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.     

Effect of methotrexate on the leukemic cell cycle in mouse ascitic leukemia L1210]

In cells of L1210 ascite leukemia cells, methotrexate inhibited H3-thymidine incorporation, blocked shortly (during 4 hours) the G1 leads to S transition, and did not affect cells in G2-phase or in the late S phase. Almost half a cell population was degenerated and cells in S- and G1-phases were affected in equal proportion. This may suggest that methotrexate is not S-phase specific for cells of leukemia L1210. A simultaneous administration of vinblastine increases the antitumour effect of methotrexate. Cells in G2-phase constitute, presumably, a significant proportion of cells recovered after methotrexate administration. A comparison of the data obtained with literature evidence shows that in the sensitive (leukemia L1210) and resistant (acute mieloid leukemia of man) forms of leukemia, methotrexate affects cells that are in S-phase, whereas cells being in G1-phase are affected only when the sensitive tumours are treated.     

Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.     

Induction of cytoplasmic respiratory deficient mutants in yeast by the folic acid analogue, methotrexate

Summary The folic acid analogue, methotrexate, was found to be an effective inducer of the cytoplasmic petite mutation in different strains of S. carlsbergensis and S. cerevisiae. The induction varies considerably from strain to strain and also with growth conditions under which the experiments are carried out. Determination of mitochondrial protein synthesis in the presence of methotrexate shows a considerable decrease in incorporation of radioactive leucine before any significant induction of petites has taken place.These results and a comparison with results from other laboratories obtained with different drugs provide the basis for a proposed mechanism of petite induction by methotrexate.     

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.     

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.     

Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: involvement of a membrane receptor and a cytosolic protein

A radioiodinated photoaffinity analogue of methotrexate, N alpha-(4-amino-4-deoxy-10-methyl-pteroyl)-N epsilon-(4-azidosalicylyl)-L- lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells [Price, E. M., & Freisheim, J. H. (1987) Biochemistry 26, 4757-4763]. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degrees C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degrees C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active immunotherapy of L1210 leukemia with neuraminidase-treated,drug-resistant L1210 sublines

Summary The effectiveness of neuraminidase-treated, drug-resistant L1210 sublines in active immunotherapy of L1210 leukemia was evaluated. Optimal conditions for the establishment of in vitro, drug-resistant cells included (a) proper drug concentration, (b) the use of logarithmic-phase cultures in fresh medium, containing 5 or 10% serum, and (c) continual exposure to drug. Active immunotherapy, after tumor burden was reduced with chemotherapy, with neuraminidase-treated cells alone was either effective or deleterious, depending upon the drug-resistant subline used for immunization. The combination of BCG and neuraminidase-treated cells was superior to treatment with chemotherapy only. Optimal response was observed with the use of parental L1210 cells, combined with BCG, in immunotherapy of parental L1210 tumor. The results emphasize that an important prerequisite to successful immunotherapy is that tumor vaccines must elicit immunologic products which are cytotoxic for residual tumor.Submitted in partial fulfillment of the requirements for the Masters of Science Degree, University of Oklahoma     

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 degrees C, inhibition was rapid (t1/2 less than 1 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 degrees C. Inactivation in this case was qualitatively similar to the corresponding process at 37 degrees 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 degrees 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.     

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.     

Immunologic heterogeneity of dihydrofolate reductase from methotrexate sensitive and resistant L1210 leukemia cells

Dihydrofolate reductase from L1210 leukemia cells which are sensitive and resistant to methotrexate has the same physical and kinetic properties and immunoreactivity with a guinea pig antiserum raised to the enzyme purified from the methotrexate resistant strain. However, a chicken antiserum to dihydrofolate reductase from methotrexate sensitive L1210 cells has greater affinity for the homologous enzyme than for the enzyme from the MTX resistant cells indicating that there is some antigenic difference in these molecules.     

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.     

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.     

Membrane protein changes in an L1210 leukemia cell line with a translocation defect in the methotrexate-tetrahydrofolate cofactor transport carrier

We report on membrane protein changes in an L1210 leukemia cell line with a highly specific defect in the function of the methotrexate (MTX)-tetrahydrofolate cofactor transport carrier. This clonal line, MTXrA, made 100-fold resistant to MTX, was derived in a single step and exhibited stable resistance over 120 generations in the absence of drug. The transport defect was associated with a 10-fold decrease in influx Vmax without a change in influx Km. There was no difference between the MTXrA and parent lines in the levels or affinities of specific cell surface binders for MTX nor in the labeling of the 44-kDa membrane protein upon treatment with the specific affinity label, N-hydroxysuccinimide ester of tritiated MTX. Consistent with impaired carrier function was the observation that trans-stimulation of MTX influx by intracellular 5-formyltetrahydrofolate observed in the parent line was not demonstrated in the MTXrA line. The transport defect was highly specific for the MTX-tetrahydrofolate cofactor transport carrier. Initial uptake rates for 5-fluoro-2'-deoxyuridine and 2-deoxyglucose were unchanged and influx and net transport of alpha-aminoisobutyric acid were, in fact, increased. There was no cross-resistance of this line to phenylalanine mustard or cytosine arabinoside, agents that utilize specific amino acid and nucleoside transport carriers, respectively. SDS-polyacrylamide gel electrophoresis of purified plasma membrane preparations stained with Coomassie Blue revealed several protein differences between the parental and MTXrA lines. Most prominent is a band at approximately 190 kDa which ran with slightly greater mobility than a lesser staining band in the parent line. [3H]Borohydride labeling of cells also identified a distinct protein peak in the MTXrA line at approximately 190 kDa eliminated by prior treatment of cells with neuraminidase. Absence of expression of protein or mRNA related to the multidrug resistance gene as well as lack of cross-resistance to daunorubicin or trimetrexate indicate that this mechanism of resistance to MTX is completely unrelated to the multidrug resistance phenomenon observed with high molecular weight heterocyclic compounds. These data represent the first demonstration of membrane protein differences in a highly resistant L1210 murine leukemia cell line with a marked unique defect in MTX transport which appears to be related to impaired mobility of the tetrahydrofolate-cofactor carrier. Further studies are now required to elucidate the possible role of one or more of these proteins in the transport defect.     

Acidic dissociation constants of folic acid, dihydrofolic acid, and methotrexate.

The acidic dissociation constants in the range HO--1.5 to pH 7 of folic acid, dihydrofolic acid, methopterin (N(10)methylfolic acid), and methotrexate have been measured by potentiometric and spectrophotometric titrations. Assignment of these dissociations was made by comparison to model compounds, by proton magnetic resonance measurements, and by examination of associated ultraviolet absorbance changes. For folic acid, the dissociation constants are as follows: N(1), pK' 2.35; N(10), pK' 0.20; N(5), pK' greater than -1.5. For dihydrofolic acid: N(5), pK' 3.84; N(1), pK' 1.38; N(10), pK' 0.28. For methotrexate: N(1), pK' 5.71; gamma-carboxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5), boxyl, pK' 4.70; alpha-carboxyl, pK' 3.36; N(10), pK' 0.50; N(5) pK' greater than -1.5. For methopterin: acidic ionization of amide, pK' 7.68; gamma-carboxyl, pK' 4.62; N(1), pK' 2.40; N(10), pK; 0.36; N(5), pK' greater than -1.5. The pK' values were determined directly for the four compounds at 25 degrees near 0.1 ionic strength, or in 0.1 to 4 M HCl for pK ln 0.1 M NaCl.