Biochemical changes associated with α-difluoromethylornithine uptake and resistance in Trypanosoma brucei

Molecular and Biochemical Parasitology, 25 (1987) 227-238

227

Elsevier MBP 00852

B i o c h e m i c a l changes associated with ct-difluoromethylornithine uptake a n d r e s i s t a n c e in

Trypanosoma brucei

Vivian Bellofatto 1, Alan H. Fairlamb 2, Graeme B. Henderson 2 and George A.M. Cross 1 Laboratories of 1Molecular Parasitology and ZMedical Biochemistry, The Rockefeller University, New York, U.S.A. Received 17 March 1987; accepted 12 May 1987)

Procyclic Trypanosoma brucei grown m semi-defined media are sensitive to a-difluoromethylornithine (DFMO) (ECs0 100 ixM), an inhibitor of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis. Organisms resistant to 5 mM DFMO (ECs0 > 20 raM) were obtained by passage in incremental amounts of drug. Resistant and wild-type cells accumulated DFMO by passive diffusion with a consequent decrease in polyamine levels, indicating inhibition of ODC in both cell types. The resistant phenotype was stable in the absence of DFMO, in which state there was no increase in ODC abundance or activity. By kinetic analysis, the ODC of resistant cells appeared normal. In wild-type and resistant cells, [3H]DFMO equally and uniquely affinitylabelled a 50 kDa polypeptide corresponding to the ODC subunit. Levels of ODC and tubulin mRNAs were elevated 4-fold in resistant cells grown in the presence of DFMO, although there was no indication of gene amplification. The intracellular concentration of dihydrotrypanothione (N1,NS-bis(glutathionyl)-spermidine), a redox intermediate unique to kinetoplastids, was unchanged in resistant cells growing in DFMO but was halved in wild-type cells exposed to DFMO for 48 h. The exceptionally elevated levels of ornithine found in DFMO-treated resistant cells most likely play a crucial role in cell survival by maintaining intracellular concentrations of dihydrotrypanothione by competing with DFMO for ODC. Key words: Trypanosoma brucei; Drug resistance; Ornithine; DFMO; N~,NS-bis(glutathionyl)-spermidine; Trypanothione

Introduction

Chemotherapy plays a major role in the control of African trypanosomiasis in livestock and humans. Chemoprophylaxis is restrained by the frequency with which this use of trypanocidal agents has resulted in the rapid and extensive spread of drug resistance [1], which can also be readily induced by therapeutic and experimental protocols [2]. e~-Difluoromethylornithine (DFMO) is an enzyme-activated, irreversible inhibitor of Correspondence address: Dr. V. Bellofano, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, U.S.A.

Abbreviations: ODC, ornithine decarboxylase; DFMO, a-difluoromethylornithine; PSG, phosphate-saline-glucose; SDSPAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; SSC, standard saline citrate; HEPES, 4-(2-hydroethyl)-l-piperazineethane sulphonic acid; HPLC, high performance liquid chromatography; kb, kilobase.

ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) [3]. It has recently been successfully used against terminal Trypanosoma brucei gambiense infections in patients considered refractory to other trypanocidal drugs [4]. Laboratory studies have shown that, as in mammalian cells, DFMO acts on trypanosomes by inhibiting ODC. This results in decreased intracellular concentrations of putrescine and spermidine, inhibition of protein and nucleic acid synthesis and gross morphological changes associated with cytostasis [5,6]. Trypanosomeg are more sensitive to DFMO than mammalian cells [3]. The underlying mechanism for this selective toxicity is unknown. It is not due to either increased uptake or greater affinity of the trypanosomatid ODC for DFMO. The curative effect of DFMO is related to drug-induced polyamine depletion because this effect is abolished by coadministration of putrescine or spermidine [6]. Trypanosomes may be

228 more sensitive to the effects of polyamine depletion because they contain the unique glutathione spermidine conjugate dihydrotrypanothione (N l,Ns-bis(glutathionyl)-spermidine) [7]. This metabolite appears to be essential for maintaining intracellular glutathione and possibly other thiol species in the correct thiol redox balance, and for the metabolism of peroxides [8]. To investigate the unique sensitivity of trypanosomes to D F M O and the mechanisms for acquired drug resistance, we have generated a DFMO-resistant T. brucei procyclic clonal cell line and examined polyamine metabolism and the mechanism of drug resistance in this phenotype. We have shown that D F M O resistance in this cell line correlates with increased O D C m R N A amounts and high intracellular ornithine concentrations. However, resistance is not due to O D C gene amplification, to advantageous alteration of the O D C active site, to alternative metabolic routes for polyamine synthesis or to changes in D F M O permeability. Evidence is presented that D F M O resistance is related to an increased capacity to maintain the intracellular concentration of N 1,NS-bis(glutathionyl)-spermidine.

and 10% (v/v) heat-inactivated fetal calf serum. The doubling time for wild-type organisms was 12-14 h and maximum cell density was approximately 5 × 10 7 ml 1. Cells were maintained in log phase by diluting into fresh medium when they approached maximal cell density. Cell number was determined using a Neubauer hemocytometer. ECs0 values refer to the concentration of DFMO that inhibited growth rate by 50%.

Selection of DFMO-resistant cells. DFMO-resistant strains of 7". brucei were obtained by a stepwise process essentially as described for Leishmania major for the generation of a methotrexateresistant cell line [11]. Cells were seeded at 5× I(P ml ~ in SDM-79 medium initially containing (1.5 mM D F M O and subcultured several times in this amount of drug until the doubling time reached that of wild-type cells grown in the absence of drug. Cells were then seeded into the next higher D F M O concentration. By cultivation in increasing concentrations of D F M O (0.5, 1, 2, 3, 4, 5 mM), cell lines resistant to 5 mM D F M O were established. Those resistant to 5 mM were cloned by limiting dilution. Clone R5 was chosen for further biochemical and molecular analysis.

Materials and Methods

Reagents. Acetone was redistilled with ninhydrin to remove amino-compounds before use. Fluorescamine (Fluram, Roche Diagnostics) was purchased from Pierce Chemical Company; Dcamphor sulfonate and methane sulfonic acid from Aldrich Chemical Company; m o n o b r o m o b i m a n e (Thiolyte Reagent) and N-2-hydroxyethyl-piperazine-N'-3-propanesulfonic acid (Ultrol brand) from Calbiochem; n-propanol (HPLC grade) from Fluka; S-adenosylmethionine from Boehringer Mannheim G m b H . Other reagents were purchased from Fisher Scientific. Cell culture. Procyclic trypanosomes were derived from variant M I T a t l . 4 (clone 117) of bloodstream T. brucei strain 427 that was transformed by culturing cells at 27°C in medium SDM79 supplemented with citrate and cis-aconitate [9]. Procyclic cultures (designated wild type) were maintained in SDM-79 at 27°C [10]. SDM-79 medium was supplemented with 7 mg ml ~ hemin

Uptake of radiolabelled DFMO into cel&. To compare the rate of drug uptake between resistant and wild-type cells it was essential to remove all D F M O present in resistant cells. Thus, resistant cultures were grown without drug for 1 week prior to experimentation. Mid-log phase cultures of wild-type and resistant cells were harvested by centrifugation and incubated (6×10 s ml -l) in SDM-79 medium with gentle shaking with 0.1 mM D F M O containing 1 ~xCi ml-1 [3H]DFMO at 27°C. Samples (1 ml) were withdrawn at intervals and washed three times with ice-cold phosphatesaline-glucose, pH 8.0 (PSG) [12] after which the pelleted cells (12000 × g, 1 min) were extracted with 0.6 N perchloric acid (30 min, 0°C). After centrifugation, the supernatants were counted in Aquasol (New England Nuclear) and, after washing with perchloric acid, the pellets were dissolved in 80% Protosol (New England Nuclear) (5 h, 55°C) and counted in Aquasol. Quenching was corrected using internal standards.

229

Cell extracts. Resistant cells, grown for five days in the absence of DFMO, and parent wild-type cells were harvested at mid-log phase (6×106 ml -l) by centrifugation (1500 × g, 6 min) and washed in PSG buffer at 4°C. Cells were resuspended (5x108 ml -l) in ODC buffer (30 mM 4(2-hydroxyethyl)-l-piperazineethanesulphonic acid (HEPES) pH 7.6, 1 mM MgC12, 5 mM dithiothreitol, 0.1 mM pyridoxal phosphate, 0.1 mM EDTA plus 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosyl lysine chloromethyl ketone, 20 ~M Aprotinin, 50 p~M Leupeptin) at 4°C and sonicated until no more cells were visible. The preparation was centrifuged (100000 x g, 60 min) and the supernatant assayed immediately or stored at -20°C. Frozen samples were thawed once and then discarded. No loss of ODC activity was found upon freezing or storage for up to 2 months. Total cellular homogenates were prepared by lysing in ODC buffer plus 0.1% Nonidet P-40 (37°C, 5 min), then diluting 5-fold with ODC buffer (0°C), aliquoting and freezing at - 20oC. Enzyme assays. ODC activity was determined by measuring [t4C]O2 release at 27°C [13], using a reaction buffer comprising 60 mM HEPES pH 7.6, 0.1 mM pyridoxal phosphate, 1.2 mM ornithine (Sigma), 1.2 p,Ci ml 1 L_[l_14C]ornithine (54 mCi mmo1-1) (New England Nuclear) and 5 mM dithiothreitol. Reactions contained 120-150 p,g protein in a final volume of 1 ml [14]. Release of [14C]02 was linear for up to 1 h. Inhibition kinetics for ODC. Time-dependent, irreversible DFMO inhibition of ODC was determined as described [14] except that the enzyme preparation was incubated at 24°C in ODC buffer with various concentrations of inhibitor. Data were analyzed as described [15,16]. Michaelis constants (KM) and maximum initial velocity (Vma×) were calculated from measurements on Hanes-Woolf plots [17]. Linear regression analysis gave r values of >0.98 in each case. [3H]DFMO labelling of ODC. Cytosolic and total lysates were labeled by incubating them at a protein concentration of 1 mg ml -~ with 60 p~Ci ml-l DL_[3,4_3H]DFMO (New England Nuclear)

at 10 p~M final concentration for various times at 27°C. Lysates were either frozen or immediately treated with sodium dodecyl sulphate (SDS) sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) [18]. After Coomassie blue staining, the gels were treated with En[3H]ance (New England Nuclear) and dried prior to fluorography at -70°C.

Nucleic acid analysis. Total parasite DNA was isolated as described [19]. Restriction endonuclease-digested DNA was electrophoresed in agarose gels and blotted onto nitrocellulose according to the technique of Southern [20]. DNA was depurinated [21] to ensure efficient transfer of large fragments. Filters were prehybridized at 42°C in 5 x standard saline citrate (SSC), 5 x Denhardt's solution [22], 50 mM sodium phosphate pH 7.0, 50% formamide and 250 ~g ml -~ sheared and denatured salmon sperm DNA. Hybridizations (42°C, 24 h) included 10 6 cpm ml 1 of probe, 5x SSC, l x Denhardt's, 20 mM sodium phosphate pH 7.0, 50% formamide and 100 ~g ml -t denatured salmon sperm DNA [21]. Blots were washed in 2 × SSC, 0.2% SDS and then in 0.2 x SSC, 0.2% SDS at room temperature. They were autoradiographed after each washing. RNA was purified from cells lysed with 4 M guanidinium thiocyanate [23] by centrifuging through a CsC1 cushion [24]. Total RNA was electrophoresed on a 1.2% agarose-formaldehyde gel [25] and transferred to nitrocellulose [26]. Filters were prehybridized in 5 x SSC, 50 mM sodium phosphate pH 7.0, 2 × Denhardt's solution, 0.1% SDS, 50% formamide, 10% Dextran sulfate and 100 Ixg m1-1 salmon sperm DNA and hybridized (24 h, 42°C) with 10 6 cpm ml 1 probe. RNA slot blots were performed according to the manufacturer's specifications (Schleicher & Schuell). The T. brucei DNA probes used in this study were an ODC cDNA clone (the genel-ous gift of Dr. C.C. Wang, University of California, San Francisco), a fragment of a rDNA clone corresponding to the 3' half of the small subunit rRNA (the generous gift of Dr. D. Campbell, University of California School of Medicine, Los Angeles), and a tandem alpha plus beta tubulin clone isolated by Mr. P. Hevezi in our laboratory. All probes were labelled with [32p]dCTP by the random oligonucleotide priming method [27].

230

Estimation of amino-compounds. Mid-log phase cells were harvested and washed twice by centrifugation in PSG at 4°C. Cells (5× 10 9) w e r e pelleted and extracted twice with 0.5 ml of 5% trichloroacetic acid in 0.01 N HC1 at 4°C. The combined supernatants were extracted five times with two volumes of ethyl acetate. Residual ethyl acetate was e v a p o r a t e d under N 2. To 1.0 ml of acid extract was added 0.5 ml 0.2 M H E P E S p H 8 and 0.05 ml 0.1 M dithiobisnitrobenzoic acid to prevent mixed disulfide formation between intracellular thiol species. The mixture was filtered through a 0.45 txm Nylon-66 centrifugal filter unit (Rainin Instruments) before analysis. A m i n o compounds were separated by reversed phase H P L C and analyzed by post-column derivatization with fluorescamine as described previously

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Estimation of thiols. Thiols were converted to fluorescent derivatives by reaction with m o n o b r o m o b i m a n e [28] before analysis. Washed organisms (108 cells) were derivatized and analyzed as described previously [35].

I

L

120 180 T i m e (h)

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240

Fig. 1. Effect of D F M O on growth of wild-type T. brucei procyclic cells in SDM-79 medium. No D F M O (e), 1 m M (o), 10 m M ( x ) , 25 m M (u). An arrow indicates when cells were diluted into fresh media containing the original a m o u n t of D F M O . A dagger shows cell death at the indicated concentration.

Results

Effects of DFMO. When wild-type cells were exposed to a range of drug concentrations from 1 to 25 raM, they grew at the normal rate for one subculture but, after reseeding into fresh media containing the initial concentrations of D F M O , had decreased growth rates or died (Fig. 1). Cultures in 25 mM D F M O initially grew poorly and died after several days. D F M O inhibited the growth of wild-type T. brucei with an apparent ECs0 of 100 txM. At initial exposure to 500 fxM D F M O , growth rate was decreased by 70% and returned to the normal rate after five subcultures. Subsequent subcultures in increased amounts of D F M O yielded similar growth profiles. A clonal population derived from a culture stabilized at a final concentration of 5 mM for 10 subcultures was designated R5 and had an ECs0 of 20 m M for D F M O . Higher drug concentrations were not tested. Thus, R5 is at least 200-fold more resistant to D F M O than the parent wild-type cells. No morphological changes were observed during the generation of resistant lines. R5 cells showed sta-

ble resistance to D F M O and were not dependent upon continuous exposure to the compound. On dilution into drug-free medium, resistant cells maintained their division time of 14 h. After cultivation in the absence of D F M O for 1-4 weeks and upon returning to 5 mM DFMO-containing medium, R5 cells grew at an unchanged rate without an initial lag.

Uptake of [3H]DFMO. Following subculture for 1 week in the absence of drug, to deplete the cells of D F M O (Table I), the rate of [3H]DFMO incorporation into R5 cells was 2.5-fold less than wild type (Fig. 2). Following subculture for 1 month in drug-free medium, resistant cells continued to show a 2.4-fold decrease in the rate of D F M O incorporation. This ratio was the same whether total D F M O incorporation, free D F M O found in the acid soluble fraction, or bound D F M O in acid precipitated material was measured. Using a published value [29] for the cell volume of T. brucei, the intracellular D F M O

231 TABLE I Intracellular concentrations of D F M O and metabolites in wild type and R5 T. brucei grown in the presence and absence of 5 m M DFMO Wt

DFMO Ornithine Arginine Putrescine Decarboxy-AdoMet Spermidine Glutathione Glutathionylspermidine Dihydrotrypanothione

0 <0.5 3.5 17 0 22 3.1 0.45 3.9

Wt+DFMO 48 h

R5 + DFMO

29 12 11 0 3.6 6 5.6 0.20 1.8

24.2 41 8.0 0.15 4.4 5.1 4.1 0.35 3.4

R5-DFMO 24 h

1 week

1.2 22 7.4 5.2 2.3 4.8 -

0 7 5.6 20 0 18 3.9 0.43 3.9

Values are in nmol (108 cells) 1 and are the average of two experiments. Column one represents wild-type (Wt) cells growing without D F M O ; column two represents wild-type cells incubated with 5 m M D F M O for 48 h. C o l u m n three represents R5 cultures growing continuously in 5 m M D F M O ; columns four and five represent R5 cultures subcultured without drug for 24 h or 1 week. All cultures were harvested at 6 x 106 cells m l - L

concentrations in wild-type and R5 cells were calculated to be 47 IxM and 20 txM respectively, after 70 rain. Despite the difference in uptake rates, the internal concentration was ultimately identical to the external concentration.

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ODC activity in wild-type and resistant T. brucei. In some cell types, stable resistance to D F M O is correlated with elevated O D C activity levels [30-33]. Therefore, O D C activity was measured in extracts of wild-type cells and R5 cells grown without D F M O for one week. U n d e r the conditions used, O D C activity was linear with time. The calculated Vmax was 720 pmol min -1 mg - t soluble protein for wild-type cells and 550 pmol min -1 mg -1 soluble protein for R5 cells. These results show that the resistant phenotype is not due to increased O D C activity. Kinetic analysis gave KM values for ornithine of 520 IxM and 550 txM for wild-type and R5 cells, respectively. Thus, the R5 phenotype does not have an O D C enzyme with a higher affinity for ornithine.

Kinetics of DFMO inhibition. The K i of D F M O b i n d i n g t o O D C was determined in wild-type and R5 cell extracts. Inhibition of O D C activity was time dependent and irreversible in both cases. Plots of half-life of enzyme activity versus the inverse concentration of D F M O were used to determine the K i for D F M O . Wild-type and resistant cells had similar K i values of 135 and 125 I~M, respectively. At infinite drug concentration, the half-lives for D F M O inactivation of wild-type and R5 O D C were essentially the same at 5.2 and 5.5 min, respectively.

232

R5

Wt

ther band intensity increased after 60 min of labelling, thus the comparison here is of total modifiable ODC. Assuming [3H]DFMO binding is a direct measure of the number of enzymatically active O D C molecules in a cell, there are similar amounts of enzyme activity in R5 and wild-type cells. When cell extracts were prepared in the presence of 0.1% NP-40 in order to extract membrane-trapped proteins, and then radiolabelled with [3H]DFMO, results similar to those shown in Fig. 3 were observed; [3H]DFMO binding reflected equal enzyme activity in both wild-type and R5 cells. The monomeric molecular weight of T. brucei O D C as determined from this and similar gels is 50000 -+ 2000. No other [3H]DFMO-radiolabelled polypeptides were seen in resistant extracts analysed on SDS-PAGE gels. Given a [3H]DFMO specific activity of 8 Ci mmol- 1 and a counting efficiency of 33%, and assuming a stoichiometry of 1:1 between DFMO and active O D C enzyme [30], T. brucei procyclic cells contain a minimum of 1 x 104 monomers of O D C per cell.

~!!!i!!~i~i~ii~i~ii~!!!!!!i~!!!i!!!!~ii!~;~i;i~ii~!!!!iiiiiiii~iiii~i!ii;i~i~i;!!!i~i~!~ 18 Fig. 3. [3H]DFMO labelling of ODC in S100 extracts of wildtype and R5 cells. Extracts containing equal amounts of enzyme activity (1.6 mg protein ml 1 for wild-type and 2.0 mg ml I for R5 lysates) were incubated with [3H]DFMO (6.5 Ci mmo1-1) for 60 min at 27°C, boiled in SDS sample buffer and electrophoresed on a denaturing 10% polyacrylamide gel. Marker sizes are in kDa. Analysis is by fluorography.

DFMO and polyamine metabolite levels in wildtype and R5 T. brucei. To assess potential mechanisms to account for the D F M O resistant phenotype of R5 cells, we determined the effect of D F M O on metabolites involved in the ODC-dependent polyamine pathway [35] in wild-type and resistant cells. In DFMO-treated wild-type cells, we observed a decrease in putrescine levels followed by a decrease in spermidine levels (Fig. 4A). Arginine, ornithine and decarboxylated S-

[3H]DFMO radiolabelling of ODC in cell lysates. Since DFMO binds irreversibly and specifically to O D C [34], titration with D F M O was used to quantify the amount of enzymatically active ODC present in extracts from wild-type and R5 cells. When extracts were incubated with [3H]DFMO and the labelled products analyzed by SDS-PAGE and autoradiographed, a single band of similar intensity was observed in each lane (Fig. 3). Nei-

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Fig. 4. Metabolite levels in wild type cells after incubation with 5 mM DFMO for increasing lengths of time at 27°C. (A) Putrescine (o) and spermidine (e) levels. (B) Arginine (V), ornithine (A) and decarboxylated S-adenosylmethionine (A) levels. (C) Dihydrotrypanothione (,,), glutathione (D) and glutathionylspermidine (+) levels.

233 2 5 4 5 6 7 8 9 I0

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O Fig. 5. DNA hybridization analysis. Total DNA was extracted from wild-type (lanes 6-10) and R5 cells grown with 5 mM DFMO (lanes 1-5), digested with Rsa II (lanes 1,6), Pst I (lanes 2,7), Hind III (lanes 3,8), Bam H1 (lanes 4,9) or uncut (lanes 5,10). DNA was electrophoresed on a 1.2% agarose gel, transferred to nitrocellulose and probed with [3zp]ODC cDNA from T. brucei. Markers are Hind III fragments of lambda DNA and values are in kilobase pairs. adenosylmethionine levels rose in a time-dependent way (Fig. 4B). Following the depletion of spermidine, an accumulation of glutathione was associated with a concomitant decrease of glutathionylspermidine and dihydrotrypanothione (Fig. 4C). Wild-type and R5 cells contained 5 and 4 m M D F M O , respectively (Table I). Thus D F M O resistance cannot be due solely to enhanced drug efflux or drug metabolism in R5 cultures. Resistant cells grown continuously with drug had markedly decreased amounts of putrescine, 4-fold lower spermidine levels and high levels of decarboxylated S-adenosylmethionine. Nonetheless, R5 cells grew at the normal rate. When rem o v e d from drug, these metabolites returned to normal levels within one week. A comparison of columns 1 and 3 in Table I shows that the intracellular concentrations of N~-mono and dihydro-

trypanothione conjugates remained at normal (wild-type) levels in the resistant cells growing in D F M O . In contrast, wild-type cells growing in drug were significantly depleted of both N l - m o n o and dihydrotrypanothione (Table I, columns 1 and 2). Significant differences were found in the intracellular concentrations of ornithine in the two cell types. One week after withdrawal of D F M O , R5 cells retained a higher concentration of ornithine, although polyamine levels were restored to normal. Finally, the arginine concentration was increased relative to the wild-type value in both R5 and wild-type cells exposed to D F M O . U p o n removal of drug in the R5 cells the arginine concentration returned to normal after one week. Analysis o f O D C gene sequences. Total cellular D N A was digested with various restriction endonucleases, electrophoresed in agarose gels and stained with ethidium bromide. In L. major, which has a sequence complexity similar to that of T. brucei [36,37], amplified D N A sequences have been seen as heavily stained bands [11]. The R5 strain does not appear to contain any amplified D N A sequences. No heavily stained areas were seen except for k D N A fragments, which were indistinguishable in resistant and wild-type strains. To test specifically for amplified O D C gene sequences, we transferred the gel to nitrocellulose and probed it with a 32p-labelled T. brucei O D C c D N A . No difference between D N A from R5 or wild-type cells (Fig. 5) was observed in the n u m b e r or intensity of any of the hybridized fragments. O D C m R N A . Equal amounts of total R N A from resistant cells growing in 5 m M D F M O and wildtype cells were fractionated by formaldehydeagarose gel electrophoresis and analyzed by transfer hybridization using 32p-labelled O D C c D N A as probe. Fig. 6 shows that the probe hybridized to an R N A of 2.3 kb in each cell line and that there was more hybridizing R N A in R5 than in wild-type cells. To confirm and quantify this difference, total R N A was serially diluted, applied to slot blots and hybridized with either the O D C probe, a tubulin probe or a r D N A probe. Equal intensity hybridization of the r D N A probe confirmed the equivalence of total R N A . Hybrid-

234

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A

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Fig. 6. Analysis of mRNA content of wild-type and resistant cells. (A) RNAs were denatured in formaldehyde-formamideand electrophoresed on a 1.2% denaturing gel. Probe was T. brucei [32p]ODCcDNA. Markers are a mixture of Bethesda Research Laboratories' RNA standards and T. brucei rRNAs and sizes are indicated in kilobases. (B-D) Slot blots containing total R5 and wild-type RNA applied in diluted amounts onto nitrocellulose and probed with T. brucei ODC cDNA (B), alpha and beta tubulin DNA (C) or rDNA (D). Microgram amounts of RNA applied to nitrocellulose are indicated. ization of the O D C probe indicated that there was approximately 4-fold more O D C m R N A in R5 cells than in wild-type procyclics. A similar difference in hybridization intensity observed using the tubulin probe demonstrated that the difference was not restricted to O D C m R N A levels but may represent a general rise in R N A polymerase II transcription. A comparison of protein extracts from R5 cells in the presence of D F M O and wild-type cultures by S D S - P A G E and Coomassie blue staining showed no differences. [3SS]Methionine pulse-labelled R5 and wild-type cultures a l s o showed equivalent banding patterns in autoradiographic analyses on S D S - P A G E . Discussion

Previous studies indicated that D F M O may be transported into trypanosomes by an energy-dependent system [38]. More recently, it was shown that D F M O uptake by T. b r u c e i bloodforms takes place by passive diffusion [39] as has been described for mammalian cells [40]. When T. b r u c e i procyclics were incubated in either 0.1 mM or 10 mM D F M O , incorporation was linear with time

for both wild-type and R5 cells (data not shown). The rate of D F M O influx in procyclic wild-type cells (1.6 nmol h-1 (106 cells) 1 at 27°C) was close to the value of 1 nmol h 1 (106 cells) 1 for bloodstream form T. b r u c e i at 37°C [39]. However, the rate of drug influx into DFMO-free R5 cells was 2.5 times lower. The reason for this consistently observed differential influx is not understood, but could perhaps be due to the presence of a transport system that promotes efflux of D F M O , similar to that reported for multidrug resistance in mammalian cells [50]. Nonetheless, when cultured continuously in the presence of 5 mM D F M O , wild-type and R5 cells contain an intracellular concentration of 5 and 4 mM D F M O , respectively. Thus, drug efflux cannot be the sole cause of resistance to D F M O since these concentrations are 30-40 times the K i for ODC. There was no evidence that loss of [3H]DFMO occurred during experimental procedures due to increased fragility of R5 cells. Analysis of intracellular [3H]DFMO present in R5 and wild-type cells after 6 h of labelling revealed no difference in [3H]DFMO concentration and no metabolism of the drug in either wild-type or R5 cells. These experiments indicate that, in both wild-type and R5

235 cell lines, D F M O enters by passive diffusion and that the 200-fold increase in drug resistance in R5 cells is not related to drug uptake. This differs from a recent publication [51] in which it was concluded that D F M O resistance in T. brucei strain 366D was due to failure of drug uptake. While this may be attributed to mutational differences it is possible that the previous findings could be the result of active efflux of D F M O . Further work is required to clarify this. Over a 48 h period, D F M O treatment of wildtype cells had characteristic effects on the intracellular concentrations of putrescine, spermidine and decarboxylated S-adenosyl methionine. Removal of D F M O from the R5 cultures resulted in the return of putrescine to the normal (wild-type) level. These results show that, in R5 cells as well as in wild-type cells, D F M O inhibits O D C activity. The low putrescine concentration in R5 also indicates that alternative biosynthetic routes to this compound [41] are unlikely to have been induced. This is supported by failure to detect agmatine in the cell extracts or arginine decarboxylase activity in extracts of either wild type or R5 cells growing in the presence or absence of DFMO (data not shown). R5 grew with a normal (wild-type) doubling time, although these cells contained very low intracellular concentrations of putrescine and spermidine. Thus, the high intracellular concentrations of these metabolites found in the wild-type T. brucei phenotype are not required for normal growth of R5. A similar result has been reported in a DFMO-resistant mammalian cell line [42]. The presence of an unidentified compensatory mechanism, necessary for cell viability and present only in resistant cells when polyamine levels are low, cannot be ruled out. The level of ornithine in wild-type cells treated with D F M O for 48 h was increased approximately 24-fold over the wild-type value. A more dramatic increase in ornithine concentration was found in R5 cells growing with D F M O ; in these cells the intracellular concentration was approximately 80 times the wild-type value. These findings are consistent with the observation that ornithine is taken up by resistant cells 14-fold faster than wild-type [51]. In R5 cells, we estimate an intracellular ornithine concentration of 7 mM

which would compete with D F M O for O D C binding in vivo and thus protect the enzyme from inactivation by D F M O . Resistance in R5 cells is not due solely to ornithine uptake as R5 grew normally in 5 mM D F M O in medium containing dialyzed serum, which was free of ornithine as determined by H P L C analysis. Thus increased ornithine synthesis may be an important factor in conferring drug resistance to R5 cells. The biosynthetic route to ornithine in T. brucei is not known but presumably this amino acid is derived from arginine [43]. Notably, arginine concentrations were significantly increased in DFMO-containing wild-type and R5 cells. The effect of O D C inhibition on the levels of the glutathione-spermidine conjugates Nl-mono (glutathionyl)spermidine and dihydrotrypanothione in the two cell types is of particular interest. T. brucei and all other trypanosomatid species do not contain glutathione reductase or glutathione peroxidase activities. Instead, these organisms possess enzymes (trypanothione reductase [44] and trypanothione peroxidase [8]) that are specific for the dihydrotrypanothione conjugate. We have shown that wild-type cells in the presence of D F M O gradually become depleted of glutathione-spermidine conjugates whereas the resistant cells do not. These results suggest that maintenance of the intracellular concentration of dihydrotrypanothione may be an important requirement for cell viability. Comparison of K M and Ki values for ornithine and D F M O , respectively, showed that R5 and wild-type O D C had almost identical affinities for these compounds. From the data in Table I, we calculated that, in the presence of D F M O , R5 cells synthesized 9 nmol putrescine (108 cells) 1 per generation. Since R5 cells have a generation time of 14 h, a net steady-state O D C activity of 10.7 pmol ornithine min -1 (108 cells) -I must be present. Thus, R5 cells maintain growth when exposed to 5 mM D F M O with a steady state concentration of active O D C that is approximately 2% of the level found in the absence of drug. The effect of D F M O on mutant cell O D C was corroborated in a [3H]DFMO-labelling experiment using R5 and wild-type cell lysates. The absence of additional radiolabelled bands indicated that other proteins do not compete for D F M O

236 binding in R5 or wild-type cells. The monomeric molecular weight of O D C was determined to be 50000 -+ 2000. The Mr of the native enzyme is 107000 [45]. Thus, O D C most likely exists in T. brucei as a homodimer. These data indicate that O D C in the R5 phenotype has similar or identical physical and chemical properties to the wildtype enzyme. The analysis of O D C gene sequences in R5 and wild type cells with a 32p-labelled T. brucei O D C cDNA probe indicated that O D C gene amplification is not a factor in R5 DFMO resistance. This is in sharp contrast to the mechanism of D F M O resistance in some mammalian cell lines, which commonly involves O D C gene amplification [33,46]. It is also in contrast to the mechanism of methotrexate resistance in the kinetoplastid L. major, which takes place by D N A amplification, resulting in the production of stable extrachromosomal circular D N A s [47]. R N A hybridization experiments using the 32p_ labelled O D C c D N A probe revealed a 4-fold increase in O D C m R N A in R5 relative to the wildtype cells. In addition, a parallel experiment using a tubulin probe unexpectedly revealed a similar difference in hybridization intensity (see below). If O D C protein synthesis is transcriptionally regulated, then R5 cells with 4-fold more O D C m R N A should show a corresponding increase in O D C protein synthesis. It is possible that a transient O D C increase in resistant cells is modulated by the decreased putrescine levels. Putrescine is known to negatively regulate O D C activity in kinetoplastids and tissue cells [31,48]. Detection of any increase of O D C occurring only in the presence of D F M O would require analysis by immunoassay. The elevated level of tubulin m R N A in the polyamine-depleted R5 cells is of particular interest and would indicate that m R N A s that encode proteins not directly involved in the polyamine pathway can be affected by changes in polyamine levels. Although roles for polyamines in overall nucleic acid synthesis have been discussed [49], an effect on tubulin m R N A has not been documented. In some cells, polyamine depletion causes gross cytoskeletal changes [49], although this does not occur in T. brucei. The elevated tubulin m R N A s in R5 cells do not give rise to increased

synthesis of tubulin proteins (data not shown). A lack of correlation between amounts of mRNAs and the proteins they encode has been observed in a comparison of O D C activity and O D C m R N A levels in some DFMO-resistant variants of $49 mouse T-lymphoma cells and can be attributed to a variety of post-transcriptional processes [31]. The effect of polyamine levels in uncoupling m R N A and protein production needs further investigation. In conclusion, the characteristics of D F M O resistance in T. brucei are considerably different from those previously described in mammalian cells. Although O D C m R N A is raised in the resistant T. brucei, this increase is not due to D N A amplification and does not lead to an increase in O D C activity. The exceptional elevation of ornithine levels in DFMO-treated cells may form the basis for the resistant phenotype. The mechanism of this elevation is unknown. Maintenance of N1,NS-bis(glutathionyl)-spermidine levels in resistant cells suggests a crucial role for this compound in cell viability and wild-type sensitivity to DFMO.

Acknowledgements The authors thank Dr. A. Pegg for providing a pure sample of decarboxylated S-adenosylmethionine and Dr. P.P. McCann of The Merrell Dow Research Institute for the generous gift of DFMO. We thank Drs. C.C. Wang and D.A. Campbell for supplying cloned D N A probes, Carrie L. Henderson and Helen Shim for expert technical assistance, and Dr. O. J/inne for suggestions concerning DFMO-labelling experiments. This work was supported by Public Health Service grants AI 21729 and AI 21429, and by the United Nations Development Program/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, and by The Rockefeller Foundation.

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