Hypoxanthine Transport Research Articles

Increased ENT2 expression and its association with altered purine metabolism in cell lines derived from different stages of colorectal cancer.

Colorectal cancer (CRC) is one of the most prevalent malignant cancer types worldwide. Although the purine metabolism pathway is vital for cancer cell survival, little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with purine metabolites. The aim of the present study was to evaluate the levels of hypoxanthine phosphoribosyl transferase (HPRT), hypoxanthine and uric acid (UA), as well as xanthine oxidase (XO) activity, and investigate their association with ENT2 expression levels in a normal human colon cell line and CRC cell lines derived from different stages of CRC. These analyses were performed using the normal colon CCD-841CoN cell line and a panel of human CRC cell lines comprising SW480, HCT15 and HCT116, which represent Dukes' B, C and D stages, respectively. Reverse transcription-quantitative PCR was performed to determine the level of ENT2 mRNA expression. In cells of all CRC stages, the levels of HPRT and hypoxanthine were significantly higher (P<0.05), while XO activity and UA levels were significantly decreased (P<0.05), compared with those in the CCD-841CoN cell line. ENT2 expression was found to be elevated in cells derived from all stages of CRC. The Dukes' D stage cell line had higher levels of HPRT and hypoxanthine, although its ENT2 level was not significantly lower than that of the Dukes' B and C stage cell lines. Increased levels of HPRT and hypoxanthine in various stages of CRC may indicate an increase in the activity of the salvage pathway. The increased expression of ENT2 implies the importance of the ENT2 protein in facilitating hypoxanthine transport, which is required for enhanced DNA synthesis via hypoxanthine recycling. In conclusion, ENT2 may have potential as a target in the development of CRC therapeutics.

Trypanosoma brucei bloodstream forms express highly specific and separate transporters for adenine and hypoxanthine; evidence for a new protozoan purine transporter family?

The transport of nucleobases and nucleosides in protozoan parasites is known to be performed by Equilibrative Nucleoside Transporter (ENT) family members, including the extensively studied P1 and P2 nucleoside transporters of T. brucei bloodstream forms. Studies with P2 knockout parasites suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Here, we deleted several ENT genes, in addition to P2, including an array comprising three genes encoding for high-affinity broad-selectivity nucleobase transporters − the longest multi-gene locus deletion in T. brucei to date. It was verified that none of them appreciably contributed to the transport of hypoxanthine in bloodstream forms grown axenically in HMI-9 medium, which was mainly performed by a previously not described hypoxanthine-specific transporter (HXT1) with a Km of 22 ± 1.7 μM and Vmax of 0.49 ± 0.06 pmol(107 cells)−1 s−1. The uptake of adenine was also assessed in the knockout cells and was performed by a highly specific adenine transporter (ADET1) with a Km of 573 ± 62 nM and Vmax of 0.23 ± 0.06 pmol(107 cells)−1 s−1. Neither HXT1 nor ADET1 displayed any affinity for other natural purines or pyrimidines and could not be completely inhibited by hypoxanthine or adenine analogues. These carriers may be the final pieces in the substantial transporter array trypanosomes can employ to fine-tune the uptake of purines from diverse environments during their life cycles, and may be encoded by genes other than those of the ENT family.

Effect of Hypoxanthine on Functional Activity of Nucleoside Transporters ENT1 and ENT2 in Caco-2 Polar Epithelial Intestinal Cells.

We studied regulation of hypoxanthine transport depending on its concentration in the culture medium. Caco-2 cells were differentiated on membrane filters to create a model of the intestine. Different hypoxanthine uptake on the apical and basolateral cell membranes was observed. The expression of SLC29 family genes encoding passive nucleoside transporters increased upon changes in hypoxanthine concentration in the medium Localization of the transporters and their influence on the effect of pharmacological preparations are discussed.

Borrelia burgdorferi Harbors a Transport System Essential for Purine Salvage and Mammalian Infection

Borrelia burgdorferi is the tick-borne bacterium that causes the multistage inflammatory disease Lyme disease. B. burgdorferi has a reduced genome and lacks the enzymes required for de novo synthesis of purines for synthesis of RNA and DNA. Therefore, this obligate pathogen is dependent upon the tick vector and mammalian host environments for salvage of purine bases for nucleic acid biosynthesis. This pathway is vital for B. burgdorferi survival throughout its infectious cycle, as key enzymes in the purine salvage pathway are essential for the ability of the spirochete to infect mice and critical for spirochete replication in the tick. The transport of preformed purines into the spirochete is the first step in the purine salvage pathway and may represent a novel therapeutic target and/or means to deliver antispirochete molecules to the pathogen. However, the transport systems critical for purine salvage by B. burgdorferi have yet to be identified. Herein, we demonstrate that the genes bbb22 and bbb23, present on B. burgdorferi's essential plasmid circular plasmid 26 (cp26), encode key purine transport proteins. BBB22 and/or BBB23 is essential for hypoxanthine transport and contributes to the transport of adenine and guanine. Furthermore, B. burgdorferi lacking bbb22-23 was noninfectious in mice up to a dose of 1 × 10(7) spirochetes. Together, our data establish that bbb22-23 encode purine permeases critical for B. burgdorferi mammalian infectivity, suggesting that this transport system may serve as a novel antimicrobial target for the treatment of Lyme disease.

Nucleobase Transport by Human Equilibrative Nucleoside Transporter 1 (hENT1)

The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter.

Uptake of purines in Plasmodium falciparum-infected human erythrocytes is mostly mediated by the human equilibrative nucleoside transporter and the human facilitative nucleobase transporter.

BackgroundPlasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside transporter (hENT1), the human facilitative nucleobase transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three transporters to the uptake of adenosine and hypoxanthine. Using the appropriate transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated.MethodsThe transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these transporters was also studied.ResultsTransport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 nucleobase transporter whereas adenosine entered predominantly through the hENT1 nucleoside transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC). It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1.ConclusionCollectively, the data obtained in this study clearly show that the endogenous host erythrocyte transporters hENT1 and hFNT1, rather than the NPP, are the major route of entry of purine into parasitized RBC. Inhibitors of hENT1 and hFNT1, as well as the NPP, should be considered in the development of anti-malarials targeted to purine transport.

Transport of purines and purine salvage pathway inhibitors by the Plasmodium falciparum equilibrative nucleoside transporter PfENT1

Plasmodium falciparum is a purine auxotroph. The transport of purine nucleosides and nucleobases from the host erythrocyte to the parasite cytoplasm is essential to support parasite growth. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a major route for purine transport across the parasite plasma membrane. Malarial parasites are sensitive to inhibitors of purine salvage pathway enzymes. The immucillin class of purine nucleoside phosphorylase inhibitors and the adenosine analog, tubercidin, block growth of P. falciparum under in vitro culture conditions. We sought to determine whether these inhibitors utilize PfENT1 to gain access to the parasite cytosol. There is considerable controversy in the literature regarding the Km and/or Ki for purine transport by PfENT1 in the Xenopus oocyte expression system. We show that oocytes metabolize adenosine but not hypoxanthine. For adenosine, metabolism is the rate limiting step in oocyte uptake assays, making hypoxanthine the preferred substrate for PfENT1 transport studies in oocytes. We demonstrate that the Ki for PfENT1 transport of hypoxanthine and adenosine is in the 300–700μM range. Effects of substrate metabolism on uptake studies may explain conflicting results in the literature regarding the PfENT1 adenosine transport Km. PfENT1 transports the tubercidin class of compounds. None of the immucillin compounds tested inhibited PfENT1 transport of [3H]hypoxanthine or [3H]adenosine. Although nucleobases are transported, modifications of the ribose ring in corresponding nucleoside analogs affect substrate recognition by PfENT1. These results provide new insights into PfENT1 and the mechanism by which purine salvage pathway inhibitors are transported into the parasite cytoplasm.

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis

In plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine–guanine–hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T-DNA insertion alleles AtAzg1-1 and AtAzg2-1 reveal a marked resistance to growth in the presence of 8-azaadenine and 8-azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8-azaadenine and 8-azaguanine. Radio-labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine–guanine transporters.

A comprehensive model of purine uptake by the malaria parasite Plasmodium falciparum: identification of four purine transport activities in intraerythrocytic parasites

Plasmodium falciparum is incapable of de novo purine biosynthesis, and is absolutely dependent on transporters to salvage purines from the environment. Only one low-affinity adenosine transporter has been characterized to date. In the present study we report a comprehensive study of purine nucleobase and nucleoside transport by intraerythrocytic P. falciparum parasites. Isolated trophozoites expressed (i) a high-affinity hypoxanthine transporter with a secondary capacity for purine nucleosides, (ii) a separate high-affinity transporter for adenine, (iii) a low-affinity adenosine transporter, and (iv) a low-affinity/high-capacity adenine carrier. Hypoxanthine was taken up with 12-fold higher efficiency than adenosine. Using a parasite clone with a disrupted PfNT1 (P. falciparum nucleoside transporter 1) gene we found that the high-affinity hypoxanthine/nucleoside transport activity was completely abolished, whereas the low-affinity adenosine transport activity was unchanged. Adenine transport was increased, presumably to partly compensate for the loss of the high-affinity hypoxanthine transporter. We thus propose a model for purine salvage in P. falciparum, based on the highly efficient uptake of hypoxanthine by PfNT1 and a high capacity for purine nucleoside uptake by a lower affinity carrier.

Cloning and functional characterization of two bacterial members of the NAT/NCS2 family inEscherichia coli

The coding potential of the genome of E. coli K-12 includes YgfO and YicE, two members of the evolutionarily conserved NAT/NCS2 transporter family that are highly homologous to each other (45% residue identity) and closely related to UapA of Aspergillus nidulans, a most extensively studied microbial member of this family. YgfO and yicE were cloned from the genome, over-expressed extrachromosomally and assayed for uptake of [(3)H]xanthine and other nucleobases, in E. coli K-12, under conditions of negligible activity of the corresponding endogenous systems. Alternative, essentially equivalent functional versions of YgfO and YicE were engineered by C-terminal tagging with an epitope from the E. coli lactose permease and a biotin-acceptor domain from Klebsiella pneumoniae. Both YgfO and YicE were shown to be present in the plasma membrane of E. coli and function as specific, high-affinity transporters for xanthine (K(m) 4.2-4.6 microM for YgfO, or 2.9-3.8 microM for YicE), in a proton motive force-dependent manner; they display no detectable transport of uracil, hypoxanthine, or uric acid at external concentrations of up to 0.1 mM. Both YgfO and YicE are inefficient in recognizing uric acid or xanthine analogues modified at position 8 of the purine ring (8-methylxanthine, 8-azaxanthine, oxypurinol, allopurinol), which distinguishes them from their fungal homologues UapA and Xut1.

Hypoxanthine transport in human glioblastoma cells and effect on cell susceptibility to methotrexate.

Cancer cells may circumvent the cytotoxic effect of antimetabolite drugs that inhibit de novo nucleotide synthesis via the uptake of extracellular preformed nucleobases or nucleosides. The goal of this study was to investigate the nucleobase transport mechanism in human U-118 glioblastoma cells and to determine whether the purine nucleobase hypoxanthine affects cell susceptibility to methotrexate. Uptake experiments were performed using 3H-labeled hypoxanthine. RT-PCR was used to determine the expression of nucleoside transporters. Methotrexate-induced apoptosis was analyzed using annexin V staining and FACScan analysis. Hypoxanthine transport in U-118 cells involved both carrier-mediated (Km = 10.5 +/- 6.3 microM, Vmax = 1.45 +/- 0.69 pmol/10(5) cells/60 s) and simple diffusion processes (Kd = 0.36 +/- 0.009 microm/10(5) cells/60 s). Uptake was sensitive to Na+ and inhibited by nucleobases but not nucleosides or nucleoside transport inhibitors. In contrast, uptake of a nucleoside, uridine, was inhibited by nucleosides but not nucleobases. RT-PCR analysis suggested the presence of hENT1, hENT2, and hCNTI nucleoside transporters in U-118 cells. In the absence of hypoxanthine, methotrexate inhibited U-118 cell proliferation and induced apoptosis. These toxic effects were diminished when hypoxanthine was present at physiologically relevant concentrations. Hypoxanthine transport in U-118 cells involves a Na+-dependent, high-affinity nucleobase transport system functionally distinct from nucleoside transporters. At physiologic concentrations, hypoxanthine protects glioblastoma cells from the cytotoxicity of methotrexate.

Na+ gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles.

The properties of hypoxanthine transport were investigated in purified brush border membrane vesicles isolated from calf proximal and distal jejunum. Hypoxanthine uptake in the vesicles was stimulated by a transmembrane Na(+) gradient and an inside negative potential resulting in a transient accumulation of intravesicular hypoxanthine, especially in the proximal jejunum. Na(+)-dependent hypoxanthine uptake at this site seemed to occur by two saturable transport systems, a high affinity (K(m)=0.33 micromol/l) and a low affinity (K(m)=165 micromol/l) transporter. Guanine, hypoxanthine, thymine and uracil inhibited intravesicular hypoxanthine uptake, whereas adenine and the nucleosides inosine and thymidine were without effect. These findings represent the first demonstration of active Na(+) gradient-dependent nucleobase transport in intestinal brush border membrane vesicles.

Functional and Molecular Characterization of Nucleobase Transport by Recombinant Human and Rat Equilibrative Nucleoside Transporters 1 and 2: CHIMERIC CONSTRUCTS REVEAL A ROLE FOR THE ENT2 HELIX 5–6 REGION IN NUCLEOBASE TRANSLOCATION

The human (h) and rat (r) equilibrative (Na(+)-independent) nucleoside transporters (ENTs) hENT1, rENT1, hENT2, and rENT2 belong to a family of integral membrane proteins with 11 transmembrane domains (TMs) and are distinguished functionally by differences in sensitivity to inhibition by nitrobenzylthioinosine and coronary vasoactive drugs. Structurally, the proteins have a large glycosylated loop between TMs 1 and 2 and a large cytoplasmic loop between TMs 6 and 7. In the present study, hENT1, rENT1, hENT2, and rENT2 were produced in Xenopus laevis oocytes and investigated for their ability to transport pyrimidine and purine nucleobases. hENT2 and rENT2 efficiently transported radiolabeled hypoxanthine, adenine, guanine, uracil, and thymine (apparent K(m) values 0.7-2.6 mm), and hENT2, but not rENT2, also transported cytosine. These findings were independently confirmed by hypoxanthine transport experiments with recombinant hENT2 produced in purine-cytosine permease (FCY2)-deficient Saccharomyces cerevisiae and provide the first direct demonstration that the ENT2 isoform is a dual mechanism for the cellular uptake of nucleosides and nucleobases, both of which are physiologically important salvage metabolites. In contrast, recombinant hENT1 and rENT1 mediated negligible oocyte fluxes of hypoxanthine relative to hENT2 and rENT2. Chimeric experiments between rENT1 and rENT2 using splice sites at rENT1 residues 99 (end of TM 2), 171 (between TMs 4 and 5), and 231 (end of TM 6) identified TMs 5-6 of rENT2 (amino acid residues 172-231) as a determinant of nucleobase transport activity, suggesting that this domain forms part(s) of the ENT2 substrate translocation channel.

Synthesis and evaluation of organosilicon inhibitors of active purine transport in human osteoblasts.

In the search for new compounds that might, once incorporated into biomaterials, stimulate the natural processes of bone regeneration, a new series of silicon-containing alkyl nucleobase analogues has been synthesized. An active hypoxanthine transport process in human osteoblasts was demonstrated, with an apparent Michaelis constant of 2.3 microM and a maximum possible rate of 0.47 pmol s(-1) x 10(6) cell. The synthesized analogues were tested for toxicity in human osteoblasts. Nontoxic analogues were tested in competition transport studies with [(14)C]hypoxanthine. Two of them were found to inhibit the active transport of hypoxanthine in human osteoblasts, with IC(50) values of 6.5 and 11.6 microM.

The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep

The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na +-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63±3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood–brain barrier, while the free diffusion is negligible. The derived value of K m (20.72±2.42 μM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61±2.28 μM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo.

Hypoxanthine transport in human tumour cell lines. Relationship to the inhibition of hypoxanthine rescue by dipyridamole

Hypoxanthine (HPX) uptake was investigated in four human tumour cell lines previously characterised as being sensitive ( ds: A549 and MCF7) or insensitive ( di: COR-L23 and T-47D) to dipyridamole (DP)-induced inhibition of HPX rescue from antipurine antifolate-induced growth inhibition. The aim of the study was to determine the mechanism underlying the differential sensitivity of HPX rescue to DP. The time–course of HPX uptake in the two ds cell lines was different in comparison to the two di cell lines. The initial rate of HPX uptake in the di cell lines was more rapid than in the ds cell lines such that at 60 sec the amount of HPX taken up by the former was 2–6 times higher than that taken up by the later. The K t and T max for HPX transport in di COR-L23 cells were 870 μM and 4.75 μM/10 6 cells/min and 1390 μM and 1.78 μM/10 6 cells/min in ds A549 cells. HPX transport was not sodium-dependent in these cells. Equilibrative nucleoside transporter 2 (ENT2)-mediated thymidine transport was also higher in di cells. DP inhibited HPX uptake into ds cell lines by ≥48% and by ≤20% in the di cell lines. Competition studies with HPX and thymidine transport via ENT2 indicated an overlap between nucleoside and nucleobase transport transporters in the breast cancer cell lines (MCF7 and T-47D). These studies showed that more rapid and extensive HPX uptake, as well as reduced sensitivity to DP inhibition, is associated with the inability of DP to prevent HPX rescue from antipurine antifolate-induced growth inhibition in certain human tumour cell lines.

Differential regulation of nucleoside and nucleobase transporters in Crithidia fasciculata and Trypanosoma brucei brucei

The regulation of the activity of purine transporters in two protozoan species, Crithidia fasciculata and Trypanosoma brucei brucei, was investigated in relation to purine availability and growth cycle. In C. fasciculata, two high-affinity purine nucleoside transporters were identified. The first, designated CfNT1, displayed a K m of 9.4±2.8 μM for adenosine and was inhibited by pyrimidine nucleosides as well as adenosine analogues; a second C. fasciculata nucleoside transporter (CfNT2) recognized inosine ( K m=0.38±0.06 μM) and guanosine but not adenosine. The activity of both transporters increased in cells at mid-logarithmic growth, as compared to cells in the stationary phase, and was also stimulated 5–15-fold following growth in purine-depleted medium. These increased rates were due to increased V max values ( K m remained unchanged) and inhibited by cycloheximide (10 μM). In the procyclic forms of T. b. brucei, adenosine transport by the P1 transporter was upregulated by purine starvation but only after 48 h, whereas hypoxanthine transport was maximally increased after 24 h. The latter effect was due to the expression of an additional hypoxanthine transporter, H2, that is normally absent from procyclic forms of T. b. brucei and was characterised by its high affinity for hypoxanthine ( K m∼0.2 μM) and its sensitivity to inhibition by guanosine. The activity of the H1 hypoxanthine transporter ( K m∼10 μM) was unchanged. These results show that regulation of the capacity of the purine transporters is common in different protozoa, and that, in T. b. brucei, various purine transporters are under differential control.

Dipyridamole potentiates the in vitro activity of MTA (LY231514) by inhibition of thymidine transport.

The novel pyrrolopyrimidine-based antifolate LY231514 (MTA), inhibits multiple folate-requiring enzymes including thymidylate synthase, glycinamide ribonucleotide formyltransferase and dihydrofolate reductase. Both thymidine and hypoxanthine are required to reverse MTA growth inhibition in leukaemia and colon cancer cells. Prevention of MTA growth inhibition by thymidine and/or hypoxanthine was investigated in two human lung (A549, COR L23) and two breast (MCF7, T47D) tumour cell lines, and the effect of the nucleoside/base transport inhibitor dipyridamole (DP) on thymidine and hypoxanthine rescue defined. MTA IC50values (continuous exposure three population doublings) were: A549–640 n M, COR L23–28 n M, MCF7–52 n M and T47D–46 n M. Thymidine (1 μM) completely prevented growth inhibition at the MTA IC50in all cell lines. At 10 × IC50, growth inhibition was only partially reversed by thymidine (≤ 10 μM); both thymidine and hypoxanthine (30 μM) being required for complete reversal, reflecting the multi-targeted nature of MTA. Growth inhibition by MTA was not affected by hypoxanthine alone. A non-toxic concentration (1 μM) of DP prevented thymidine/hypoxanthine rescue of MTA indicating that DP may potentiate MTA activity by preventing nucleoside and/or base salvage. Thymidine transport was inhibited by ≥ 89% by 1 μM DP in all cell lines, whereas hypoxanthine transport was inhibited only in A549 and MCF7 cells. Therefore, prevention of end-product reversal of MTA-induced growth inhibition by DP can be explained by inhibition of thymidine transport alone. © 2000 Cancer Research Campaign

Nucleobase transport in opossum kidney epithelial cells and Xenopus laevis oocytes: the characterisation, structure–activity relationship of uracil analogues and oocyte expression studies of sodium-dependent and -independent hypoxanthine uptake

The characteristics of hypoxanthine transport were examined in opossum kidney (OK) epithelial cells and Xenopus laevis oocytes. In both cell types hypoxanthine influx was mediated by two distinct transport systems: a high-affinity Na +-dependent system and a Na +-independent transporter. Na +-dependent hypoxanthine transport in OK cells was saturable ( K m 0.78±0.29 μM) and was inhibited by guanine, uracil, thymine and 5-fluorouracil ( K i values 0.5–7 μM), whereas adenine had no effect. Substitutions at the 2- and 4-position had a marked effect on the ability of uracil to inhibit Na +/hypoxanthine influx by OK cells revealing that an oxo group at both the 2- and 4-positions of uracil is required for interacting with the transporter. The properties of Na +-dependent hypoxanthine influx in oocytes were similar to those observed in OK cells. In particular, xanthine and oxypurinol inhibited hypoxanthine influx, a characteristic not observed previously for the Na +/nucleobase carrier in pig LLC-PK 1 renal cells. Na +-independent hypoxanthine influx in OK cells and oocytes was of a lower affinity ( K m 90–180 μM). Adenine and guanine inhibited Na +-independent hypoxanthine flux in OK cells, but had no effect in oocytes. Injection of LLC-PK 1 mRNA into oocytes resulted in a 1.5-fold stimulation of Na +/hypoxanthine flux over water-injected oocytes. These results reveal further heterogeneity in Na +/nucleobase cotransporters.

Crithidia luciliae: Functional Expression of Nucleoside and Nucleobase Transporters inXenopus laevisOocytes

Hall, S. T., Penny, J. I., Gero, A. M., and Krishna, S. 1998.Crithidia luciliae: Functional expression of nucleoside and nucleobase transporters inXenopus laevisoocytes.Experimental Parasitology90, 181–188. The expression of purine-specific nucleoside and base transporters ofCrithidia luciliaehas been demonstrated inXenopus laevisoocytes. Poly(A)+-mRNA fromC. luciliae, cultured in either purine-replete or purine-starved conditions, was microinjected intoX. laevisoocytes. For “purine-replete” mRNA, expression of adenosine and hypoxanthine uptake in microinjectedX. laevisoocytes was increased on average 9- and 3-fold above water-injected controls, respectively. Expression of adenosine and hypoxanthine uptake in oocytes microinjected with “purine-starved” mRNA was 8 and 3-fold above water-injected controls, respectively. Substrate competition indicated an adenosine/deoxyadenosine transporter and a separate base transporter specific for hypoxanthine. In contrast toC. luciliae in vivo, where the level of activity of adenosine and hypoxanthine transport was regulated by the level of purines in the medium, the heterologous expression of these transporters (from both purine replete and deplete cultures) inX. laevisoocytes was independent of the extracellular purine concentration. These results may suggest that the presence of specific transporter message is independent of the extracellular purine content, indicating that the regulation of activation and expression of these transporters inC. luciliaemay not be under transcriptional control.