Formation Of Methotrexate Polyglutamates Research Articles

Optimizing antimetabolite-based chemotherapy for the treatment of childhood acute lymphoblastic leukaemia.

Optimizing antimetabolite-based chemotherapy for the treatment of childhood acute lymphoblastic leukaemia.

Time course of methotrexate polyglutamate formation and degradation in the pre-B-leukaemia cell line Nalm6 and in lymphoblasts from children with leukaemia.

With the aim of investigation, the mechanisms of resistance to methotrexate (MTX) in children refractory to leukaemia-treatment, we established a method of analysing MTX metabolism in Nalm6 cells (human pre-B). The optimal extracellular concentration for MTX uptake and MTX polyglutamate (MTXPG2-6) formation at a density of 5 x 10(6) cells/ml was 1 microM 3H-MTX. After 15 h incubation at this concentration, a plateau of 5 pmol/10(6) cells of total MTX accumulated in the form of equal amounts of polyglutamates 3, 4 and 5 and low amounts of MTX and polyglutamates 2 and 6. MTX preloaded cells rapidly lost MTX and MTXPG2 in MTX-free medium, while MTXPG5 was still formed and then degraded very slowly. After 8 h in medium without MTX, 40% of total MTXPG was lost, after 24 h, 70%. The method is feasible for patient blasts. The number of blasts isolated from bone marrow after diagnosis is enough to perform small kinetic studies. The uptake of MTX into patient blasts is about 1/10 of that in Nalm6 cells.

Characterization of cross-resistance to methotrexate in a human breast cancer cell line selected for resistance to melphalan

We have demonstrated previously decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan (MelR MCF-7). Cross-resistance studies of MelR MCF-7 cells revealed that this cell line was 6.7-fold cross-resistant to methotrexate, but only 2-fold resistant to trimetrexate. Methotrexate transport studies in MelR MCF-7 cells showed a 2-fold decrease in initial methotrexate uptake and a 2-fold decrease in the V max for methotrexate uptake in the resistant cells. Methotrexate resistance in MelR MCF-7 cells was also associated with a decrease in non-effluxable methotrexate following incubation with radiolabeled drug for 24 hr. Characterization of intracellular methotrexate after accumulation for 24 hr demonstrated decreased levels of free methotrexate in MelR MCF-7 cells. Analysis of methotrexate polyglutamate formation in MelR MCF-7 cells indicated that the decrease in non-effluxable, non-protein-bound methotrexate was associated with a 3-fold decrease in higher order methotrexate polyglutamate formation. No difference was noted in folylpolyglutamate synthetase activity between the resistant and parental cell lines. Therefore, the observed decrease in methotrexate polyglutamate formation in MelR MCF-7 cells appeared to result from decreased availability of substrate. There was no evidence of any alteration in the amount of the catalytic activity of dihydrofolate reductase in MelR MCF-7 cells compared with parental MCF-7 (WT MCF-7) cells; moreover, the binding affinity of dihydrofolate reductase for methotrexate and the percentage of protein-bound methotrexate were similar in both cell lines. In addition, the total amounts of thymidylate synthase protein and thymidylate synthase catalytic activity in MelR MCF-7 cells were unchanged. Thus, acquired methotrexate resistance in MCF-7 cells selected for resistance to melphalan appears to result from down-regulation of methotrexate uptake.

Methotrexate osteopathy in infants with tumors of the central nervous system

Methotrexate osteopathy, previously reported as a complication of maintenance-therapy for acute lymphoblastic leukemia, is characterized by osteopenia, particularly involving the lower extremities, thick, dense provisional zones of calcification, growth arrest lines, and corner fractures resembling scurvy. In attempts to postpone radiotherapy in infants under three years of age, the multicentric German therapy protocol for childhood central nervous system tumors (HIT-89 protocol) has employed high cumulative methotrexate doses. Here we describe osteopathy in three patients as a toxic side effect after administration of cumulative methotrexate doses of 20 g/m2, 80 g/m2 and 135 g/m2. The high prevalence of this adverse effect in infants with tumors of the central nervous system may be attributed to the long-term therapy with high cumulative methotrexate-doses. Both factors may favor intracellular accumulation of methotrexate and formation of methotrexate-polyglutamates and may be responsible for bone toxicity. Apparently the susceptibility of the rapidly growing skeletal structures of infants under three years of age to this toxic side effect of methotrexate is remarkably high.

Methotrexate metabolism in mutant chinese hamster ovary cells lacking dihydrofolate reductase

To study the influence of the level of dihydrofolate reductase (DHFR) on methotrexate (MTX) metabolism, the formation of methotrexate polyglutamates (MTXPGs) and the retention of the drug were examined in Chinese hamster ovary cells (DUKXB11) lacking DHFR and in control cells (CHO-UTC). Both cells accumulated MTXPGs poorly. After a 24-hr incubation with 1.0 μM [ 3H]MTX, the level of total MTX in DUKXB11 cells was 40% of that in CHO-UTC cells, reflecting the lack of DHFR-bound MTX and MTXPGs in the mutant cells. MTXPGs accounted for a higher proportion of the intracellular MTX in DUKXB11 than in CHO-UTC cells (25 vs 18%). Following exposure to 3.0 μm MTX for 24 hr, total drug levels were similar in both cell lines, and MTXPGs constituted even more of the intracellular drug in DUKXB11 cells compared to CHO-UTC cells (34 vs 23%). DUKXB11 cells accumulated longer MTXPGs (MTXG1u 3,4) compared to CHO-UTC cells (MTXGlu 2,3), following exposure to both 1.0 and 3.0μM MTX. The longer MTXPGs in the mutant cells may have resulted from the lack of DHFR in them. Binding of MTXPGs to DHFR in CHO-UTC may interfere with their further polyglutamylation. When cells were resuspended in drug-free buffer for 1 hr following a 24-hr incubation with MTX, the retention of drug was less in DUKXB11 cells (46%) than in CHO-UTC cells (78%), due mainly to a greater loss of unmetabolized MTX in the mutant cells (89%) than in control cells (26%). Nevertheless, the amount of non-exchangeable unmetabolized MTX retained in DUKXB11 cells following exposure to 3.0 μm MTX exceeded the MTX-binding capacity. These studies demonstrate that DHFR-deficient cells accumulated more and longer MTXPGs than control cells. In addition, they suggest that some unmetabolized MTX was retained in cells not bound to DHFR.

The effects on 4-aminoantifolates on 5-formyltetrahydrofolate metabolism in L1210 cells. A biochemical basis of the selectivity of leucovorin rescue.

This report describes studies designed to evaluate possible inhibitory effects of diaminoantifolates on folate-dependent biosynthetic enzymes in intact L1210 leukemia cells. A novel approach is described which involves an assessment of the metabolism of and biosynthetic flux of the one-carbon moiety from (6S)5-formyltetrahydrofolate in folate-depleted cells. Pretreatment with methotrexate (10 microM), resulting in the formation of methotrexate polyglutamates, or continuous incubation with trimetrexate (1 microM) inhibited growth of folate-depleted L1210 cells in the presence of folic acid or 5-formyltetrahydrolate. In both control and drug-treated cells, double-labeled (6S)-5-[14C]formyl[3H]tetrahydrofolate was rapidly metabolized with the loss of the [14C]formyl group. Under all conditions, the predominant metabolite was 10-formyl[3H]tetrahydrofolate, detectable both intracellularly and extracellularly. In drug-treated cells, there was a remarkably small decrease in the level of 10-formyl[3H]tetrahydrofolate (approximately 30%) and a 10-fold rise in the level of [3H]dihydrofolate to less than 20% of the total folate pool. The incorporation of [14C]formyl group from 5-[14C]formyltetrahydrofolate into thymidylate, serine, and methionine was unaffected by the presence of 1 microM trimetrexate, consistent with the generation of sufficient 5,10-[14C]methylenetetrahydrofolate to drive these reactions. Similarly, the presence of methotrexate polyglutamates had no effect at the level of amino acid synthesis; however, carbon transfer into thymidylate was markedly inhibited. Even though 10-formyltetrahydrofolate was readily formed from 5-formyltetrahydrofolate in this model, the net incorporation of 14C from 5-[14C]formyltetrahydrofolate into purine nucleotides was inhibited by both methotrexate and trimetrexate treatments. Similar findings were obtained when [14C]glycine incorporation into purine nucleotides was monitored in cells incubated with unlabeled 5-formyltetrahydrofolate. Finally, in antifolate-treated cells incubated with unlabeled 5-formyl-tetrahydrofolate, transfer of 14C from [14C]formate or [14C]serine into biosynthetic products or incorporation of [3H]deoxyuridine into nucleic acids was potently inhibited. These results suggest that insufficient levels of tetrahydrofolate and 5, 10-methylenetetrahydrofolate were formed to drive these reactions despite the presence of high levels of 10-formyltetrahydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)

Methotrexate metabolism analysis in blood and liver of rheumatoid arthritis patients: Association with hepatic folate deficiency and formation of polyglutamates

Serum and red blood cell methotrexate (MTX) levels, as well as hepatic levels of MTX and folate, were analyzed in 24 patients who had received long-term oral MTX weekly for the treatment of rheumatoid arthritis. The serum MTX level peaked rapidly and was insignificant after 24 hours. The red blood cell MTX level was not related to the interval from the last MTX dose. In hepatic tissue obtained by liver biopsy, MTX was found in a predominantly polyglutamated form with depleted hepatic folate stores when compared with baseline specimens. A brief period of therapy with oral folinic acid repleted hepatic folate. It is possible that MTX hepatotoxicity is related to reduced hepatic folate levels and formation of MTX polyglutamates.

Formation of methotrexate polyglutamates in purified myeloid precursor cells from normal human bone marrow.

Immature myeloid precursor cells were preferentially selected from normal human bone marrow by using immune rosette techniques that employed monoclonal antibodies against mature granulocytes, monocytes, T and B lymphocytes, and erythroid precursors (Mo5, M3, OKT3, B1, and EP1, respectively). We examined the formation, retention, and cytotoxic effects of methotrexate (MTX) polyglutamates (MTX-PGs) in these purified myeloid precursor cells. After 1- and 24-h exposures to MTX, with thymidine and deoxyinosine as rescue, the intracellular MTX-PG profile was examined by high-pressure liquid chromatography. Efflux patterns of MTX-PGs were also studied after additional 1- and 24-h incubations in drug-free media. Cytotoxic effects of retained MTX-PGs on bone marrow myeloid precursors were examined by colony formation in drug-free semisolid agar. Normal myeloid precursor cells converted MTX to MTX-PGs in a concentration- and time-dependent manner, preferentially retaining MTX-PGs with three to five glutamyl moieties. At low concentrations of MTX (1 microM), MTX-PG formation was insufficient to maintain saturation of the target enzyme dihydrofolate reductase after removal of drug from the incubation medium, and there was no decrease in myeloid colony formation. At higher concentrations of MTX (10 microM), formation of higher molecular weight polyglutamates was sufficient to allow for 24-h saturation of intracellular binding capacity after removal of extracellular drug and resulted in a 35% reduction in the formation of colony-forming units in culture. Comparison of MTX metabolism in normal bone marrow cells and the MTX-sensitive HL-60 human leukemia cell line showed twofold greater PG formation by these tumor cells after 24-h exposure to 1 or 10 microM MTX, and a marked (greater than 30-fold) increase in cytotoxicity for the HL-60 cells as compared with normal myeloid precursors, suggesting that the MTX polyglutamation may be important to its selective antitumor action.

A methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates.

Methotrexate (MTX)-resistant human breast cancer cells (MTXR ZR-75) were obtained following serial passage of the wild-type ZR-75-1 cells (wild-type ZR-75) in MTX. The resistant cell line contains neither quantitative nor qualitative changes in dihydrofolate reductase compared to the parental line. Resistance is associated with a 3-fold decrease in MTX transport into MTXR ZR-75 cells as well as a 3-fold decrease in the activity of thymidylate synthetase in the resistant subline. Moreover, marked differences were observed between the wild-type and MTXR ZR-75 cells in their ability to convert MTX to its polyglutamate derivatives. Wild-type ZR-75 cells accumulate significant intracellular levels of antifolates during prolonged (24 h) exposure to 2 microM MTX, due to the formation of MTX polyglutamates. In contrast, essentially no polyglutamates are formed in the MTXR cells even during conditions which result in a vast excess of free intracellular drug in these cells. This defect is not associated with any apparent change in the activity of the enzyme folylpolyglutamyl synthetase, nor is there any alteration in the apparent Km of this enzyme for MTX in the resistant cells. Further studies demonstrate that the MTXR ZR-75 cells are cross-resistant to antifolate analogues which can be converted to polyglutamate derivatives (aminopterin and dichloromethotrexate), yet they are relatively sensitive to antifolate analogues such as 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, triazinate, and trimetrexate, which cannot be converted to polyglutamate forms. These studies identify a new mechanism (diminished accumulation of MTX polyglutamates) associated with resistance to MTX and lend additional support to the hypothesis that the formation of these derivatives is an important determinant of MTX cytotoxicity.

Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells

The glutamylation of methotrexate has been evaluated in H35 hepatoma cells in vitro as a function of the conditions of culture. Glutamylation yields methotrexate polyglutamate with two to five additional glutamate residues and is a saturable process. The rate of glutamylation increases little above 10 μ m extracellular methotrexate which corresponds to an intracellular concentration of approximately 4 μ m. The rate of glutamylation measured over a 6-h period was stimulated by a reduction in cellular folates and prior incubation of the cells with insulin. Glutamylation was also more rapid in dividing cultures than in confluent cells. The combination of insulin inclusion and folate reduction, which was additive, caused approximately a fourfold increase in the rate of glutamylation over control cells under the conditions tested. The maximal rate of methotrexate glutamylation, which was 100 nmol/g/h, occurred in folate-depleted, insulin-supplemented cells. Supplementing folate-depleted cells with reduced folate coenzymes caused the glutamylation to be reduced by more than 90%. The turnover of methotrexate polyglutamates in cells saturated with these derivatives occurred at approximately one-half the rate of net synthesis and was stimulated to nearly the same extent by folate depletion and insulin. In addition to showing that folates can modify the rates of methotrexate polyglutamate formation, data are presented suggesting that methotrexate polyglutamates can regulate their own synthesis. The consequences of the formation of these retained forms of methotrexate in H35 hepatoma cells ( M. Balinska, J. Galivan, and J. K. Coward (1981) Cancer Res. 41, 2751–2756 ) and the effects of potential regulators of this process are discussed in terms of the glutamylation of folates in the cells and the chemotherapeutic effects of antifolates.

The kinetics of methotrexate polyglutamate formation and efflux in a human breast cancer cell line (MDA.MB.436): The effect of insulin

The rate and extent of the formation of methotrexate poly-γ-glutamates has been studied in a human breast cancer cell line (MDA.MB.436). Cells were exposed to medium containing 10 −7 M radiolabelled methotrexate (MTX) for various periods. Cell extracts were subjected to gel filtration on Bio-Gel P2 and radioactivity in each fraction determined. This has allowed quantitation of the relative amounts of MTX and its polyglutamate derivatives. It has been proposed that MTX polyglutamates may act as storage forms of the drug which could result in prolonged cytotoxicity. This possibility was investigated by determining the ability of the cell line to retain MTX and its olyglutamate derivatives for periods of up to 48 hr after removal of MTX from the incubation medium. Our results show that the lower mol. wt polyglutamates (< three extra glutamic acid residues) rapidly efflux from the cell, while the higher mol. wt species (> three glutamic acid residues) are extensively retained, having an efflux half-life of approximately 30 hr. It has been shown that insulin may potentiate the cytotoxicity of MTX to breast cancer cells in vitro. Our results indicate that in the MDA.MB.436 cell line the total intracellular drug at the steady state is unaffected by insulin (10 −6 M). However insulin does result in a 30% increase in the contribution of the higher mol. wt polyglutamates to the total intracellular drug. Our data therefore suggest that the ability of insulin to potentiate the cytotoxic effects of MTX may be related to the hormone's ability to modulate the synthesis of MTX polyglutamates.

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH 2-10-CH 3PteGlu 4). When the time-dependent synthesis of MTX polyglutamates (4-NH 2-10-CH 3PteGlu 2 and higher) at extracellular concentrations of 10 and 100 μ m methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μ m is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μ m intracellular methotrexate, respectively, after 24 h in the presence of 100 μ m extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μ m with both cell types which corresponded to 10 to 12 μ m intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu 4–5) are more avidly retained by the cells than the shorter ones (Glu 2–3).

Factors controlling the concentrations of methotrexate in cultured hepatic cells

The polyglutamate metabolites of methotrexate are as inhibitory to the target enzyme dihydrofolate reductase as is methotrexate. Because of their greater retention they have a longer half-life within the cells and thus a greater potential for cytotoxicity. These metabolites have been found in numerous cells and tissues and are extensively synthesized in cultured hepatic cells. Uptake of methotrexate by primary cultures of rat hepatocytes occurs by a pathway which is independent of the folate coenzymes but appears to be related in some way to cholic acid and organic anion uptake. The evidence for the commonality of these pathways is (a) an instability of both uptake systems in the absence of hormones in the culture medium, (b) nearly equal inhibition of uptake by PCMS and NEM, and (c) cross competition of cholic acid and methotrexate for entry into the cells. Cholic acid and BSP can also selectively inhibit methotrexate polyglutamate formation in hepatocytes. Methotrexate entry into H35 hepatoma cells is mediated by the transport system which is shared by folate coenzymes and is not inhibited by cholic acid, BSP or sulfhydryl reagents. At concentrations of cholic acid or BSP which inhibit methotrexate polyglutamate formation in hepatocytes there is little or no loss of polyglutamate formation in H35 cells, possibly because BSP and cholic acid are taken up less by H35 cells than by hepatocytes.