Introduction

Top Abstract Introduction Materials & Methods Results Discussion References

Mammalian cells possess a variety of mechanisms for accumulating nucleosides from the extracellular milieu, including both nonconcentrative (equilibrative) Na⁺-independent mechanisms and concentrative Na⁺-dependent transporters (Griffith and Jarvis, 1996; Cass, 1995; Belt et al., 1993). Isoforms of the equilibrative systems can be identified by their sensitivities to inhibition by the NBMPR and dipyridamole. Many cell types, including the Ehrlich ascites tumor cell line used in the present study (Hammond, 1991), coexpress the NBMPR-sensitive (es² isoform) and NBMPR-resistant (ei isoform) equilibrative transporters. However, dipyridamole sensitivity is species-dependent with mouse and rat transporters exhibiting about a 10- to 100-fold lower sensitivity to the inhibitor, respectively, compared with human transporters (Ogbunude and Baer, 1990; Shank and Baldy, 1990; Plagemann and Woffendin, 1988; Hammond and Clanachan, 1985). The Na⁺-dependent concentrative nucleoside transporters are subclassified according to substrate specificity into purine-selective (cif), pyrimidine-selective (cif) and nonselective (cib) isoforms (Cass, 1995; Belt et al., 1993). All of the aforementioned Na⁺-dependent transporters are resistant to inhibition by NBMPR. An additional isoform designated cs or N5, that is sensitive to NBMPR, has been identified recently in freshly isolated human leukemia cells (Paterson et al., 1993); the substrate specificity and biological prevalence of the cs isoform have not been defined.

Nucleoside transporters are involved in the control of the extracellular and intracellular levels of adenosine. Adenosine is widely recognized as an important regulator of cell function (Phillis, 1991), particularly in cardiovascular and neuronal systems (Jacobson et al., 1992; Rongen et al., 1997; Williams, 1989, 1987). Functional nucleoside transporters are also required for the cellular accumulation, and hence the cytotoxicity, of several nucleoside analogues used in cancer chemotherapy (e.g., cytosine arabinoside, 2-chlorodeoxyadenosine) (reviewed by Cass, 1995). There is also evidence that 2',2'-difluorodeoxycytidine (gemcitabine), a new nucleoside analogue with potential for the treatment of solid tumors (Hui and Reitz, 1997; Moore, 1996), enters cells via nucleoside transporters (Griffiths et al., 1997, Fang et al., 1996; Jansen et al., 1995). Identification of the relative activities of the various subtypes of nucleoside transporters is essential for the rational use of nucleoside drugs that rely on these transporters to enter cells. Differential distribution of the transporter subtypes among normal and tumor cells may lead to the development of therapeutic protocols involving combinations of nucleoside drugs and selective transport inhibitors to enhance tumor cell sensitivity and reduce side-effects. However, our understanding of the distribution and cellular regulation of the various transporter isoforms and their relative substrate selectivities, information critical to the realization of the clinical potential described above, remains limited.

The equilibrative, Na⁺-independent transporters of Ehrlich cells have been characterized extensively in our laboratory, and this cell line has proven useful for the study of transporter substrate and inhibitor selectivities (Hammond, 1992, 1991). Previous studies using [³H]uridine as a substrate have also established the existence of a minor Na⁺-dependent nucleoside uptake component operating in Ehrlich cells (Hammond, 1991). However, because analysis of the cellular transport of [³H]uridine required the cells to be ATP-depleted (to minimize uridine metabolism) further characterization of this system could not be undertaken using uridine as the substrate. We have now used [³H]formycin B, a poorly metabolized inosine analogue (Plagemann and Woffendin, 1989), as a substrate to characterize both the equilibrative and concentrative nucleoside transporters in ATP-replete Ehrlich ascites tumor cells. The effects of gemcitabine on formycin B uptake by these cells were then examined to determine the relative roles of the Na⁺-dependent and -independent nucleoside transport systems in the cytotoxic efficacy of this new chemotherapeutic agent.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Materials. [G-³H]Formycin B (14 Ci/mmol, radiochemical purity 99.8%) and [5,6-³H]Uridine (35-50 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA) and ICN Biomedicals, Inc. (Costa Mesa, CA), respectively. [³H]Water (1 mCi/g) and [carboxyl-¹⁴C]-dextran-carboxyl (0.58 mCi/g) were purchased from Du Pont Canada Inc (Markham, Ontario, Canada). Gemcitabine was a gift from Eli Lilly Inc. (Scarborough, Ontario, Canada). Other nucleosides, NBMPR and dipyridamole (2,6-bis(diethanolamino)-4,8-dipiperidinopyrimido-[5,4-d]pyrimidine) were supplied by Sigma Chemical Co. (St. Louis, MO). All other compounds were of reagent grade.

Cultivation and isolation of Ehrlich ascites tumor cells. Cells were propagated by i.p. transplantation of ascitic fluid in mice (Swiss, male, 30 g), and transferred weekly to new hosts by i.p. inoculation with 0.3 ml of undiluted ascites fluid. Five to seven days after inoculation, cells were harvested and washed at least three times in isotonic saline (0.15 M NaCl) to remove contaminating erythrocytes. The cell pellet was resuspended in PBS (pH 7.35; 137 mM NaCl, 6.3 mM Na₂HPO₄, 2.7 mM KCl, 1.5 mM KH₂PO₄, 0.5 mM MgCl₂, 0.9 mM CaCl₂) or an iso-osmotic Li⁺ buffer (nominally Na⁺-free, pH 7.35; 135 mM LiCl, 6.3 mM K₂HPO₄, 2.7 mM KCl, 1.5 mM KH₂PO₄, 0.5 mM MgCl₂, 0.9 mM CaCl₂), as appropriate. It has been shown in a variety of systems that Li⁺ is unable to substitute for Na⁺ at Na⁺-dependent nucleoside transporters, and hence differences in [³H]nucleoside uptake observed in Na⁺ vs. Li⁺ medium have generally been taken to represent the operation of Na⁺-dependent nucleoside transporters (Doherty and Jarvis, 1993; Baer et al., 1992; Dagnino et al., 1991; Williams and Jarvis, 1991; Plagemann and Aran, 1990; Plagemann et al., 1990; Baer and Moorji, 1990; Williams et al., 1989; Jarvis, 1989; Spector and Huntoon, 1984). For experiments involving [³H]uridine uptake, cells were depleted of ATP by sequential incubation with rotenone (20 ng/ml; 15 min at 37°C) and 2-deoxyglucose (2 mM; 15 min at 37°C). This procedure has been shown to reduce the ATP content of these cells by 95% and prevent [³H]uridine metabolism over the time course of these studies (Hammond and Johnstone, 1989).

[³H]nucleoside uptake. All assays were conducted at room temperature (22°C). Uptake was initiated by addition of cell suspension (1 × 10⁷ cells/ml) to [³H]substrate layered over a 200-µl cushion of silicone oil/mineral oil (21:4 v/v) in 1.5-ml microcentrifuge tubes. Assays were terminated after a defined incubation time (minimum 5 sec, including the 2-sec pelleting time) by centrifugation of cells through the oil for 10 sec at 12,000 × g. The supernatant and oil were removed and the cell pellets were digested with 1 M NaOH for approximately 16 hr at room temperature. The digest was analyzed for [³H] content by standard liquid scintillation counting techniques in 5 ml of scintillation cocktail. The estimated time required to pellet the cells through the oil layer (2 sec, determined from detailed nonlinear analyses of uptake time courses) is included in all reported incubation times.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Formycin B interaction with equilibrative nucleoside transporters. Initial studies examined the capacity of formycin B to inhibit the total uptake and the NBMPR-resistant uptake of [³H]uridine by ATP-depleted cells in Li⁺ medium (fig. 1A). Under these conditions, uptake was mediated solely by the equilibrative (Na⁺-independent) nucleoside transporters. Formycin B inhibited the total transporter-mediated uptake of 10 µM [³H]uridine with an IC₅₀ of 188 ± 38 µM and a pseudo Hill coefficient not significantly different from unity. When similar studies were conducted in the presence of 100 nM NBMPR to selectively inhibit the es transporter, formycin B exhibited a significantly lower IC₅₀ of 50 ± 29 µM (Student's t test, P < .05) for inhibition of the remaining ei-transporter-mediated component. Using K_m values for [³H]uridine uptake by Ehrlich cells determined previously (Hammond, 1991), it was calculated that formycin B had a 6-fold higher affinity for the ei transporter (K_l = 48 µM) than for the es transporter (K_l = 277 µM). In the converse experiments, where uridine was tested as an inhibitor of 10 µM [³H]formycin B influx (fig. 1B), uridine did not distinguish between the es and ei-mediated influx of [³H]formycin B. 

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Competitive interactions of uridine and formycin B with the equilibrative nucleoside transporters of Ehrlich ascites tumour cells. The transporter-mediated uptake (20 sec) of 10 µM [3H]uridine (A) and 10 µM [3H]formycin B (B) by ATP-depleted cells was assessed under nominally Na+-free conditions (Li+ replacement) in the presence of a range of concentrations of nonradiolabeled formycin B (A) or uridine (B), respectively. Parallel assays were conducted in the absence (, total influx) and presence (, ei-mediated influx) of 100 nM NBMPR, and the difference was defined as the es transporter-mediated uptake at each concentration of inhibitor (data not shown). Results are presented as a percentage of the accumulation observed in the absence of test inhibitor (control). Each point represents the mean ± S.E. from five experiments. Inhibition constants (Kl), shown as insets, were calculated using the IC50 values derived from these plots and the Km values reported in table 2.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]uridine () and [3H]formycin B () influx by NBMPR. ATP-depleted cells were incubated in Li+ medium with the indicated concentrations of NBMPR for at least 15 min and then exposed to [3H]substrate (5 µM) for 20 sec. The results are presented as a percentage of the transporter-mediated accumulation observed in the absence of NBMPR (control). The dotted lines show the relative amount of NBMPR-resistant uptake of each [3H]substrate. Each point represents the mean ± S.E. from at least five experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Time course of 10 µM [3H]formycin B accumulation by ATP-replete Ehrlich cells in the absence (A; iso-osmotic replacement with Li+) and presence (B) of Na+. Cells were incubated with 10 µM [3H]formycin B in the absence () and presence of 100 nM NBMPR () or 10 µM dipyridamole/NBMPR (, nonmediated influx), for the times indicated (abscissa), and the assays were then terminated as described in the text. The dotted line represents the expected steady-state intracellular concentration of [3H]formycin B based solely on the activity of a nonconcentrative facilitated diffusion system. Each point represents the mean ± S.E. from four experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of transporter-mediated [3H]formycin B accumulation by Ehrlich cells. Time courses for the cellular accumulation of a range of concentrations of [3H]formycin B were constructed as shown in figure 3, using ATP-replete cells in the absence (total uptake) and presence of 100 nM NBMPR (NBMPR-resistant uptake) and 10 µM NBMPR/dipyridamole (nonmediated uptake). NBMPR-sensitive transporter-mediated uptake was defined as the total accumulation minus that measured in the presence of 100 nM NBMPR. Initial rates of influx (ordinate) were estimated from the initial portions of hyperbolic curves fitted to the transporter-mediated uptake time courses (corrected for non-mediated uptake) and plotted against the [3H]formycin B concentration (abscissa) to obtain the hyperbolic relationships shown (total, ; NBMPR-sensitive, ; NBMPR-resistant, ). Each point represents the mean ± S.E. from three to five experiments. Michaelis Menten constants derived from these experimental data by computer-assisted analysis of the nonlinear curve fits are shown in table 2.

Na⁺-dependent nucleoside transport in Ehrlich cells. Once the uptake of [³H]formycin B by the equilibrative es and ei transporters of Ehrlich cells was characterized, similar experiments were done in the presence of Na⁺ to assess the contribution of Na⁺-dependent concentrative transporters to the cellular uptake of [³H]formycin B.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of Na+ removal (Li+ substitution) on [3H]formycin B uptake by the NBMPR-sensitive and NBMPR-resistant nucleoside transporters of Ehrlich cells. Time courses of uptake of 10 µM [3H]formycin B (± Na+) were constructed in the presence and absence of 100 nM NBMPR and 10 µM NBMPR/dipyridamole as shown in figure 3. NBMPR-sensitive transporter-mediated uptake was defined as the total accumulation of [3H]formycin B minus that seen in the presence of 100 nM NBMPR. NBMPR-resistant transporter-mediated uptake was calculated as the accumulation of [3H]formycin B in the presence of 100 nM NBMPR minus uptake in the presence of 10 µM NBMPR/dipyridamole. Data (mean, n = 4) obtained in the Li+ medium was subtracted from that determined in the presence of Na+ (ordinate) for incubation times ranging from 5 to 62 sec (abscissa). Values of less than zero represent an enhancement of [3H]formycin B uptake upon replacement of Na+ with Li+.

Gemcitabine inhibition of [³H]formycin B uptake. Initial studies were conducted to examine the ability of gemcitabine to inhibit the NBMPR-resistant uptake of 10 µM formycin B by cells equilibrated in both normal PBS buffer and Li⁺ medium (fig. 6; table 3). A concentration of 50 µM gemcitabine had minimal effect (<10% inhibition) on the equilibrative transporter-mediated uptake of [³H]formycin B when using the Li⁺ medium (fig. 6A). However, in the presence of Na⁺, 50 µM gemcitabine significantly inhibited (~36%) the NBMPR-resistant cellular accumulation of [³H]formycin B at all incubation times tested (fig. 6B).

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of gemcitabine on the NBMPR-resistant transport of [3H]formycin B by Ehrlich cells in the absence (A; iso-osmotic replacement with Li+) and presence (B) of Na+. Cells were incubated with 100 nM NBMPR (NBMPR-resistant uptake) or 10 µM dipyridamole/NBMPR (non-mediated uptake) for at least 15 min, and then exposed to 10 µM [3H]formycin B for the times indicated (abscissa) in the presence and absence of 50 µM gemcitabine. Results are plotted as the transporter-mediated accumulation of [3H]formycin B (NBMPR-resistant minus nonmediated uptake) in the presence () and absence () of gemcitabine. Each point represents the mean ± S.E. from five experiments. Gemcitabine had no significant effect on the NBMPR-resistant uptake [3H]formycin B in the Li+ medium. In the presence of Na+, gemcitabine had a significant inhibitory effect at all time points with the exception of 22 sec (Student's t test, P < .05).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Concentration-dependence of gemcitabine inhibition of [3H]formycin B uptake by the nucleoside transporters of Ehrlich cells. The transporter-mediated uptake of 10 µM [3H]formycin B was assessed using cells equilibrated in Li+ medium in the absence of NBMPR (, total equilibrative influx), and in cells preincubated with 100 nM NBMPR (NBMPR-resistant influx) in both normal PBS (Na+, ) and Li+ medium (). A 22-sec incubation time with [3H]formycin B was used for studies done in the Li+ medium (), and a 5-min incubation time was used for those conducted in the presence of NBMPR and Na+ (). Results are shown as a percentage of the accumulation observed under each condition in the absence of gemcitabine (control). Each point represents the mean ± S.E. from five experiments. IC50 values derived from these plots are shown in table 4.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

Formycin B has been used extensively for the analysis of nucleoside transport systems of mammalian cells (e.g., Conant and Jarvis, 1994; Crawford et al., 1990; Plagemann and Aran, 1990; Plagemann et al., 1990; and see reviews by Griffith and Jarvis, 1996; Cass, 1995; Belt et al., 1993). It has been shown in a variety of cell types that formycin B is poorly metabolized (phosphorylated), relative to endogenous nucleosides such as adenosine and uridine, over the time periods typically used for these types of studies (<5 min) (Roovers and Meckling-Gill, 1996; Crawford and Belt, 1991; Plagemann and Woffendin, 1989; Jakobs and Paterson, 1986). This means that ATP-replete cells may be used, enabling the study of formycin B uptake by energy-dependent processes. Formycin B has been established as a substrate for both the es and ei subtypes of equilibrative transporters, and the cif and cib subtypes of Na⁺-dependent transporter (Griffith and Jarvis, 1996; Cass, 1995). Previous studies from our laboratory have shown that ATP-depleted Ehrlich cells accumulate [³H]uridine to intracellular levels exceeding those present in the initial incubation media (Hammond, 1991), and part of this uptake was dependent on the presence of Na⁺. However, because all studies with [³H]uridine required the use of ATP-depleted cells to prevent the cellular trapping of uridine as its polyphosphates, the magnitude and integrity of the plasma membrane Na⁺ gradient was questionable making analysis of the Na⁺-dependent transport component problematic under these conditions. Using [³H]formycin B, we have now confirmed the presence of a Na⁺-dependent nucleoside transporter in Ehrlich cells. The influence of this concentrative transporter on the cellular accumulation of [³H]formycin B was more apparent when studies were conducted in the presence of 100 nM NBMPR. These results are compatible with the coincident operation of an NBMPR-resistant uni-directional concentrative transporter and an NBMPR-sensitive bi-directional equilibrative transporter (i.e., the es transporter). Both the cif and cib subtypes of Na-dependent transporter have been shown to transport formycin B in other systems. These particular transporter subtypes have also been shown to accept guanosine as a substrate (Plagemann and Aran, 1990; Williams and Jarvis, 1991; Vijayalakshmi and Belt, 1988; also see reviews by Griffith and Jarvis, 1996 and Cass, 1995). However, we have shown previously that [³H]guanosine uptake by Ehrlich cells is not Na⁺ dependent (Hammond, 1992). Therefore, the Na⁺-dependent formycin B uptake observed in Ehrlich cells may be mediated by a novel form of concentrative nucleoside transporter.

Substitution of Na⁺ by Li⁺ resulted in a significant reduction in the capacity of cells to accumulate [³H]formycin B. Nevertheless, even in the Li⁺ medium, Ehrlich cells still accumulated [³H]formycin B to levels about 40% higher than the concentration in the initial incubation medium. A similar Na⁺-independent concentrative effect has been observed by others using formycin B and has been attributed to binding to intracellular components (Plagemann and Woffendin, 1989).

[³H]Formycin B has a 6-fold higher affinity for the ei subtype of equilibrative transporter than for the es transporter of Ehrlich cells. This was evident from data on formycin B inhibition of [³H]uridine influx (fig. 1), as well as the results of experiments examining the Na⁺-independent cellular accumulation of a range of concentrations of [³H]formycin in the presence and absence of 100 nM NBMPR (table 2). Formycin B selectivity for the ei transporter is also compatible with data showing that half of the uptake of 10 µM [³H]formycin B was resistant to inhibition by NBMPR compared with only 25% of 10 µM [³H]uridine influx (fig. 2); [³H]uridine has similar affinities for both forms of equilibrative transporter expressed by Ehrlich cells (Hammond, 1991). Formycin B is similar in this regard to 2-chloroadenosine and soluflazine that are also selective for the ei transporter (Hammond, 1991; Griffith et al., 1990).

When assays were conducted in the presence of Na⁺, the V_max values for [³H]formycin B uptake by both the NBMPR-sensitive and NBMPR-resistant transporters of Ehrlich cells were similar to those determined previously using either [³H]uridine (Hammond, 1991) or [³H]guanosine (Hammond, 1992) as substrates. An unexpected result of the present study, however, was the finding that the rate of [³H]formycin B influx in the Li⁺ medium was significantly greater (~10-fold increase in V_max) than that observed in the presence of Na⁺ (table 2). This phenomenon was evident at all [³H]formycin B concentrations studied (see fig. 4). In the absence of complicating factors the opposite effect would be expected; i.e., the initial rate of substrate influx should decrease when one class of contributing transporter is inactivated. Indeed, this is what has been observed by other investigators when using [³H]formycin B to study cellular nucleoside transport mechanisms (Borgland and Parkinson, 1997; Roden et al., 1991; Crawford et al., 1990; Dagnino and Paterson, 1990; Plagemann et al., 1990; Jakobs and Paterson, 1986). The difference in influx rate (Na⁺ vs. Li⁺ medium) was most apparent under conditions where the inward-directed formycin B concentration gradient was greater than 2, and, as such, the time course of this phenomenon was more prolonged in the presence of 100 nM NBMPR than in its absence. It is possible that Na⁺ stimulated an, as yet undefined, formycin B efflux mechanism, or had a direct inhibitory effect on the equilibrative transporters. Alternatively, incubation of Ehrlich cells in a relatively Na⁺-free medium may have resulted in elevated intracellular levels of adenosine arising from enhanced ATP metabolism, possibly coupled with decreased ATP formation due to reduced adenosine kinase activity (Parkinson and Geiger, 1996). This in turn, could lead to a trans-acceleration of the cellular uptake of [³H]formycin B via the equilibrative transporters. Trans-acceleration of nucleoside flux has been observed in various systems (Jarvis, 1986), and has recently been invoked to explain a paradoxical increase of [³H]formycin release from L1210 cells on removal of the Na⁺ gradient (Borgland and Parkinson, 1997). This would also explain why the greatest difference (± Na⁺) in our study was seen at the early time points (see fig. 5); the outwardly directed adenosine gradient would be expected to decline with incubation time. It should be noted that, although the absolute rate of influx (V_max) was lower in the Na⁺ medium than in the Li⁺ medium, the efficiency of the transporters (defined as V_max/K_m) did not change significantly (see table 2) and is similar to that reported for [³H]formycin B uptake by human erythrocytes (V_max/K_m ~ 0.12 sec¹; see Griffith and Jarvis, 1996). Further delineation of the mechanism(s) underlying this phenomenon awaits detailed kinetic studies on the ion-dependence of [³H]formycin B uptake by Ehrlich cells.

Gemcitabine is a pyrimidine antimetabolite, structurally related to cytosine arabinoside, that has significant potential for the treatment of a variety of solid tumors (Hui and Reitz, 1997; Moore, 1996). The first step in the clinical activity of gemcitabine is its uptake into the target cells, where it is subsequently metabolized by deoxycytidine kinase to its triphosphate derivative and incorporated into DNA (Guchelaar et al., 1996; Plunkett et al., 1995). High concentrations of gemcitabine (>1 mM) have been reported to inhibit [³H]uridine uptake by recombinant es transporters (hENT1) expressed in Xenopus oocytes (Griffiths et al., 1997), and a recombinant pyrimidine-selective concentrative transporter (rCNT1) expressed in COS-1 cells (Fang et al., 1996). Furthermore, the nucleoside transport inhibitor dipyridamole and its congener BIBW22BS have been shown to inhibit the antiproliferative activity of gemcitabine in various cancer cell lines (Jansen et al., 1995). These results suggest that the cellular uptake of gemcitabine is mediated by nucleoside transporters, but information on the affinities of gemcitabine for the various transporter subtypes is lacking.

Our study used the well characterized nucleoside transport system of Ehrlich cells to assess the capacity of gemcitabine to interact with es, ei and Na⁺-dependent transporters. Gemcitabine inhibited [³H]formycin B uptake by the equilibrative transporters of Ehrlich cells with a potency similar to that seen for a number of endogenous nucleosides (K_i ~ 400 µM), including its parent compound deoxycytidine (Hammond, 1991), and is likely a substrate for these transporters. However, the concentrations of gemcitabine required for interaction with the equilibrative transporters are 10,000-fold higher than those associated with its cytotoxic activity (~40 nM) (Shewach and Lawrence, 1995; Ross and Cuddy, 1994), suggesting that these particular transporters may play only a minor role in the cellular accumulation of gemcitabine in the clinical environment.

In contrast to that seen with the equilibrative transporters, gemcitabine was an exceptionally potent inhibitor of Na⁺-dependent [³H]formycin B influx in Ehrlich cells. In the presence of Na⁺, gemcitabine inhibited about 50% of the NBMPR-resistant uptake of [³H]formycin B with an IC₅₀ = 17 nM. The remaining uptake required >10 µM gemcitabine for inhibition and this likely represented the activity of the ei transporter. These data suggest that gemcitabine may prove useful as a tool to identify the relative proportions of Na⁺-dependent and -independent NBMPR-resistant transporters in a cell. The potency of gemcitabine for inhibition of the Na⁺-dependent nucleoside transporter is comparable to its cytotoxic activity (LC₅₀ ~ 30 nM) in a number of model systems (Shewach and Lawrence, 1995; Ross and Cuddy, 1994), indicating that these transporters are likely involved in the cellular uptake of gemcitabine that is required for its clinical efficacy.

In summary, we have established that Ehrlich cells possess a Na⁺-dependent transporter that accepts formycin B (but not guanosine; Hammond, 1992) as a substrate. These cells also express both the es and ei subtypes of equilibrative transporters, and formycin B was found to be relatively selective for the ei subtype. Gemcitabine was identified as a potent and selective inhibitor of the Na⁺-dependent transporter of Ehrlich cells, suggesting that the cellular expression/activity of Na⁺-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.