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PubMed1985

Effect of depletion of cellular glutathione on methotrexate influx, efflux and retention in hepatocytes.

In isolated hepatocytes the influence of cellular glutathione (GSH) on initial influx, net uptake and efflux of methotrexate (MTX) was determined. Endogenous glutathione in rat liver cells was depleted by either fasting of rats or by in vivo administration of phorone prior to cell preparation. The initial rate of influx of MTX was found to be higher in hepatocytes of fasted and phorone-treated rats than in those of untreated, fed control rats. The Km values for the methotrexate influx in GSH-deficient hepatocytes were up to 3 times lower than in normal cells, whereas Vmax remained unchanged. These results disclose an increased efficiency of the MTX transport system in cells with diminished cellular GSH levels. On the other hand, titration of external membrane SH groups by 203Hg p-CMBS revealed up to three times higher amounts of free SH groups on cells from starved and phorone-treated rats than on hepatocytes of fed rats. Increased efficiency of the MTX transport system in GSH-deficient cells may, therefore, be interpreted as increased capacity of the MTX transport carrier for which free membrane SH groups are known to be essential. Despite activation of initial transport of MTX here, later net accumulation of MTX became smaller than in cells with normal GSH levels. Efflux of MTX from liver cells was not influenced by fasting or phorone treatment of rats, however, the "nonexchangeable" pool of MTX was found to be decreased, which indicates inhibition of formation of MTX polyglutamates here. This inhibition was most likely responsible for the decreased amounts of MTX finally accumulated in GSH-deficient hepatocytes.

4-Chloromercuribenzenesulfonate↗
PubMed1987

Metabolism of methotrexate and gamma-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 gamma-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 microM) in the medium. This treatment provided free intracellular MTX in greater than 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 microM 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. Km values were 8-fold lower for TBM than for MTX in assays using an enzyme preparation from rabbit liver, and Vmax 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.

Aldehyde Oxidase↗
PubMed1987

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 microM [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 microM 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 (MTXG1u3,4) compared to CHO-UTC cells (MTXG1u2,3), following exposure to both 1.0 and 3.0 microM 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 microM 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.

Animals↗
PubMed1988

Effects of methotrexate on purine and pyrimidine metabolism and cell-kinetic parameters in human malignant lymphoblasts of different lineages.

MOLT-4 (T-), RAJI (B-), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 microM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depending on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 microM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 microM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 microM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 microM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Cycle↗
PubMed1993

Retentiveness of methotrexate polyglutamates in cultured L1210 cells. Evidence against a role for mediated plasma membrane transport outward.

In the present report, studies are described examining the issue of methotrexate (MTX) polyglutamate retentiveness in cultured L1210 cells. Measurements made in intact L1210 cells showed that the rate of egress of [3H]MTX and [3H]MTX+G1 was different depending upon whether [3H]-MTX and its polyglutamates were accumulated intracellularly following biosynthesis during growth (3 hr) in the presence of [3H]MTX or when cells were pulse loaded (5 min) with [3H]MTX or [3H]-MTX+G1 just prior to measurement of egress. In the former case, [3H]MTX egressed with a T1/2 of 15 +/- 2 min, while [3H]MTX+G1 egressed with a T1/2 of 50 +/- 9 min. In pulse loaded cells, both [3H]MTX and [3H]MTX+G1 egressed with a T1/2 of 3.5 +/- 0.5 min. The same rapid egress of [3H]MTX and [3H]MTX+G1 seen following pulse loading was also documented in cells grown for 3 hr in the presence of nonradioactive MTX to normalize conditions with respect to the intracellular accumulation of [3H]MTX polyglutamates seen during exposure of cells to [3H]MTX during growth. In light of these results, MTX, MTX+G1, MTX+G2 and MTX+G4 were examined directly as permeants for the outwardly-directed ATP-dependent pump (Schlemmer SR and Sirotnak FM, J Biol Chem 267: 14746-14752, 1992) mediating most of MTX efflux in intact L1210 cells using inside-out plasma membrane vesicles isolated from these cells. ATP-dependent efflux of [3H]MTX and [3H]MTX+G1 exhibited values for Km of 46-50 microM and values for Vmax of 102-106 pmol/min/mg protein. As competitive inhibitors of [3H]MTX and [3H]MTX+G1 efflux, MTX+G1 and MTX, respectively, exhibited Ki values of 43-47 microM, that is, Km approximately Ki for both permeants. Also, values for Ki of 45-48 microM were obtained with MTX+G2 and MTX+G4 as competitive inhibitors of [3H]MTX efflux. From these results, we conclude that MTX and its polyglutamates are equivalent as copermeants for ATP-dependent efflux through the plasma membrane and that retentiveness of MTX polyglutamates is not determined at this level in these cells.

Adenosine Triphosphate↗
PubMed1996

Comparison of methotrexate polyglutamylation in L1210 leukemia cells when influx is mediated by the reduced folate carrier or the folate receptor. Lack of evidence for influx route-specific effects.

We previously described a methotrexate-resistant L1210 cell line (MTXrA) that lacks a functional reduced folate carrier and does not appreciably express the folate receptor. In the present study, we utilized MTXrA cell lines stably transfected with cDNAs encoding either the folate receptor or the reduced folate carrier to investigate the influence of the route of folate influx on the rate and extent of methotrexate polyglutamylation. At an extracellular methotrexate concentration of 0.1 microM, influx in the folate receptor transfectant (MTXrA-TF1) and in the reduced folate carrier transfectant (MTXrA-R1) was equal and methotrexate polyglutamates accumulated at an identical rate, but the onset was delayed until dihydrofolate reductase was saturated with the monoglutamate (approxmately 3 hr). The onset of polyglutamate formation was immediate and identical among the lines in cells pretreated with the lipophilic dihydrofolate reductase inhibitor trimetrexate to block methotrexate binding to dihydrofolate reductase. The spectra of individual methotrexate polyglutamates that accumulated were similar, with the tetraglutamate present as the predominant form. A 100-fold higher methotrexate concentration was required to detect methotrexate uptake and polyglutamylation in the transport defective parent MTXrA line, demonstrating that diffusion or an unidentified low affinity route also supports polyglutamylation. Since the folate receptor and the reduced folate carrier achieve nearly identical rates of polyglutamylation despite very different mechanisms of methotrexate delivery, the data suggest that transport-mediated substrate channeling to folylpolyglutamate synthetase is unlikely to play a role in tetrahydrofolate metabolism. This study supports the notion that it is the intracellular concentration of methotrexate achieved within the cell that drives polyglutamylation irrespective of its route of entry.

Animals↗
PubMed1984

An unusual suppression of immune response: pretreatment with Poly(Glu60,Phe40) suppresses response to poly(Glu60,Phe40).

The synthetic random copolymer poly( Glu60 , Phe40 ) ( GPhe ) is an excellent immunogen in SWR/J (H-2q) mice. The intravenous injection of soluble GPhe by itself led to antibody production and GPhe -specific T-lymphocyte proliferation. The proliferating lymphocyte was sensitive to anti-Thy 1.2 and anti-Ly 1 antisera. A subsequent immunization with GPhe should therefore lead to an enhanced response. Yet, a single intravenous injection of an aqueous solution of GPhe suppressed the immune response to a subsequent immunization with GPhe in CFA as measured by GPhe -specific plaque-forming colonies, ELISA, T-cell proliferation, and delayed-type hyper-sensitivity. The suppression was not transferable from pretreated mice into normal or irradiated syngeneic recipients with either sera or lymphoid cells. The antibody generated from the pretreatment could not be responsible for the suppression as injection of SWR anti- GPhe antibody into SWR/J mice enhanced, rather than suppressed, the response to the subsequent immunization with GPhe . Pretreatment of mice with a rabbit anti-idiotypic antiserum produced against (SWR anti- GPhe antisera) had no effect on the immune response to GPhe . Thus, the suppression cannot be explained by a simple B-cell tolerance mechanism. This type of unusual suppression was observed only with mice of H-2q haplotype and not with mice of H-2 haplotypes a and k which are also responders to GPhe .

Animals↗
PubMed1990

Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. I. GPhe-stimulated enhancement of antigen-dependent proliferative responses.

Following interaction of the random polymer (Glu60,Phe40)n (GPhe) with antigen-presenting accessory cells (APC), unusual costimulatory activities were noted in several murine T cell systems. When GPhe, in contrast with other random copolymers (GT,GL), was added during "inhibition" and T cell "repertoire" studies as a (negative) control to GLA-reactive nonclonal T cell lines of haplotypes H-2d (DCL-2) or H-2bm12, augmentation of T cell proliferation ([3H]thymidine incorporation ([3HT]) to homologous antigen was observed. Augmentation by GPhe was also observed in the response of a GLPhe-reactive (H-2s X H-2d)F1 T cell line and the allogeneic response of the clonal T cell line D10.G4.1. This augmentation was critically dependent on the concentration of adherent accessory cells. Although the mechanism of action of GPhe remains, as yet, undefined, the GPhe-mediated enhancement of DCL-2 (a TH2, H-2d anti-GLA, T cell line) proliferation was not dependent upon the production of either IL-1 or IL-6 by accessory cells. In addition, enhanced DCL-2 proliferation was not accompanied by a significant increase in detectable IL-4 release.

Animals↗
PubMed1990

Enhanced proliferation of murine T cell lines following interaction of poly(Glu60,Phe40) (GPhe) and antigen-presenting accessory cells. II. The role of accessory cells and cytokines in the activity of GPhe on antigen-independent proliferation.

Our previous study (1) demonstrated the "cytokine-like" activity of poly(Glu60,Phe40)(GPhe) in augmenting the antigen-dependent proliferation of a variety of long-term murine T cell lines, particularly the bulk, BALB/c anti-poly (Glu36,Lys24,Ala40) (GLA), interleukin-4-producing, DCL-2 T cell line. GPhe was found to also augment the antigen-independent proliferation of DCL-2 in response to exogenous cytokines ([interleukin(IL)-2 +/- IL-1] in most experiments). Such exogenous cytokine-driven proliferative responses of DCL-2 were used to investigate further the role of accessory cells and of various soluble factors in the action of GPhe. GPhe did not act as a direct mitogen for T cells, rather it acted in a costimulatory fashion, requiring the presence of plastic-adherent accessory cells and a T cell growth factor (either IL-2 or IL-4). In the presence of accessory cells and exogenous IL-2, augmentation of antigen-independent DCL-2 proliferation by GPhe or by IL-1 depended upon the induction of autocrine IL-4 production. However, GPhe also augmented the response of these cells in the presence of exogenous IL-4 (+/- IL-2, +/- IL-1), and exogenous IL-4 added in combination with exogenous IL-2 (+/- IL-1) failed to mimic the GPhe effect, suggesting that another signal was involved in the mechanism of action of GPhe. The ability of allogeneic accessory cells to interact with GPhe to augment proliferative responses suggested that either a soluble factor or an unusual non-MHC-restricted cell-cell interaction provided this signal. In the presence of uv-irradiated accessory cells, DCL-2 proliferation was enhanced over that observed in the presence of non-uv-treated accessory cells, mimicking the GPhe effect, and interaction of GPhe with uv-irradiated accessory cells did not result in further enhancement of DCL-2 cytokine-driven proliferation. Using monoclonal antibodies which could block the function of IA or CD4 molecules, these cell-surface "adhesion" molecules were shown not to participate in the activity of GPhe. By the addition of recombinant cytokines, neutralizing antibodies, or indomethacin, the mechanism of action of GPhe was also shown not to be dependent upon IL-1, IL-6, IL-7, TNF alpha, or prostaglandin production by accessory cells. However, the presence, individually, of some of these factors (IL-1, IL-7, or prostaglandins) could influence to a variable degree the magnitude of the GPhe effect.

Animals↗
PubMed1990

Enhanced proliferation of murine T cell lines following interaction of Poly(Glu60, Phe40) (GPhe) and antigen-presenting accessory cells. III. Possible mechanisms responsible for activity of GPhe.

The random copolymers (Glu80, Phe20)n (GPhe20), (Glu60, Phe40)n (GPhe), and (Glu50, Phe50)n (GPhe50) were compared for the capacity to augment proliferation of antigen-reactive murine T cell lines. GPhe20, GPhe, and GPhe50 showed "augmenting" activity in order of increasing potency. Phenylalanyl residues constituted a significant portion of the "active" determinant(s) in the GPhe polymers tested. High titer murine anti-GPhe (ascites fluid) inhibited augmentation by GPhe of exogenous (IL-1 + rat-conditioned media (RCM] driven T cell proliferation, indicating that (a) the antibodies by binding to specific active determinant(s) in GPhe may have prevented critical GPhe-APC membrane interaction, and/or (b) "GPhe-anti-GPhe" complexes interfered with necessary "processing" of GPhe by APCs. Time course studies demonstrated that the appearance of increased T cell proliferation after GPhe addition occurred after proliferation to (a) nominal antigen or (b) exogenous (IL-1 + RCM) had reached peak [3H]thymidine incorporation ([3HT]). This suggested that more than GPhe-APC membrane interaction was necessary for GPhe activity. Leupeptin, a lysosomal protease inhibitor, inhibited the augmentation of T cell proliferation by GPhe, which led to the conclusion that GPhe must be "processed" by APCs to exhibit activity.

Animals↗
PubMed1988

Studies of fluorescence depolarization at high pressures of dimyristoylphosphatidylcholine liposomes containing poly(gamma-benzyl-L-glutamate).

The behaviors of four kinds of poly(gamma-benzyl-L-glutamate) (PBLG) molecules and their locations in dimyristoyl-phophatidylcholine (DMPC) liposomes were studied by fluorescence techniques at high pressures of up to 981 bar. The fluorescent substances 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-aminonaphthalene-8-sulfonate (ANS) and PBLGs labeled with a dansyl group were used as probes of hydrophobic and hydrophilic spheres and the motions of polypeptides, respectively. The changes in mobilities of the PBLG molecules depended on their concentrations, degrees of polymerization and the lengths of hydrocarbon chains attached to their terminals. The molecular motions of the PBLGs were altered by increase in their contents, caused by squeezing the PBLG molecules out of the membranes. Models of the membranes and the behavior of polypeptides in the membranes at high pressures are discussed.

Dimyristoylphosphatidylcholine↗
PubMed1988

Synthesis of taurine- and/or acetyl group-conjugated poly(D-Glu, D-Lys) derivatives and their effects on fibrinolysis.

A series of C-sulfoethylated and/or N-acetylated poly(D-Glu, D-Lys) (polyGL) derivatives was synthesized and their effects on tissue-type plasminogen activator (t-PA)-induced fibrinolysis were investigated. These derivatives accelerated t-PA-induced plasma (fibrin) clot lysis and t-PA-catalyzed plasminogen activation with the following order of potency: C-sulfoethylated and N-acetylated polyGL greater than N-acetylated polyGL greater than C-sulfoethylated polyGL greater than polyGL. The most potent stimulator C-sulfoethylated and N-acetylated polyGL associated with both t-PA and plasminogen under low ionic conditions, whereas it did not prevent the bindings of t-PA and plasminogen to fibrin. These results suggest that t-PA and plasminogen preferentially bind to sulfated or carboxylated polyanions, which may be required to possess certain neutral groups, and such complex formation may improve the plasminogen activation kinetics in a different manner from that of fibrin.

Acetylation↗