Haskins Laboratories1 and Department of Chemistry and Physical Sciences2, Pace University, 41 Park Row, New York, NY 10038, USA
Department of Biology, St Francis College, 180 Remsen Street, Brooklyn, NY 11201, USA3
Grace Cancer Drug Center, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA4
Author for correspondence: Nigel Yarlett. Tel: +1 212 346 1246. Fax: +1 212 346 1586. e-mail: nyarlett{at}pace.edu
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ABSTRACT |
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Keywords: Polyamines, Trichomonas, polyamine oxidation, acetylated polyamines
Abbreviations: DENSpm, di(ethyl)norspermine; ODC, ornithine decarboxylase; SSAT, spermidine:spermine N1-acetyltransferase
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INTRODUCTION |
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The urogenital parasite Trichomonas vaginalis differs significantly from other eukaryotes in several aspects of its polyamine metabolism. The parasite produces and excretes large amounts of putrescine via an energy-generating arginine dihydrolase pathway (Linstead & Cranshaw, 1983 ; Yarlett et al., 1996
). The putrescine excreted is used to drive the uptake of spermine via a putrescine:spermine antiporter that selectively transports 1 mol spermine into the cell while exporting 2 mol putrescine, effectively balancing the counterion charge (Yarlett & Bacchi, 1994
). It appears therefore, that T. vaginalis relies upon exogenously supplied polyamines to satisfy its needs for these molecules. Trypanosoma cruzi epimastigotes have also been shown to depend upon the uptake of putrescine to satisfy their polyamine requirement (Ariyanayagan & Fairlamb, 1997
), hence polyamine (or diamine) auxotrophy may be a common adaptation amongst parasites.
In this study we are able to demonstrate that the spermine taken up by the trichomonads is backconverted to spermidine, via a spermidine:spermine N1-acetyltransferase (SSAT)/polyamine oxidase coupled pathway. SSAT catalyses the transfer of an acetyl group from acetyl-CoA to a terminal aminopropyl nitrogen of spermine or spermidine forming N1-acetyl spermine or N1-acetyl spermidine, respectively, which in turn are metabolized to spermidine and putrescine by the action of polyamine oxidases (Fig. 1). In mammalian cells these enzymes are constitutively present at low levels, but are highly inducible by certain agents such as thioacetamide and certain polyamine analogues such as di(ethyl)spermidine and di(ethyl)norspermine (DENSpm) (Erwin & Pegg, 1986
; Libby et al., 1989
). It has been hypothesized that bis(alkyl) polyamine analogues inhibit the growth of mammalian cells by increasing backconversion and export of the natural polyamines, and by acting as functionless equivalents of the natural polyamines (Erwin & Pegg, 1986
; Libby et al., 1989
). We demonstrate in this study that growth of T. vaginalis with DENSpm also results in polyamine depletion and significant inhibition of growth by a mechanism that involves competition with the transport of spermine and inhibition of SSAT effectively blocking spermidine synthesis.
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METHODS |
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Subcellular fractionation.
Cell pellets were resuspended in 10 mM Tris/HCl buffer (pH 7·4) containing 225 mM sucrose, 1 mM CaCl2 and 1 mM MgCl2, and broken by 35 strokes in a Potter-Elvehjem tissue homogenizer at 4 °C. The broken cells were diluted in isotonic buffer and centrifuged at 400 g for 10 min, resulting in nuclear and cell-free extract fractions. The cell-free extract was further fractioned by centrifugation, yielding a large granular fraction (2200 g, 10 min), a small granular fraction (28000 g, 30 min) and a final cytosolic fraction (Lindmark & Müller, 1973). The fractions were characterized by use of the following marker enzymes: malic enzyme (LGF), acid phosphatase (SGF) and lactate dehydrogenase (cytosol), which were assayed as described by Lindmark & Muller (1973). The integrity of the particles was demonstrated using 0·1% Triton X-100.
Enzyme analysis.
SSAT and spermidine N1-acetyltransferase activities were determined by measuring the incorporation of radioactivity from labelled acetyl-CoA into monoacetylspermine or monoacetylspermidine, respectively. The radiolabelled monoacetylpolyamine product was dried on cellulose phosphate paper discs and exhaustively washed to remove unused [14C]acetyl-CoA. The assay was performed in incubations containing 0·5 mM Bicine (pH 8·0), 16·5 µM [1-14C]acetyl-CoA (60 µCi mmol-1), 1 mM acetyl-CoA, 0·0510 mM spermidine or spermine and 25 µl dialysed cell-free extract (50100 µg protein) in a final volume of 200 µl for 60 min at 37 °C. The reaction was stopped with 20 µl ice cold 0·5 M hydroxylamine, boiled for 3 min, microfuged (12500 g for 1 min) and applied to cellulose phosphate discs (Whatman P81). The filters were washed thoroughly with distilled water and finally flushed with methanol prior to drying. Radioactivity was determined using a Beckman TriCarb 1600 CA liquid scintillation counter (Hewlett Packard).
Polyamine oxidase activity was determined by two separate methods; the first measured the spermine-dependent formation of hydrogen peroxide. The peroxide released was detected by measuring the production of tetraguaiacol from guaiacol (2-methoxyphenol) at 420 nm. The assay contained 10 mM glycine (pH 8·0), 10 mM guaiacol, 5 U horseradish peroxidase and 50100 µg dialysed T. vaginalis protein; 1 mM N1-acetylspermine or 1 mM spermine with 0·5 mM CoA was added to start the reaction. The second method employed measuring the absorbance change at 450 nm in a buffer containing 10 mM glycine (pH 8·0), 50 µM FAD and 50100 µg dialysed T. vaginalis protein. The reaction was started by adding 1 mM N1-acetylspermine or 1 mM spermine with 0·5 mM CoA. Protein was determined by the Lowry method.
Inhibition of polyamine biosynthesis.
The effect of DENSpm on growth and polyamine biosynthesis was determined by inoculating 1·5x106 cells into 10 ml fresh culture media containing 50 µM of the compound. After 16 h, the cells were counted using a haemocytometer and compared to the growth of control cultures containing no DENSpm. The cells were harvested by centrifugation at 920 g for 5 min at room temperature and washed with phosphate-buffered salts solution. The cell pellets were lysed with 6% trichloroacetic acid and stored at -80 °C until analysed by HPLC. Polyamines were separated on a C-18 10 µm column (4·5x250 mm) using a series LC 410 pump (Perkin-Elmer) fitted with a rheodyne 50 µl loop injector at a flow rate of 1 ml min-1. Standards and samples were precolumn derivatized by mixing with 2 vols o-phthaldialdehyde and separated using a discontinuous gradient of 12 mM lithium citrate containing 1 mM octanesulfonic acid (pH 2·65) and eluted using acetonitrile (Yarlett & Bacchi, 1988 ). Samples and standards were analysed using a fluorescence monitor (excitation wavelength 320 nm, emission wavelength 455 nm) (Perkin-Elmer). Areas under the peak were determined using a ß-Ram computer software program (IN/US Systems).
Polyamine uptake and metabolism.
The ability of T. vaginalis to transport and metabolize ornithine, putrescine and spermine was determined in 10 ml semi-defined growth medium (Yarlett & Bacchi, 1988 ), containing 5 µM [2,3-3H]ornithine (10 µCi), 0·18 µM [1,4-14C]putrescine (1 µCi) or 0·09 µM [4,7-14C]spermine (0·5 µCi). Cultures were inoculated with 1·5x106 parasites, incubated for 16 h, and the cells counted and harvested by centrifugation at 1000 g for 5 min (Sorvall RTB centrifuge; DuPont). The cells were washed once in phosphate-buffered salts solution and lysed with 6% TCA at 4 °C. Denatured protein was removed by centrifugation and the supernatant filtered through a 0·45 µm Acrodisc filter (Gelman) prior to analysis. Polyamines were separated by ion-paired reverse-phase HPLC using a Perkin Elmer series 410 pump fitted with a Rheodyne 50 µl loop injector. Samples and standards were separated on a C-18 Percosil column (4·6x250 mm) using a linear gradient consisting of 0·1 M NaH2PO4, 2% acetonitrile and 8 mM octanesulfonic acid (pH 2·65). Polyamines were eluted with 150 mM NaH2PO4, 26% acetonitrile and 8 mM octanesulfonic acid (pH 3·25) (Yarlett et al., 1994
). Samples were analysed using a flow-through model 1B Radiometric detector (IN/US Systems) which mixed three parts scintillant (INFLOW ES) to one part sample. Signals were integrated using ß-Ram computer software version 1·62 (IN/US Systems).
Calculation of data.
MichaelisMenten kinetics were used to analyse the enzymesubstrate interactions (Segal, 1976 ). Enzyme activities and Km values were derived using HanesWoolf analysis of substrate plots using Grafit computer software (Erithacus Software). HanesWoolf analysis was selected because it enables a more accurate determination of Km in a crude homogenate (Segal, 1976
). The Km is defined as the substrate concentration at which the initial reaction velocity is half maximal. The inhibition of SSAT by DENSpm was analysed by EadieHofstee plots and the Ki determined from analysis of variation in Km versus increasing inhibitor concentration. This analysis results in a more accurate approximation of Ki as Km is unaffected by protein content (Segal, 1976
). The results are presented as the mean±sample standard deviation (SD) of the coordinate values in the matrix.
Chemicals.
Radioactive substrates were obtained from DuPont. DENSpm was a gift from the National Cancer Institute. All other chemicals were from Sigma.
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RESULTS |
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Polyamine oxidase activity was also determined in crude extracts by measuring the formation of peroxide, a product of the oxidase, in incubations containing guaiacol and horseradish peroxidase. The assay measures the formation of tetraguaiacol (A420) from guaiacol, which occurs in the presence of Ox.BOLDHPOINT; liberated from H2O2 by the action of peroxidase. The formation of tetraguaiacol from guaiacol by the trichomonad polyamine oxidase was dependent upon the addition of CoA and spermine. In the absence of added peroxidase a minor endogenous rate (<10% of that with added peroxidase) was obtained, suggesting the presence of a minor amount of endogenous peroxidase activity in T. vaginalis. Interference due to the presence of catalase can be ruled out as this enzyme has previously been shown to be absent from this parasite (Müller, 1989 ). Addition of spermine or CoA alone resulted in minor rates [0·14 and 0·09 nmol min-1 (mg protein)-1, respectively] until both were added to the reaction mixture [0·47 nmol min-1 (mg protein)-1].
Polyamine interconversion
This series of experiments investigated the uptake and metabolism of radiolabelled polyamines and polyamine precursors. T. vaginalis was grown for 16 h in a semi-defined medium containing 5 µM [2,3-3H]ornithine, 0·18 µM [1,4-14C]putrescine or 0·09 µM [4,7-14C]spermine and analysed for radiolabelled polyamine content. HPLC analysis of acid extracts of cells grown in the presence of radiolabelled ornithine shows that ornithine is taken up (250 pmol per 107 cells), and is converted by ODC to putrescine (340 pmol per 107 cells). However, putrescine was not further metabolized to spermidine or spermine (Fig. 3a). The presence of 5 mM difluoromethylornithine, a suicide inhibitor of ODC, reduced the intracellular amount of putrescine to 70 pmol per 107 cells (79% inhibition; Fig. 3b
). Likewise, putrescine was readily taken up by T. vaginalis (Fig. 3d
), reaching 70 pmol per 107 cells, and again no conversion to the higher polyamines was detected. These data are in agreement with previous results which failed to detect S-adenosyl-L-methionine decarboxylase activity and a forward-directed polyamine biosynthetic pathway in this parasite (Yarlett, 1988
). In contrast, radiolabelled spermine was taken up (26 pmol per 107 cells) and converted to spermidine (6 pmol per 107 cells) and putrescine (1·4 pmol per 107 cells; Fig. 3e
). The presence of 50 µM DENSpm reduced the amount of radiolabelled spermine taken up by 95% (1·4 pmol per 107 cells) and completely blocked backconversion to spermidine and putrescine (Fig. 3f
). These results demonstrate that polyamine uptake and backconversion is a constitutive feature of these cells.
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DISCUSSION |
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The subcellular distribution of SSAT and polyamine oxidase indicates that they are in separate subcellular compartments. The predominantly cytosolic location of SSAT would enable spermine, taken up by the putrescine:spermine antiporter system (Yarlett & Bacchi, 1994 ), to be acetylated. Acetylation of polyamines neutralizes the charge on the polyamine and enhances transport. This processing of spermine may be necessary to enable entry of spermine into subcellular compartments such as nuclei where it may then be deacetylated, liberating free spermine. The predominantly hydrogenosomal location of the polyamine oxidase suggests that acetylation of spermine is also necessary for entry of spermine into this organelle, where it is further metabolized by the polyamine oxidase, releasing spermidine. That acetylation has a significant role in hydrogenosome metabolism is corroborated by the significant ultrastructural damage that occurs to these organelles when polyamine metabolism is blocked by use of polyamine analogues, such as diaminobutanone or DENSpm (Reis et al., 1999
; Santoro et al., 1999
). Previous localization studies with mammalian cells have demonstrated that polyamine oxidase is also localized in subcellular compartments (Pavlov et al., 1991
; Holta, 1977
).
SSAT is the first and rate-limiting step in the backconversion pathway of polyamine metabolism (Casero & Pegg, 1993 ; Woster, 1993
). The enzyme acetylates the aminopropyl end of spermine or spermidine, thus reducing the charge on the polyamine. In mammalian cells, SSAT has been characterized as being highly substrate specific, rapidly inducible and having a short half-life (Woster, 1993
). The enzyme is rapidly induced in mammalian cells by a variety of factors including polyamine analogues, such as DENSpm (Porter et al., 1991
). Acetylated polyamines can then act as a substrate for polyamine oxidase or can be excreted (Woster, 1993
; Wallace, 1987
) which also results in downregulation of polyamine biosynthetic enzymes and suppression of polyamine transport. The net effect of the bis(alkyl) polyamine analogues is to deplete intracellular polyamines and thereby inhibit cell growth (Porter et al., 1991
; Casero et al., 1989
; Pegg et al., 1989
). In contrast to mammalian cells, where an SSAT induction of 200- to 1000-fold was observed (Porter et al., 1991
; Casero et al., 1989
; Pegg et al., 1989
), the trichomonad enzyme was not induced by 16 h in vitro culture with 50 µM DENSpm.
Acetylation, particularly N1-acetylspermidine, may enhance polyamine excretion since acetylated polyamines are typically found outside of the cell, not within it (Wallace, 1987 ). Growth inhibition in mammalian cell lines by DENSpm is believed to be the result of super induction of SSAT, which results in increased N1-acetylspermidine production which is then apparently excreted into the medium (Pegg et al., 1989
); thus SSAT may be a determinant in polyamine export (Porter et al., 1991
). Consistent with this hypothesis is the observation that murine L1210 and B16 melanoma cell lines which do not superinduce SSAT maintain a nitrogen (or charge) equivalent balance between analogue uptake and polyamine depletion (Libby et al., 1989
; Bergeron et al., 1989
). In this study it was found that DENSpm caused a significant reduction of trichomonad intracellular polyamines. Based upon a mean intracellular volume of 4760 µl per 108 cells (Knodler et al., 1994
), it can be calculated that the intracellular concentration of DENSpm reaches 7590 µM. This is approximately 1·52 times the extracellular concentration, and well above the Ki for SSAT. These findings suggest that T. vaginalis is able to concentrate polyamines (and their analogues) against a concentration gradient. The mode of action of DENSpm in T. vaginalis appears to involve competition with spermine for transport into the cell (Yarlett & Bacchi, 1994
), and, once internalized, to block backconversion of spermine to spermidine by inhibition of SSAT. This is borne out by the observation that the total tetra-amine pool size (spermine plus DENSpm) of treated and untreated cells are essentially unchanged. Hence, consistent with the proposed mode of action, the greatest effect of DENSpm is on the trichomonad spermidine pool, which is decreased 68% in treated cells. These results clearly demonstrate the potential of this pathway as a rational target for the future design of antitrichomonad agents.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 February 2000;
revised 20 June 2000;
accepted 18 July 2000.