Lines of Leishmania major Are Independent of R or H

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY (01987 hy The American Society for Biochemistry and Molecular Biology, Inc. Vol. 262, No. 28, Issue of October 5, pp ,1987 Printed in {J.S.A. Reductions in Methotrexate and Folate Influx in Methotrexateresistant Lines of Leishmania major Are Independent of R or H Region Amplification* (Received for publication, February 2, 1987) Thomas E. Ellenberger and Stephen M. Beverley$ From the Department of Pharmacology, Harvard Medical School, Boston, Massachusetts The methotrexate (MTX) and folate transport prop- cellular target of MTX, the novel bifunctional dihydrofolate erties of five MTX-resistant lines of Leishmania major reductase-thymidylate synthase present in protists (Ferone have been examined. These resistant lines all show a and Roland, 1980; Garrett et al., 1984), has been characterized decreased V,,, for MTX influx, with no change in by biochemical and molecular approaches (Beverley et al., apparent affinity (Kt). The V,,, of folate influx is also 1986; Meek et al., 1985; Grumont et al., 1986). Molecular proportionately decreased without alteration in Kt, studies of highly MTX-resistant Leishmania have revealed supporting our proposal that there is a single carrier mediating influx of both ligands. Amplifications of two amplification of two different regions of wild-type DNA: (i) the 30-kilobase R region, including the dihydrofolate reducregions of DNA, the R region (encoding dihydrofolate tase-thymidylate synthase gene the amplification of which reductase-thymidylate synthase) and the H region leads to dihydrofolate reductase-thymidylate synthase over- (Beverley, S. M., Coderre, J. A., Santi, D. V., and production, and (ii) the 48-kilobase H region, of unknown Schimke, R. T. (1984) Cell 38, ), were also coding potential (Beverley et al., 1984). Current data suggest observed. In a given line, the amplifications occurred that the H region encodes a MTX resistance determinant. singly, in combination, or not at all. No other regions These amplified DNAs are present in the form of extrachroof amplification were detected. The phenotype of re- mosomal circular DNAs (Beverley et al., 1984). The isolation duced MTX transport was moderately stable in the of resistance elements in Leishmania as well as the extensive highly resistant RlOOO line, being retained for more literature concerning folate metabolism and antifolate resisthan 200 generations in the absence of MTX in vitro tance in prokaryotes, protozoa, and cultured mammalian cells and during one passage through an infected mouse; in are powerful resources for addressing the problem of drug contrast, R- and H-amplified DNA were less stable. resistance in trypanosomatid parasites. The lack of correlation of R and H amplification with Decreased MTX accumulation is a common mechanism of reduced MTX transport suggests that alterations in MTX resistance in cultured mammalian cells (Fischer, 1962; transport are not causally mediated by gene amplifi- Huennekens et al., 1978; Kamen et al., 1980; Sirotnak et al., cation in Leishmania, but are a separate mode of MTX 1981; Frei et al., 1984) and bacteria (Sirotnak et al., 1967; resistance. The onset of decreased MTX transport was Mandelbaum-Shavit, 1976). Dewes et al. (1986) recently realso examined; wild-type Leishmania developed a re- ported decreased MTX accumulation in MTX-resistant duced v,,, of MTX influx within 24 h following ex- Crithidia fasciculata, a nonpathogenic protozoan related to posure to 1 p~ MTX, which is extremely unstable in Leishmania, as well as decreased MTX accumulation and the absence of drug pressure. A comparable decrease gene amplification in every MTX-resistant line of Leishmania in the V,,, of influx is seen in cells exposed to MTX in major examined. Because amplification of a locus in cultured media in which cytotoxicity is eliminated. As the folate] mammalian cells can mediate drug resistance by decreasing MTX transporter is regulated by exogenous folate, cellular accumulation of many structurally unrelated drugs these data suggest that the initial rapid decrease in (Biedler et al., 1983; Riordan and Ling, 1985; Fojo et at., 1985; MTX transport may be a cellular regulatory response, Baskin et al., 1981; Hamada and Tsuruo, 1986; Van der Bliek in contrast to that found within the RlOOO line which et al., 1986), we sought to establish whether or not amplifiresembles a more stable genetic mutation. cation of the R and/or H regions in Leishmania resulted in The prevalence of protozoan parasites resistant to commonly used anti-parasitic agents (Wang, 1982; Peters, 1985) emphasizes the need to understand how drug resistance emerges, spreads, and maintains itself in the natural parasite populations. We have studied mechanisms of methotrexate resistance in the trypanosomatid parasite Leishmania. The - * This work was supported by National Institutes of Health Training Grant GM07306-llA1 for Pharmacological Sciences and National Institutes of Health Grant AI The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Burroughs-Wellcome Scholar in Molecular Parasitology changes in MTX transport. Our results indicate that decreased MTX influx is independent of these two amplifications. EXPERIMENTAL PROCEDURES Materials-MTX, folate, and thymidine were purchased from Sigma. [3H]MTX and [3H]folate(Amersham Corp.) were purified as described (Ellenberger and Beverley, 1987). Growth and Plating of Cells--All cell lines are derivatives of the LT252 line of Leishmania (Ebrahimzadeh and Jones, 1983), identified in our laboratory as Leishmania major (Beverley et af., 1987). Cells were propagated in M199 medium, and their growth was monitored as described by Ellenberger and Beverley (1987). The MTX EC, for - The abbreviations used are: MTX, methotrexate; Kt, apparent affinity for the folate/mtx influx activity. T. E. Ellenberger, and S. M. Beverley, manuscript in preparation.

2 13502 MTXIFolate Transport in MTX-resistant Leishmania each line is defined as the concentration of MTX that decreased the rate of growth by 50%. Colonies of Leishmania promastigotes were obtained following growth on 1% agar plates (60 mm) containing M199 medium (Iovannisci and Ullman, 1983) with 2 pg/ml biopterin. The uninoculated plates were dried for 48 h at room temperature and then equilibrated for 60 min in 95% air/5% CO,. Each plate was seeded with 200 promastigotes, wrapped tightly with Parafilm, and incubated at 26 C for 7-12 days. Plating efficiency is expressed as the number of macroscopic colonies of drug-treated cells relative to untreated cells plated simultaneously. Description of Cell Lines-The RlOOO line, selected for resistance to 1 mm MTX, has been described previously (Beverley et al., 1984; Coderre et al., 1983). Revertant lines of the RlOOO line were obtained by serial passage (approximately six cell doublings per passage) in the absence of MTX. Clones 10 and 19 were obtained by G. Kapler in our laboratory from single colonies of the LT252 line. MTXresistant derivatives of these clones, Clone 10-R10 and Clone 19-R50, were obtained by successive propagation in 2, 5, and 10 p~ and 2, 5, 10, 20, and 50 pm MTX, respectively. The HTN-R10 line was obtained from the LT252 line by G. Kapler as follows. Cells were suspended in 25 pg/ml vinblastine plus 10 pg of RlOOO DNA, subjected to electroshock, and then spread at a density of 2 X IO6 promastigotes on 60-mm M199 agar plates containing 10 p~ MTX. After several weeks, single colonies (one of which is referred to as the HTN-R10 line) were picked and grown in liquid medium containing 10 p~ MTX. The clonal POJ-1 line (Garvey et al., 1985) is derived from the LT252 line and was obtained from Buddy Ullman (Oregon Health Sciences University, Portland, OR). The LM-R1 line was obtained after a single passage of wild-type cells in 1 p~ MTX, as described under Results. The MU-10 line resulted from passage of the RlOOO line through a mouse.3 10 RlOOO promastigotes in 0.1 ml of Hanks balanced salt solution were injected into the dorsal aspect of the hind foot of a BALB/c mouse. Amastigotes were harvested from the foot lesion 25 weeks later and transformed into promastigotes by culture in M199 medium, yielding the MU-10 line. Adaptation of Wild-type LT252 Cells to 1.0 WM MTX-Log phase LT252 cells were washed once with drug-free medium, resuspended at 5 x lo5 cells/ml in 100 ml of medium alone or containing 1 pm MTX, and examined at the specified times. At each time point, 3 aliquots of cells were taken: (i) 0.5 ml of cells was vortexed to disperse cell clumps and counted: (ii) another 0.5 ml was diluted, vortexed, and plated as described above; (iii) cells from a 10-ml aliquot were washed and grown in drug-free medium for 7-8 doublings to provide sufficient numbers for transport assays. Transport Assay-Transport of MTX and folate was measured as described by Ellenberger and Beverley (1987). MTX-resistant lines were washed once with drug-free medium and grown for 4-5 cell doublings in the absence of MTX before assay of transport. Kinetic parameters of MTX and folate influx were measured over concentrations of g~ or p ~ respectively., Steady-state accumulation of MTX was determined following 40 min in 5 pm MTX, when the intracellular ligand concentration appeared maximal and constant. Quantitation of Amplified DNAs-R and H region amplification was quantified by cell blot hybridization (Gadler, 1983) as modified in our lab.3 Briefly, 2-fold dilutions of each cell line were applied to nitrocellulose membranes (Schleicher & Schuell), lysed and fixed to the membrane with NaOH, and hybridized to radiolabeled probes specific for the R or H region (Beverley et al., 1984). The R region probes consisted of either plts-5-s45 (Beverley et al., 1986) or the 1.0-kilobase EcoRI-BglII fragment from this plasmid inserted into PUCT,~ a vector derived from puc8 (Vieira and Messing, 1982) by replacement of the polylinker with that of TVX (Maniatis et al., 1982). The H region probe consisted of X LTM-HC3 (Beverley et al., 1984). The intensity of hybridization for resistant cells was evaluated relative to wild-type cells, following normalization to the end point obtained by subsequent rehybridization of the membranes to the p- tubulin probe plt-1 (Huang et al., 1984). Amplification of other regions of DNA was monitored by restriction enzyme digestion and electrophoresis of total genomic DNA as described (Coderre et al., 1983). B. J. Sina, G. M. Kapler, and S. M. Beverley, manuscript in preparation. pucn provided by M. Fromm, Stanford University. RESULTS Decreased MTX and Folate Influx in MTX-resistant Lines-Five MTX-resistant lines of Leishmania were examined, obtained after varying periods of selection in MI99 medium as described under Experimental Procedures. Levels of MTX resistance ranged from approximately 3-fold to more than 5000-fold relative to wild type (Table I). Uptake and accumulation in MTX-resistant lines were examined by measuring the V,,, and Kt for folate and MTX influx and the steady-state levels of intracellular MTX (Table I). A significant reduction in V,,, of MTX influx was seen in every MTX-resistant line, with no change in Kt (Table I). This includes the RlOOO (27% of wild type), HTN-R10 (37% of wild type), Clone 19-R50 (37% of wild type), Clone 10-R10 (24% of wild type), and LM-Rl lines (46% of wild type). Decreases in the Vmax of MTX influx were not correlated with the degree of MTX resistance of each line, as exemplified by the comparison of the Clone 10-RlO (EC, = 27 p ~ and ) RlOOO lines (ECS0 = 3.2 mm) which exhibit comparable reductions in v,.. However, as shown below, at least two other resistance mechanisms may occur within these lines, which might obscure any correlation if present. Steady-state accumulation of MTX was also generally decreased in the resistant lines (Table I). Examination of folate transport revealed parallel altera- tions in folate influx for these lines, without exception. These alterations consisted of decreases in the V,,, of folate influx proportional to the decreases of the V,,, of MTX influx, with no change in Kt (Table I). Gene Amplification in MTX-resistant Leishmania-Genomic DNA from the MTX-resistant lines was examined for evidence of R and H region amplification using the cell blot hybridization method (Table I). Amplification of the R region is observed only in the RlOOO (100-fold) and Clone 19-R50 lines (30-fold), and amplification of the H region is observed only in the RlOOO (50-fold) and HTN-10 lines (200-fold). The absence of R or H amplification was confirmed by hybridization of probes specific for either region of DNA to blots of either restriction enzyme-digested DNA or chromosomes resolved by pulsed field gradient electrophoresis (data not shown). No other DNA amplifications were evident after electrophoresis of restriction enzyme-digested genomic DNA, a method capable of detecting amplifications in Leishmania of more than 5-10 fold (Coderre et al., 1983). Comparison of the MTX transport and amplification data in Table I reveals that while all MTX-resistant lines exhibit decreased rates of MTX influx, these lines exhibit any and all possible combinations of R and H amplification. Both R and H, only H, only R, or neither R nor H amplification may be found in combination with decreased MTX uptake. There is no simple association between decreased MTX uptake and either H or R region amplification. Rapid Induction of Decreased MTX Uptake in Leishmania-Following the observations of Dewes et al., (1986), we examined the onset of decreased MTX transport following exposure to a low concentration of MTX. After inoculation into medium containing 1 PM MTX, the cells stopped dividing with 12 h and thereafter exhibited clumping and altered morphology, as described previously for Leishmania and Crithidia (Coderre et al., 1983; Dewes et al., 1986). More than 90% of the cells remained motile, and growth resumed after 2-3 days at an initially reduced rate (generation time of h uersus 8-10 h in drug-free medium). At varying times during this period, cells were harvested, washed, and grown seven to eight cell doublings in the absence of drug before the assay of transport. In this manner, the MTX transport prop-

3 MTXIFolate Transport in MTX-resistant Leishmania TABLE I MTX and folate uptake by wild-type (LT252) and drug-resistant L. major promastigotes The MTX values and levels of R and H region amplification were measured as described under Experimental Procedures. Uptake and steady-state labeling experiments were performed as described under Experimental Procedures and by Ellenberger and Beverley (1987). Values are means f s. D. Copy number Methotrexate Folate MTX R H ECm K* V W Steady-state Kt V,. (n) (n) labeling (n) P U p~ pmol/min/mg pmol/min/mg LT k f 7.6 (6) 42 f 8.9 (4) 0.7 f f 5.7 (5) HTN-R f f 5.2 (2) 24 f 5.2 (2) 0.6 f f 6.2 (2) C19-R k f 5.8 (5) 38 f 12 (4) 0.5 * f 10 (2) C10-R f f 1.3 (2) 11 f 3.9 (2) 0.4f k 1.9 (2) RlOOO f f 2.9 (2) 16 f 9.8 (2) 0.4f f 3.3 (2) LM-R f f 4.3 (2) 20 f 4.2 (2) NDd NDd -~ pmol of MTX/mg following a 40-min labeling in 5.0 pm MTX. Significantly different from LT252 ( p < 0.01). e Significantly different from LT252 ( p < 0.05). ND, not determined. erties of the viable cells remaining at each time were determined. The Vmax for MTX influx rapidly declines to approximately 60% of the wild-type rate after 24 h of exposure to 1 p~ MTX (Fig. 1). At this time, the viability of the population is 17% as measured by colony formation. The Vmax of MTX influx declines somewhat further during the next 20 days, while the viable cell population declines to 5% and then eventually recovers and expands (this latter phase is not shown in Fig. 1). Throughout this experiment, the Kt for MTX transport was unaffected (data not shown). We have repeated this experiment three times, with similar results (Fig. 1). One of the cell populations that eventually grew out of 1 p~ MTX was termed the LM-R1 line. This line grows with a doubling time of 9.1 h in 1 p~ MTX and exhibits 3-fold resistance. Interestingly, the transport properties of this line are similar to those observed in the wild-type cell population after 24 or more h exposure in 1 pm MTX (Fig. l), suggesting that no further alteration in MTX transport occurred in the surviving cells. IOOL {loo Modulation of MTX Transporter Activity by Exogenous MTX-The activity of the folate/mtx transporter is modulated by exogenous folate, declining in culture medium containing 5 p~ folate (Ellenberger and Beverley, 1987). The rapid decline in the V,,, of MTX influx following MTX exposure suggested the possibility that MTX could similarly modulate the activity of the transporter. To evaluate this effect in the absence of cytotoxicity, cells were grown in culture medium supplemented with 5 mm thymidine (standard culture medium contains 100 p~ adenine, as Leishmania is auxotrophic for purines). Wild-type cells grow at the normal rate in this medium and exhibit only a slight (11%) reduction in growth rate in 1 p~ MTX; at higher MTX concentrations, the growth rate is progressively reduced (Table 11). Cells grown in the supplemented medium containing 1 p~ MTX exhibit a 48% reduction in V,,, for MTX influx (Table 11). Leishmania grown in this medium plus 5 p~ folate exhibit a 78% reduction in the V,,, of MTX influx, as expected (Table 11). Interestingly, the 48% reduction in VmaX for MTX influx in supplemented medium containing 1 p~ MTX is comparable to that exhibited by the LM-R1 line (54% reduction; Table I) and by cell populations undergoing adaptation to growth in 1 p~ MTX (25-60% reduction; Fig. 1). Stability of the Decreased MTX Influx-The initial decline in the VmaX of MTX influx observed after one passage in 1 pm MTX is markedly unstable in the absence of drug pressure. Following four passages (24 cell doublings) in drug-free medium, the V,,, for MTX influx of the LM-R1 line increased from 46% of the wild-type rate (Table I) to 83% (V,,, = 34 pmol/min/mg). Similar results were obtained from clonal lines, obtained by plating the 24-h population from one ex- Hours FIG. 1. Induction of decreased MTX transport by exogenous MTX. Wild-type promastigotes were exposed to 1.0 pm MTX as described under Experimental Procedures. At the indicated times, cells were removed and washed free of MTX, and viability was determined by plating cell aliquots on agar plates. The plating efficiency relative to log phase cells growing in drug-free medium is shown (B). The remaining washed cells were grown seven to eight generations in drug-free medium before determination of Kt and Vmax as described (Ellenberger and Beverley, 1987). The Vmax for MTX uptake, expressed as the percentage of the Vmax of the starting population, is shown for three experiments (0, 0, A). The apparent affinity (KJ for MTX remained indistinguishable from the wild-type value at 1.2 k 0.3 p~ (n = 18). TABLE I1 Regulation of MTX transport activity by exogenous MTX and folate in wild-type Leishmania The LT252 line was grown for four to five doublings in M199 medium containing 5 mm thymidine with the addition of folate or MTX as indicated, and log phase cells were examined for MTX transport activity as described under Experimental Procedures. h pmollminlmg % None 8.1 f f (2) 5 p~ folate 8.3 f f (2) 1 pm MTX 9.1 f f (2) 5 pm MTX 9.8 ND ND a ND, not determined.

4 MTXIFolate Transport in loot - I 80 - a B MTX-resistant Leishmania 1 C 7 PI03 FIG. 2. Stability of decreased MTX transport in MTX-resistant Leishmania. The copy number of the R (solid bars) and H (hatched bars) regions was determined as described under Experimental Procedures. Accumulation of MTX was also measured for each line following a 40-min incubation in 5 pm MTX (open bars). Data is shown for the RlOOO line (Coderre et al., 1983) and reversion derivatives (P35, P53, P103) obtained following35,53, and 103 passages in the absence of MTX. The MU-IO line was derived from amastigotes obtained after injection of the RlOOO line into susceptible mice. periment shown in Fig. 1 on drug-free plates; approximately 30 cell doublings were required for the initial viable cell to grow to sufficient numbers for transport experiments. The Vmaxfor MTX influx for three of these clonal lines was 8090% of the wild-type rate (data notshown) uersus 60% in the parental 24-h population (Fig. 1). The RlOOO line, selected for growth in 1 mm MTX by stepwise selection over a period of many months (Coderre et al., 1983), exhibited decreased MTX accumulation, which was much more stable. MTX accumulation was measured in a series ofrlooo revertant lines, generated by growth in the absence of MTX; changes in accumulation presumably reflect alterations in influx, as MTXefflux is not detectably altered in the RlOOO line (datanot shown; see Ellenberger and Beverley, 1987, for methods). The reduction in MTX accumulation was maintained for more than 200 cell doublings, and approximately 620 cell doublings in the absence of drug were required to attainwild-type levels of accumulation (Fig. 2). R and H region amplifications are also lost during propagation in the absence of drug (Beverley et al., 1984). However, R amplification was lost most rapidly, followed by H amplification, and lastly by the reduction in MTX accumulation (Fig. 2). The stabilities of reduced MTX accumulation and gene amplification were also examined following one passage of RlOOO through an infected mouse. The MU-10 line, derived from lesion amastigotes 25 weeks after inoculation, lacks R region amplification andretains intermediate levels of H amplification while exhibiting reduced MTX accumulation comparable to thatof the parentalrlooo line (Fig. 2). Frequency of H Amplification and Decreased MTX Uptake in Different Lines of Leishmania-We have examined numerous MTX-resistant derivatives of the original LT252 line of L. major or clonal derivatives thereof and find amplification of the H region in only a fraction of these lines5 (Table I). In contrast, Dewes et al. (1986) found amplification of an XbaI fragment characteristic of the H region5in every line selected from the POJ-1 subline of the LT252 line (Garvey et al., 1985).Examination of DNA from the POJ-1line reveals novel DNA fragments that are characteristic of the H region amplification found within the RlOOO line (Fig. 3; Beverley et al., 1984). These novel restriction fragments are not found in S. M. Beverley, unpublished data. A B i I -R /,I 2.3-; FIG. 3. Detection of H region amplification rearrangements in POJ-1 DNA by blot hybridization analysis. Genomic DNA digested with BglII waselectrophoresed on an agarose gel and blotted to a Genescreen Plus membrane (New England Nuclear -DuPont) as described previously (Beverley et al., 1984). The blot was hybridized with 32P-labeledDNA corresponding to the leftmost portion of the wild-type H region, which includes the site of rearrangement of the H amplification in the RlOOO line (Beverley et al., 1984). Specifically, the 13-kilobase EcoRI fragment of LTS-113 (Beverley et al., 1984) was used. Lane A, 4 pg of LT252 DNA; lane B, 4 pg of POJ-1 DNA; lane C, 0.05 pg RlOOO DNA. DNA size markers (kilobase) are shown to the.!.eft of the figure. Fragments characteristic of the wild-type H region (+) and the rearranged, amplified H region ( R ) are shown (Beverley et al., 1984). POJ-1 DNA was provided by L. Simpson. the LT252 line (Fig. 3) or in 16 clonal derivatives thereof (data notshown). When the POJ-1line or other lines bearing low levels of pre-existing amplified DNA are exposed to MTX, elevated levels of amplified DNA rapidly appear2 (Coderre et al., 1983). In contrast, the initial selection of the wild-type LT252 line with MTX frequently does not yield amplification of either the R or H regions: as exemplified by the LM-R1 line (Table I). The origins of the pre-existing amplified DNA in the POJ-1line are unknown. DISCUSSION We have shown that MTX transport is reduced in five lines of L. major selected for resistance to MTX. Reductions in the Vmaxof MTX influx are observed, in theabsence of alterations in apparent affinity (Kt)for MTX (Table I). Cultured mammalian cells exhibiting reduced MTX uptake have been described that only exhibit alterations in the V,, of influx, although alterations in the apparent affinity for MTX are also found (Huennekens et al., 1978; Sirotnak et al., 1981).

5 MTXIFolate Steady-state accumulation of MTX is reduced in the resistant Leishmania; however, accumulation is not simply related to the degree of MTX resistance (Table I). This finding suggests that other mechanisms may contribute to cellular resistance, as discussed below. The MTX-resistant Leishmania also exhibit reductions in the V,,, of folate influx that are proportional to the reductions in MTX influx, again in the absence of alterations in apparent affinity for folate influx. The parallel alterations in MTX and folate uptake in these lines are consistent with the hypothesis that a single carrier mediates high affinity influx of both folate and MTX in L. major (Ellenberger and Beverley, 1987). Exposure of wild-type Leishmania to 1 p~ MTX in the absence of cytotoxicity reduces the V, of MTX transport (Table 11) in a manner similar to the regulation of the folate/ MTX carrier by exogenous folate (Ellenberger and Beverley, 1987). Under cytotoxic conditions, Leishmania exhibit a 40% reduction in the V,,, of MTX influx after 24 h of exposure to 1 p~ MTX (Fig. 1). The decreased rate of influx is maintained while a MTX-resistant population emerges, such as the LM-R1 line which exhibits a 52% reduction in the V,,, of influx. The observation that Leishmania exposed to 1 pm MTX on either a short term or chronic basis exhibit similar decreases in MTX uptake suggests that a large fraction of the viable cells remaining after 24 h may have developed reduced MTX uptake. This proposal is consistent with our finding that MTX can regulate the activity of the transporter. The notion of a regulated initial response to MTX is also supported by the fact that 1 p~ MTX causes the same decrease in transport regardless of toxicity or the duration of exposure (Tables I and 11; Fig. 1). This phenomenon, in which MTX may reduce its own toxicity by inducing a decrease in transport, has not been reported in studies of cultured mammalian cells or bacteria. The ability of Leishmania to modulate MTX transport and the observation that all five MTX-resistant lines exhibit altered MTX transport suggest that every MTXresistant line of Leishmania will exhibit decreased MTX influx. Dewes et al. (1986) have presented data that are consistent with the hypothesis of a rapidly induced response to MTX in a related trypanosomatid. In C. fasciculata, MTX resistance arises in a high proportion of cells exposed to low concentrations of MTX (2 p ~ ) although, the MTX transport properties of these 2 pm-resistant cells were not examined. Interestingly, these cells rapidly adapt to growth in 1 mm MTX, and once adapted, the cells accumulated MTX to only 10% of the wildtype level. Leishmania exposed to or selected with 1 p~ MTX exhibit decreased MTX uptake that is extremely unstable in the absence of drug pressure, which is consistent with the hypothesis of the initial decrease being mediated by a regulatory response. In contrast, the RlOOO line of Leishmania, selected in a stepwise manner for high levels of MTX resistance (Coderre et al., 1983), exhibits decreased MTX accumulation that is moderately stable, persisting for 200 cell doublings in the absence of MTX and requiring more than 600 cell doublings to regain normal MTX accumulation (Fig. 3). Reduced MTX accumulation is also retained after passage through the amastigote state, a period spanning 25 weeks in the absence of drug pressure. The stability of decreased MTX uptake in the RlOOO line suggests that a genetic mutation could be responsible for this phenotype, which slowly reverts during growth in the absence of drug. It is possible that different mechanisms lead to decreased MTX uptake in the RlOOO and LM-R1 lines, although both phenotypes consist of alterations solely in the Vmax of influx. Ultimately, the mechanism(s) by Transport in MTX-resistant Leishmania which the Vmax of influx is reduced following short term or chronic MTX treatment may be determined by biochemical purification of the folate/mtx transporter followed by molecular characterization, as has been reported for the folate carrier of Lactobacillus (Henderson et al., 1977). Independence of R and H Region Amplification and Decreased MTX Transport-Our data strongly suggest that amplification of the R and/or H region(s) is not causally related to the phenotype of decreased MTX transport observed in MTX-resistant lines of Leishmania. Decreased MTX trans- port is seen in all of our MTX-resistant lines (Table I), including those showing R amplification (Clone 19-R50), H amplification (HTN-RlO), both amplifications (RlOOO), or neither amplification (Clone 10-R10). Clone 10-R10 exhibits the most pronounced reduction in MTX transport, yet fails to exhibit detectable amplification of the R or H regions (Table I). The differing stabilities of R amplification, H amplification, and decreased MTX accumulation observed in the RlOOO line grown in the absence of drug (Fig. 2) further support the hypothesis that gene amplification and MTX transport alterations are not causally associated in Leishmania. In addition, we have derived H-amplified lines by stepwise selection of LT252 for resistance to two other drugs, and both lines exhibit normal influx and accumulation of MTX and folate and normal MTX efflux.' Thus, it is clear that neither H nor R amplification mediates decreased MTX accumulation in Leishmania. Our data also suggest that the simultaneous occurrence of gene (H) amplification and decreased MTX transport reported by Dewes et al. (1986) in the POJ-1 line of Leishmania probably represents the simultaneous occurrence of the two independent mechanisms. A sensitive method capable of detecting 10-fold or greater amplification of any new locus (Coderre et al., 1983) has not revealed any amplifications other than those of the R or H in the five MTX-resistant lines. As the reductions in MTX accumulation in all lines are modest, at most 4-fold, it is conceivable that a several-fold amplification undetectable by our methods could mediate decreased accumulation. Multiple Mechanisms of MTX Resistance in Leishmania and Other Species-Bacteria and cultured mammalian cells can exhibit diverse mechanisms of antifolate resistance, including (i) overproduction of dihydrofolate reductase, mediated by gene amplification in mammalian cells (Schimke, 1982); (ii) decreased MTX accumulation, mediated by alterations in transport systems or MTX polyglutamylation (Fischer, 1962; Sirotnak et al., 1967; Henderson et al., 1977; Huennekens et al., 1978; Kamen et al., 1980; Sirotnak et al., 1981; Cowan and Jolivet, 1984); or (iii) evolution of a MTXinsensitive dihydrofolate reductase (Flintoff and Essani, 1980; Haber et al., 1981; Bertino et al., 1983). These resistance mechanisms may occur independently or in combination with one another (Haber et al., 1981; Sirotnak et al., 1981; Bertino et al., 1983; Frei et al., 1984; Ohnuma et al., 1985). Leishmania may exhibit a comparably diverse set of independent MTX resistance mechanisms, employing dihydrofolate reductasethymidylate synthase overproduction mediated by R region amplification, decreased MTX uptake, the resistance determinant encoded by the H region amplification,2 and possibly other mechanisms.' Our results therefore highlight the diversity of resistance mechanisms potentially available to protozoan parasites undergoing chemotherapeutic attack. Acknowledgments-We thank G. Kapler for developing and providing many of the lines described in this work. We thank John Maggio for extensive use of his high performance liquid chromatography system, B. Ullman for providing the POJ-1 line of L. major, L. Simpson for providing POJ-1 DNA, M. Petrillo-Peixoto, B. J. Sina,

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