Microcalorimetric evaluation of the effects of methotrexate and 6-thioguanine on sensitive T-lymphoma cells and on a methotrexate-resistant subline.

Isothermal microcalorimetry was used in order to continuously monitor and quantitatively assess the action of two antineoplastic drugs, methotrexate (MTX) and 6-thioguanine (6-TG), on a human T-lymphoma cell line, CCRF-CEM. The results with MTX were compared with data from experiments with a MTX-resistant subline, CEM/MTX. The slope of the power-time curve after drug injection relative to that obtained during unperturbed growth, was used to construct dose-response curves. The normal cell line was characterized by a D50 value (i.e., the dose producing half the maximal response) of 0.05 microM for MTX and 0.38 microM for 6-TG. For the MTX-resistant subline the D50 value was 8 microM of MTX. Comparisons of the continuous power-time curves showed the inhibitory effect of 6-TG to be faster than that of MTX.     

Microcalorimetric evaluation of the effects of methotrexate and 6-thioguanine on sensitive T-lymphoma cells and on a methotrexate-resistant subline

Isothermal microcalorimetry was used in order to continuously monitor and quantitatively assess the action of two antineoplastic drugs, methotrexate (MTX) and 6-thioguanine (6-TG), on a human T-lymphoma cell line, CCRF-CEM. The results with MTX were compared with data from experiments with a MTX-resistant subline, CEM/MTX. The slope of the power-time curve after drug injection relative to that obtained during unperturbed growth, was used to construct dose-response curves. The normal cell line was characterized by aD ₅₀ value (i.e., the dose producing half the maximal response) of 0.05 μM for MTX and 0.38 μM for 6-TG. For the MTX-resistant subline theD ₅₀ value was 8 μM of MTX. Comparisons of the continuous power-time curves showed the inhibitory effect of 6-TG to be faster than that of MTX.     

Endogenous thymidine and hypoxanthine are a source of error in evaluating methotrexate cytotoxicity by clonogenic assays using undialyzed fetal bovine serum

None of 13 fresh human tumor samples of various histology cloned in a two-layer agar culture system with 20% undialyzed fetal bovine serum (FBS) showed sensitivity to three antifolates, methotrexate (MTX), trimetrexate and 5,8-dideazaisofolic acid (IAHQ), even after continuous exposure to the highest concentrations (100 microM) for 21 days. In order to investigate this lack of antifolate drug effect, we compared the toxicity of continuous MTX exposure in the human colon carcinoma cell line HCT-8, cloned in a thymidineless medium (RPMI 1640) supplemented with 10% horse serum (HS), 10% fetal bovine serum (FBS), 20% FBS or 20% dialyzed FBS. In the presence of native FBS, when the minimum clone size was set at 30 cells/colony, the survival of HCT-8 cells reached a plateau at approximately 60% of untreated control after exposure to MTX concentrations between 0.1 microM and 100 microM. Only when the minimum clone size was set at 2 X 10(3) cells/colony was the sensitivity of HCT-8 cells to the antimetabolite comparable to that obtained in HS or dialyzed FBS (ED50 values in the range of 0.01 microM). MTX protection experiments indicated that even very small concentrations of thymidine and hypoxanthine together were sufficient to reproduce the pattern of sensitivity to MTX observed under culture conditions with undialyzed FBS. We conclude that for a proper evaluation of MTX cytotoxicity in clonogenic assays, dialyzed FBS and thymidine-less media should be employed; if native FBS is an absolute requirement for growth, only very large colonies (at least 10 cell divisions) should be scored.     

甲氨蝶呤联合羟基氯喹对类风湿关节炎合并糖尿病患者糖化血红蛋白水平的影响

目的:观察类风湿关节炎(rheumatoid arthritis,RA)合并糖尿病(diabetes mellitus,DM)患者使用甲氨蝶呤(methotrexate,MTX)联合羟基氯喹(hydroxychloroquine,HCQ)治疗前后糖化血红蛋白(glycosylated hemoglobin,HbA1C)水平的变化。方法:通过回顾性分析,选取联合使用MTX+HCQ或单独使用MTX治疗的RA合并DM患者,且在治疗前及开始治疗12个月内分别至少有1次HbA1C值,记录其年龄、性别、诊断、体重指数、使用糖皮质激素情况。计算用药前到用药后12个月内HbA1C最低值的变化。结果:40例使用HCQ+MTX和45例使用MTX患者符合入选标准。两组间年龄、性别、体重指数、用药前HbA1C水平相似,MTX+HCQ组糖皮质激素使用比例(40.00%)比HCQ组更多(26.67%)(P=0.25)。MTX+HCQ组治疗后HbA1C有明显下降(0.42%,P=0.00),且MTX+HCQ组HbA1C降低幅度高于MTX组(0.42%,0.12%,P=0.02)。结论:与单独使用MTX相比,RA合并DM患者联合使用HCQ+MTX可明显降低HbA1C水平。     

甲氨蝶呤对大鼠尿液蛋白质组的影响

甲氨蝶呤是常见的免疫抑制剂,曾被大剂量用于治疗癌症,近年来其被小剂量用于治疗类风湿性关节炎。文中尝试研究甲氨蝶呤对大鼠尿蛋白质组的影响。大鼠口服甲氨蝶呤构造用药模型,再收集大鼠10 h内的尿液,并且采用液相色谱串联质谱(liquid chromatography tandem mass spectrometry,LC-MS/MS)分析大鼠的尿蛋白。总共鉴定到31个差异蛋白,其中7个蛋白与甲氨蝶呤药物作用和类风湿性关节炎症状有关。部分大鼠的生物学过程反映了甲氨蝶呤对机体谷胱甘肽代谢以及对JAK/STAT信号通路的影响。结果显示,尿蛋白具有反映甲氨蝶呤对大鼠机体影响的能力。对单个大鼠个体差异蛋白的分析体现出不同个体对该药物的反应有较大差异。     

Two-site autoinhibition of the ADP-ribosylating mosquitocidal toxin (MTX) from Bacillus sphaericus by its 70-kDa ricin-like binding domain

The mosquitocidal toxin (MTX) from Bacillus sphaericus SSII-1 is an approximately 97-kDa arginine-specific ADP-ribosyltransferase that is activated by proteolytic cleavage, thereby releasing the active 27-kDa enzyme (MTX(30-264)) and a 70-kDa C-terminal fragment (MTX(265-870)). In solution, the cleaved 70-kDa fragment is still a potent inhibitor of the ADP-ribosyltransferase activity of MTX. Here we studied the interaction of the 70-kDa fragment with the enzyme domain of MTX. Several C-terminal deletions of the 70-kDa fragment inhibited the enzymatic activity of MTX(30-264). However, the IC(50) values were about 2 orders of magnitude higher for the deletions than for the 70-kDa fragment. A peptide covering amino acid residues 265-285 of the holotoxin exhibited the same inhibitory potency as the C-terminal deletions of the 70-kDa fragment. MTX(265-285) contains several acidic residues, of which D273 and D275 were found to be essential for the inhibitory effect. Exchange of these residues in the 70-kDa fragment (MTX(265-870)) reduced its inhibitory potency. Kinetic analysis showed that the peptide MTX(265-285) had no effect on the V(max) of MTX(30-264) but increased the K(m) for NAD. By contrast, the 70-kDa fragment deleted of residues Ile265 through Asn285 inhibited the enzyme activity of MTX(30-264) mainly by decreasing the V(max) of the enzyme. A second binding site for interaction of MTX(265-870) with MTX(30-264) was localized to the C-terminus within the region of residues 750-870. The data support a two-site binding model for inhibition of the ADP-ribosyltransferase activity of MTX(30-264) by the 70-kDa fragment MTX(265-870) with an interaction of amino acid residues 265-285 at the active site and an allosteric inhibition by the C-terminal part of the 70-kDa fragment.     

Activity of dendrimer-methotrexate conjugates on methotrexate-sensitive and -resistant cell lines

Dendritic nanostructures can play a key role in drug delivery, due to the high density and variety of surface functional groups that can facilitate and modulate the delivery process. We have investigated the effect of dendrimer end-functionality on the activity of polyamido amine (PAMAM) dendrimer-methotrexate (MTX) conjugates in MTX-sensitive and MTX-resistant human acute lymphoblastoid leukemia (CCRF-CEM) and Chinese hamster ovary (CHO) cell lines. Two amide-bonded PAMAM dendrimer-MTX conjugates were prepared using a dicyclohexylcarbodiimide (DCC) coupling reaction: one between a carboxylic acid-terminated G2.5 dendrimer and the amine groups of the MTX (conjugate A) and another between an amine-terminated G3 dendrimer and the carboxylic acid group of the MTX (conjugate B). Our studies suggest that conjugate A showed an increased drug activity compared to an equimolar amount of free MTX toward both sensitive and resistant cell lines, whereas conjugate B did not show significant activity on any of the cell lines. Despite substantially impaired MTX transport by MTX-resistant CEM/MTX and RII cells, conjugate A showed sensitivity increases of approximately 8- and 24-fold (based on IC50 values), respectively, compared to free MTX. Co-incubation of the cells with adenosine and thymidine along with either conjugate A or MTX resulted in almost complete protection, suggesting that the conjugate achieves its effect on dihyrofolate reductase (DHFR) enzyme through the same mechanism as that of MTX. The differences in cytotoxicity of these amide-bonded conjugates may be indicative of differences in the intracellular drug release from the cationic dendrimer (conjugate B) versus the anionic dendrimer (conjugate A), perhaps due to the differences in lysosomal residence times dictated by the surface functionality. These findings demonstrate the feasibility of using dendrimers as drug delivery vehicles for achieving higher therapeutic effects in chemotherapy, especially in drug-resistant cells.     

Interaction of anticancer drug mitoxantrone with DNA analyzed by electrochemical and spectroscopic methods

Cyclic voltammetry coupled with different spectroscopic (UV/Vis, fluorescence and Raman) techniques were used to study the interaction of mitoxantrone (MTX), an antitumor drug, with calf thymus DNA in acetate buffer solutions (pH 4.5). The interaction of MTX with DNA could result a considerable decrease in the MTX peak currents and a hypochromic and bathochromic shift in the maximum adsorption bands of MTX as well as the emission quenching in the MTX fluorescence spectra. The variations in the electrochemical and spectral characteristics of MTX indicated MTX bind to DNA by an intercalative mode. This conclusion was reinforced by Raman data. The merely particular vibrations were affected in Raman, suggesting that only a portion of the chromophore of MTX was involved in the intercalation into DNA duplex. These studies are valuable for a better understanding the detailed mode of MTX-DNA interaction, which should be important in deeper insight into the therapeutic efficacy of MTX and design of new DNA targeted drug.     

The antileukemic activity of modified fibrinogen–methotrexate conjugate

Background

The search for new, innovative methods to treat all types of diseases, especially cancer-related ones, is a challenge taken by pharmaceutical companies and academic institutions. The use of conjugates containing widely-known and widely-used bioactive substances is one of the ways to solve this problem. Research into drug binding with macromolecular carrier systems has joined the search for new therapeutic strategies.

Methods

The main goal of this paper is the potential offered by the use of fibrinogen derivatives as an antileukemic drug carrier. Physicochemical properties of the obtained conjugate were analyzed, characterizing alterations in relation to the starting carrier and analyzing biological implications. The intraperitoneally (i.p.) inoculated P388 mouse leukemia model for in vivo studies was used.

Results and conclusions

Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug were developed. Carrier preparation and a conjugate synthesis in aqueous solution were formulated, as well as purification of the conjugate was performed. The study showed that the survival of leukemia mice treated with FH–MTX conjugate was indeed significantly longer than survival in both untreated animals (control) and mice treated with unbound MTX. A significant increase in the antileukemic activity of MTX conjugated with hydrolysed fibrinogen was observed as compared with the unconjugated drug. Reported data suggest that hydrolysed fibrinogen can serve as a carrier molecule for the MTX drug with the aim of enhancing its antileukemic activity.

General significance

Conjugates consisting of a fibrinogen derivative with a covalently bound anticancer drug seem to be a promising anticancer drug.     

Early medication use in new-onset rheumatoid arthritis may delay joint replacement: results of a large population-based study

IntroductionUse of disease-modifying anti-rheumatic drugs (DMARDs) in rheumatoid arthritis (RA) may prevent joint damage and potentially reduce joint replacement surgeries. We assessed the association between RA drug use and joint replacement in Quebec, Canada.MethodsA cohort of new-onset RA patients was identified from Quebec’s physician billing and hospitalization databases from 2002–2011. The outcome was defined using procedure codes submitted by orthopedic surgeons. Medication use was obtained from pharmacy databases. We used alternative Cox regression models with time-dependent variables measuring the cumulative effects of past use during different time windows (one model focussing on the first year after cohort entry) for methotrexate (MTX), and other DMARDs. Models were adjusted for baseline sociodemographics, co-morbidity and prior health service use, time-dependent cumulative use of other drugs (anti-tumor necrosis factor [anti-TNF] agents, other biologics, cyclooxygenase-2 inhibitors [COXIBs], nonselective nonsteroidal antiinflammatory drugs [NSAIDs], and systemic steroids), and markers of disease severity.ResultsDuring follow-up, 608 joint replacements occurred among 11,333 patients (median follow-up: 4.6 years). The best-fitting model relied on the cumulative early use (within the first year after cohort entry) of MTX and of other DMARDs, with an interaction between MTX and other DMARDs. In this model, greater exposure within the first year, to either MTX (adjusted hazard ratio, HR = 0.95 per 1 month, 95 % confidence interval, 95 % CI 0.93-0.97) or other DMARDs (HR = 0.97, 95 % CI 0.95-0.99) was associated with longer time to joint replacement.ConclusionsOur results suggest that longer exposure to either methotrexate (MTX) or other DMARDs within the first year after RA diagnosis is associated with longer time to joint replacement surgery.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0713-3) contains supplementary material, which is available to authorized users.     

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/JEM.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 interaction of the steroidal antagonist faslodex and methotrexate.

Faslodex (FAS, ICI 182, 780), a novel steroidal estrogen antagonist decreased high-dose methotrexate (MTX) cytotoxicity in MCF-7 breast cancer cells. When FAS is given at least 24 hr prior to MTX, the resultant interaction is antagonistic. However, when breast cancer cells are exposed to FAS 24 hr after MTX, the interaction between FAS and MTX is not antagonistic. The proliferation of cells exposed to 0.1 microM FAS and 10 microM MTX alone or in combination with FAS 24 hr prior to MTX was in the following order: FAS>FAS 24 hr prior to MTX>MTX. MTX administration 24 hr prior to FAS had the following inhibitory effects on the growth of cells: MTX 24 hr prior to FAS >MTX>FAS 24 hr prior to MTX>FAS>control (no drug exposure). To determine if the antagonistic interaction between FAS and MTX was a function of sequence and time, cells were exposed to FAS 24 hr and 36 hr prior to MTX exposure. The percentages of control rates were 42.70 +/- 4.60% and 57.89 +/- 0.55%, respectively, from a 24 hr and 36 hr exposure of FAS prior to MTX. The growth rates after 24 and 36 hr exposures to MTX alone were 30.30 +/- 0.61% and 33.11 +/- 2.57% of control rates, respectively. These studies suggest that: a) the interactions between FAS and MTX are sequence-dependent; b) FAS antagonizes the effect of MTX when FAS administration precedes MTX, and c) FAS antagonism to MTX is a function of time.     

Promoted electron transfer of mitoxantrone binding with DNA by cytochrome c

A promoted electron transfer of an antitumor drug, mitoxantrone (MTX), intercalating into DNA duplex was successfully obtained upon addition of cytochromes c (cyt. c) in NaAc-HAc buffer solution (pH 4.5). The experimental results suggested that co-existence of MTX and cyt. c in the DNA helix is an important factor for accelerated electron transfer of MTX, where the promoter, cyt. c, operated smoothly through the DNA bridge. The UV/Vis spectroscopic experiments further confirmed the interaction process. Furthermore, a possible mechanism of such reaction was also discussed in this paper.     

Enhancement of 6-thioguanine cytotoxic activity with methotrexate

Studies were completed to characterize the cytotoxic and biochemical interaction of methotrexate (MTX) and 6-thioguanine (6-TG). Pretreatment of L1210 leukemia cells for 3 hr with MTX substantially enhanced the cytotoxicity of 6-TG. The LD₉₀ of 6-TG in cells pretreated with 1μM MTX was 0.9pM, compared to an LD₉₀ of 800 pM when the two drugs were given concurrently and an LD₉₀ of 30 pM resulted with 6-TG alone. HPLC analysis of intracellular metabolites demonstrated an increased conversion of 6-TG to 6-TG-nucleotides in cells pretreated with MTX. A marked enhancement of 6-TG incorporation into RNA also was noted (MTX→6-TG: 350 fmol/μg RNA vs 6-TG: 98 fmol/μg RNA). However, there was a suppression of 6-TG incorporation into DNA when cells were pretreated with MTX (MTX→6-TG: 170 fmol/μg DNA vs 6-TG: 690 fmol/μg DNA). These results suggest that: 1) an enhancement of 6-TG antileukemic activity can be obtained with MTX pretreatment, and 2) the enhancement of 6-TG cytotoxicity following MTX exposure is not associated with 6-TG incorporation into DNA, but rather with incorporation of 6-TG into RNA. This drug sequence may be beneficial in the clinical treatment of leukemia.     

Methotrexate binding causes structural and functional changes in lung cystatin

Regulation of cysteine proteinases and their inhibitors is of utmost importance in diseases like lung cancer, chronic inflammatory conditions such as asthma, emphysema, and idiopathic pulmonary fibrosis. Protease-antiprotease imbalance accelerates disease progression. In the present study, the effect of antineoplastic and antirheumatic drug methotrexate (MTX) on lung cystatin (a cysteine protease inhibitor) was studied to explore drug induced changes in functional and structural integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of MTX with goat lung cystatin (GLC-I). Stern-Volmer analysis of MTX-GLC-I system at different temperatures indicates the presence of static component in the quenching mechanism. The thermodynamic parameters ΔH? and ΔS? were -3.8 kJ/mol and 94.97 J?mol?1?K?1, respectively, indicating that both hydrogen bonds and hydrophobic interactions played a major role in the binding of MTX to GLC-I. Methotrexate (7 μM) caused complete inactivation of lung cystatin after 6 hours. The results of FT-IR spectroscopy reflect perturbation of the goat lung cystatin on interaction with MTX. Methotrexate induced loss of function change in the inhibitor could provide a rationale for the off target tissue injury caused by the drug and for the design of agents against such an injury.     

Interference of 7-hydroxymethotrexate with the determination of methotrexate in plasma samples from children with acute lymphoblastic leukemia employing routine clinical assays

The accuracy of two clinical assays, the enzyme-multiplied immunoassay (EMIT) and fluorescence polarization immunoassay (FPIA2), universally employed for measurement of plasma levels of methotrexate (MTX) in children administered a high dose of this drug for treatment of acute lymphoblastic leukemia was evaluated here. Because of its superior specificity, sensitivity, and precision, high performance liquid chromatography (HPLC) was selected as the reference method with which the other two procedures were compared using approximately 420 different plasma samples for method comparison. 7-Hydroxymethotrexate (7-OHMTX), the major plasma metabolite of MTX, that can be detected in plasma at relatively high concentrations for long periods following infusion of a high dose of MTX, was also quantitated by HPLC. Forty-two and 66 h after infusion, the plasma level of MTX was overestimated in 2% and 3% of the samples by the FPIA2 procedure in 5% and 31% by the EMIT assay. The overall correlation coefficients (r2) for the values obtained by FPIA2 or EMIT versus those based on HPLC were 0.989 and 0.663, respectively. The presence of 7-OHMTX exerted a highly significant influence (p=0.0007 as determined by the unpaired t-test) on MTX measurement by the EMIT assay. We conclude that the rapid automated procedures routinely used at present and in particular EMIT, suffer from cross-reactivity with metabolites of MTX. Thus, the relatively high percentage of samples in which the level of MTX is overestimated at check-points by EMIT may result in longer periods of hospitalization, higher costs and prolonged administration of elevated doses of "rescue" leucovorin with an increased risk for relapse.     

Synovial membrane immunohistology in early-untreated rheumatoid arthritis reveals high expression of catabolic bone markers that is modulated by methotrexate

Introduction

We aimed to investigate the expression and therapeutic modulation of the receptor activator of the NF-κB ligand (RANKL) system in early-untreated rheumatoid arthritis (RA).

Methods

In this study, 15 patients with newly diagnosed RA (median symptom duration 7 months) were started on methotrexate (MTX) 20 mg weekly. Synovial biopsies were obtained by needle arthroscopy at baseline and 8 weeks after initiation of therapy. X-rays of the hands and feet were obtained at baseline and 1 year after diagnosis. Immunohistochemistry was performed to detect RANKL, receptor activator of nuclear factor-κB (RANK) and osteoprotegerin (OPG) in the synovial biopsies. The in vitro effect of MTX was tested on RA-derived primary fibroblasts and the osteoblasts-like osteosarcoma cell line (rtPCR, Western blot and ELISA) and in osteoclasts (tartrate-resistant acid phosphatase staining and dentine pit formation assay).

Results

MTX decreased synovial cellularity as well as RANK expression and the RANKL/OPG ratio. We confirmed this effect by a decrease of the mRNA and protein RANKL/OPG ratio in synovial-derived fibroblasts and osteoblasts-like tumoral cells exposed in vitro to methotrexate. Supernatants from MTX treated osteoblasts-like tumoral cells prevented pre-osteoclast formation in the absence of exogenous RANKL. Furthermore, MTX blocked osteoclastogenesis from peripheral blood mononuclear cells despite the presence of macrophage colony stimulating factor and RANKL, which indicates that MTX directly inhibits osteoclastogenesis.

Conclusions

The synovial membrane of early-untreated RA is characterized by a high RANKL/OPG ratio that can be reversed by methotrexate.     

Calorimetric determination of inactivation parameters of micro-organisms

AIMS: This study aimed to apply differential scanning calorimetry (DSC) to evaluate the thermal inactivation kinetics of bacteria. METHODS AND RESULTS: The apparent enthalpy (DeltaH) of Escherichia coli cells was evaluated by a temperature scan in a DSC after thermal pretreatment in the calorimeter to various temperatures between 56 and 80 degrees C. Conventional semilogarithmic survival curve analysis was combined with a linearly increasing temperature protocol. Calorimetrically determined D and z values were compared to those obtained from plate count data collected under isothermal conditions to validate the new approach. CONCLUSIONS: The calculated D values using both apparent enthalpy and viability data for cells heat treated in the DSC were similar to the D values obtained from isothermal treatment. Temperatures for 1 through 10-log microbial population reductions, calculated from plate count and enthalpy data, were in agreement within 0.5-2.4 degrees C at a 4 degrees C min-1 heating rate. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel calorimetric method provides an approach to obtain accurate and reproducible kinetic parameters for inactivation. The calorimetric method here described is time efficient and is conducted under conditions similar to food processing conditions.     

Reductions in methotrexate and folate influx in methotrexate-resistant lines of Leishmania major are independent of R or H region amplification

The methotrexate (MTX) and folate transport properties of five MTX-resistant lines of Leishmania major have been examined. These resistant lines all show a decreased Vmax for MTX influx, with no change in apparent affinity (Kt). The Vmax of folate influx is also proportionately decreased without alteration in Kt, supporting our proposal that there is a single carrier mediating influx of both ligands. Amplifications of two regions of DNA, the R region (encoding dihydrofolate reductase-thymidylate synthase) and the H region (Beverley, S.M., Coderre, J.A., Santi, D.V., and Schimke, R.T. (1984) Cell 38, 431-439), were also observed. In a given line, the amplifications occurred singly, in combination, or not at all. No other regions of amplification were detected. The phenotype of reduced MTX transport was moderately stable in the highly resistant R1000 line, being retained for more than 200 generations in the absence of MTX in vitro and during one passage through an infected mouse; in contrast, R- and H-amplified DNA were less stable. The lack of correlation of R and H amplification with reduced MTX transport suggests that alterations in transport are not causally mediated by gene amplification in Leishmania, but are a separate mode of MTX resistance. The onset of decreased MTX transport was also examined; wild-type Leishmania developed a reduced Vmax of MTX influx within 24 h following exposure to 1 microM MTX, which is extremely unstable in the absence of drug pressure. A comparable decrease in the Vmax of influx is seen in cells exposed to MTX in media in which cytotoxicity is eliminated. As the folate/MTX transporter is regulated by exogenous folate, these data suggest that the initial rapid decrease in MTX transport may be a cellular regulatory response, in contrast to that found within the R1000 line which resembles a more stable genetic mutation.