Intracellular Methotrexate Research Articles

Intrinsic Resistance of Cervical Squamous Cell Carcinoma Cell Lines to Methotrexate (MTX) as a Result of Decreased Accumulation of Intracellular MTX Polyglutamates

The causes of intrinsic of intrinsic MTX resistance in four human cervical squamous cell carcinoma cell lines (SiHa, C-33A, ME-180, C-41) with different sensitivities to methotrexate (MTX) were determined. The in situ or whole-cell assay for thymidylate synthase (TS) was used to screen for known causes of MTX resistance, including increased dihydrofolate reductase (DHFR), altered DHFR, impaired MTX uptake, and decreased formation of MTX polyglutamates. While all four cell lines displayed initial sensitivity to MTX as demonstrated by a TS activity of <20% following a 3-hr incubation with MTX, TS activity recovered in three of the four cell lines following a 4-hr incubation in drug-free media. Determination of MTX-polyglutamate accumulation in the four cell lines following a 24-hr incubation with 10 μ M [ 3H]MTX revealed that a higher total intracellular level of MTX polyglutamates was achieved in the sensitive cell line (SiHa), with 54% occurring as long-chain tri-, tetra-, or pentaglutamates. The three resistant cell lines were found to contain less total MTX polyglutamates, with only 38, 14, and 13% occurring as long-chain MTX polyglutamates. Intrinsic MTX resistance in some cervical squamous cell carcinoma cell lines appears to be associated with a diminished accumulation of long-chain MTX polyglutamates, which are preferentially retained intracellularly.

Mechanism of the Discrepant Effect of a Combination of Methotrexate plus Dipyridamole on Human Hematologic Cell Lines

Mechanisms of the discrepant effect of methotrexate and dipyridamole on human hematologic cultured cell lines were investigated by analyzing intracellular methotrexate levels and thymidine incorporation through the salvage pathway, since the combination of methotrexate and dipyridamole has different effects according to cell type: additive effects on ML-1 and THP-1 (myelo-monocytoid cells); reduced effects on MOLT-3, SKW-3, P32/ish and BL-TH (lymphoid cells). Dipyridamole reduced the toxicity of methotrexate by diminishing intracellular methotrexate levels in MOLT-3 and BL-TH (lymphoid cells), in which the reduction of intracellular methotrexate affected more than just the blocking of the salvage pathway required for growth by dipyridamole. On the other hand, dipyridamole enhanced the toxicity of the combination by blocking the salvage pathway in an ML-1 (myelo-monocytoid cell) and in a methotrexate-resistant subline of BL-TH/MTX (lymphoid cell), in which the salvage pathways were considered activated. Dipyridamole could prove to be a useful drug for reversing the drug resistance caused by the activation of the salvage pathway.

Methotrexate Resistance in Datura innoxia (Uptake and Metabolism of Methotrexate in Wild-Type and Resistant Cell Lines).

A wild-type Datura innoxia cell line (Px4) was used to select methotrexate-resistant cells through a stepwise procedure. Two independently selected cell lines, MTX161 and MTX132, were stable and shown to be 5 to 15 times more resistant to methotrexate than wild type. These methotrexate-resistant cells were similar to the wild-type cells in levels and kinetic properties of dihydrofolate reductase, the sensitivity of dihydrofolate reductase to methotrexate, the binding of [3H]methotrexate to soluble proteins, and the formation of methotrexate polyglutamate derivatives. High performance liquid chromatographic analyses indicated that methotrexate polyglutamylation is only slight and may not be significant in the toxicity of methotrexate to Datura cells. The uptake of methotrexate was also investigated in the wild-type and resistant cells. The Px4 cells exhibited a linear uptake that lasted for 1 to 7 h. The uptake was saturable, pH and energy dependent, and had a Km of 65.6 nM and a Vmax of 12.5 nmol h-1g-1 fresh weight. Neither MTX161 nor MTX132 exhibited the sustained uptake of methotrexate shown by the Px4 cells. As a result, there were greatly reduced concentrations of intracellular methotrexate in resistant cells. Resistant cell lines had 2- to 3-fold higher Km values for methotrexate uptake compared with Px4 cells. It is proposed that these cells became resistant as a result of a stable change in the membrane transport system for methotrexate.

Interactions of vinblastine and vincristine with methotrexate transport in isolated rat hepatocytes.

The accumulation of methotrexate (MTX) in the presence of vinblastine (VBL) and vincristine (VCR) was studied in isolated rat hepatocytes. In accordance with our recent study on vindesine (VDS), we found VBL and VCR to reduce net MTX accumulation significantly at 15 min after MTX addition. Drug concentrations of 100 microM VBL and 500 microM VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 microM VBL, the accumulation data demonstrate that the major effect of VBL is on MTX influx. Dixon-plot analyses are suggestive of competitive inhibition of the MTX influx, yielding inhibition constants (Ki values) of 55 microM for VBL and 110 microM for VCR. Since the Ki values correspond grossly to plasma levels obtained in humans shortly after the infusion of therapeutic doses of the vinca alkaloids studied herein, the interaction with MTX uptake could serve to diminish the toxicity of MTX to nonmalignant cells.

Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate

Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei and this induction of micronuclei was enhanced by multiple treatments with the drug (Yamamoto et al., 1981; Hayashi et al., 1984; CSGMT/JEMS · MMS, 1990). More micronuclei and chromosomal aberrations in mouse bone marrow cells were induced by multiple than by single treatment. The MTX level in mouse plasma and bone marrow showed little (or no) differences between single and quadruple treatments several hours after the injection(s). On the other hand, the DHFR activity in bone marrow cells 3 h after one and four injections was decreased to approximately 38 and 0%, respectively, of that in non-treated mice. Furthermore, the intracellular MTX level in the bone marrow cells (but not in total bone marrow) after four injections was about 10-fold higher than that after one injection. The amount of MTX bound to protein 3 h after four injections, as assayed by gel filtration (Sephadex G-25), was approximately 8-fold greater than after one injection. Therefore, the multiple-dose effects of MTX on the induction of micronuclei and chromosomal aberrations may be explained by the intracellular accumulation of MTX resulting in an enhancement of enzyme inhibition.

The effect of vindesine on methotrexate hydroxylation in the rat

The effect of vindesine (VDS) on methotrexate (MTX) disposition was studied in biledrained rats administered VDS prior to [ 3H]MTX, and in isolated rat hepatocytes and rat liver homogenate concomitantly incubated with MTX and VDS at 37°. In vivo, 7-hydroxylation was reduced by 0.65 mg/kg VDS. In VDS-treated animals, biliary recovery of the MTX dose (50mg/kg) as 7-hydroxymethotrexate (7-OH-MTX) (1.75 ± 0.2%, mean ± SEM) was significantly reduced compared to controls (2.83 ± 0.57%). In vitro, hydroxylation of MTX (10–200 μM) in hepatocytes was reduced by 14.3 and 66.4% (means) at 12.5 and 100 μM VDS, respectively. With increasing VDS concentrations up to 100 μM, a reduction in intracellular MTX accumulation could account for the decreased MTX hydroxylation. Experiments in a cell free system gave no evidence of inhibition of 7-OH-MTX formation by VDS. In vitro MTX transport studies demonstrated that VDS inhibited the hepatocellular influx of MTX, as (1) the accumulation of MTX corresponded inversely to increasing VDS concentrations and (2) the MTX efflux was not increased by VDS. The apparent K i for VDS inhibition of MTX influx was 57 μM. We suggest that VDS, by reducing the 7-OH-MTX formation in liver cells, may have implications for combination chemotherapy regimens which include MTX.

A phase II study of combined methotrexate and teniposide infusions prior to reinduction therapy in relapsed childhood acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Teniposide (VM-26) can increase intracellular methotrexate (MTX) and its polyglutamate derivatives in vitro and thus has the potential to improve the therapeutic index of regimens containing MTX. In this phase II study, children and adolescents with acute lymphoblastic leukemia (ALL) in first or second marrow relapse were randomly assigned to receive either simultaneous (n = 11) or sequential (n = 12) continuous infusions of MTX and VM-26 prior to reinduction. Infusions of VM-26 were begun 12 hours after completion of MTX infusion in the sequential group. Dosages were individually adjusted to maintain plasma concentration levels of 10 microns for MTX and 15 microns for VM-26; total infusion times were 24 and 72 hours, respectively. Significant toxicity in the first six patients who received the scheduled 72-hour VM-26 infusion (including one drug-related death) prompted a 50% reduction in infusion duration. The reduced dose was associated with similar but more manageable toxicity. Examination of bone marrow aspirates 10 days after therapy was begun showed one complete and two partial marrow remissions; a fourth patient who had an aplastic marrow on day 10 received no further chemotherapy and had a complete remission (CR) documented on day 31. There was no obvious clinical advantage associated with either infusion schedule, although small sample sizes preclude definitive conclusions. The 17% response rate to the MTX/VM-26 therapeutic window in patients with refractory disease suggests the need for further investigation to evaluate alternative schedules and concomitant therapy for this drug combination.

Reversal of Methotrexate Cytotoxicity to Human Bone Marrow Cells and Leukemic K562 Cells by Leucovorin: Methotrexate Polyglutamates Formation as a Possible Important Factor

Methotrexate (MIX) is metabolized intracellularly to MTX‐polyglutamates (MTX‐PGs), which markedly inhibit several folate‐dependent enzymes. Polyglutamation defect, therefore, is one of the important factors in drug resistance. In this study, reversal of MTX cytotoxicity by l‐leucovorin (l‐LV) was investigated using normal human bone marrow granulocyte progenitor cells (G‐CFCs), and MTX‐sensitive and ‐resistant leukemic K562 cell lines; the latter showed diminished polyglutamation. Cytotoxicity of 10‐7M MTX to G‐CFCs was completely reversed by an equimolar concentration of l‐LV, but with higher MTX concentrations, relatively more l‐LV was required. The reversal of MTX cytotoxicity by l‐LV was more effective against bone marrow cells than MTX‐sensitive K562 cells; this reversal seemed to be correlated to the total intracellular MTX levels as well as MTX‐PG formation (low in bone marrow cells and high in K562 cells). When MTX‐sensitive and ‐resistant K562 cells were incubated with MTX under conditions in which the total intracellular MTX levels of both cells were similar, successful reversal of MTX toxicity by l‐LV was demonstrated in MTX‐resistant cells, but not in MTX‐sensitive cells, suggesting that an increase of MTX‐PG formation in MTX‐sensitive cells may explain the failure of l‐LV to overcome MTX cytotoxicity. In addition to competitive reversal of MTX cytotoxicity by LV, noncompetitive reversal relating to variable formation of MTX‐PGs is suggested to be another important factor in the mechanism of the reversal of MTX cytotoxicity by LV.

Impairment of Methotrexate (MTX)‐Polyglutamate Formation of MTX‐resistant K562 Cell Lines

We examined the mechanism of methotrexate (MTX) resistance in five K562 cell subclones resistant to MTX. Based on a clonogenic assay, the IC50s of these MTX‐resistant clones were 10 to 40μMMTX, indicating 2,000 to 5,000‐fold resistance as compared to that of the parent cell line. The doubling times of these MTX‐resistant K562 cell lines are longer (27–60 hr) than that of the parent K562 cell line (24 hr). One‐hour MTX accumulation in the resistant cells was 70–80% of that in parent cells. To investigate the formation of MTX‐polyglutamates (MTX‐PGs), resistant cells were incubated with 3H‐MTX (1 or 1OμM) for 24 hr in the presence of thymidine and deoxyinosine to prevent cytotoxicity. MTX (‐Glu1) and the polyglutamate metabolites (MTX‐Glu2, ‐Glu3, ‐Glu4 and ‐Glu5) were analyzed by a high‐pressure liquid chromatography (HPLC) technique. After a 24‐hr incubation with 10μM MTX, the total concentration of intracellular MTX reached 39 to 89 nmol/g protein, only 20 to 40% of the MTX level of the parent K562 cells. The HPLC analysis revealed that less than 2% of intracellular MTX was in the form of high‐molecular MTX‐PGs (MTX‐Glu3, ‐Glu4 and ‐Glu5) in the five MTX‐resistant K562 cell lines, while in the parent cells MTX‐Glu3–5 comprised 46% of the total intracellular MTX. These data indicate the possibility that impairment of MTX‐PG formation, with transport alteration, may be a special mechanism for the high level of resistance to this agent in human leukemic cells.

High-dose methotrexate therapy with leucovorin rescue: in vitro investigations on human osteosarcoma cell lines.

High-dose methotrexate (MTX) therapy with subsequent leucovorin (LV) rescue (HDMTX-LV) in the treatment of osteosarcoma is based on the assumption that this tumor has a deficient uptake system for MTX and reduced folates. To simulate features of HDMTX-LV therapy protocols in vitro, sensitive and MTX-resistant human osteosarcoma cell lines and a lymphoblastoid cell line were exposed to MTX and/or LV at various dosages and time schedules and the effects on DNA metabolism and on cell growth were evaluated. The data show that in osteosarcoma cells and in lymphoblasts the cytotoxic effects of 10(-6) M to 10(-7) M MTX can be substantially reversed by LV if the antidote is applied within the first 12 h of MTX exposure. The results are not consistent with the assumption mentioned above and should be taken into consideration when designing new therapeutic regimens. An alternative hypothesis for the efficacy of HDMTX-LV is discussed. It is concluded that HDMTX-LV therapy may be effective in the treatment of osteosarcoma, even when subpopulations of the tumor cells exhibit different mechanisms of resistance to MTX, such as elevated levels of dihydrofolate reductase or a deficient transport system for MTX, if high doses of MTX are applied long enough to ensure lethal intracellular MTX levels and low-dose LV schedules instituted after a long delay are used.

Metabolism of methotrexate and γ-tert-butyl methotrexate by human leukemic cells in culture and by hepatic aldehyde oxidase in vitro

The cellular uptake and metabolism of methotrexate (MTX) and γ- tert-butyl methotrexate (TBM) were compared in CEM human leukemic lymphoblasts and a highly MTX-resistant subline (CEM/MTX) in which MTX uptake is defective. The CEM/MTX cells were found previously to be as sensitive as the parent line to TBM. While MTX was polyglutamylated extensively in the CEM cells, giving abundant levels of non-effluxing conjugates, polyglutamylation in CEM/MTX cells was reduced severely, even after exposure to a high MTX concentration (100 μm) in the medium. This treatment provided free intracellular MTX in >100-fold excess over the dihydrofolate reductase level. In contrast to MTX, the ester TBM was unmetabolized in either cell line. Uptake levels after incubation of CEM and CEM/MTX cells with 2 μM TBM for 24 hr were 17 and 15 pmol/mg protein respectively. Thus, TBM accumulated equally in both cells and was well retained despite the lack of polyglutamylation. These results, together with the previously observed affinity of the drug for dihydrofolate reductase, provide a plausible rationale for the comparable sensitivity of CEM and CEM/MTX cells to TBM. Experiments were also performed to determine the susceptibility of TBM to metabolic detoxification by hepatic aldehyde oxidase. K m , values were 8-fold lower for TBM than for MTX in assays using an enzyme preparation from rabbit liver, and V max values were 8-fold higher. Neither MTX nor TBM was oxidized to its 7-hydroxy derivative in intact CEM or CEM/MTX cells. Because TBM is capable of overcoming at least one of the modalities of MTX resistance, defective polyglutamylation, and may be more efficiently detoxified than MTX by the action of hepatic aldehyde oxidase, it has the potential to be a useful agent for the treatment of MTX-resistant tumors.

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.

Kinetics of Methotrexate and Its Metabolites in Red Blood Cells

It has been suggested that the intra-erythrocyte levels of methotrexate (MTX) and its polyglutamized derivatives could offer a method for evaluating intracellular MTX accumulation and its metabolism in children with acute lymphoblastic leukemia. We were interested in measuring the intra-erythrocyte levels of MTX in patients with solid tumors receiving high-dose MTX treatment. After a first incorporation stage occurring during infusion, MTX concentrations subsequently increased 9-12 days after the treatment as polyglutamized derivatives. Thirty days after the infusion, MTX and its polyglutamates were still measurable in erythrocytes. The percentage of polyglutamization varied on an individual basis, but two groups of patients could be separated according to their ability to form polyglutamates. We also noted the presence of 7-hydroxy-methotrexate (7-OH-MTX) appearing 48 hours after the beginning of the infusion which was still present in 17/20 samples 30 days after the treatment.

Glutamylation of methotrexate in hepatoma cells in vitro: Regulation and the development of specific inhibitors

Methotrexate is glutamylated in cultured hepatoma cells to derivatives that contain a total of 2 to 5 γ-glutamyl residues. The rate of polyglutamate formation and extent of accumulation are saturable with respect to both medium concentration of methotrexate and time. Maximal rates of glutamylation and accumulation of methotrexate polyglutamates at steady state occur at approximately 10 μ m extracellular methotrexate. Inclusion of physiologic concentrations of insulin or removal of folate from the medium each cause a doubling of the rate of glutamylation, and these effects are additive. Insulin and folate restriction also enhance the accumulation of methotrexate polyglutamates. In combination they result in a doubling in the intracellular methotrexate polyglutamate pool at steady state and a shift in the polyglutamate distribution to longer-chain-length species. The importance of the longer-chain-length polyglutamates is apparent from the 6-hr retention of the polyglutamate species: Glu 2, 15%; Glu 3, 21%; Glu 4, 50%; and Glu 5, 83%. In probing the glutamylation reaction, a new series of inhibitors have been initiated. These are based upon replacing the incoming glutamate with 4-fluoroglutamate or synthesizing methotrexate with the glutamate replaced by 4-fluoroglutamate. The 4-fluoroglutamyl analogs of methotrexate are effective inhibitors of dihydrofolate reductase but cannot be glutamylated. They will be utilized to probe the role of glutamylation in antifolate activity and folate metabolism.

Rate-limiting steps in the interactions of fluoropyrimidines and methotrexate

Rate-limiting steps are defined between methotrexate (MTX) and 5-fluorouracil (FU) or 5-fluorodeoxyuridine (FUdR) and [ 14C]-formate incorporation into RNA, DNA and protein as a function of the basal rate of dTMP synthesis. When Ehrlich cells are incubated with 0.1 μM FUdR, 1 μM FU and 50 μM MTX for 1–35 min, [ 3H]-deoxyuridine (UdR) incorporation into DNA is maximally inhibited within 1, 10 and 15 min respectively. The delay in suppression of [ 3H]-UdR incorporation into MTX-exposed cells compared to cells exposed to FU or FUdR is related to the slow transport of MTX and the increasing free intracellular MTX levels. Influx of MTX is 4 and 10 times slower than FU and FUdR respectively. At 2.5, 5, 10 and 15 min the free intracellular MTX levels (nmol/g dry wt) are 5.8, 7.4, 8.7 and 8.8 respectively. Free intracellular FdUMP is identified 1 min after exposure of cells to FU and FUdR. Antagonism to MTX-suppression of [ 14C]-formate incorporation into RNA, DNA and protein occurs when cells are simultaneously exposed to MTX and FU or FUdR. However, [ 14C]-formate incorporation into RNA, DNA and protein is maximally inhibited when Ehrlich tumor cells are incubated with 50 μM MTX for 10 min and then exposed to 1 μM FU for 1 min (a time in which free intracellular MTX is maximal and [ 3H]-UdR incorporation is maximally suppressed). Hence the sequence and time of administration of FU or FUdR and MTX inhibition of formate incorporation into RNA, DNA and protein is related to the rate of (a) FU, FUdR and MTX transport, (b) FU and FUdR metabolism to FdUMP and (c) generation of maximal free intracellular MTX.

Hormonal alteration of methotrexate and folate polyglutamate formation in cultured hepatoma cells

Glutamylation of the antifolate methotrexate in H35 hepatoma cells was stimulated by physiologic concentrations of insulin and dexamethasone. At saturating concentrations of the hormone a 2.7-fold stimulation could be obtained with insulin (65 n m, 16-h exposure) and a 1.8-fold stimulation with dexamethasone (100 n m, 16-h exposure). The increases in glutamylation caused by the hormones were not additive, and both were inhibited by actinomycin D and cycloheximide. N 6, O 2′-dibutyryl cAMP and theophylline caused a modest reduction of glutamylation in control and dexamethasone-treated cultures, but repressed the stimulation caused by insulin by approximately one-third. Enhancement of synthesis by dexamethasone and insulin was associated with increases in the tri-, tetra-, and pentaglutamate derivatives of methotrexate, with little change in intracellular methotrexate and methotrexate diglutamate. When the conversion of folinic acid into the folylpolyglutamate pool was examined in folate-depleted H35 cells, insulin and dexamethasone had similar effects. The results suggest that these hormones play a role in the glutamylation of the folate coenzymes in a liver-derived transformed cell line in culture and that these effects are also reflected in the interaction of the cells with antifolates such as methotrexate.

Characteristics of the accumulation of methotrexate polyglutamate derivatives in Ehrlich ascites tumor cells and isolated rat hepatocytes.

The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate and their interactions with H2 folate reductase was evaluated in the Ehrlich ascites tumor cell and the isolated rat hepatocyte. Methotrexate polyglutamates were detected within 15 minutes in hepatocytes exposed to 1 microM methotrexate, and continued to accumulate for at least 60 minutes producing a large transmembrane gradient. These derivatives appeared to be preferentially retained within the cell even under conditions where release of intracellular methotrexate was induced by dibutyryl cyclic AMP or isobutyl methyl xanthine. Deoxycholate and bromosulfophthalein, compounds which inhibited methotrexate influx into hepatocytes, reduced the ratio of methotrexate polyglutamates to methotrexate, suggesting that these agents also inhibit the metabolism of methotrexate. In studies with the Ehrlich ascites tumor accumulation of methotrexate polyglutamates was increased over 5-fold by the addition of 5 mM L-glutamine or L-glutamate and exhibited a positive correlation with the extracellular concentration of methotrexate. Vincristine and probenecid, agents that increase the intracellular levels of methotrexate by inhibiting efflux, also produced a marked increase in methotrexate polyglutamates. When Ehrlich ascites tumor cells were exposed to 10 microM methotrexate and 5 mM L-glutamine intracellular polyglutamates were detected within 10 minutes and their levels increased linearly over 4 hours. As these derivatives accumulated, there was a decline in intracellular methotrexate due at least in part to a replacement of methotrexate on H2 folate reductase by polyglutamates and subsequent efflux of the previously bound methotrexate from the cell. When ppolyglutamate derivatives were in excess of the H2 folate reductase binding capacity and extracellular methotrexate removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the major portion of the enzyme. These studies indicate that (1) intracellular methotrexate is rapidly converted to polygammaglutamyl derivatives, (2) these metabolites effectively compete with methotrexate for binding sites on H2 folate reductase, (3) these derivatives are retained within the cell more effectively than methotrexate, and (4) vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells.

Reduction of the anti-metabolic and anti-proliferative effects of methotrexate by 17β-oestradiol in a human breast carcinoma cell line, MDA-MB-436

We have investigated the modifying influence of 17β-oestradiol on the anti-metabolic and growth inhibitory actions of methotrexate (MTX) in a human breast cancer cell line, MDA-MB-436. This cell line contains detectable oestrogen receptors but is progesterone receptor-negative. Furthermore, 17β-oestradiol (10 −10−10 −6 M) failed to influence DNA synthetic rate as assessed by [ 3H] -TdR or [ 3H] -UdR incorporation and cell proliferative rate was similarly unaffected. Although by these criteria 17β-oestradiol failed to elicit a biological response in the MDA-MB-436 cell line, 10 −6M 17 β-oestradiol significantly reduced the anti-metabolic and anti-proliferative actions of MTX. In the presence of 17β-oestradiol approximately twice the concentration of MTX was required to inhibit cell proliferation to the same extent as was observed following exposure to MTX alone. This partial reversal of MTX effects was accompanied by a 20% reduction in the steady-state intracellular MTX concentration when cells were exposed to the drug in the presence of 10 −6 M 17 β-oestradiol.

Synthesis, binding and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells.

Synthesis, binding, and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells were investigated by gel filtration and high-pressure liquid chromatography to separate methotrexate from its metabolites. MCF-7, ZR-75-1, and MDA-231 human breast cancer cells were found to readily convert methotrexate to higher polyglutamates during a 24-hr incubation period, although at differing rates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase revealed that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. Similarly, methotrexate polyglutamates accumulated free in the cytosol and when cells were suspended in drug-free medium, were retained intracellularly both bound to dihydrofolate reductase and in the unbound fraction, indicating their slow passage through the cell membrane. Studies of methotrexate polyglutamate binding to purified bacterial dihydrofolate reductase revealed high affinity binding for compounds with up to 6 additional glutamyl residues. These studies demonstrate that methotrexate polyglutamates are readily formed in human breast cancer cells, bind intracellularly to dihydrofolate reductase, and are selectively retained both bound to the enzyme and free in the cell cytosol.

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).