Characterization of a Novel Na+-dependent, Guanosine-specific, Nitrobenzylthioinosine-sensitive Transporter in Acute Promyelocytic Leukemia Cells*

NB4 cells are the only bona fide in vitro model of human acute promyelocytic leukemia. We have examined cytidine and guanosine transport in this cell line and characterized a novel guanosine-specific transporter. Cytidine transport occurred predominately by equilibrative nitrobenzylthioinosine (NBMPR)-sensitive (es) transport. In the presence of Na+, guanosine at various concentrations accumulated at least 6-fold above equilibrium. The initial rate of guanosine transport in Na+ buffer decreased by 75% with the addition of 1 μm NBMPR and the IC50 for NBMPR inhibition was 0.7 ± 0.1 nm. Replacement of Na+ with choline also resulted in a 75% decrease in total guanosine transport. The potent inhibition of guanosine transport by NBMPR and the loss of transport in choline suggested that a Na+-dependent NBMPR-sensitive transporter was responsible for the majority of guanosine uptake. This concentrative, sensitive transporter is Na+ dependent with a stoichiometric coupling ratio of 1:1. This novel transporter, referred to as csg, is guanosine-specific with total guanosine transport inhibited by only 50% in the presence of 1 mmcompeting nucleosides. HL-60, acute myelocytic leukemia cells, do not exhibit csg activity while L1210, murine acute lymphocytic leukemia cells, exhibit csg transport. The presence of thecsg transporter suggests an important role for guanosine in particular forms of leukemia and may provide a new target for cytotoxic therapy.

Purine and pyrimidine nucleotides, and their related metabolic products, participate in numerous biological processes. Nucleotides can be synthesized endogenously via de novo synthetic pathways and, as a result, are not considered essential nutrients (1,2). However, a number of tissues including hematopoietic and intestinal epithelial cells are deficient in de novo nucleotide synthetic pathways and rely on the salvage of exogenous nucleosides to maintain nucleotide pools and to meet their metabolic demands (3). Nucleoside analogs have important clinical applications in the therapy of neoplastic and viral diseases (4). Most nucleosides and their analogs execute their biological activity intracellularly, but due to their hydrophilic nature do not readily permeate the lipid bilayer. Therefore, the uptake or release of nucleosides and/or nucleoside analogs in mammalian cells is mediated by multiple distinct transport proteins (5,6). Currently, seven functionally distinct nucleoside transport (NT) processes have been identified. The classification of NT processes is based on functional and pharmacological characteristics including: transport mechanism, e ϭ equilibrative, c ϭ concentrative; sensitivity to nitrobenzylthioinosine (NBMPR), 1 s ϭ sensitive, and i ϭ insensitive; and permeant selectivity.
In general, equilibrative processes are widely distributed among mammalian cells and tissues and exhibit broad permeant selectivity (6 -9). There are two discrete equilibrative transporters, NBMPR-sensitive (es) and -insensitive (ei), that have similar kinetic properties, but differ markedly in sensitivity to NBMPR (10 -12). Both equilibrative transporters are inhibited by low concentrations of dipyridamole and dilazep (9). The es transporter is inhibited at low concentrations (Յ1 nM) (10) as a direct result of interaction of NBMPR with high affinity binding sites (K d ϭ 0.1-1 nM) (13,14), while the ei transporter is not affected by NBMPR up to Ͼ 10 M (10,12,13).
Concentrative nucleoside transport processes have been identified in numerous specialized mammalian cells (6). These transporters are insensitive to NBMPR and dipyridamole at concentrations up to 10 M (9, 15). There is evidence for five subclasses of concentrative transporters that display a complex pattern of overlapping permeant selectivities (5). Due to overlapping permeant selectivities, a complex phenotype can result whereby one nucleoside may be simultaneously transported by more than one process within one cell or tissue. The nucleosides are transported against a concentration gradient into the cell, coupled with the movement of Na ϩ down a concentration gradient (16). Thus, these processes are described as inwardly directed Na ϩ /nucleoside symporters (17).
There are four major Na ϩ dependent, concentrative, NBMPR-insensitive (ci) transport systems with varying substrate specificities. cif prefers purines and formycin B is the model substrate (18). Two distinct systems prefer pyrimidines and are both referred to as cit. These similar processes are distinguished by the ability to transport guanosine (15,19), and thymidine is the model substrate for both systems. The fourth ci transporter exhibits broad permeant selectivity and is referred to as cib (6,20,21). Both adenosine and uridine are substrates for all four ci transporter types (2). A preliminary report suggested the presence of a concentrative NBMPR-sensitive (cs) transporter in freshly isolated leukemia cells (22), however, this transport process remains to be characterized.
There are large differences in NT activities from one cell type to another (23). Some cell types such as human erythrocytes express only a singe NT system, es (24), whereas, both es and ei are present in various cell lines, including BeWo human choriocarcinoma cells (25). Other cell lines such as L1210 murine leukemia cells and IEC-6 mouse intestinal epithelial cells, express multiple transporters, including equilibrative and concentrative transporters, simultaneously (18,15). As well, NT activities within the same cell type have been observed to change with changes in growth state (26,27), differentiation (21,(32)(33)(34)(35)(36)(37), and neoplastic transformation (28). For example, induction of monocytic (21,35) or neutrophilic differentiation (33,34) of HL-60 human leukemia cells leads to a decrease in es nucleoside transport activity and increased Na ϩ -dependent nucleoside transport activity (33)(34)(35).
NB4 cells were originally established from the leukemia cells of a patient with acute promyelocytic leukemia (APL) (29). This cell line expresses the specific t [15;17(q22,q11-12)] chromosomal translocation of APL, thus providing a better model for APL than HL-60 cells which were derived from a patient with acute myelocytic leukemia (3) and lack the characteristic chromosomal translocation. In our laboratory, examination of the NT processes present in NB4 cells has revealed a nucleoside transport phenotype different from that of HL-60 cells (31). Analysis of adenosine, thymidine, and uridine transport unveiled the presence of both ei and es transporter systems, and most significantly the presence of a cif transport system (31). This observation was the first report of the presence of cif in a human cell line. To further establish the nucleoside transporter phenotype of NB4 cells, this study directly examined the characteristics of both cytidine and guanosine transport in this cell line. Here we describe the complete characterization of an NBMPR-sensitive, Na ϩ -dependent guanosine-specific transporter (csg), the first to be described in eukaryotic cells.

MATERIALS AND METHODS
Cell Culture-NB4 cells were originally isolated and characterized from a human patient with APL (29). The cells were maintained in Iscove's modified Dulbecco's medium (Life Technologies, Inc., Burlington, ON, Canada) with 10% fetal bovine serum (ICN Biochemicals, Inc., Mississauga, ON, Canada), supplemented with penicillin and streptomycin (50 units/ml) and maintained at 37°C in a humidified atmosphere of 5% CO 2 . The cells were routinely passed in tissue culture flasks (Fisher Scientific, Whitby, ON, Canada) from passage 5 to 40. Cell counts were determined using a Coulter Counter (Model ZM) and cell volume with a Coulter Channelizer 256 (Coulter, Hialeah, FL). Cell viability was determined by a trypan blue dye exclusion assay and cell viability was Ͼ85% in all experiments.
Employing rapid assay technology, the uptake of [ 3 H]guanosine and [ 3 H]cytidine was determined using an inhibitor and oil stop procedure at 22°C (38). A 100-l volume of transport buffer, containing Na ϩ or choline buffer with or without 1 M NBMPR, a known concentration of cold permeant and radioactive permeant was layered over 125 l of oil (16% Fisher 0121 light paraffin oil and 84% Dow-Corning (Mississauga, ON, Canada) 550 silicone fluid, with a final density of 1.032 g/ml) in a 1.5-ml Eppendorf microcentrifuge tube. The uptake intervals were initiated by the addition of 100 l of cells incubated in choline or Na ϩ buffer with or without NBMPR to the microcentrifuge tube, allowing cells to mix with the transport buffer solution for a specific time interval (0, 3, 5, 7, 10, 15, 30, 120, and 300 s) and the interval was ended by the addition of 200 l of 200 M cold dilazep, a nucleoside transport inhibitor (a gift from Hoffman La-Roche and Co., Basel, Switzerland). The cells were then pelleted through the oil layer by centrifugation at 14,000 rpm for 15 s using an Eppendorf model 5415C centrifuge. Each time point was performed in triplicate. The transport buffer was aspirated off the oil layer and the tubes were washed twice with water to remove radioactive tracer while preserving the cell pellets. The cell pellets were solubilized by the addition of 300 l of 5% Triton X-100 for 1 h at 22°C. The contents of the microcentrifuge tubes were then transferred to scintillation vials and 5 ml of ScintiVerse (Fisher Scientific, Whitby, ON, Canada) was added. Radioactivity was determined using a Beckman (Fullerton, CA) LS 7800 liquid-scintillation spectrometer. Time 0 values for uptake were determined by first adding dilazep followed closely by the cell suspension and immediate centrifugation. Cell number and volume were measured immediately after the transport assay was complete. The intracellular volume of NB4 cells was determined from the cell volume distributions recorded by the Coulter Channelizer 256 (39) and was consistently 1.2-1.4 l. Initial rates were estimated from computer generated (TableCurve, Jandel Scientific, Corte Madera, CA) best-fit equations for Na ϩ , Na ϩ -NBMPR, choline, and choline NBMPR treatments. The best-fit equation was chosen and produced a linear response over the first 4 or 5 time points (0, 3, 5, 7, and 10 s). The linear tangent drawn between 0 and 1 s was used as the initial rate estimate for all time courses.
Nucleoside transport processes were distinguished by exploiting the use of NBMPR, an inhibitor of es and cs transport, and by the use of a Na ϩ replacement choline buffer. Transport measured in Na ϩ buffer alone is representative of total transport (A) and measurements made in choline buffer represent total equilibrative transport (es ϩ ei) (C). Transport measured in Na ϩ -NBMPR reveals total insensitive transport (ei ϩ ci) (B), while measurements made in choline NBMPR measure ei (D) only. By subtraction, the individual nucleoside transport processes can be identified. Fig. 1.
Kinetic Studies-The kinetic parameters of transport, K m and V max , were estimated by measuring uptake at various substrate concentrations at a time point that was on the linear portion of the time course curve and subtracting time 0 uptake. This time point was 7 s for cytidine and 5 s for guanosine. Uptake versus concentration for Na ϩ , Na ϩ -NBMPR, choline, and choline-NBMPR time courses was expressed graphically. By subtraction between curves at each concentration point, as shown in Fig. 1, the ci, cs, ei, and es curves were generated over the same concentration. The untransformed data was fitted to a one-site binding model (GraphPad Prism) and the computer generated K m and V max for each nucleoside were recorded.
Statistical Analysis-ANOVA followed by the Dunnett's test was used to compare uptake of [ 3 H]guanosine in the presence of competitors to [ 3 H]guanosine uptake without competition (control) using Instat (GraphPad, San Diego, CA) for personal computers. Significance was reached when p Ͻ 0.05.

Time Courses and Initial
Rates of Nucleoside Transport-Initial transport rates were estimated from computer-generated best-fit equations (see "Materials and Methods"). The presence of es, ei, cs, and ci transporters were resolved by subtraction of the appropriate time courses. Time courses were performed in Na ϩ and Na ϩ free (choline substituted) buffers in the absence or presence of NBMPR to determine the nucleoside transport process profile for each nucleoside studied (Fig. 1). The proportions of each NT system for cytidine and guanosine are given in Table I.
Cytidine transport in NB4 cells occurred almost exclusively via es (Table I). The elimination of the Na ϩ gradient by replacement with choline had no effect on cytidine uptake (Fig. 2, A  and B). At 50 M, cytidine uptake reached equilibrium at ϳ45 s ( Fig. 2A) while at 5 M cytidine reached equilibrium at ϳ25 s and continued to accumulate (Fig. 2B). A small amount of cytidine transport, 5% of total (Table I), occurred in the choline ϩ NBMPR treatment group. However, this process was unsaturable and likely representative of diffusion.
Direct examination of [ 3 H]guanosine transport in NB4 cells revealed the presence of a concentrative, NBMPR-sensitive Na ϩ -dependent transporter. At 5, 50, and 100 M guanosine, transport in the presence of a Na ϩ gradient was almost entirely abrogated by the addition of 1 M NBMPR (Fig. 3, Table I). The replacement of Na ϩ with choline led to a 70 -80% decrease in total uptake. Guanosine reached equilibrium and accumulated 6 -7-fold at 50 and 100 M, and 15-fold at 5 M over the 5-min time course. Initial rates of ci transport, as determined by subtraction of choline NBMPR from Na ϩ -NBMPR, were consistently small and whether the rates were representative of facilitated transport was not clear. To measure ci directly, i.e. and without subtraction, dipyridamole was added to the Na ϩ medium. Accumulation above equilibrium did not occur at 50 or 5 M guanosine (data not shown). The majority of Na ϩ -dependent concentrative uptake was inhibitable by NBMPR, suggesting that concentrative sensitive (cs) transport was responsible for the preponderance of total guanosine transport observed in this cell line. We have named this transporter csg.
In a previous study by our group, two high affinity NBMPRbinding sites were identified on NB4 cells (31). To help elucidate whether one of these sites could represent the csg transporter we determined the IC 50 of the csg transporter for NBMPR. Cells were preincubated in graded concentrations of NBMPR (0.01-1000 M) and uptake of guanosine was measured at 5 s. The IC 50 for NBMPR inhibition was determined from a plot of guanosine uptake (pmol/l/s) versus NBMPR concentration. The transporter was found to be highly sensitive to NBMPR with an IC 50 of 0.70 Ϯ 0.7 nM (data not shown).
Kinetics of Transport-To further characterize the transport of cytidine and guanosine in NB4 cells, kinetic studies were performed. The kinetic parameters of transport were measured at nucleoside concentrations ranging from 1.9 to 500 M for cytidine and from 1.9 to 125 M for guanosine. Below 125 M guanosine the equilibrative system accounted for a small portion of uptake and began to plateau. Beyond 125 M guanosine, however, uptake via this route began to climb steeply and exceeded the cs system. This potential second binding site was not analyzed due to a lack of data points beyond 500 M, limited by guanosine solubility, and the lack of physiological relevance. The K m value for the ei transporter was 58 M for guanosine, and the K m values for the es transporter were 112 and 52 M for cytidine and guanosine, respectively. The transport affinities of both equilibrative systems for guanosine were similar to the K m values we previously reported for adenosine, uridine, and thymidine (31), while those for cytidine were approximately 2-fold lower. The K m of the csg transporter was estimated at 49 M, similar to the affinities for both the es and ei transporters. The csg transporter showed the greatest capacity to transport guanosine into NB4 cells, with a V max of 1.6 Ϯ 0.2 pmol/l/s while the equilibrative processes contributed much less at 0.4 Ϯ 0.04 and 0.3 Ϯ 0.04 pmol/l/s for es and ei, respectively. Although the affinity of the equilibrative systems for cytidine was low, the es transporter showed a high capacity to transport this substrate as reflected in the high V max value. The es V max value of 9.58 Ϯ 0.4 for cytidine is about 2-fold greater than the V max value of both equilibrative processes for adenosine (31).
Cation Dependence of the csg Transporter-The cation specificity of the csg transporter was investigated by substituting Na ϩ for other monovalent cations. Time courses were generated using 5 M guanosine. The uptake of [ 3 H]guanosine in the presence of 140 mM gradients of the chlorine salts, K ϩ Cl and Li ϩ Cl, and in choline was compared with the uptake demonstrated in a Na ϩ Cl gradient. Concentrative transport was reduced by about 10-fold when extracellular Na ϩ was replaced with K ϩ , Li ϩ , or a choline gradient, indicating high Na ϩ specificity of the transporter.
Effect of Na ϩ on Guanosine Uptake-To investigate the stoichiometric coupling ratio associated with the csg transporter the initial rates (flux) of guanosine at a concentration of 100 M, as a function of the extracellular Na ϩ concentration (0 -140 mM) was measured. The relationship of the flux of guanosine versus sodium concentration produced a hyperbolic curve (Fig.  4A), suggesting a ratio of sodium:guanosine of 1:1. This data was further analyzed using the following Hill equation (40), Flux ϭ V max ͓Na ϩ ͔ n /͑K Na ϩ n ϩ ͓Na ϩ ͔ n ͒ (Eq. 1) where K Na ϩ n is the concentration of Na ϩ that is able to produce one-half of the maximum rate of nucleoside flux, V max refers to the maximum rate of nucleoside flux at saturating concentra-

TABLE I Initial transport rates for nucleoside transport in NB4 cells
Transport assays were carried out as described under "Materials and Methods" and Fig. 1. Initial transport rates were estimated from computer-generated best-fit equations over the linear portion of the time course curve and transport processes were resolved by subtraction (as explained in Fig. 1). The data presented are the means Ϯ S.E. of at least three separate experiments performed in triplicate. The numbers in parentheses represent the percentage of total initial transport rates that each individual transport process comprises. tions of Na ϩ , and n is the Hill coefficient. A plot of flux/[Na ϩ ] n against flux for the correct value of n will yield a straight line. Fig. 4B displays the results of this type of plot of the data in Fig. 4A. A straight line was observed when n ϭ 1, K Na ϩ ϭ 14.6 Ϯ 3.8 mM, and V max ϭ 2.57 Ϯ 0.2 pmol/l/s, indicating the binding site on the carrier requires the presence of one Na ϩ molecule.
Substrate Specificity of the Na ϩ -dependent Concentrativesensitive Transporter-The substrate specificity of this novel csg transporter in NB4 cells was investigated by measuring the transport of 100 M guanosine in the presence of increasing concentrations of other nucleosides, a nucleobase, a nucleoside analog, and nucleoside transport inhibitors. At concentrations of 1 mM, 2Ј-deoxyguanosine, guanine, uridine, hypoxanthine, adenosine, and inosine significantly inhibited the Na ϩ -dependent cs uptake of guanosine at 5 s (p Ͻ 0.05) (Fig. 5) by 35-50%. The other compounds tested including thymidine, xanthine, and ganciclovir, at concentrations of 1 mM, were unable to significantly inhibit Na ϩ -dependent cs guanosine transport. NBMPR and dipyridamole at 1 and 20 M, respectively, markedly inhibited Na ϩ -dependent cs transport of guanosine by ϳ90%. When cold guanosine was present at 500 M (pH 7.1) uptake of [ 3 H]guanosine was inhibited by ϳ70%. Concentrations greater than 500 M were unattainable due to guanosine's low solubility at physiological pH.
Existence of Na ϩ -dependent cs Transport in Other Cell Lines-To assess whether other leukemia cell lines exhibit this novel Na ϩ -dependent concentrative-sensitive transporter for guanosine or csg, we examined two commonly studied cell lines, HL-60 and L1210 cells. Time courses were performed for guanosine uptake at 50 and 100 M (Table II and data not  shown). The initial transport rates were estimated from computer-generated best-fit equations over the linear portion of the time course curve and individual processes were resolved as discussed earlier. Guanosine entered HL-60 cells by both es and ei at 50 and 100 M, but there was no evidence of concentrative-sensitive transport. L1210 cells exhibited Na ϩ -dependent cs guanosine transport at 50 M guanosine, but this transport process was not easily detected at higher concentrations (Table II). DISCUSSION In this study, the direct examination of guanosine transport revealed the presence of an NBMPR and dipyridamole-sensitive Na ϩ -dependent, guanosine-specific transporter (csg), the first to be described in eukaryotic cells. Concentrative-sensitive transport (cs) has been previously described in abstract form (22) in freshly isolated human leukemia cells from chronic lymphocytic and acute myelogenous leukemia patients. The characteristics of the csg transporter identified in NB4 cells are different from those of the cs transporter described in freshly isolated leukemia cells. Only adenosine analogs were per- meants for the previously described cs transporter, however, the csg transporter does not accept adenosine directly (31) and only 50% of guanosine transport via this system is inhibited in the presence of a 10-fold greater concentration of adenosine. It seems likely that with the differences between this csg transporter and the previously described cs process that other cs processes will be discovered.
NB4 cells exhibit two high affinity NBMPR-binding sites (31). Both binding sites had K d values in the nanomolar range, 0.1 and 0.35 nM, consistent with the high affinity systems previously described (9). In this study, the NBMPR inhibition of guanosine transport via the csg transporter was 0.7 Ϯ 0.1 nM, suggesting that one of the two NBMPR-binding sites may be that of the csg transporter.
The csg transporter was Na ϩ dependent, K ϩ is likely too large, and Li ϩ too small to emulate Na ϩ in the csg system. The stoichiometric coupling ratio was determined to be 1:1, analogous to the concentrative insensitive (ci) formycin B selective transporter (cif) and both ci thymidine selective processes (cit) (5).
To provide insight into the structure of this NT membrane protein, permeant selectivity was assessed. In general, the transporter appeared to be highly guanosine specific. Guanosine transport was inhibited by only 55% in the presence of 1 mM competing nucleosides. Other concentrative systems prefer purines (18), pyrimidines (15,19), or accept both purines and pyrimidines (2). As well, other concentrative systems experience greater than 50% inhibition in the presence of competing nucleosides at concentrations equal to (43) or as little as 2-fold greater (44) than the model nucleoside in question. It appears that the substrate specificity of this novel csg transporter was dependent on both the presence of an intact ribose ring and the existence of particular functional groups. Ganciclovir, although resembling guanosine in its base structure, has a disrupted ribose ring and is unable to compete with guanosine for the transporter. Although there were no statistically significant differences between 2Ј-deoxyguanosine, inosine, guanine, hypoxanthine, adenosine, or uridine inhibition, 2Ј-deoxyguanosine appeared to be the most successful competitor of guanosine transport possibly due to the presence of an intact ribose ring and functional groups almost identical to those of guanosine. Guanine, with identical functional groups to guanosine, but without the ribose ring, appeared to compete with less success than 2Ј-deoxyguanosine. Studies of the es transporter have shown the transporter to be highly stereoselective strongly preferring the D-enantiomer over the L-enantiomer (45) with the ability of a molecule to be transported greatly decreased or elevated by loss or substitution of the 3Јhydroxyl residue (46). Therefore, manipulation of molecule structure has the potential to alter transport capacity significantly.
We examined two commonly studied leukemia cell lines, HL-60 and L1210 cells: only L1210 cells exhibited concentrative-sensitive transport. From these experiments it appears that concentrative-sensitive guanosine transport is not unique to human leukemia cell lines. HL-60 cells were isolated from a patient with acute myelocytic leukemia rather than APL (NB4) (30). Several differences between HL-60 cells and NB4 cells have already been reported by this laboratory (31,47). The csg transporter appears to be another distinguishing feature of the NB4 cell line.
Why do NB4 cells express this guanosine-specific transporter? Guanylate ribonucleotides are involved in a variety of biological roles including, RNA and DNA synthesis, and are required for the function of small GTP-binding proteins (such as Ras) and heterotrimeric G proteins involved in signal transduction (2,48,49). dGTP pools appear to be rate-limiting in DNA biosynthesis in rat liver and hepatomas (48). NB4 cells were observed to transport less guanosine when harvested in FIG. 4. Sodium-dependent guanosine flux in relation to sodium concentration. The uptake of guanosine (100 M) was measured in the presence of various extracellular Na ϩ concentrations (0 -140 mM). To obtain the different sodium concentrations, choline was partially substituted to give an osmolarity of 300 Ϯ 10 osmole. Uptake was measured over a full time course (0, 3, 5, 7, 10, 15, 30, 120, and 300 s) and flux at 1 s was determined. A is a plot of guanosine uptake versus Na ϩ concentration. B is a plot of flux/[Na ϩ ] n versus flux when n ϭ 1. One sodium ion is transported per one guanosine molecule as suggested by the linearity of this plot.
FIG. 5. Substrate specificity of the cs transport in NB4 cells. Transport of 100 M guanosine was measured in the presence of 1 mM competing nucleosides, a nucleobase, a nucleoside analog, or nucleoside transport inhibitors. Uptake was measured after a 5-s incubation of the cells with the transport buffer (Na ϩ buffer, 100 M guanosine, and the competitor). The flux of guanosine observed in the presence of 1 mM concentration of the various competitors is expressed as a percentage of guanosine flux measured in the absence of competing compounds. 2Ј-Deoxyguanosine, inosine, hypoxanthine, guanine, adenosine, uridine, NBMPR, and dipyridamole significantly inhibited guan uptake. The bars represent the mean Ϯ S.E. of data obtained in at least three separate experiments. Ganciclovir A, 1 mM ganciclovir ϩ 100 M guanosine; ganciclovir B, 1 mM ganciclovir ϩ 5 M guanosine. plateau phases (data not shown) and therefore it is conceivable that the csg transporter provides a means of ensuring that an adequate supply of dGTP is available to the cell to sustain DNA replication and excessive proliferation. In a hematopoietic cell, such as the NB4 cell, de novo synthesis is limiting and the salvage via membrane transporters is likely to be critical.
Nucleoside transport processes appear to be regulated during cellular differentiation (21,33). HL-60 cells induced to differentiate to neutrophils by treatment with dimethyl sulfoxide (53), retinoic acid, or tiazofurin (an IMP dehydrogenase inhibitor) show a decrease in intracellular GTP and GDP pools (49,50,53) and an increase in the salvage of guanosine and guanine (50). Providing guanosine and/or guanine at 1-100 M (concentrations used in this study) to the medium, prevented the GTP depletion and inhibited myeloid maturation (49,50) in a dose-dependent manner (53). Thus, the salvage and/or nucleoside transport capabilities of a cell are important when considering the efficacy of intracellular targeted cytotoxic agents. Differentiation has also been observed in vivo in acute myelocytic leukemia patients receiving tiazofurin (51). There has been some suggestion for the involvement of a G-protein modulated signal transduction pathway in the neutrophilic differentiation of HL-60 cells (33). NB4 cells can be terminally differentiated to granulocytes with retinoic acid (29,47) and to monocytes with the combination of 12-O-tetradecanolyphorbol-13-acetate and 1,25-dihydroxyvitamin D 3 (52). It will be enlightening to see if when NB4 cells are differentiated to neutrophils or monocytes, the csg is differentially regulated as a result of differences in the requirement for G-proteins and/or guanine ribonucleotides.
Cytidine enters NB4 cells via Na ϩ -independent, presumably equilibrative, routes only, resembling thymidine transport (31). At a physiological concentration of 5 M (7, 41), cytidine accumulated 7-fold above equilibrium suggesting rapid intracellular metabolism of the substrate. When growing cells are exposed to physiological concentrations of nucleosides, the intracellular phosphorylation of the nucleoside often exceeds the nucleoside influx. Thus, the intracellular free nucleoside concentration attains a very slow steady state (7). At 50 M cytidine, very little accumulation of the nucleoside occurred, suggesting that the intracellular phosphorylation reaction was saturated at this concentration.
In general, dNTP and NTP levels are higher in actively dividing tumor cells than in untransformed dividing cells (41). For example, proliferating rat hepatoma 3924A cells in cultured monolayers have a 6.5-fold greater dCTP pool size over normal regenerating rat liver (42). The large accumulation of cytidine in the NB4 leukemia cell line may be a reflection of the increased requirement for dCTP and CTP to perform DNA replication thereby allowing for rapid and extensive proliferation.
In conclusion, we have identified a new Na ϩ -dependent guanosine-specific nucleoside transporter that may play a role in leukemia development, cell proliferation, and myeloid differentiation. This transporter strongly prefers guanosine over other physiological permeants and may provide a new target for chemotherapy.

TABLE II
Initial transport rates for guanosine transport in various leukemia cell lines Initial transport rates for guanosine at 50 M were estimated from computer-generated best-fit equations over the linear portion of the time course curve and transport processes were resolved by subtraction (as explained in Fig. 1). The data presented are the means Ϯ S.E. of at least three separate experiments performed in triplicate. The numbers in parentheses represent the percentage of total initial transport rates that each individual transport process comprises.