DNA amplification in arsenite-resistant Leishmania

Experimental

DNA Amplification S. DETKE, Department

Cell Research 180 (1989) A61--17

in Arsenite-Resistant K. KATAKURA,

of Microbiology

and K.-P. CHANG’

and Immunology, UHSlThe Chicago Medical 3333 Green Bay Road, Norah Chicago, Zifinois 60064

School,

Arsenite-resistant variants of a trypanosomatid protozoan, Leishmania mexicana amazonensis, were selected in vitro by stepwise increases of sodium arsenite concentrations up to 30 @4 in the culture medium. These variants were found to contain amplified DNA as extrachromosomal supercoiled molecules of about 69 kb. They originate from a single chromosome in the wild-type cells. There is evidence of chromosomal changes in these cells associated with the selection for arsenite resistance. The apparent absence of these circular molecules in the wild type and their loss from the drug-sensitive revertants suggest amplification of chromosomal DNA into these extrachromosomal circles as the mechanism of arsenite resistance. 0 1989 Academic Press, 1~

DNA amplification is a well-documented phenomenon as a mechanism of dmgresistance [l-3]. The amplified genes overproduce proteins specific to the drugs used for the selection of resistance, thereby overcoming their toxic or inhibitory effects ~ This phenomenon has been demonstrated experimentally to occur in trypanosomatid protozoa, Leishmania. These parasites normally live in the digestive tract of the vector sandfly as extracellular promastigotes and in the macrophages of th.e mammalian host as intracellular amastigotes. Acclimatization of promastigotes in vitro to increasing concentrations of methotrexate [4] or @B 3713 [5] or tunicamycin [6] rendered them drug-resistant. The target enzymes of these inhibitors, i.e., dihydrofolate reductase-thymidylate synthetase and ~-acetylg~~c~sa~ minyltransferase, are overproduced, resulting from amplification of certain mosomal DNA as extrachromosomal circles with the genes encoding enzymes in these cells. This unicellular protozoan is thus a useful model for studying DNA amplification as a mechanism of drug resistance. Tunicamycin-resistant variants of Leishmania also express virulent phenotypes, suggesting that the amplified DNA sequence, especially the ~-ace~y~g~~c~sammyltransferase gene in these cells, may mediate their virulence [6]. The finding of this correlation prompted us to produce other drug-resistant cells wit extrachromosomal circular DNA amplified from different chromosomal t-e of the compounds used is sodium arsenite-a trivalent arsenical and classic ydryl-binding agent [7] cytotoxic in vitro to both mammalian cells [8, 9] and parasites [lo]. A brief exposure of promastigotes to arsenite induced their expression of stress proteins [ll], as found with other cells [B2]. Prolonged exposure to other trivalent arsenicals killed trypanosomatids [ 131probably by inhibiting their ’ To whom reprint requests should be addressed. 161

Copyright 0 1989 by Academic Press, Inc. All tights of reproductmn in any form reserved 0014-4827189 %Oi.OG

162 Detke, Katakura,

and Chang

thiol enzymes in the intermediary metabolism [14] or their trypanothione-a novel glutathione-spermidine conjugate crucial for intracellular thiol redox balance and detoxification of oxidative metabolites [ 151.Resistance of an undefined mechanism to arsenicals has long been known in their clinical use for chemotherapy of parasitic diseases [lo]. Thus, the production of arsenite-resistant variants is also of value for studying arsenic stress, toxicity, and resistance at the cellular level. We report here our success of producing arsenite-resistant Leishmania. In contrast to tunicamycin-resistant cells, these variants do not differ from their parental wild type in virulence. We observed in these variants multiple copies of extrachromosomal supercoiled DNA with properties suggestive of gene amplification as the mechanism of arsenite resistance, although the gene products responsible for this remain unknown.

METHODS amazonensis (LV 78) promastigotes were grown at 27°C in Medium 199 with 10% (v/v) heat-inactivated fetal bovine serum and 25 m&f Hepes, pH 7.4 [16]. Sodium arsenite-resistant variants were selected by growing wild-type cells in this medium with increasing drug concentrations to 30 N. Murine macrophages of the J774G8 line were infected at 35°C with wild-type or amen&e-resistant promastigotes in RPM1 1640 with 20% of the same serum and 50 m&f Hepes. Leishmanial differentiation in these macrophages was assessed as described previously [16]. Revertants of arsenite-resistant cells were obtained by allowing them to differentiate first in these macrophages into amastigotes and then back into promastigotes in drug-free medium. DNA isolation. Total DNA was isolated from promastigotes grown to stationary phase by phenol/chloroform extractions combined with proteinase K and ribonuclease A digestions [6]. Extrachromosomal DNA was isolated from about 5x10" cells by the alkaline lysis method [17] and further purified by equilibrium ultracentrifugation in CsCl and ethidium bromide as before [6]. The purified DNA was digested with restriction endonucleases under conditions as suggested by the suppliers and subjected to agarose gel electrophoresis. Orthogonalfield (OFAGE) andfield inversion (FIAGE) agarose gel electrophoresis. Samples used for OFAGE include arsenite-resistant cells, their extrachromosomal DNAs isolated as described, and their revertants and parental wild-type cells. Agarose blocks containing these materials were prepared to give either lo7 cells or 1 ug DNA per block and processed as before [6] for OFAGE [20] at 300 V for 20 h at 13-15°C with a pulse duration of 35-40 s. BamHI digested DNA samples were studied by FIAGE at 200 V for 12 h at 12”C, the forward and reverse times being 1.5 and 0.5 s, respectively. Hybridization. Hybridizations were done ip the presence of heparin and fnters were washed as described by Singh and Jones [18]. DNA was ‘*P-labeled by the random hexanucleotide primer method [ 191. Cells. Leishmania

mexicana

RESULTS Selection and Stability

of Arsenite Resistance

The EDSoof sodium arsenite for the wild type of L. m. amazonensis was about 7-8 uM, as determined in vitro based on growth inhibition of promastigotes (Fig. 1). Viable cells were seen in wild-type culture treated with arsenite at a concentration of 4.5 uJ4 or lower. These cells were subsequently passaged every 3 to 7 days in medium with an arsenite concentration 10 to 50 % greater than that in the previous culture. Cells were made resistant to arsenite at 30 pM in about 1 month.

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NaAsOz (PM) Fig. 1. Arsenite sensitivity of wild-type and arsenite-resistant Leishmania mexicana amazonensis. Promastigotes grown to late-log phase were treated at 5~ lo6 ceils/ml in culture media with different concentrations of the drug. After 5-7 days, cells grown in cultures were counted and the percentage of survival was calculated. q , 6, Wild-type cells; 0, +, two separate lines of cells resistant to 30 @4 sodium arsenite.

Several cultures of arsenite-resistant variants were prepared and all could be passaged continuously in vitro. The arsenite-resistant cells often required a large inoculum during their routine subculturing and had a short stationary phase. The ED50of arsenite-resistant variants was increased to about 95 The arsenite-resistant phenotype of the variants appeared when they were maintained as such than when they were allowed to differentiate into amastigotes. The arsenite-resistant phenotype of the variants remaine unchanged after releasing the drug pressure for up to 8 months, as shown by arsenite treatment of the variants grown in drug-free medium continuously as promastigotes. However, most of the cells were found to revert to arsenitesensitive phenotype when the promastigotes released from drug pressure for 4 months were allowed to differentiate at 35°C in macrophages, grown as amastigotes therein for 1 to 2 weeks and permitted to differentiate back at 27°C into promastigotes. In these experiments, we observed no difference between t arsenite-resistant and the wild-type cells in their infectivity to macrophages.

164

Detke, Katakura,

and Chang

Fig. 2. BamHI digestion of DNA from wild-type and arsenite-resistant Leishmania. One microgram of DNA was digested with BamHI and then electrophoresed in a 0.3 % agarose gel at 1 V/cm for 16 h. Hind111 digested I-DNA was used as molecular weight standard. (A) Ethidium bromide stained gel. Lane 1, Hind111 digested I-DNA; lane 2, DNA from wild-type cells; lane 3, DNA from arseniteresistant cells; lane 4, X/z01 digested I-DNA. (B) Hybridization with DNA isolated from arseniteresistant cells by alkaline lysis method, 6-h autoradiography. Lane 1, DNA from wild-type cells; lane 2, DNA from arsenite-resistant cells. (C) Same as (B) except autoradiography increased to 24 h.

DNA Amplification

of Arsenite-Resistant

Cells

As shown by agarose gel electrophoresis followed by ethidium bromide staining, complete digestions of variant DNA by various endonucleases produced discrete bands, which were inapparent in the wild-type and revertant DNAs prepared and studied simultaneously. BamHI digestion of the variant DNA yielded four discrete bands: a doublet of 9.2 and 9.4 kb, and another doublet banded at about 25 kb (Fig. 2A). Both sets of the doublets showed intensive hybridization (Fig. 2B) when probed with the DNA isolated from the variants by alkaline lysis (see below). After prolonged exposure, this probe was also seen to hybridize weakly with BamHI fragments at 9- and 25-kb regions of the wild-type DNA (Fig. 2 a. The DNA samples isolated by CsCl-ethidium bromide ultracentrifugation from two different stocks of the arsenite-resistant cells gave identical BamHI restriction fragments of 9- and 25-kb doublets (Fig. 3). The Structure of Amplicons Arsenite-Resistant Cells

and Chromosomal

Changes in

As shown by OFAGE followed by ethidium bromide staining, the arseniteresistant cells contained unusual structures (Figs. 4A and 5 A, lanes 1 and 2), which were absent from revertants (Fig. 4A, lane 4; Fig. 5A, lane 3) and wildtype cells (Fig. 5 A, lane 4). The variant-specific DNA migrated off the main path of the chromosomal DNA bands (Figs. 4 and 5, small arrows). DNAs isolated

17.1 I.4 i.6

Fig. 3. BamHI digestion patterns of DNA isolated from arsenite-resistant Leishmania. See legend to Fig. 2 for experimental conditions. Lanes 1 and 2, samples isolated by CsCl-ethidium bromide ultracentrifugation from two different stocks of arsenite-resistant cells: lane 3: NindIII digested i.DNA.

from variants by alkaline lysis or by equilibrium ultracentrifugation in CsCLet dium bromide migrated in OFAGE similarly (Fig. 4, lane 3) and hybridized to the abnormally migrating DNA of the arsenite-resistant cells (Fig. 4, lanes B-3). la overexposed blots (Fig. 5 B), the probe lit up the amplified DNA trailing from t

Fig. 4. Orthogonal field agarose gel electrophoresis of DNA from arsenite-resistant Leishmanin and revertants. The total DNA from 10’ cells or 1 ug amplified DNA was electrophoresed at 300 \I for 20 h with a pulse duration of 35 s for each pair of electrodes. Lanes 1 and 2, two different stocks of arsenite-resistant cells; lane 3, DNA isolated from these cells by CsCl-ethidium bromide ultracentrifugation; lane 4, revertants. Arrows point to anomalously migrating DNA in arsenite-resistant cells and their extrachromosomal amplified DNA isolated. (A) Ethidium bromide staining; (B) hybridization with DNA isolated from drug-resistant cells by CsCl-ethidium bromide ultracentrifugation.

166 Detke, Katakura,

and Chang

Fig. 5. Orthogonal field agarose gel electrophoresis of DNA from Leishmania. See legend to Fig. 4 for experimental conditions. Lanes 1 and 2, arsenite-resistant cells; lane 3, revertants; lane 4, wildtype cells. (A) Ethidium bromide staining; (B) hybridization with amplified DNA probe and overexposed. Large arrows, region of chromosomal changes; small arrows, anomalously migrating circular DNA.

slot and some linear chromosomal DNA, indicating clearly that they shared homology, but migrated along different paths during OFAGE. The chromosomal DNA lit up by the probe appeared as two bands of about equal intensity in arsenite-resistant cells (Fig. 5B, lanes 1 and 2), as a broad band in the revertants (Fig. 5B, lane 3) and as a sharp band in the wild-type cells (Fig. 5 B, lane 4). The hybridization and ethidium bromide-staining patterns of these chromosomal bands were very comparable, except for those in the revertants (see region between large arrows in Figs. 5A and 5 B). The revertants resembled arseniteresistant cells in having two ethidium bromide stained bands, but only a single

Fig. 6. Field inversion gel electrophoresis of partially digested amplified DNA. Approximately 500 ng of DNA isolated from arsenite-resistant cells by the alkaline lysis method was digested with 0.1 unit ofBamH1 for the following time periods: lane 1,O min; lane 2, 1 min; lane 3, 5 min; lane 4, 10 min; lane 5, 20 min; lane 6, 30 min; lane 7, 30 min with 5 units BumHI.

Arsenite-resistant Leishmunia

167

hybridization signal, as found in the wild-type cells. These chromosomal changes described were consistent observations on three separate occasions and were independent of running conditions used for OFAGE. The chromosomal DNA bands in this region were not precisely sized, but appeared to be within the range 600-650 kb, as estimated by comparison with A-DNA ladder as standard. The circular DNA samples isolated were further studied by FIAGE after BamHI digestions for determining the numbers and sizes of the fragments. Undigested DNA gave a single slow-migrating band (Fig. 6, lane 1). A brie1 digestion of this sample with 0.1 U of BamHI for 1 min produced an additional fast-migrating band of almost equal intensity (Fig. 6, lane 2). Upon further digestions for up to 30 min, the intensity of the slow-migrating band gradually diminished with a concomitant increase in that of the fast-migrating band (Fig. 6. lanes 34). Seven to eight additional bands began to emerge 5 min after BarnHI digestion and their sizes appeared to range from 9 to 59 kb (Fig. 6, lanes 4-6). Complete digestion of the sample with 5 U of the enzyme for 30 min yielded two broad bands at about 9 and 25 kb (Fig. 6, lane 7), comparable to those seen in Figs. 2 and 3. DISCUSSION In the present study, we have produced arsenite-resistant variants of a trypanosomatid protozoan, L. m. amazonensis. There appears to be no previous report for the production of arsenical-resistant eukaryotes, although such variants of bacteria have long been known [21, 221. Prior exposure of murine fibroblasts to a sublethal dose of arsenite renders them somewhat more arsenite-resistant 191,but no attempt was made to produce drug-resistant lines. Our variants are about 12fold more resistant to arsenite than their parental wild type, as indicated by EDSO’sfor inhibiting their growth in vitro as promastigotes. The rapid loss of arsenite resistance after passing the variants through one cycle of leishmanial differentiation is a finding in contrast to our previous observations with tunicamytin-resistant Leishmania prepared by the same method [6]. The instability of arsenite resistance in relation to leishmanial differentiation is of interest for further study. We present evidence of DNA amplification in the arsenite-resistant variants produced. Arsenite may now be added to a number of drugs, which have been shown previously to select for variants of eukaryotic cells with DNA amplification associated with drug resistance [l-3]. DNA amplification is demonstrated in arsenite-resistant cells on the basis of several criteria used previously [4-6]. The amplified DNA was initially noted in our variants as discrete BamHI fragments of two doublets at about 9 and 25 kb. This was further shown by their intense signals in Southern hybridization with amplified DNA probes isolated by alkaline lysis or CsCl equilibrium ultracentrifugation from the variants. The fact that the amplified DNA can be isolated by these methods is indicative of their being extrachromosomal supercoiled circular molecules. They indeed migrate exactly as such in OFAGE as separate entities independent of the linear chromosomal ladder (Figs.

168 Detke, Katakura,

and Chang

4 and 5), as found previously for extrachromosomal circular DNA [4-61. We further studied the amplified DNA by FIAGE-a useful technique to analyze simultaneously linear and circular DNA molecules with a wide range of size distribution [23, 241. A single band appears after FIAGE of the amplified DNA isolated by alkaline lysis, suggesting a single size or form of the circular amplicons. This is strongly supported by the emergence of only one additional band with increased mobility after a very brief digestion with BamHI (Fig. 6, lane 2). Such migration behavior of DNA in FIAGE has been reported for the conversion of circles into the linear forms. We estimate the size of the linear DNA band, as representing that of each circle, to be within the range 65 to 70 kb. This estimate is consistent with the sum (=69 kb) of the individual BamHI fragments of the amplified DNA seen as two doublets of 9 and 25 kb in the agarose gel-Southern blots (Figs. 2A, 2B and 3) and as two broad bands of about these sizes with equal fluorescent intensity after FIAGE (Fig. 6, lane 7). The precise order of linkage among the four BamHI fragments to form each circle remains to be mapped. However, it appears that the two fragments of comparable size must be linked together within the circle in order to produce the ladder of seven to eight bands seen after its partial BamHI digestion (Fig. 6, lanes 4-6). Fewer bands would be expected as the partial digest pattern, if each circle were to contain the four fragments arranged in a different order (alternating 9- and 25kb fragments) or to contain fewer numbers of BamHI fragments. The amplified circular DNA of arsenite-resistant cells originate from their chromosome, as found previously with Leishmania made resistant to other drugs [4-61. This is indicated in the present study by the hybridization of the circular DNA to chromosomal bands of about 600 to 650 kb not only in arsenite-resistant cells but also in their parental wild-type cells and revertants. Previously, extrachromosomal circular DNA molecules have been shown to originate from a very large chromosome(s) unresolvable by OFAGE in tunicamycin-resistant cells of the same species [6] and from chromosomes of apparently different sizes in methotrexate-resistant L. major [4, 51. In all these variants, different chromosomal origin of the circular DNAs suggests that they contain different genes responsible for the resistance to different drugs. Indeed, we observed no hybridization between extrachromosomal circular DNAs from arsenite-resistant cells and those from tunicamycin-resistant cells (unpublished). Of interest are the chromosomal changes seen here with the development and loss of arsenite resistance, as shown by hybridization with the extrachromosomal circular DNA. Hybridization of the extrachromosomal circles to a single band in the wild-type Leishmania points to their origin from a single chromosome resolvable by OFAGE. The hybridization of two OFAGE bands in arsenite-resistant cells with the same probe is indicative of chromosomal changes, resulting possibly from abnormal duplication of the chromosome coupled with its sequence deletion or amplification or duplicationtranslocation-events frequently encountered during DNA amplification in other drug-resistant cells [l-3]. The precise explanations for these changes and their reversion during the development and loss of arsenite-resistance await further study.

The arsenite resistance of our variants is apparently due to DNA arnp~i~cati~~ seen in these cells. Consistent with this suggestion is the simu~taueo~s erne~ge~c~ of both events, as found previously in ~ei~~~~~i~ and other cells with gene am~~i~cation proven as the mechanism of their drug resistance [l-3]. T ties of the amplified genes and their products remai unknown in our resistant variants. Known biological activities of arsenicals would suggest that the amplified DNA might contain a gene(s) encoding one or more of the following: arsenate-s~eci~c stress proteins [12], meta~~oth~on~ne[25,26], drug tr~§~~~er [21, 221, enzymes in intermediary metabolism [14] or in arsenic detox via methylation [9, 271, or trypanothione [15]. The possibility of the he metallothionine, and multidrug receptor gene amphfication in the variants seems unlikely on the basis of our preliminary studies, which showed no $~~~ta~t~a~ d~~ere~ces between the variants and their parental wildcells in then- sensitivity to thermal stress, to heavy metals (e.g., Cd2’ and a~ti~eis~maui~ drugs (e.g., sodium stibogluconate and drfi (unpublished data). Work is under way to further exa possibilities listed. DNA amplification reported here appears to be a novel mechanism of arsenite resistance in ~e~~~~a~ia and possibly in other ~~ka~y~t~~ cells as well. We thank Dorothy Pinneo and Pauline Kuta for typing this manuscript. This research was supported by NH-I-NIAID Grant AI-.20486 to K.-P. Chang.

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170 Detke, Katakura and Chang 23. Levene, S. D., and Zimm, B. H. (1987) Proc. Nutl. Acad. Sci. USA 84,4054-4057. 24. Carle, G. F., Frank, M., and Olson, M. V. (1987) Science 232, 65-68. 25. Beach, L. R., and Palmiter, R. D. (1981) Proc. Natl. Acad. Sci. USA 78, 2110-2114. 26. Hamer, D. H. (1986) Annu. Rev. Biochem. 55, 913-951. 27. Willhite, C. C., and Fern, V. H. (1984) Adv. Exp. Med. Biol. 177, 205-228. Received April 15, 1988 Revised version received July 4, 1988

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