Presence Of Methotrexate Research Articles

A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting.

Cytochrome b2 is sorted into the intermembrane space of mitochondria by a bipartite N-terminal targeting and sorting presequence. In an attempt to define the sorting pathway we have identified an as yet unknown import intermediate. Cytochrome b2-dihydrofolate reductase (DHFR) fusion proteins were arrested in the presence of methotrexate (MTX) so that the DHFR domain was at the surface of the outer membrane while the N-terminus reached into the intermembrane space where the sorting signal was removed. This membrane-spanning, mature-sized species was efficiently chased into the mitochondria upon removal of MTX. Thus, an intermediate was generated which was exposed to the intermembrane space but was still associated with the inner membrane. This intermediate was also found upon direct import of cytochrome b2 and derived fusion proteins. These membrane-bound mature-sized cytochrome b2 species loop through the matrix and could be recovered in a complex with mt-Hsp70 and the inner membrane MIM44/ISP45, a component of the inner membrane import apparatus. This novel sorting intermediate can only be explained by a pathway in which cytochrome b2 passes through the matrix. The existence of such an intermediate is inconsistent with a pathway by which entrance of the mature part of cytochrome b2 into the matrix is stopped by the sorting sequence; however, its presence is fully consistent with the conservative sorting pathway.

Chloroplast Protein Import

Proteins have to be at least partially unfolded upon passage through the biological membranes. Previous studies with a dihydrofolate reductase fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by binding of a ligand, methotrexate, failed to block its translocation across the envelopes, suggesting that chloroplast envelopes have strong activity to unfold proteins [America, T., Hageman, J., Guéra, A., Rook, F., Archer, K., Keegstra, K. & Weisbeek, P. (1994) Plant Mol. Biol. 24, 283-294]. In the present study, we have analyzed in vitro translocation of a fusion protein consisting of the entire plastocyanin precursor and dihydrofolate reductase across the chloroplast envelope membranes and the thylakoid membrane. In the presence of methotrexate, the fusion protein was imported into the stroma but its translocation across the thylakoid membrane was blocked. The fusion protein that bound to the envelope became susceptible to digestion by thermolysin. These results suggest that, while the envelope membranes can unfold the methotrexate-bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re-bound to methotrexate in the stroma.

MK-801 and memantine protect cultured neurons from glutamate toxicity induced by glutamate carboxypeptidase-mediated cleavage of methotrexate

Cleavage of methotrexate into glutamate and diaminomethylpteroate by intrathecal glutamate carboxypeptidase is a new approach to the treatment of acute methotrexate neurotoxicity. The simulation of glutamate carboxypeptidase rescue from high-dose methotrexate in neuron astrocyte cocultures of rat cerebellum or cerebral cortex resulted in a selective, concentration-dependent neurotoxicity. The neurotoxicity was caused by the enzymatic release of glutamate from methotrexate at lower concentrations of methotrexate, and by both glutamate and diaminomethylpteroate at concentrations of methotrexate exceeding 200 μM. The good neuroprotection afforded by MK-801 and memantine suggested that glutamate toxicity was mediated by N-methyl-D-aspartate receptors. Methotrexate alone was not toxic to astrocytes, neurons, or the neurite networking. [ 3H]thymidine and [ 3H]deoxyuridine incorporation studies showed that astrocyte proliferation in the presence of methotrexate was maintained by the reutilization of pyrimidine bases for DNA synthesis. N-methyl-D-aspartate receptor antagonists should be coadministered in future experimental and clinical trials examining intrathecal glutamate carboxypeptidase rescue of methotrexate toxicity.

A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes.

Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes.

Production of the enzyme dihydrofolate reductase by methotrexate-resistant bacteria isolated from soil.

Six bacterial cultures isolated from soil were capable of growing in the presence of methotrexate (MTX). Two strains, PFR-1 and 3, developed resistance to 500 micrograms cm-3 MTX in the medium and produced elevated levels of the enzyme dihydrofolate reductase (EC 1.5.1.3): 2580 and 2702 U dm-3 compared to the sensitive parent strains (28 and 35 U dm-3). Isolate PFR-3 showed maximum enzyme production (4950 U dm-3, specific activity 12.56 U mg-1 in flasks and 5737 U dm-3, specific activity 14.80 U mg-1 in 5-dm3 fermenter) during exponential phase of growth (6 h) at 37 degrees C and pH 7.0.

Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle.

Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.

The phenotype of a dihydrofolate reductase mutant of Saccharomyces cerevisiae

We have constructed a dihydrofolate reductase mutant ( dfr1) of Saccharomyces cerevisiae. The mutant has auxotrophic growth requirements for the C1 metabolites dTMP, adenine, histidine and methionine, similar to those of wild-type (wt) strains grown in the presence of methotrexate (MTX). However, unlike wt strains treated with MTX, the growth requirements of the dfr1 mutant are not satisfied by exogenous 5-formyltetrahydrofolic acid (FA; folinic acid) in complex (YEPD) medium. This result is surprising, as yeast cells treated with MTX are expected to be phenocopies of dfr1 mutants. The inability of the mutants to metabolize FA suggests that the DFR1 gene product may have a role in folate metabolism in addition to its well-characterized function in the reduction of dihydrofolate. From dfr1 strains, we have isolated secondary mutants whose growth can be supported by FA in YEPD medium. This FA-utilizing phenotype is attributable to recessive mutations which we have designated fou. In addition to their inability to metabolize FA, the dfr1 strains are unable to grow on medium containing the non-fermentable carbon source glycerol, suggesting that the DFR1 gene product is also required for mitochondrial function. In order to overcome this lack of respiratory activity in the dfr1 mutants, we isolated strains containing a dominant mutation, DIR, which allows growth on glycerol in the presence of antifolate drugs. When crossed into dfr1 strains, the DIR mutation conferred respiratory competence. These strains should be useful in a variety of studies on the genetics and biochemistry of folate metabolism in this simple eukaryote.

Assay for expression of methotrexate-resistant dihydrofolate reductase activity in the presence of drug-sensitive enzyme

A simple, continuous spectrophotometric assay for dihydrofolate reductase (DHFR) activity was adapted for determination of drug-resistant enzyme activity expressed from transfected genes in cells containing drug-sensitive enzyme. Methotrexate inihibition characteristics in this assay system were assessed for the murine wild-type (WT) enzyme as well as variant genes encoding amino acid substitutions at codon positions 22 (arg22) or 31 (trp31) expressed in DHFR-deficient Chinese hamster ovary (CHO) cells and in mouse 3T# cells. Methotrexate concentrations were thus identified which maximized inhibition of the wild-type enzyme while maintaining substantial arg22 or trp31 activity. Mixing experiments were conducted to determine the minimum amount of drug-resistant enzyme distinguishable from a constant amount of wild-type enzyme in the presence of methotrexate. Mixtures of enzymes from a variety of sources (WT, arg22, or trp31 expressed in CHO or 3T3 cells) demonstrated a detection limit of 0.03 to 0.06 nmol/min. Assay of methotrexate-resistant arg22 DHFR appeared to be limited by the low level of activity associated with this enzyme variant, whereas assay of the trp31 variant was limited by enzyme inhibition at lower concentrations of methotrexate. The assay was thus applicable to two quite diverse DHFR variants and may be useful for assaying the expression of other drug-resistant DHFR genes as well after introduction into cells containing drug-sensitive enzyme.

Amplification of plasmid (L factor) DNA and increased production of a plasmid gene product (γ-interferon) in mouse L cells

It has previously been reported that composite DNAs derived from L factor, a polyoma-related mammalian plasmid, can be established in several mouse cell lines after transfection. Here, we report that the copy number of a plasmid composite DNA consisting of L factor, pBR DNA, dihydrofolate reductase ( dhfr) gene, and γ-interferon (γ-IFN) gene was increased more than 10-fold after two successive adaptations of the plasmid-bearing mouse L cells to increasing concentrations of methotrexate (MTX), an inhibitor of dhfr. The structure of the amplified L factor plasmid remained intact during prolonged cell culture, but the copy number remained to be amplified only when the selective pressure (presence of MTX in the medium) has been exerted during the culture. Cells bearing the amplified plasmid produced a higher level of γ-IFN compared with the original clone, which was likely to be derived from the plasmid γ-IFN gene amplified along with L factor and the dhfr gene.

Monoclonal antibody targeting of methotrexate (MTX) against MTX-resistant tumour cell lines.

Several Methotrexate (MTX)-resistant sublines of the osteogenic sarcoma cell line 791T were derived by continuous selection in the presence of MTX and 12-O-tetradecanoylphorbol-13-acetate (TPA). Studies including assays of the uptake and binding of [3H]MTX and fluoresceinated-MTX, determined that these sublines showed diminished MTX transport, and that none of them appeared to overproduce the MTX-target enzyme dihydrofolate reductase. Conjugates of the anti-791T monoclonal antibody 791T/36 linked to MTX via human serum albumin (HSA) were prepared by Dr M.C. Garnett. These were cytotoxic selectively for cells bearing the 791T/36-defined antigen (gp72), and were found to be as cytotoxic to most of the MTX-resistant 791T sublines as they were to parental 791T cells. Furthermore, an anti-MTX/anti-gp72 bispecific antibody 516 augmented the cytotoxicity of HSA-MTX conjugate to the MTX-resistant 791T variant R120 apparently as efficiently as for parental 791T cells. It is suggested that acquired drug resistance caused by deficient transport mechanisms may be partially or wholly overcome by targeting the drug to a readily-internalised cell surface antigen.

The influence of methotrexate on radiation-induced damage to different lengths of the rat spinal cord

An experimental model in the rat has been used to assess the possible enhancement of damage to the spinal cord when radiation is given in the presence of methotrexate (MTX). The dose of MTX used, 4 mg/kg, was the maximum dose that could be given to the rat, when administered into the cerebral spinal fluid circulation, without risk of serious neurological effects. Lengths of 4, 8 and 16 mm of the cervical spine were irradiated with single doses of X rays. For animals that developed paralysis within 30 weeks, caused predominantly by the presence of white matter necrosis, there was no evidence to indicate that MTX enhanced the radiation response of the rat spinal cord, at least at a more clinically relevant level of effect i.e. a low incidence of paralysis. However, for the doses associated with the 50% level of effect (ED50) to an 8 mm long field a significant (p less than 0.005) enhancement of the response was seen, suggesting a dose modification factor of 1.19 +/- 0.07. This was interpreted in terms of an hypothesis to explain the well documented volume effect for very small fields in the rat spinal cord which is based on the migration of viable cells in the periphery of the irradiated site. The apparent smaller effect seen when only 4 mm of the spine was irradiated might be related to the nature of the lesion induced at doses required to produce paralysis in these small fields; the lesions were not restricted to white matter but were more severe and also involved the grey matter and nerve roots after a slightly shorter latent period.

Reduced drug accumulation as the mechanism of extreme clinical resistance to methotrexate in the human T-cell leukemia xenograft, LALW-2

The mechanisms were examined that underlie the extreme resistance to methotrexate (MTX) by near diploid leukemic T-cells (LALW-2) exposed to the drug only during the course of therapy administered to the patient of origin. Despite the LALW-2 cells being highly resistant to MTX (inhibitory dose for 50% of cells, more than 10(-3) mol/l), southern blot analysis did not show any amplification of the dihydrofolate reductase gene, nor was there any evidence, by comparison with drug-sensitive CCRF-CEM cells, that the gene was overexpressed. Kinetic analysis of dihydrofolate reductase activity in the presence of MTX provided no basis for attributing resistance in LALW-2 cells to a change in enzyme structure. By contrast, studies of MTX accumulation revealed that the LALW-2 cells accumulated significantly less drug than either CCRF-CEM cells or a MTX-resistant CCRF-CEM subline with a characterized transport defect. These data suggest that extreme MTX resistance in LALW-2 cells is mediated by reduced drug accumulation in the absence of any effect on the target enzyme.

Mechanism of leucovorin reversal of methotrexate cytotoxicity in human MCF-7 breast cancer cells

Previous studies have suggested that metabolic inhibition by methotrexate (MTX) is multifactorial and that cytotoxicity can be reversed by the reduced folate leucovorin. In this report we investigated the mechanism of leucovorin rescue in the MCF-7 human breast cancer cell line. Cells were exposed to various concentrations of MTX (0.5, 1.0, 3.0, and 10.0 μM) for 24 hr followed by rescue with labelled leucovorin (0.5 to 50 μM). The changes in the intracellular folate pools 24 hr following the addition of leucovorin were quantitated by high-pressure liquid Chromatographie methods. The changes in the folate pools during rescue were compared with the ability of various concentrations of leucovorin to affect cellular rescue from MTX using a cloning assay. Our studies show that the total labelled intracellular folate pools increased in a log-linear fashion with respect to leucovorin exposure concentrations up to 100 μM. The degree of accumulation at a given leucovorin concentration was not significantly different in the absence or presence of MTX over the concentration range of 0.5 to 10 μM. Individual folate pool levels (tetrahydrofolate, 10-formyl tetrahydrofolate, 5-formyl tetrahydrofolate, 5-methyl tetrahydrofolate, and 5,10-methylene tetrahydrofolate) reached those present in cells not exposed to MTX at concentrations of leucovorin that were not adequate to rescue the MTX-treated cells. With exposure to concentrations of leucovorin capable of rescue, the individual folate pool levels were up to twelve times greater than those found in untreated cells, consistent with competition for catalytic activity at folate-dependent enzymes in addition to dihydrofolate reductase. The dihydrofolate pool also increased with increasing leucovorin concentration; but, unlike the reduced folates, this oxidized folate reached a maximal level that was dependent on the MTX concentration to which the cells had been exposed. This suggests that competition between MTX and leucovorin occurs at the level of dihydrofolate reductase via a competitive interaction with dihydrofolate in this intact cell system. The ability of leucovorin and its metabolites to compete with direct inhibitors of dihydrofolate reductase and other metabolically important folate-dependent enzymes appears to be associated with leucovorin rescue.

Refolding of Escherichia coli dihydrofolate reductase: sequential formation of substrate binding sites.

The kinetics of refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3) have been examined upon dilution of unfolded enzyme in 4.5 M urea to 1.29 M urea in 0.02 M phosphate buffer (pH 7.2) at 10 degrees C. Changes in the intrinsic protein fluorescence on refolding are characterized by four phases. Based on changes in the amplitudes of these phases, as a consequence of quenching of the intrinsic fluorescence by ligands, it is possible to determine the step at which a ligand binds during the refolding process. The results show that either NADP or NADPH binds to the last species formed in a sequence involving three intermediates between the unfolded and native states. Dihydrofolate, on the other hand, binds during the formation of the second observed intermediate. When refolding is performed in the presence of methotrexate, an analogue of dihydrofolate, and NADPH, NADPH binds, as determined from changes in NADPH fluorescence, to the third observed intermediate rather than the last (fourth) species formed. Measurements of the recovery of enzymatic activity during refolding suggest that dihydrofolate also induces NADPH binding prior to the final observed folding phase. These results define more closely the formation of structural domains during the folding of dihydrofolate reductase.

Methotrexate-resistant mechanisms in human choriocarcinoma cells

Choriocarcinoma cells grown in the presence of methotrexate (MTX) developed resistance in two ways. HCCM-derived sublines (relatively high MTX resistance) produced enhanced levels of dihydrofolate reductase (DHFR) and had impaired transport of MTX. Altered transport was the primary determinant of response in CC1-derived sublines (low MTX resistance). Since the selection procedures used were identical, it was assumed that altered MTX transport was insufficient to account entirely for the various degrees of resistance. An increased level of DHFR activity was necessary for the development of high MTX resistance. The overproduction of DHFR was the consequence of amplification of the DHFR gene sequence. The incidence of double minutes (DMs) in metaphase paralleled the degree of resistance. However, DMs were also present in cells not showing DHFR gene amplification. Mechanisms other than DHFR gene multiplication were responsible for the de novo synthesis of DMs.

Methotrexate-resistant mechanisms in human choriocarcinoma cells

Choriocarcinoma cells grown in the presence of methotrexate (MTX) developed resistance in two ways. HCCM-derived sublines (relatively high MTX resistance) produced enhanced levels of dihydrofolate reductase (DHFR) and had impaired transport of MTX. Altered transport was the primary determinant of response in CC1-derived sublines (low MTX resistance). Since the selection procedures used were identical, it was assumed that altered MTX transport was insufficient to account entirely for the various degrees of resistance. An increased level of DHFR activity was necessary for the development of high MTX resistance. The overproduction of DHFR was the consequence of amplification of the DHFR gene sequence. The incidence of double minutes (DMs) in metaphase paralleled the degree of resistance. However, DMs were also present in cells not showing DHFR gene amplification. Mechanisms other than DHFR gene multiplication were responsible for the de novo synthesis of DMs.

Antiproliferative effects of methotrexate on peripheral blood mononuclear cells.

Methotrexate was added to cultured mononuclear cells from the peripheral blood of normal individuals and patients with rheumatoid arthritis (RA) to study the drug's effects on mononuclear cell proliferation and antibody synthesis. In the presence of methotrexate, marked antiproliferative effects (to levels less than 15% of baseline) were seen with 3H-deoxyuridine, but not with 3H-thymidine, as the marker of cell division. This difference was not due to altered kinetics of proliferation or the presence of salvage nucleotides in the culture medium. The absence of suppression of antibody production preactivated by pokeweed mitogen in vitro and the low levels of suppression of spontaneous IgM rheumatoid factor production by blood mononuclear cells from RA patients suggested a relative resistance of activated cells to the effects of methotrexate. The effects of methotrexate on both cell proliferation and antibody synthesis were completely reversed by the addition of high concentrations of exogenous folinic acid. The results suggest that methotrexate has effects on immunocompetent cells that may contribute to the efficacy of this drug in the treatment of RA and other autoimmune diseases.

Characterization by flow cytometry of fluorescein-methotrexate transport in Chinese hamster ovary cells.

We have studied by flow cytometry the transport of fluorescein-methotrexate in Chinese hamster ovary cells. Fluorescein-methotrexate appears to enter cells via a mechanism different from the carrier-mediated system for methotrexate. This conclusion is supported by the following observations: 1) Fluorescein-methotrexate is transported equally well into normal and mutant cells defective in the inward methotrexate uptake. 2) Folic acid and its reduced states, which competitively inhibit methotrexate uptake, do not alter fluorescein-methotrexate transport. 3) Fluorescein-methotrexate accumulation exhibits a low temperature coefficient (Q10 = 1.6) compared with the influx of methotrexate (Q10 = 6-8). 4) Initial rates of fluorescein-methotrexate uptake are concentration dependent but are not saturable. 5) Fluorescein-methotrexate uptake is very slow and reaches steady state after 8 h, whereas at an equimolar concentration methotrexate reaches saturation after 20 min. 6) Initial influx rates of fluorescein-methotrexate are not affected by the presence of methotrexate. 7) Sulfhydryl-reactive mercurials, which block methotrexate transport, do not reduce fluorescein-methotrexate influx, but rather stimulate it. Thus, based on the nonsaturability of fluorescein-methotrexate inward transport, its low temperature coefficient, and lack of inhibition with structural analogs, we conclude that fluorescein-methotrexate is accumulated in hamster cells by a passive diffusion process.

An alternative method for assaying cerebrospinal fluid protein in the presence of methotrexate.

Therapeutic concentrations of methotrexate can cause significant positive interference in cerebrospinal fluid (CSF) protein values when assayed in the Du Pont aca. Conversely, our modified turbidimetric method, in which trichloroacetic acid (TCA) plus a sample blank containing dilute hydrochloric acid is used in place of TCA, exhibits little or no interference from methotrexate. This was verified by assaying solutions that contained a constant amount of protein (approximately 430 mg/L) and various amounts of methotrexate (0.0-2.3 x 10(-4) mol/L) by both the Du Pont aca and the manual turbidimetric method. As expected, the aca results showed increasing protein values with increasing methotrexate, whereas the manual method gave results approximating the expected protein value irrespective of the methotrexate concentration.

Cytological evidence of defective template in the fragile X chromosome

In cells of fragile X patients, the changed X segment may appear as a poorly staining region or a gap, or as a deletion, involving one or both chromatids. To find out whether the fragile site represents an incompletely replicated DNA sequence, as has been suggested recently, we analyzed the four chromatids of methotrexate-induced endoreduplicated fragile X chromosomes. Our main observations were: (1) a deleted chromatid was never internal to a poorly staining one; (2) an endoreduplicated X chromosome with a fragile site never included a normal chromatid. These results can be explained by assuming that DNA at the fragile site, when replicated in the presence of methotrexate, may undergo defective replication and give rise to improperly packaged chromatin, appearing as a chromatid with a poorly staining region or a gap in the following metaphase. The same DNA may fail to function as a template in the following S-phase and give rise to a chromatid with a single-stranded segment, appearing as a deleted chromatid in the following metaphase.