addressed. Cell Cultures. Human leukemic CCRF-CEM cells and MTX trans 1The abbreviations used are: TS, thymidylate synthase: MTX. mctholrexale; port-defective CEM/MTX cells (20, 21) were grown as a suspension CB3717, A"Â°-propargyl-5,8-dideazafolic acid: ICI-198,583,2-desamino-2-melhyl- A"Â°-propargyl-5.8-dideazafolic acid; 10-EdAM, 10-ethyl-IO-deazaaminopterin; culture in RPMI 1640 (with folie acid) supplemented with either 10% horse serum or fetal calf serum, 2 HIMglutamine, penicillin (100 units/ NHS-MT.X, /V-hydroxysuccinimide ester of MTX; HBSS, 4-<2-hydroxyelhyl)-l- ml), and streptomycin (100 fig/ml) at 37"C in a 5% CO2 humidified pipcrazincelhanesulfonic acid buffered saline solution; FBP. folate binding pro tein; DHFR, dihydrofolate reducÃase. atmosphere. In addition, CEM/MTX cells were maintained in the 7544

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1990 American Association for Cancer Research. MEMBRANE TRANSPORT OF FOLATE-BASED TS INHIBITORS continuous presence of 1 UM MTX. Prior to growth inhibition or The clear supernatant was applied to a DE52 minicolumn to separate transport studies CEM/MTX cells were maintained for at least 4 monoglutamate from polyglutamate forms of ['HJICI-198,583 accord transfer generations in the absence of MTX. CEM-7A cells, a subline ing to methods described by McGuire et al. (32). The column was of CCRF-CEM with a 30-fold overproduction of the reduced folate/ washed with 35 ml 10 mM Tris-HCl (pH 7.5), 125 mM NaCl, and 2.5 MTX carrier (22) were grown in folate-free RPMI 1640 supplemented mM dithiothreitol. Polyglutamates were eluted with 3 ml 0.1 N HC1. with 10% dialyzed fetal calf serum, 2 mM glutamine, and antibiotics as described above, and 0.25 nM 5-formyltetrahydrofolate as the sole folate source. CEM-FBP cells, a subline of CEM/MTX (21), were grown in RESULTS folate-free RPMI 1640, 10% dialyzed horse serum, glutamine, and antibiotics as described above and 0.5-1.0 nM of either folie acid or 5- Growth Inhibition Studies. Table 1 shows the growth-inhibi formyltetrahydrofolate as the sole folate source. tory effects of MTX, CB3717, and ICI-198,583 on CEM cells Growth Inhibition Assay. Cells in the logarithmic phase of growth and three different sublines. CCRF-CEM cells were 41-fold were plated at an initial density of 7.5 x 104/ml into the individual more sensitive to ICI-198,583 than for CB3717. CEM/MTX wells of a 24-well tissue culture plate. Before this, the medium was cells, lacking the reduced folate/MTX carrier present in CCRF- changed to folate-free RPMI medium supplemented with 10% dialyzed CEM cells, were 240-fold resistant to MTX. Cross-resistance, fetal calf serum and 1 nM 5-formyltetrahydrofolate as the sole folate 10- and 36-fold, was noted for CB3717 and ICI-198,583, re source (unless otherwise indicated). Appropriate drug concentrations spectively. CEM-7A cells, overexpressing the reduced folate were added at the time of initiation of the cultures. After 72 h contin uous exposure to the drugs, cells were counted by a Sysmex CC-110 carrier, were more sensitive to MTX (6.7-fold), CB3717 (20- microcell counter, and cell viability was determined microscopically by fold), and ICI-198,583 (7.7-fold) than parental CCRF-CEM trypan blue exclusion. cells. However, the greatest sensitivity for CB3717 and ICI- Transport Studies. Uptake of ['HJMTX and [3HJICI-198,583 (specific 198,583 was noted for CEM-FBP cells. On 50% inhibitory activity, 0.2-0.5 Ci/mmol) in CEM cells expressing the reduced folate concentration basis, CEM-FBP cells were 640-fold more sen carrier was determined at 37"C with 15 x 10" (CCRF-CEM, CEM/ sitive to CB3717 and 61-fold more sensitive to ICI-198,583 MTX) or 5 x 10" (CEM-7A) cells suspended in 1 ml of HBSS contain when compared to CCRF-CEM cells. As shown in Table 1, ing 107 mM NaCl, 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesul- protection from growth inhibition by MTX, CB3717, and ICI- fonic acid, 26.2 mM NaHCO3, 5.3 mM KCI, 1.9 mM CaCl2, 1.0 mM 198,583 was observed when CEM-FBP cells were grown with MgCl2, and 7.0 mM o-glucose, pH 7.4, with NaOH. Uptake was stopped at selected time intervals by the addition of 9 volumes of ice- 20 nM folie acid as the sole folate source instead of 1 nM folie cold transport buffer. After centrifugation for 5 min (800 x g, 4Â°C),the acid. Fig. 1 demonstrates in more detail that 20 nM folie acid, cell pellet was washed once more with ice-cold transport buffer. The unlike 20 nM 5-formyltetrahydrofolate, can provide a (partial) final pellet was resuspended in 0.5 ml H2O and analyzed for radioactiv protective effect upon growth inhibition of CEM-FBP cells by ity in Optifluor scintillation fluid (United Technologies Packard, Brus CB3717 and ICI-198,583. On the other hand, thymidine (20 sels, Belgium) and an Isocap-300 scintillation counter with a counting ÃŸM)gives complete protection from growth inhibition. efficiency for "H of 53%. In other experiments the HBSS buffer was Carrier Transport of I'HJMTX and |-'H]ICI-198,583. A role replaced by an anion-deficient buffer, (28) containing 20 mM 4-(2- for the reduced folate/MTX carrier in the transport of ['H]ICI- hydroxyethyl)-l-piperazineethanesulfonic acid and 225 mM sucrose, 198,583 is supported by data shown in Fig. 2 and Table 2. Fig. pH 7.4, with MgO. Uptake of ['HJICI-198,583 by CEM-FBP cells was 2 shows a time course for the uptake of ['HJMTX and ['H]ICI- determined as follows. CEM-FBP cells (5 x IO6)were incubated in 10 198,583 by CCRF-CEM, CEM/MTX, and CEM-7A cells. ml folate-free RPMI medium supplemented with 10% dialyzed horse CEM-MTX cells which have a transport defect for MTX serum in the presence of 10-50 nM pHJICI-198,583. At the indicated showed an impairment for transport of [3H]ICI-198,583 as well. times, cells were centrifuged for 5 min at 800 x g. The supernatant was On the other hand, CEM-7A cells, overproducing the reduced removed by suction and the residual fluid was removed with cotton tissues. To distinguish between cell surface-bound and intracellular folate carrier, showed a concomitant increase in the uptake of ['HJ-ICI-198,583, cells were rinsed for 1 min at 4"C in 1 ml acidic ['HJMTX and ['HJICI-198,583. Although under certain cir saline, pH 3.5 (21, 26, 29, 30). After centrifugation (1 min, 13,000 x cumstances (21 ) an /V-hydroxysuccinimide ester of MTX (NHS- g) in an Eppendorf centrifuge, radioactivity was determined in the MTX) can be used as inhibitor of the FBP system, the active supernatant fraction (containing cell surface-bound ['HJICI-198,583 ester is known to be one of the most potent inhibitors of the which is stripped off at pH 3.5) and the cell pellet (containing "acid- reduced folate/MTX carrier system (27). Treatment of CEM- resistant" intracellular ['HJICI-198,583 (26, 29, 30). 7A cells with 50 nM NHS-MTX resulted in a 94% inhibition Binding Studies. Binding of ['HJfolic acid or ['HJICI-198,583 was of{JH]ICI-198,583 influx (results not shown). Finally, Table 2 determined (unless otherwise indicated) in 1-ml assay mixtures contain shows that CB3717 and ICI-198,583 were inhibitors of ['HJ- ing 3 x IO6CEM-FBP cells in HBSS, pH 7.4, and 50 pmol ['HJfolic acid or ['HJICI-198,583 (specific activity, 0.5 Ci/mmol). After 15 min MTX transport in CCRF-CEM and CEM-7A cells. ICI- incubation at 4Â°C,cells were centrifuged in an Eppendorf microcentri- 198,583, however, is a 20-25-fold better inhibitor of ['HJMTX fuge (l min, 13,000 x g). The supernatant was removed by suction and transport than CB3717, with a 50% inhibitory concentration residual fluid was removed with cotton tissues. Cell pellets were resus pended in H2O and analyzed for radioactivity. Nonspecific binding of Table 1 Growth inhibition of human CCRF-CEM leukemia cell line by MTX, CB3717. andlCI-198,583 label was determined by measuring radioactivity in the presence of a 1000-fold molar excess of nonlabeled folie acid. Polyglutamylation of |3H|ICI-198,583. CEM-FBP cells (20 x 10") CelllineCCRF-CEM 198,58317 were incubated in 40 ml folate-free RPMI medium supplemented with 10% dialyzed horse serum in the presence of 25 nM ['HJICI-198,583. CEM/MTX 7750 605 CEM-7A 1.2 36 2.2 After 24 incubation, cells were centrifuged and washed with 10 ml CEM-FBP* HBSS, pH 7.4. Membrane-bound ['HJICI-198,583 was removed by 19.4 1.1 0.2874 CEM-FBP1MTX8.119501400CB3717705 24ICI- rinsing the cells in 1 ml of acidic saline, pH 3.5, as described above. Â°Cell growth was determined after 72 h incubation. IC50 is defined as the The final cell pellet was resuspended in 1 ml 50 mM sodium phosphate concentration of drug required to inhibit cell growth by 50% of controls. buffer, pH 5.5, and boiled for 5 min (31). Cellular debris was removed * Cells grown at I nM folie acid as the sole folate source. by centrifugation for 1 min at 13,000 x g in an Eppendorf centrifuge. ' Cells grown at 20 nM folie acid as the sole folate source. 7545

J2 50-

* 50-

25 CB 37171 nM ) ICI- 198.583 {nM ) =T=*=1 Fig. I. Growth inhibition ut CEM-FBP cells b> CB3717 (left) or 1CM98.583 25 50 (right) in the absence (A) or presence of 20 nM 5-formyltetrahydrofolate (A), 20 nM [3H]-IC1-198.583 nM folie acid (O) or 20 JIM thymidinc (â€¢).Drugs and additions were added to the cultures simultaneously, followed by a 72-h incubation period. Further conditions rig. 3. Binding of |'H]ICM98.583 to CEM-FBP cells as a function of the for growth inhibition are described in "Materials and Methods." extracellular |'H|K'I 198.583 concentration. C'EM-FBP cells (2 x 10" in 10 ml HBSS. pH 7.4) were incubated for 15 min at 4Â°Cwith the indicated concentrations of [3H|ICI 198.583 in the absence (O) or presence (â€¢)ofa 1000-fold molar excess of folie acid. After incubation, cells were centrifuged (800 x #. 5 min. 4Â°C)and further processed as described in "Materials and Methods."

Table 3 Relative affinities of the l-'BPJrom i'EM-h'BP cells for CB37I7, H'1-19X,5N3, and other (antisolate compound* CEM-FBP cells (3 x lOVml in HBSS. pH 7.4) were incubated for 10 min at 4Â°Cwith 50 pmol of | 'H|lblic acid and increasing concentrations of the indicated folate compound. The inverse molar ratio of compound required to displace 50% of |3H|folic acid from the FBP is depicted as relative affinity.

: 100 compoundFolie(Antisolate affinity10.370.33" Â¡2BÃŽ acidCB37I75-Methvltetrahvdrotolatc

ICI198.5835-Forrmltelrali\drofolate 0.30o.i r0.009"0.008"" MTX10- EdA MRelative Data from Ref. 21. Fig. 2. Time course of accumulation at 37"C of [3H]MTX (left) and [3H|ICI- 198,583 (righi) by CCRF-CEM cells (O), CEM/MTX cells (A), and CEM-7A cells (â€¢).Uptake buffer. HBSS (pH 7.4): extracellular concentration of (3H|M l'\ 40- and |'H| 1C1-198.583 was 2 MM.

[3H]iCI-196.583[3H]MIXn_4 fable 2 Inhibition U//V//A//A influx by CBÃŒ7I7.ICI-198,583, and other folate analogues in human Ã•'CRF-Ã•'EM and CEM-7A leukemia celli r- 30- I'HjMTX influx in CCRF-CEM cells and CEM-7A cells was determined oxer a 3-min and a I min period, respectively. Transport buffer, HBSS (pH 7.4). Extracellular concentration of [3H]-MT.\. 5 n\i. Further details are described in "Materials and Methods." 1C',,,represents the concentration of (anti)folate com pound required to inhibit [3H]MTX influx b\ 50't. Actual influx rates of [3H|- 20. MTX in CX'RF-CEM and C'EM-7A cells were 3.3 and 221 pmol/min/IO1 cells, |L. respectively.

FolatecompoundIO-EdAM1CI-198.5835-Formvlletrah\drofolateMTXCB37

2k 1 24 hoursâ€” 17Folie Fig. 4. Internali/ation of |'H]ICI-198.583 and |'H|MTX by CEM-FBP cells. acidICâ€ž(,CCRF-CEM2.22.84.59.574540:M)CEM-7A1.62.34.510.857505 C'EM-FBP cells were incubated for 1.4. and 24 h at an extracellular concentration of 10 n.M |'H|1C1-198.583 (G) and 100 nM |'H|M1.\ (U) in the absence or close to those of a folate analogue like 10-EdAM. presence (â€¢)ofa 10-fold molar excess of folie acid. Distinction between internal ized [3H]ICT-198.583 and cell surface-bound [3H]ICI-I98.583 (40-45 pmol/101 7/8Binding Studies. Fig. 3 shows that CEM-FBP cells exhibit a cells, not shown) was done by suspending cells in acidic saline pH 3.5 as described substantial cell surface-binding capacity for ['H]1C1-198,583 in "Materials and Methods." (45 pmol/107 cells). Half-maximal binding of ['H]ICI-198,583 was at a 2-3 nM extracellular concentration. For comparison, substantially higher than for the DHFR inhibitors MTX or 10- half-maximal binding of ['Hjfolic acid and ['HjMTX is at EdAM. approximately 1 and 100 nM extracellular concentration (data pHJICI-198,583/|'H|MTX Uptake by CEM-FBP Cells. Up not shown). The high affinity binding of the FBP for 1C1- take of l'HIICI-198,583 by CEM-FBP cells is shown in Fig. 4. 198,583 is also illustrated in Table 3. The FBP has an affinity At a 10 nM extracellular concentration, CEM-FBP cells accu for CB3717 and ICI-198,583 that is only 3-fold lower than for mulated 15 and 34 pmol ['H]ICI-198,583/10' cells after 4 and folie acid and close to the affinity of 5-methyltetrahydrofolate. 24 h incubation, respectively. From a cell volume of 0.55 x The affinity of the FBP for the TS inhibitors, however, is 10 l2 liter/cell (calculated from a mean cell diameter of 10.2 Â± 7546

Ãœ.7tiin), this latter value corresponds with a 600-fold concen- of binding affinity (Table 3). The relative affinities of the FBP trative uptake of ['HjICI-198,583 from the medium. After a for folie acid and 5-formyltetrahydrofolate compared to 24-h incubation of CEM-FBP cells with ['HJICI-198,583, 65% CB3717 and ICI-198,583 (Table 3) may be the explanation why of the total intracellular ['HJICI 198.583 was found to be folie acid, unlike 5-formyltetrahydrofolate, provided a substan metabolized to polyglutamate forms (results not shown). Fig. 4 tial protection against the cytotoxic effects of the TS inhibitors shows that also |'H]MTX can be internalized via the FBP. (Fig. 1). The affinity of the FBP for folie acid is 3-fold higher However, since the affinity of the FBP for MTX is significantly than for CB3717 or ICI-198,583 which implies that displace lower than for ICI-198,583 (Table 3), much higher extracellular ment of folie acid from the FBP requires a higher molar ratio concentrations of ['H]MTX (=100 nivi)are required to observe of CB3717 or ICI-198,583. In contrast, the lower affinity of levels of ['H]MTX internalization that approach levels of ('H]- the FBP for 5-formyltetrahydrofolate relative to CB3717 and ICI-198,583 internalizaron at an extracellular concentration of ICI-198,583 may be the basis for why this reduced folate lOnM. compound does not protect cells from the cytotoxic effects of these agents as illustrated in Fig. 1. In addition to the capacity to bind, CEM-FBP cells were able to internalize (Fig. 4) and to DISCUSSION polyglutamate [3H]ICI-I98,583. Similarly, in L1210 cells, The role of inhibitors of folate metabolism with targets other CB3717 (33) and ICI-198,583 (14) were found to be polyglu- than dihydrofolate reducÃase in the treatment of neoplastic tamated as well. It should be noted that although CEM-FBP cells internalize 34 pmol ['H]ICI-198,583/107 cells over a 24-h diseases in an area of considerable current interest. In particu lar, inhibitors of thymidylate synthase (folate analogues and period, this amount is still lower than the total cell surface- fluoropyrimidines) have been shown to be clinically active (1- binding capacity of these cells (Fig. 3). This suggests that either 3, 5-14). This study has addressed the issue of membrane internalization proceeds at a rather low efficiency [less than one transport of two folate analogue TS inhibitors (CB3717 and transport cycle per binding site in 24 h] or that only part of the 1CI-198,583) in human CCRF-CEM leukemia cells either lack available binding sites is actively involved in the internalization process. The mechanism of internalization of [1H]ICI-198,583 ing (20, 21) or overexpressing (22) the reduced foIate/MTX carrier system present in parental cells, or expressing a mem [and other (anti)folate compounds] by the FBP probably pro brane-associated folate-binding protein (21) instead of the re ceeds via receptor-mediated endocytosis as suggested by studies duced folate carrier. of Kamen et al, (30) and by our laboratory (34). A recent study A role for the reduced folate carrier in transport of CB3717 by Rothberg et al. 35), however, suggested that the internali (and related compounds) has been suggested by others (5, 15), zation process does not involve the clathrin-coated pit endocytic but the absence of appreciable cross-resistance in cells with pathway. Although the internalization process of ['HjICI- transport-related MTX resistance (14-19) seems to be incon 198,583 by the FBP (Fig. 4) is certainly much slower than via sistent with these observations. The present transport studies the reduced folate carrier (Fig. 2), it is highly concentrative with radiolabeled ICI-198,583 (Fig. 2), however, demonstrate even at nanomolar extracellular concentrations. that this compound can be transported via the reduced folate The role of the FBP in transport of folate analogues may not carrier. Å’M/MTX cells, with defective MTX transport, and have been recognized in the past since cytotoxicity studies are CEM-7A cells, up-regulated for reduced folate transport, generally carried out in media containing high levels of folate. showed a concomitant decrease and increase, respectively, in These high folate levels (e.g., 2-10 UM folie acid) could, in the uptake of ['HJMTX and [3H]ICI-198,583 compared to general, result in a down-regulation of synthesis of the FBP CCRF-CEM cells. Likewise, cytotoxicity studies (Table 1) are (26). Adapting cells to folate levels in the nanomolar range may compatible with these observations, except that the degree of lead to an up-regulation in the expression of the FBP (21, 26, cross-resistance of CEM/MTX cells for MTX (240-fold), 36). The FBP in CEM-FBP cells is expressed to a level 45 CB3717 (10-fold), and ICI-198,583 (36-fold) is not identical. pmol/107 cells; the level of FBP in other tumors or normal This is consistent with cross-resistance studies with LI210 tissues is unclear. leukemia cell lines (14) or H35 hepatoma cell lines (15) lacking In summary, this paper demonstrates that human CCRF- the reduced folate carrier. Regardless of the target enzyme level, CEM leukemia cells transport folate-based inhibitors of thy the lesser cross-resistance for CB3717 may be due to the fact midylate synthase (CB3717 and ICI-198,583) by at least two that the reduced folate carrier has a much lower affinity for processes. These include the reduced folate/MTX carrier and a CB3717 than for MTX and ICI-198,583 (Table 2). For this second route that mediates transport of folie acid and may be reason, ICI-198,583 is more active than CB3717. associated with a high level of FBP. Transport of CB3737 and This study, however, suggests that another route for uptake ICI-198,583 via the FBP is consistent with the high affinity of of CB3717 and ICI-198,583 besides the reduced folate carrier the FBP for these compounds. This is in contrast to the low can be of importance. CEM cells that express a membrane- affinity of the FBP for inhibitors of DHFR such as MTX. In associated FBP were found to be highly sensitive to the TS view of these observations, future studies should consider the inhibitors (Table 1). This was associated with a high affinity of importance of multiple transport systems for the uptake of the FBP for both CB3717 and ICI-198,583 (Table 3). By folate analogues with a variety of target enzymes in folate contrast, the FBP has a low affinity for the ÃœHFRinhibitors, metabolism, i.e., DHFR, TS, or glycinamide ribonueleotide MTX and 10-EdAM, suggesting that this may be the basis for transformylase (37), in drug actions. the resistance of CEM-FBP cells to these compounds (21). At a chemical level, these results suggest that the 4-hydroxy group ACKNOWLEDGMENTS is important in the high affinity binding of the FBP to folates, The authors are extremely grateful to ICI Pharmaceuticals, Alderley or quinazoline folate analogues like CB3717 and ICI-198,583. Park, Cheshire, United Kingdom, for their generous funding of the Replacement of the 4-hydroxy group by a 4-amino group, as in synthesis of the ['HJICI-198,583. I. Kathmann is thanked for her MTX or 10-EdAM, apparently results in a significant reduction excellent teehnical assistance. 7547

REFERENCES (TMQ). Proc. Am. Assoc. Cancer Res., 26: 230, 1985. 20. Rosowsky, A., Lazarus, H.. Yuan, G. C., Bcltz, W. R., Mangini, L., Abelson, 1. Harrap. K. R.. Jackman, A. L., Newell, D. R., Taylor, G. A., Hughes, L. R., H. T., Modest, E. J., and Frei, E., III. Effects of methotrexate esters and and Calvert, A. H. Thymidylate synlhase: a target for anticancer drug design. other lipophilic antifolates on methotrexatc-resistant human leukemic lym- Adv. Enzyme Regul., 29: 161-179, 1989. phoblasts. Biochem. Pharmacol., 29: 648-652, 1980. 2. Moran, R. G. Leucovorin enhancement of the effects of the fluoropyrimidines 21. Jansen, G., Westerhof, G. R., Katmann, I., Rademaker. B. C., Rijksen, G., on thymidylate synthase. Cancer (Phila.), 63: 1008-1012, 1989. and Schornagel. J. H. Identification of a membrane-associated folate binding 3. Pinedo, H. M., and Peters, G. J. Fluorouracil: biochemistry and pharmacol protein in human leukemic CCRF-CEM cells with transport-related metho ogy. J. Clin. Oncol., 6: 1653-1664. 1988. trexate resistance. Cancer Res., 49: 2455-2459, 1989. 4. Galivan, J., Nimec, Z., and Rhee, M. Synergistic growth inhibition of rat 22. Jansen, G.. Jarmuszewski, M. J. A., Westerhof. G. R.. Kathmann. I.. Rijksen. hepatoma cells exposed in vitro to A/'Â°-propargyl-5,8-dideazafolate with G., and Schornagel, J. H. Regulation of methotrexate transport in human methotrexate or the lipophilic antifolates trimetrexate or metoprine. Cancer leukemic CCRF-CEM cells up-rcgulatcd for folate (analogue) transport Res., 47: 5256-5260, 1987. inward. Proc. Am. Assoc. Cancer Res., 30: 479. 1989. 5. Jones, T. R., Calvert, A. H., Jackman, A. L., Brown, S. J., Jones, M., and 23. Henderson, G. B., Tsuji, J. M., and Kumar, H. P. Characterisation of the Harrap, K. H. A potent antitumor quinazoline inhibitor of thymidylate individual transport routes that mediate the influx and efflux of methotrexate synthetase: synthesis, biological properties and therapeutic results in mice. in CCRF-CEM human lymphoblastic cells. Cancer Res., 46: 1633-1638. Eur. J. Cancer., 17: 11-19, 1981. 1986. 24. Sirotnak, F. M. Obligate genetic expression in tumor cells of a fetal mem 6. Jackman, A. L., Taylor, G. A., Moran, R. G., Bishop. J. A. M., Bisset, G., brane property mediating "folate" transport: biological significance and Pawalczak. K., Balmanno, K., Hughes, L. R., and Calvert, A. H. Biological properties of 2-desamino-2-substituted-5,8-dideazafolates that inhibit thy implications for improved therapy of human cancer. Cancer Res., 45: 3992- midylate synthase. Proc. Am. Assoc. Cancer Res.. 29: 287, 1988. 4000, 1985. 7. Hughes, L. R., Marsham, P. R., Oldfield, J., Jones, T. R., O'Connor, B. M.. 25. Van der Veer, L. J.. Westerhof, G. R., Rijksen. G., Schornagel. J. H., and Bishop, J. A. M., Calvert, A. H., and Jackman, A. L. Thymidylate synthase Jansen, G. Cytotoxicity of methotrexate and trimetrexate and its reversal by inhibitory and cytotoxic activity of a series of C2substituted -5,8-deazafolates. folinic acid in human leukemic CCRF-CEM cells with carrier-mediated and Proc. Am. Assoc. Cancer Res., 29: 286, 1988. receptor-mediated folate uptake. Leuk. Res.. 13: 981-987. 1989. 8. Cantwell, B. M., Earnshaw, M., and Harris. A. L. Phase II study of a novel 26. Jansen. G.. Kathmann. I., Rademaker. B. C.. Braakhuis. B. J. M.. Westerhof, antifolate. A"Â°-propargyl-5.8-dideazafolic acid (CB3717). in malignant meso- G. R., Rijksen. G., and Schornagel, J. H. Expression of a folate binding thelioma. Cancer Treat. Rep.. 70: 1335-1338, 1986. protein in L1210 cells grown in low folate medium. Cancer Res.. 49: 1959- 9. Bassendine, M. F., Curtin, N. J., Loose. H., Harris. A. L., and James, O. F. 1963, 1989. W. Induction of remission in hepatocellular carcinoma with a new thymidyl 27. Henderson, G. B.. and Montague-Wilkie, B. Irreversible inhibitors of meth ate synthase inhibitor CB3717: a phase II study. J. Hepatol., 4: 349-356, otrexate transport in L1210 cells. Characteristics of inhibition by an /V- 1987. hydroxysuccinimidc ester of methotrexate. Biochim. Biophys. Acta, 735: 10. Calvert, A. H., Newell, D. R., Jackman. A. L.. Gumbrell, L. A., Sikora, E., 123-130, 1983. Grzelakowska-Sztabert, G., Bishop. J. A. M.. Judson, I. R., Harland, S. J.. 28. Henderson, G. B., and Zevely, E. M. Use of non-physiological buffer systems in the analysis of methotrexate transport in I 1.'HI cells. Biochem. Int.. 6: and Harrap, K. R. Recent preclinical and clinical studies with the thymidylate synthase inhibitor A"Â°-propargyl-5,8-dideazafolic acid (CB3717). Nati. Can 507-515, 1983. cer Inst. Monogr., 5: 213-218, 1987. 29. Kamen, B. A., and Capdevila, A. Receptor-mediated folate accumulation is 11. Vest, S., Bork, E., and Hansen, H. H. A phase I evaluation of A"Â°-propargyl- regulated by the cellular folate content. Proc. Nati. Acad. Sci. USA, 83: 5,8-dideazafolic acid. Eur. J. Cancer Clin. Oncol., 24: 201-204, 1988. 5983-5987, 1986. 12. Cantwell, B. M. J., Macaulay, V., Harris, A. L., Kaye, S. B.. Smith, I. E., 30. Kamen. B. A.. Wang. M. T., Streckfuss. A. J., Pcryea, X.. and Anderson, R. Milsted, R. A. V., and Calvert, A. H. Phase II study of the antifolate A"Â°- G. W. Delivery of folates to the cytoplasm of MA 104 cells is mediated by a propargyl-5,8-dideazafolic acid (CB3717) in advanced breast cancer. Eur. J. surface membrane receptor that recycles. J. Biul. Chem., 263:13602-13609, Cancer Clin. Oncol., 24: 733-736, 1988. 1988. 13. Sessa, C. Zuccheti, M., Ginier, M., Willems, Y., D'Incaici. M., and Cavalli. 31. McGuire, J. J.. Mini. E.. Hseih, P., and Bertino. J. R. Role of mcthotrexate F. Phase I study of the antifolate A"Â°-propargyl-5,8-dideazafolic acid. polyglutamates in methotrexate- and sequential methotrexate-5-fluorouracil- CB37I7. Eur. J. Cancer Clin. Oncol., 24: 769-775, 1988. mediatcd cell kill. Cancer Res., 45: 6395-6400, 1985. 14. Jackman. A. L., Newell, D. R., Jodrell, D. I., Taylor, G. A., Bishop, J. A. 32. McGuire, J. J., Hseih, P., Coward, J. K., and Bertino, J. R. Enzymatic M., Hughes, L. R., and Calvert, A. H. In vitro and in vim studies with 2- synthesis of folylpolyglutamates. Characterization of the reaction and its desamino-2-CH,-A'10-propargyl-5,8-dideazafolate (ICI 198583), an inhibitor products. J. Biol. Chem., 255: 5776-5788, 1980. of thymidylate synthase. In: H. C. Curtius, S. Gishla. and N. Blau (eds.). 33. Sikora. E.. Jackman, A. L., Newell, D. R., and Calvcrl, A. H. Formation and Chemistry and Biology of Pteridines 1989. pp. 1023-1026. Berlin: Walter retention and biological activity of yv'Â°-propargyl-5,8-dideazafolic acid (CB3717) polyglutamales in L1210 cells in vitro. Biochem. I'harmacol., 3?: de Gruyter & Co., 1990. 15. Patii, S. D., Jones, C., Nair, M. G., Galivan, J., Maley, F.. Kisliuk, R. L., 4047-4054, 1988. Gaumont, Y., Duch, D., and Perone. D. Folate analogues. 32. Synthesis and 34. Westerhof, G. R., Jansen, G., Kathmann, G. A. M., Rijksen, G., and biological evaluation of 2-desamino-2-methyl-/V'Â°-propargyl-5,8-dideazafolic Schornagel, J. H. Characterization of receptor-mediated (anti)folate uptake acid and related compounds. J. Med. Chem., 32: 1284-1289, 1989. in human CCRF-CEM cells. In: H. C. Curtius, S. Gishla, and N. Blau (eds.). 16. Diddens, H.. Niethammer, D., and Jackson, R. C. Patterns of cross-resistance Chemistry and Biology of Pteridines 1989. pp. 1284-1287. Berlin: Walter to the antifolate drugs trimetrexate, metoprine. homofolate, and CB3717 in de Gruyter & Co., 1990. human lymphoma and osteosarcoma cells resistant to methotrexate. Cancer 35. Rothberg, K. G.. Ying, Y., Kolhouse. J. F., Kamen, B. A., and Anderson, R. Res., 43: 5286-5292, 1983. G. W. The glycophospholipid-linked folate receptor internalizes folate with 17. Jackson, R. C., Besserer, J. A., and Fry. D. W. Effects of extracellular out entering the clathrin-coated pit endocytic pathway J. Cell Biol., 110: concentration of folate or leucovorin on cellular sensitivity to trimetrexate, 637-649, 1990. CB3717 or methotrexate. Fed. Proc., 43: 950. 1984. 36. Kane. M. A., Elwood, P. C., Portillo, R. M.. Antony, A. C.. Najfeld, V., 18. Jackson, R. C. Unresolved issues in the biochemical pharmacology of anti Finley, A.. Waxman, S., and Kolhouse, J. F. Influence on immunoreactive folates. Nail. Cancer Inst. Monogr., 5:9-15, 1987. folatc-binding proteins of extracellular folate concentration in cultured hu 19. Takemura. Y., Ohnuma. T., Arkin. H.. Holland. J. F., and Calvert, A. H. man cells. J. Clin. Invest.. 81: 1398-1406. 1988. Biological effects of A'-(4-(AI((2-amino-4-hydroxyl-6-quinazonyl)methyl) 37. Beardslcy. G. P., Moroson, B. A., Taylor, E. C.. and Moran. R. G. A new prop-2-ynylamino)benzoyl)-i.-glutamic acid (CB3717) on human acute lym- folate antimetabolite, 5,10-dideaza-5,6,7,8-tetrahydrofolate is a potent inhib phocytic leukemia cell lines resistant to methotrcxale (MTX) or trimetrexate itor of de novo purine synthesis. J. Biol. Chem., 264: 328-333, 1989.

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Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1990 American Association for Cancer Research. Multiple Membrane Transport Systems for the Uptake of Folate-based Thymidylate Synthase Inhibitors

Gerrit Jansen, Jan H. Schornagel, G. Robbin Westerhof, et al.

Cancer Res 1990;50:7544-7548.

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