Irreversible inhibitors of methotrexate transport in L1210 cells: Characteristics of inhibition by an N-hydroxysuccinimide ester of methotrexate

Methotrexate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide react to form an activated ester of methotrexate which is a potent irreversible inhibitor of methotrexate transport in L1210 cells. In cells treated with the reagent at 37°C, inhibition was rapid ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{< 1}\text{min}$), optimal at pH 6.8, half-maximal at an inhibitor concentration of 20 nM, and complete at high levels of the reagent. Specificity was indicated by the fact that excess methotrexate added during the pretreatment step protected the transport system against inactivation. Irreversible inhibition was also observed in cells exposed to the reagent at 4°C. Inactivation in this case was qualitatively similar to the corresponding process at 37°C; it appeared rapidly, was half-maximal at 20 nM, and could be prevented by the addition of high concentrations of the substrate. The extent of the inhibition, however, reached a maximum of only 75%, even in samples containing excess or multiple additions of reagent. The latter findings suggest that at 4°C the transport protein exists in two forms, one (75% of the total) containing binding sites which are accessible to the active ester, and the other (25% of the total) with inaccessible sites. The identity of these sites is suggested to be transport proteins which have outward and inward orientations, respectively.     

Affinity labeling of the 5-methyltetrahydrofolate/methotrexate transport protein of L1210 cells by treatment with an N-hydroxysuccinimide ester of [3H]methotrexate

An N-hydroxysuccinimide ester of [3H]methotrexate has been employed to covalently label a specific binding protein that resides in the plasma membrane of L1210 cells. Incorporation of radioactivity into this protein accounted for 55% of total cellular labeling, was half-maximal at a reagent concentration of 27 nM, and was blocked either by prior exposure to unlabeled reagent or by the addition of excess methotrexate. A role for this protein in methotrexate transport was supported by the observations that: (a) similar concentrations of reagent were required for both labeling of the binding protein and irreversible inhibition of transport; (b) the amount of labeled binding protein was comparable to observed levels of transport protein; (c) protection against labeling was afforded by thiamin pyrophosphate, a potent competitive inhibitor of methotrexate transport; and (d) labeling of the binding protein was not observed in a subline of L1210 cells that has a defect in the ability to transport methotrexate. The binding protein could be solubilized from the membrane by various ionic and non-ionic detergents and the covalent bond between the incorporated [3H]methotrexate and the protein was stable to a variety of conditions, including high concentrations of mercaptoethanol and hydroxylamine and extremes of pH. The labeled protein fractionated as a nearly symmetrical peak on Sephacryl S-300 and it appeared as a single band (Mr = 36,000) after electrophoresis in polyacrylamide gel containing sodium dodecyl sulfate.     

Photoinactivation of the methotrexate transport system of L1210 cells by 8-azidoadenosin 5'-monophosphate.

Methotrexate transport in L1210 cells is mediated by a carrier protein that can bind organic and inorganic phosphate compounds in addition to the various folate substrates. The photoaffinity labeling agent, 8-azidoadenosine 5'-monophosphate (8-azido-AMP), also interactis (Ki = 140 microM) with the receptor site for this transport system, and upon irradiation with ultraviolet light, irreversibly inhibits methotrexate uptake. Protection against this inactivation is afforded by either a substrate (methotrexate) or a competitive inhibitor (inorganic phosphate). The light-induced reaction proceeds rapidly (t1/2 = 2 min at 23 degrees C under the conditions described) and produces half-maximal reduction in the transport rate when the 8-azido-AMP concentration is 65 microM. complete photoinactivation of methotrexate transport could not be obtained from a single exposure to 8-azido-AMP (up to 1.0 mM), but it could be achieved by the repetitive illumination of cells in a fresh medium. The phosphate and folate/adenine transport systems of L1210 cells are not affected by irradiation in the presence of 8-azido-AMP.     

Transport of methotrexate in L1210 cells: Mechanism for inhibition by p-chloromercuriphenylsulfonate and N-ethylmaleimide

Methotrexate transport in L1210 cells is highly sensitive to inhibition by p-chloromercuriphenylsulfonate (CMPS) and, to a lesser extent, by N-ethylmaleimide. A 50% reduction in the methotrexate influx rate occurred upon exposure of cells to 3 μM CMPS or 175 μM N-ethylmaleimide, while complete inhibition was achieved at higher levels of these agents. Dithiothreitol reversed the inhibition by CMPS, suggesting that a sulfhydryl residue is involved. This residue is apparently not located at the substrate binding site of the transport protein, since methotrexate failed to protect the system from inactivation by either CMPS or N-ethylmaleimide, and the transport protein retained the ability to bind substrate (at 4°C) after exposure to these inhibitors (at 37°C). Methotrexate efflux was also inhibited by CMPS (50% at 4 μM), indicating that both the uptake and efflux of methotrexate in L1210 cells occur via the same transport system. High concentrations of CMPS (greater than 20 μM) increased the efflux rate, apparently by damaging the cell membrane and allowing the passive diffusion of methotrexate out of the cell.     

Folate uptake in L1210 cells: mediation by an adenine transport system.

Uptake of folate by L1210 cells in mediated by a transport system whose primary substrate is adenine. This conclusion is based upon the following evidence: (a) Folate uptake is inhibited competitively by adenine; (b) The K_t for folate transport (430 μM) is comparable to the K_i (450 μM) for folate inhibition of adenine transport; (c) The K_t for adenine transport (21 μM) agrees with the K_i (17 μM) for inhibition of folate transport by adenine; (d) The adenine analogs, 1-methyl-3-isobutylxanthine and 6-mercapto-purine, each inhibit folate and adenine transport to a comparable degree; and (e) Rates of folate and adenine uptake vary in parallel fashion during growth of L1210 cells.     

Structural requirements for anion substrates of the methotrexate transport system in L1210 cells

A broad spectrum of structurally diverse anions reversibly inhibits the influx of methotrexate in L1210 cells. Several of the more effective anions and their respective inhibition constants (K_i values) were: 5-methyltetrahydrofolate (0.3 μm), bromosulfophthalein (2 μm), thiamine pyrophosphate (3 μm), 8-anilino-1-naphthalene sulfonate (7 μm), phthalate (20 μm), and AMP (50 μm). Moderate inhibition was observed with P_i (K_i = 400 μm) and other divalent inorganic anions, while small monovalent anions such as Cl^? (K_i = 30 mm) were the least effective. When these same anions were tested for an effect on methotrexate efflux, stimulation was observed with some anions, while others had no effect. Enhancement was produced by folate compounds and p-aminobenzoylglutamate, small monovalent (e.g., Cl^?, acetate, and lactate) and divalent (e.g., phosphate and succinate) anions, a few nucleotides (e.g., AMP), and thiamine pyrophosphate, while little or no effect was associated with trivalent anions (e.g., citrate), most nucleotides, and large organic anions (e.g., bromosulfophthalein, NAD, and NADP). Anions with the ability to promote methotrexate efflux in control cells lost this capacity upon exposure of the cells to an irreversible inhibitor of methotrexate influx. These results support the hypothesis that methotrexate transport proceeds via an anion-exchange mechanism and moreover provide evidence that anion substrates for this system can be identified by their ability to promote methotrexate efflux. Anions which appear most likely to participate in this exchange cycle in vivo are P_i and AMP.     

Binding properties of the 5-methyltetrahydrofolate/methotrexate transport system in L1210 cells

A binding component with a high affinity for 5-methyltetrahydrofolate (K_D = 0.11μm) is present on the external surface of L1210 cells. The amount of binder (1 pmol/mg protein) corresponds to 8 × 10⁴ sites per cell. The participation of this component in the high-affinity 5-methyltetrahydrofolate/methotrexate transport system is supported by similarities in the K_D values for 5-methyltetrahydrofolate and methotrexate binding and the K_t values of these compounds for transport. Relative affinities for other folate substrates (aminopterin, 5-formyltetrahydrofolate, and folate) and various competitive inhibitors (thiamine pyrophosphate, ADP, AMP, arsenate, and phosphate) are also similar for both the binding component and the transport system. The measured binding activity does not represent low-temperature transport of substrate into cells, since it is readily saturable with time and is eliminated by either washing the cells with buffer or by the addition of excess unlabeled substrate.     

Use of non-physiological buffer systems in the analysis of methotrexate transport in L1210 cells

Methotrexate transport parameters have been compared in L1210 cells suspended in a series of HEPES buffer systems of varying ionic compositions. While no effect was observed on the Vmax for methotrexate influx, the Kt for half-maximal influx, the steady-state level of methotrexate, and the efflux rate each varied substantially and to an extent which could be correlated directly to the anionic composition of the external medium. Buffer composition also affected the membrane potential, the ATP level of the cells, and, in one instance, the cell volume, but these changes did not exert a significant effect on the transport process. These results suggest that the integrity of L1210 cells is not adversely affected by either the presence of HEPES in the suspending medium or by the absence of certain physiological ions, and, moreover, that methotrexate transport parameters measured under these conditions, although not necessarily indicative of the quantitative events that might occur in vivo, can nevertheless provide meaningful information on the properties and mechanism of this transport system.     

Intracellular phosphate and its possible role as an exchange anion for active transport of methotrexate in L1210 cells

L1210 cells transport P_i in the absence of added Na⁺. Uptake shows saturation kinetics (K_t = 1.7 mM), is temperature-dependent, and can be reduced 80% by high levels of unlabeled P_i, and thus has the characteristics of a carrier-mediated process. This transport process is also inhibited by methotrexate. The methotrexate-sensitive component constitutes half of total P_i uptake, and is reduced by 50% at a concentration of methotrexate (2 μM) that is comparable to its K_t (1.5 μM) for transport into the cells. An impermeable fluorescent analog of methotrexate and an irreversible inhibitor of the methotrexate transport system (carbodiimide-activated methotrexate) also inhibit this same P_i uptake component. It is concluded that methotrexate and P_i can be transported by the same carrier system. The basis for this shared uptake is suggested to be that the methotrexate carrier protein facilitates the obligatory exchange of extracellular folate compounds for intracellular divalent anions, and that a primary exchange anion is P_i. A principal energy source for active transport of methotrexate might then be the concentration gradient for P_i that is maintained by the Na⁺-dependent, P_i transport system of these cells.     

Effects of inhibition of N-linked glycosylation by tunicamycin on nucleoside transport polypeptides of L1210 leukemia cells

Membrane polypeptides (relative mass (Mr) 48,000--55,000) associated with the equilibrative transport of nucleosides were identified in cultured murine leukemia (L1210/C2) cells by site-specific photolabeling with [3H]nitrobenzylthioinosine ([3H]NBMPR). Growth of cells in the presence of tunicamycin resulted in the gradual conversion of 3H-labeled polypeptides to a form that migrated more rapidly (Mr 42,000--47,000) during sodium dodecyl sulfate (SDS)--polyacrylamide gel electrophoresis. When plasma membrane fractions were photolabeled and incubated with O-glycanase or endoglycosidase F, the [3H]NBMPR-labeled polypeptides migrated in SDS-polyacrylamide gels with the same mobility as native NBMPR-binding polypeptides, whereas incubation with either N-glycanase or trifluoromethane sulfonic acid converted [3H]NBMPR-labeled polypeptides to the more rapidly migrating form (Mr 41,000--48,000). These observations are consistent with the presence of N-linked oligosaccharides of the complex type on the NBMPR-binding polypeptides of L1210/C2 cells. Tunicamycin exposures that reduced incorporation of [3H]mannose into plasma membrane fractions by greater than 95% had little, if any, effect on either the affinity (Kd values, 0.1-0.2 nM) or abundance (Bmax values, 200,000--220,000 sites/cell) of NBMPR-binding sites, whereas uridine transport kinetics at 37 degrees C were altered in a complex way. Thus, although N-linked glycosylation is not required for insertion of the NBMPR-binding protein into the plasma membrane or for interaction of NBMPR with the high-affinity binding sites, it is important for function of at least one of the three nucleoside transporters expressed by L1210/C2 cells.     

Sodium-dependent nucleoside transport in mouse leukemia L1210 cells

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.     

Characterization of the methotrexate transport defect in a resistant L1210 lymphoma cell line

L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg²⁺ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells was well below the intracellular level of dihydrofolate reductase (45 nmol/10⁹ cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{min}$). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). K_t and V values of 2 × 10^?5m and 0.31 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells/min, respectively, were determined for the apparent influx in $\text{L}\text{1210}\text{R}\text{81}$ cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment.     

A fluorescent derivative of methotrexate as an intracellular marker for dihydrofolate reductase in L1210 cells

Fluorescein isothiocyanate coupled via a diaminopentyl-linking group to methotrexate (G.R. Gapski, J. M. Whiteley, J. I. Rader, P. L. Cramer, G. B. Henderson, V. Neef, and F. M. Huennekens, 1975, J. Med. Chem.18, 526–528) produces a fluorescent compound which is a strong inhibitor of dihydrofolate reductase (K_i = 60 nM) purified from L1210 murine leukemia cells. The fluorescent methotrexate derivative is preferentially taken up by methotrexate-resistant rather than wild-type L1210 cells grown in culture and acts as a visual marker for dihydrofolate reductase (K_D = 50 nM) during both purification and polyacrylamide electrophoresis. Uptake, which is proportional to the level of dihydrofolate reductase (often an indicator of the degree of acquired cellular methotrexate resistance), occurs slowly and via a route that is distinct from the carrier-mediated system utilized by these cells to transport methotrexate.     

Transport routes utilized by L1210 cells for the influx and efflux of methotrexate

Routes which contribute to the transport of methotrexate across the plasma membrane of L1210 cells have been evaluated. A single high affinity transport system was found to be the only route for methotrexate uptake. This conclusion was derived from the observations that influx at high substrate concentrations (up to 50 microM) both reaches a single maximum value and can be inhibited by greater than 98% either by treatment of the cells with an active ester of methotrexate or by the direct addition of excess amounts of competitive inhibitors. Efflux, conversely, could be separated into three components. One of these routes was dependent upon extracellular anions and could be blocked by active ester treatment and, therefore, appeared to be the same transport system which mediates methotrexate influx. A second route was identified by its sensitivity to bromosulfophthalein, while a third component was insensitive to both active ester treatment and to bromosulfophthalein. When these efflux routes were quantitated in a buffered saline medium, the methotrexate influx carrier was found to account for the major portion (71%) of total efflux. The inhibitor-insensitive component contributed an additional 23%, while the remaining 6% was attributable to the bromosulfophthalein-sensitive route. The addition of glucose increased total efflux by 3-fold and caused a substantial change in the proportion of efflux that occurred via each of the three components. The major portion of efflux (46%) now occurred via the bromosulfophthalein-sensitive route, while the influx carrier contributed only 29% of the total. The inhibitor-insensitive route accounted for the remaining 25%. The opposite result was obtained with metabolic inhibitors which decreased total efflux but increased the contribution by the influx carrier to greater than 80%. The demonstration of multiple routes for methotrexate efflux and their differential sensitivities to alterations in energy metabolism thus provides a basis for explaining previously described asymmetries between the influx and efflux of methotrexate in mouse leukemia cells.     

Characterization of the multiple transport routes for methotrexate in L1210 cells using phthalate as a model anion substrate

Summary o-Phthalate is actively transported into L1210 cells and the primary route for cell entry is the same transport system which mediates the influx of methotrexate and other folate compounds. The identity of the influx route has been established by the following observations: (A) Phthalate influx is competitively inhibited by methotrexate and the inhibition constant (K _i ) is comparable to theK _i for half-maximal influx of methotrexate; (B) Various anions inhibit the influx of phthalate and methotrexate with comparableK _i values; (C) The influx of phthalate and methotrexate both fluctuate in parallel with changes in the anionic composition of the external medium; and (D) A specific covalent inhibitor of the methotrexate transport system (NHS-methotrexate) also blocks the transport of phthalate. In contrast, the efflux of phthalate does not occur via the methotrexate influx carrier, but rather by two separate processes which can be distinguished by their sensitivities to bromosulfophthalein. Efflux via the bromosulfophthalein-sensitive route constitutes 75% of total efflux and is enhanced by glucose and inhibited by oligomycin. The inability of phthalate to exit via the methotrexate influx carrier is due to competing intracellular anions which prevent phthalate from interacting with the methotrexate binding site at the inner membrane surface.     

Irreversible inhibition of a monoclonal antibody by a nitrophenyl ester

TEPC-15 is a phosphorylcholine-binding mouse myeloma protein which reacts with an ester-containing phosphorylcholine, thep-nitrophenyl ester of 6-(phosphorylcholine)hexanoic acid (PEPCH). The rate of nitrophenolate release mediated by the antibody ispH-dependent and increases with increasingpH. The antibody becomes inactive during the reaction with the ester. The inactive antibody is not reactivated even after treatment with hydroxylamine. Antibody activity is associated with the Fab' fragment. These observations together with thepH profile of the reaction suggest that the ester acylates a lysine side chain near the antibody-binding site.A preliminary discussion of this work was presented at the 78th Annual Meeting of the American Society of Biological Chemists, Philadelphia, Pennsylvania, 1987.     

The influence of methotrexate pretreatment on 5-fluorouracil metabolism in L1210 cells

Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 microM methotrexate. The nucleotide derivatives of FUra, including a 5-fluoro-2'-deoxyuridylate, and 5-fluorouridine-5'-triphosphate were also increased nearly 5-fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-pyrophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrexate between 0.1 microM and 10 microM. Both the increase in 5-phosphoribosyl-1-pyrophosphate and FUra accumulation could be prevented by the addition of Leucovorin (N5-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrexate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de novo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl-1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate pools were reduced following methotrexate pretreatment by the addition to the cultures of hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for the conversion to IMP, the intracellular accumulation of FUra was not enhanced. Also, if the inhibitor of 5-phosphoribosyl-1-pyrophosphate synthetase, 7-deazaadenosine, was given to cultures with methotrexate, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of FUra accumulation. In addition, when 5-fluoro-2'-deoxyuridine was added with the methotrexate to cell cultures, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of intracellular FUra accumulation. These results indicate that the ability of methotrexate to enhance FUra accumulation was probably the consequence of the antipurine effect of methotrexate which resulted in a reduction of the complex feedback inhibition on 5-phosphoribosyl-1-pyrophosphate synthesis and utilization. The resultant increased 5-phosphoribosyl-1-pyrophosphate pools were then capable of being utilized for the conversion of FUra to 5-fluorouridylate, the possible rate-limiting step in FUra intracellular metabolism and the major determinant of the rate of intracellular FUra accumulation. When methotrexate preceded FUra, there was synergistic cell killing as determined by soft agar cloning. The exact mechanism of this sequential synergistic antitumor activity may be the result of the enhanced incorporation of FUra into RNA, since the increased 5-fluoro-2'-deoxyuridylate which is formed is unlikely to increase substantially the inhibition of dTMP synthesis induced by methotrexate pretreatment.     

Nucleoside transport in L1210 murine leukemia cells. Evidence for three transporters

L1210 murine leukemia cells have two nucleoside transport activities that differ in their sensitivity to nitrobenzylmercaptopurine riboside (NBMPR). This study re-examines NBMPR-insensitive nucleoside transport in these cells and finds that it is mediated by two components, one Na(+)-dependent and the other Na(+)-independent. A mutant selected previously for loss of NBMPR-insensitive transport lacks only the Na(+)-independent activity. When NBMPR is used to block efflux via the NBMPR-sensitive transporter, uptake of formycin B (a nonmetabolized analog of inosine) is concentrative in both the parental and mutant cells, but the intracellular concentration of the nucleoside is 5-fold lower in the parental cells. Decreased accumulation of formycin B in the parental cells is due to efflux of the nucleoside via the NBMPR-insensitive, Na(+)-independent transporter that the mutant lacks. The Na(+)-dependent transporter appears to accept most purine, but not pyrimidine, nucleosides as substrates. Two exceptions are uridine, a good substrate, and 7-deazaadenosine, a poor substrate. In contrast, all of the nucleosides tested are substrates for the Na(+)-independent transporter. We conclude that L1210 cells have three distinct nucleoside transporters and that the specificity of the Na(+)-dependent transporter is similar to that of one of the two Na(+)-dependent nucleoside transporters seen in mouse intestinal epithelial cells.