Intracellular Methotrexate Research Articles

Anion exchange mechanism for transport of methotrexate in L1210 cells

Structurally diverse anions (folate, 5-formyltetrahydrofolate, AMP, ADP, thiamine pyrophosphate, phosphate, sulfate, and chloride) that are competitive inhibitors of methotrexate influx in L1210 cells also enhance the efflux of methotrexate from these cells. The increase in efflux reaches a maximum of 2- to 4-fold depending upon the anion employed, and the anion concentrations required for half-maximal stimulation of efflux are similar to their K i values for inhibition of methotrexate influx. A competitive inhibitor of methotrexate uptake (fluorescein-diaminopentane-methotrexate) that is not transported by this system, does not increase methotrexate efflux. These results suggest that the efflux of intracellular methotrexate is coupled to the concomitant uptake of an extracellular anion.

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.

Marrow cell kinetics in patients treated with methotrexate and citrovorum factor.

Methotrexate (MTX) was administered by continual intravenous infusion for 24 hours in doses ranging from 200-800 mg/m2 to 16 patients who had metastatic cancers and normal bone marrows and 11 patients with acute leukemia. Citrovorum factor (CF) was administered every 6 hours for 12-15 doses beginning 36 hours after the start of the MTX infusion. Plasma and urine were collected to measure MTX concentration. Daily bone marrow aspirates were obtained for measuring intracellular MTX concentration, labelling index (LI), mitotic index (MI), grain count distribution, cellular DNA distribution by flow cytometry (FCM), and marrow morphology. The plasma MTX concentration proved to be a function of dose and creatinine clearance. There was a positive correlation between the creatinine clearance and MTX clearance. The intracellular MTX concentration correlated highly with the plasma concentration. The LI, grain count, and proportion of cells in S phase increased on days 2 and 3. The magnitude of the changes on days 2 and 3 was dose related. The MI fell on day 2 and recovered by day 4. Transient megaloblastic changes occurred. The cell cycle perturbations in leukemic marrow were less pronounced than those in normal marrows. These observations are consistent with a transient, dose related, S phase delay.

Transport of methotrexate by primary cultures of rat hepatocytes: Stimulation of uptake in vitro by the presence of hormones in the medium

Rat hepatocytes in primary monolayer culture exhibit an energy-dependent, concentrative accumulation of methotrexate. This transport system appears not to be involved in the uptake of the reduced folate derivatives. The capacity to accumulate methotrexate is rapidly diminished during culture and several lines of evidence indicate that this is due to the nonphysiologic environment provided by the culture system. The uptake system is partially stabilized by including glucagon, hydrocortisone, and tocopherol in the medium; these factors increase both the rate and extent of accumulation of methotrexate and result in intracellular levels that are approximately 250% of control (4 h incubation) after 24 and 48 h in culture. The site of hormonal stimulation of uptake is not completely understood, but several features of this process are apparent. Including the additives in the medium does not alter either the metabolism of methotrexate to polyglutamates or its intracellular binding. Thus, the main difference between the control and treated hepatocytes appears to be the capacity to accumulate free intracellular methotrexate. These observations suggest that the hormones act in some manner to stabilize the plasma membrane, and more specifically, the transport system for methotrexate. A similar effect has been observed with the hepatic transport system for asialoglycoproteins. ( A. L. Tarentino and J. H. Galivan, 1980, In Vitro, 16, 833–846). Some understanding of the mechanism of the factors in stimulating methotrexate uptake can be achieved from these studies. Glucagon appears to be acting at least in part through adenosine 3′:5′-monophosphate since it can be replaced by either isoproterenol and theophylline or N 6,O 2′ -dibutyryl adenosine 3′:5′-monophosphate. Dexamethasone can replace hydrocortisone, showing that the metabolism of hydrocortisone is not necessary for it to be active. Tocopherol may be acting as an antioxidant since it can be totally replaced by ascorbic acid. None of the data indicate whether these factors act directly on the plasma membrane or by altering the level of various intracellular metabolites which in turn stabilize the membrane. They do suggest that losses in function that are observed in the transfer of hepatocytes from the intact organ to culture may be prevented by adding factors, particularly hormones, that the cells normally encounter in vivo, and that the restoration of function may in part reflect a physiological function of the hormones.

K +-induced alterations of energetics and exchange diffusion in the carrier-mediated transport of the folic acid analog, methotrexate, in ehrlich ascites tumor cells

The bidirectional fluxes and energetics of methotrexate transport in Ehrlich ascites tumor cells were profoundly altered in a high [K +], low [Na +] buffer (K + buffer). Incubation of cells for 30 min in K + buffer reduced influx by 27% and the efflux rate constant by 53%. This asymmetrical inhibition of bidirectional fluxes increased the net exchangeable intracellular methotrexate level per cell, but the actual intracellular methotrexate concentration at the steady state was similar to that in Na + buffer, since the high [K +] caused an increase in intracellular water. Because cells exposed to K + buffer were depolarized, the apparent electrochemical potential difference for methotrexate was markedly reduced. However, the steady-state intracellular methotrexate level was still related to the extracellular concentration by an absorption isotherm, indicating asymmetry in the bidirectional fluxes similar to that observed in Na + buffer and thus predicting that transmembrane gradients would be produced at very low extracellular methotrexate concentrations. Glucose, which had little effect on bidirectional fluxes and reduced the steady-state level of methotrexate in Na + buffer, stimulated influx, inhibited efflux and rapidly increased the steady state in K + buffer similar to the effects of glucose in the presence of iodoacetate in Na + buffer. Finally, cells exposed to K + buffer exhibited trans-stimulation of [ 3H]methotrexate influx when loaded with non-labeled methotrexate, a phenomenon not observed in Na + buffer. The results indicate that although methotrexate transport is not affected by transient changes in the cationic composition of the extracellular compartment, prolonged exposure of cells to a high [K +], low [Na +] environment markedly alters the physical properties of the cells and the transport parameters for methotrexate and reveals characteristics of the methotrexate carrier system that are not evident in other buffer systems.

5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates.

Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolated:dUMP C-methyltransferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and poly(gamma-glutamyl) conjugates of pteroate and methotrexate (MTX) has been examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(gamma-glutamate)s within 4 hr of exposure to 1 microM MTX. MTX and MTX(gamma-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and I-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to two sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(gamma-glutamate), 7,8-dihydropteroylpentaglutamate, and I-5-10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, < 10, and < 10 nM, respectively, by equilibrium dialysis. We propose that the sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.

Fluoropyrimidine-induced antagonism to free and tightly bound methotrexate: Suppression of [ 14C] formate incorporation into RNA and protein

The inhibitory effect of methotrexate (MTX) on [ 14C] formate incorporation into RNA and protein in Ehrlich ascites tumor cells is decreased by pretreatment with fluoropyrimidines. When the basal rate of [ 3H] deoxyuridine (UdR) incorporation into DNA is decreased to 26, 64 and 95% by 5-fluorodeoxyuridine (FUdR), the inhibitory effect of MTX on [ 14C] formate incorporation into (a) RNA is decreased to 40, 19 and 3%, and (b) protein is decreased to 52, 25 and 7%. MTX alone, however, suppressed [ 14C] formate incorporation into RNA and protein 56 and 67%, respectively. Free intracellular MTX is required for maximal suppression of [ 14C] formate incorporation into RNA and protein after pretreatment of cells with FUdR. When high-affinity binding sites for MTX are > 95% saturated with MTX, but free or exchangeable MTX is not present, [ 14C] formate incorporation into RNA and protein is stimulated in the presence of FUdR. However, as the intracellular levels of MTX in excess of the tightly bound fraction is increased with 1 μM and 10 μM MTX, there is progressive inhibition of [ 14C] formate incorporation into RNA and protein in cells exposed to FUdR. Theses data support the concept that fluoropyrimidine-induced antagonism to MTX is related, in part, to levels of reduced folates available for purine and protein synthesis.

Methotrexate polyglutamate synthesis by cultured human breast cancer cells.

We studied the conversion of methotrexate to poly-gamma-glutamyl derivatives by cultured human breast cancer cells. After incubation with 2 micro M [3',5',9-3H]methotrexate, MCF-7 cells were washed free of extracellular drug and were boiled to lyse cells and to release drug bound to dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The supernatant fraction was chromatographed on Sephadex G-15 to separate parent drug from polyglutamate forms. These cells rapidly and quantitatively converted methotrexate to polyglutamates, such that after 24 hr of incubation, 70 +/- (SEM) 3% of intracellular methotrexate existed as polyglutamates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase indicated that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. These studies demonstrate that methotrexate polyglutamates are readily formed in human tumor cells and bind to dihydrofolate reductase. Because these forms of the drug may be selectively retained within the cell, they may be important determinants of the duration of action and, ultimately, the cytotoxicity of methotrexate in human solid tumors.

Mechanism of synergistic cell killing when methotrexate precedes cytosine arabinoside: study of L1210 and human leukemic cells.

Synergistic killing of L1210 cells occurs when methotrexate (MTX) is administered just before 1-beta-D-arabinofuranosylcytosine (Ara-C). This pehnomenon is dependent upon both the dose and time of exposure to MTX. Such increased killing of cells can be explained by the enhanced intracellular accumulation of Ara-C in cells exposed to MTX. This enhancement of Ara-C entry into cells was only observed when the dose of MTX was high enough (1, 10, and 100 muM) to result in free intracellular nondihydrofolate reductase-bound MTX. At the highest doses of MTX (10 and 100 muM) Ara-C triphosphate was increased eightfold and deoxycytidine triphosphate was decreased by 50%. Therefore, the maximum synergistic cell kill when MTX precedes Ara-C may be the consequence of greater inhibition of DNA polymerase by th;e increased Ara-C triphosphate in the presence of the decreasing natural substrate of this enzyme, deoxycytidine triphosphate. Enhanced Ara-C accumulation after administration of MTX was also observed in human acute myelogenous leukemia cells.

654 TEMPORAL RELATIONSHIP BETWEEN ACCUMULATION OF METHOTREXATE POLYSLUTAMATES AND PROLONGED INHIBITION OF DNA SYNTHESIS

Human diploid fibroblasts grown in the presence of methotrexate (MTX) accumulated the drug and metabolized it to poly-γ-glutamyl metabolites (polyglutamates). After 15 min incubation in 1 μM MTX, the non-diffusable component of MTX in these cells was 58 pg MTX equivalent/mg cell protein, none of which consisted of polyglutamates. Accumulation of polyglutamates was linear with time so that after 6 h the cells contained 285 pg/mg of which 45 pg/mg was MTX, 162 pg/mg was MTX monoglutamate and 78 pg/mg was MTX diglutamate. It has been suggested that the continued presence of a component of intracellular MTX in excess of that bound to intracellular binders is required for the inhibition by MTX of deoxyuridine (dU) incorporation into DNA. When fibroblasts were preincubated for 1 h in 1 μM MTX-containing medium and then transferred into MTX-free medium, there was complete recovery of their ability to incorporate dU into DNA. However when the pre-incubation was increased to 4 and 6 h, the 24 h incorporation of dU decreased from 65.5 nM dU/gm cell protein to 17.5 and 4.9 nM/gm respectively. Thus inhibition by MTX of dU incorporation into DNA after the longer incubations was not dependent on the continued presence of MTX in the culture medium and was associated with a rise both in total intracellular MTX derivatives and most particularly in MTX polyglutamates.

In vitro correlates of clinical response to methotrexate in acute leukaemia and Burkitt's lymphoma

The response of drug-resistant patients with acute leukaemia and Burkitt's lymphoma to treatment with a 24 h infusion of methotrexate (MTX) followed, in some cases,by cytosine arabinoside was correlated with in vitro measurements of total intracellular MTX, exchangeable intracellular MTX, and suppressibility of deoxyuridine (UdR) incorporation in isolated marrow blast cells at extracellular MTX concentrations of 10(-8)M, 10(-7)M, 10(-6)M and 10(-5)M. Total intracellular MTX levels and exchangeable intracellular MTX levels were not significantly different in responding or non-responding patients at any MTX concentration, but increased four-fold for every ten-fold concentration increment studied. Extracellular MTX levels in excess of 10(-7)M appeared necessary to allow accumulation of exchangeable intracellular MTX. UdR incorporation at 10(-6)M and 10(-5)M differed significantly between responding and non-responding patients, with responders having less than 20% of control values and non-responders having greater than 40% of control values. Further, increasing the extracellular MTX concentration from 10(-6)M to 10(-5)M produced no significant decrease in UdR incorporation in either group. The therapeutic implications of this apparent threshold are discussed.

Relationship between intracellular dihydrofolate reductase and tightly bound intracellular methotrexate in human neoplastic cells

Relationship between intracellular dihydrofolate reductase and tightly bound intracellular methotrexate in human neoplastic cells

Characteristics of the Vincristine-induced Augmentation of Methotrexate Uptake in Ehrlich Ascites Tumor Cells

Abstract Studies were undertaken to characterize the effect of vincristine on methotrexate uptake in Ehrlich ascites tumor cells and to define the mechanism of this interaction. Vincristine (10 µm) does not alter the unidirectional influx of methotrexate but slows the unidirectional efflux of methotrexate and leads to a large increase in the steady state level of exchangeable intracellular methotrexate. These effects of vincristine are rapidly, but not completely, reversible. Vincristine stimulation of net methotrexate uptake occurs within 4 min of exposure of cells to this agent and increases over an extracellular vincristine concentration range of at least 5 to 50 µm. Vincristine does not alter the intracellular water content nor the chloride distribution ratio. The addition of glucose to the medium partially reverses the effect of vincristine and completely reverses the stimulatory effect of sodium azide on the net uptake of methotrexate. Vincristine (10 µm) increases net uptake of methotrexate in L1210 leukemia cells but does not have a significant effect on net methotrexate uptake in the L-cell mouse fibroblast over a 2½-hour interval of exposure even though replication of the L-cell is completely arrested. The similarity between the effects of vincristine and metabolic poisons on methotrexate uptake and the partial reversal of the effect of vincristine by glucose suggest that this agent enhances methotrexate uptake by the inhibition of cellular energy metabolism with the consequent inhibition of an energy-dependent process which limits methotrexate accumulation within the cell. The data suggest further that this effect of vincristine represents an interaction with a cellular element(s) that is different from the interaction which results in the arrest of cell division in metaphase.

Transport Energetics of the Folic Acid Analogue, Methotrexate, in L1210 Leukemia Cells: Enhanced accumulation by metabolic inhibitors

Energy-dependent transport of the folic acid analogue, methotrexate, was studied in L1210 murine leukemia cells. A number of metabolic inhibitors were found to enhance the unidirectional influx and accumulation of methotrexate. This phenomenon was studied in detail with sodium azide. The azide-induced increase in the unidirectional influx of methotrexate was associated with a rise in both the maximum transport velocity and the influx Michaelis constant. The effect of azide on influx was not related to an increase in the passive diffusion of methotrexate. Addition of pyruvate, an energy substrate for those metabolic processes inhibited by azide, was found to depress methotrexate influx in the presence and absence of azide. The unidirectional efflux of methotrexate was slowed by azide. Efflux from cells loaded to high levels of methotrexate in the presence of azide was a two-component process with the major part of intracellular methotrexate exiting at a slow and constant velocity. The data suggested that azide reduces the maximum transport velocity and the Michaelis constant for the unidirectional efflux process. In the absence of azide the electrochemical potential for intracellular methotrexate exceeds that for extracellular methotrexate. The data indicate that azide further increases this electrochemical potential difference. These studies therefore suggest that energy-dependent transport of methotrexate is enhanced in the presence of azide in the L1210 leukemia cell.

Carrier-mediated Transport of the Folic Acid Analogue, Methotrexate, in the L1210 Leukemia Cell

Abstract Transport of methotrexate (4-amino-10-methylpteroyl-glutamic acid), a structural analogue of folic acid, was studied in L1210 mouse leukemia cells grown in tissue culture. Intracellular methotrexate was present in two states. (a) There was an essentially nonexchangeable intracellular fraction probably bound to the enzyme dihydrofolate reductase. (b) The remaining intracellular methotrexate was rapidly exchangeable and likely present in the cell water in an osmotically active state. In addition, there was a small rapidly exchanging component of methotrexate associated with the cells which had the characteristics of a loose adsorption on or near to the cell membrane. Methotrexate was shown to be negligibly metabolized by this cell system. Because of the presence of tight intracellular binding sites for methotrexate, prior to saturation of these sites, free intracellular methotrexate does not appear and consequently efflux is negligible. This permits the accurate measurement of unidirectional uptake velocities. The major portion of the unidirectional influx process demonstrated Michaelis-Menten kinetics with a high degree of structural specificity, competitive inhibition by folic and folinic acids, and marked temperature dependence. Addition of these competing substances in excess to cells at the steady state with extracellular methotrexate leads to countertransport of intracellular methotrexate. Electrochemical potential gradients for free intracellular methotrexate were demonstrated at low levels of extracellular methotrexate and intracellular methotrexate concentrations at equilibrium exhibited Michaelis-Menten kinetics. Efflux of intracellular methotrexate was not first order and appeared to be partially saturated. The Michaelis constant and maximum velocity for the unidirectional efflux process were estimated, and when related to these parameters for the unidirectional influx, were compatible with an accumulating system. These studies indicate that methotrexate transport in the L1210 cell is carrier-mediated and uphill. Methotrexate exhibits several qualities which make it a valuable model substrate for the study of the transport characteristics of folic acid and its naturally occurring derivatives. The implication of these findings with respect to the development of more effective folic acid antagonists is considered.