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 V(max) for MTX influx, with no change in apparent affinity (K(t)). The V(max) of folate influx is also proportionately decreased without alteration in K(t), 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 amplification 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 V(max) of MTX influx within 24 h following exposure to 1 μM MTX, which is extremely unstable in the absence of drug pressure. A comparable decrease in the V(max) of influx is seen in cells exposed to MTX in media in which cytotoxicity is eliminated. As to 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.