Abstract
The uptake of [3H]formycin B by Ehrlich ascites tumor cells was examined in both normal Na+ buffer (physiological) and nominally Na+-free buffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subtypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nucleoside analogues by these cells. Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure transport kinetics in energetically competent cells. Initial studies established that formycin B inhibited [3H]uridine uptake by the ei (equilibrative inhibitor-insensitive) and es (equilibrative inhibitor-sensitive) transporters of Ehrlich cells withKi values of 48 ± 28 and 277 ± 25 μM, respectively. Similarly, [3H]formycin B hadKm values of 111 ± 52 and 635 ± 147 μM for uptake by the ei and es transporters, respectively. When assays were conducted in the presence of Na+, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es transporters, the intracellular concentration of [3H]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the activity of a Na+-dependent transporter. Interestingly, the initial rate of uptake of [3H]formycin B was significantly higher in the Li+ buffer (es-mediated Vmax = 65 ± 10 pmol/μl · sec) than in the Na+buffer (Vmax = 8.4 ± 0.9 pmol/μl · sec); this may reflect trans-acceleration of [3H]formycin B uptake by elevated intracellular adenosine levels resulting from the low Na+ environment. This model was then used to assess the interaction of gemcitabine (2′,2′-difluorodeoxycytidine) with the equilibrative and concentrative nucleoside transporters. Gemcitabine, which has shown considerable potential for the treatment of solid tumors, was a relatively poor inhibitor of [3H]formycin B uptake via the equilibrative transporters (IC50 ∼ 400 μM). In contrast, gemcitabine was a potent inhibitor of the Na+-dependent nucleoside transporter of Ehrlich cells (IC50 = 17 ± 5 nM). These results suggest that the cellular expression/activity of Na+-dependent nucleoside transporters may be an important determinant in gemcitabine cytotoxicity and clinical efficacy.
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 (es2isoform) 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 (Jacobsonet 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
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.
Uptake data are presented as intracellular [3H]substrate concentrations (pmol/μl intracellular volume; μM) after correction for the amount of [3H]label present in the extracellular space of the cell pellet. Intracellular and extracellular water volumes of the cell pellets were determined by incubating cells with a combination of [carboxy-14C]dextran (cell impermeant) and [3H]water for 3 min and then processing the samples as described above. All kinetic values and inhibition constants were derived from the “transporter-mediated” (total uptake minus nonmediated uptake) accumulation of [3H]substrate, unless otherwise indicated. Nonmediated uptake was defined as the cellular accumulation of [3H]substrate in the presence of 10 μM dipyridamole + 10 μM NBMPR. Uptake that was not inhibited by 100 nM NBMPR but was sensitive to 10 μM NBMPR/dipyridamole, was defined as that mediated by NBMPR-resistant transporters (see Hammond, 1991). Initial rates (Vi) of [3H]formycin B flux were estimated as the uptake at 1 sec determined by extrapolation of hyperbolic curves fitted (computer-generated; GraphPad Prism v2.01) to time course data. For inhibition studies, cells were either incubated with inhibitor for 30 min before exposure to [3H]substrate or exposed to inhibitor concurrently with substrate, as specified in “Results.” Most inhibition assays involved a 20-sec incubation of cells with [3H]substrate, the exception being those involving inhibition of NBMPR-resistant [3H]formycin B influx by gemcitabine, where cells were exposed to [3H]formycin B and gemcitabine concurrently for 5 min. Inhibitor Kl values (inhibition constants) were calculated from the relationshipKl = IC50/(1 + [S]/Km ), where [S] is the assay concentration of substrate, Km is the Michaelis-Menten constant for substrate transport and IC50is the concentration of inhibitor required to block influx by 50%, assuming competitive inhibition kinetics. Results are presented as mean ± S.E. of replicate (n) experiments conducted in duplicate. Statistical significance was assessed using the Student’st test (two-tailed, P < .05) for unpaired or paired samples, as appropriate.
Results
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. UsingKm 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 theei transporter (Kl = 48 μM) than for the es transporter (Kl = 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 andei-mediated influx of [3H]formycin B.
The concentration dependence of NBMPR inhibition of [3H]uridine and [3H]formycin B influx (5 μM, 22-sec incubation) was assessed under nominally Na+-free conditions (Li+ medium) to compare the relative contribution of the es and eitransporters to the cellular accumulation of these substrates (fig.2). The inhibition profile obtained using either substrate was clearly biphasic, and similar IC50values were observed for NBMPR inhibition of [3H]formycin B (3.3 ± 0.7 nM) and [3H]uridine (3.6 ± 0.1 nM) uptake. In both cases, 100 nM NBMPR was sufficient to inhibit alles-mediated uptake. However, a significantly larger proportion of the [3H]formycin B uptake (49 ± 3%) was resistant to inhibition by NBMPR compared to that observed using [3H]uridine (23 ± 1%). A similar proportion of NBMPR-resistant to NBMPR-sensitive [3H]formycin B influx (∼50%) was observed when comparable assays were done using normal Na+ buffer (data not shown).
A full time-course for the uptake of 10 μM [3H]formycin B (± 100 nM NBMPR) was constructed (fig.3A). The maximum intracellular concentration of [3H]formycin B achieved in Li+ medium was approximately 14 μM, in both the presence and absence of NBMPR (table 1). Nonmediated uptake represented less than 5% of the total uptake over the first 30 sec of incubation. The initial rate of transporter-mediated uptake of 10 μM [3H]formycin B was 1.54 ± 0.07 pmol/μl · sec in the absence of NBMPR; this was reduced to 0.38 ± 0.05 pmol/μl · sec in the presence of 100 nM NBMPR.
Similar time-courses were defined for a range of concentrations of formycin B, and initial rates of influx (unidirectional, inward flux of permeant) were plotted against formycin B concentration (fig.4A) to determine the Michaelis Menten kinetic constants for [3H]formycin B uptake by Ehrlich cells (table 2). In Li+medium, the Km of [3H]formycin B for the ei transporter (111 ± 52 μM) was more than 6-fold lower than that determined for the es transporter (635 ± 147 μM). The ei transporter mediated about 7% (Vmax = 6 ± 2 pmol/μl · sec) of the total uptake of [3H]formycin B (Vmax = 93 ± 21 pmol/μl · sec). That component of uptake defined as non-mediated (determined in the presence of 10 μM NBMPR and 10 μM dipyridamole) was linear with [3H]formycin B concentration. Comparative data obtained previously using [3H]uridine (Hammond, 1991) and [3H]guanosine (Hammond, 1992) as substrates 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.
Time courses of 10 μM [3H]formycin B uptake were constructed in the presence and absence of 100 nM NBMPR (fig. 3B, table1). The maximum intracellular concentration of [3H]formycin B achieved in the absence of NBMPR was 23 ± 3 μM with an initial rate of transporter-mediated influx of 0.79 ± 0.08 pmol/μl · sec. In the presence of 100 nM NBMPR, Ehrlich cells concentrated the [3H]formycin B to an even greater extent (39 ± 8 μM) but at a lower rate (Vi = 0.19 ± 0.02 pmol/μl · sec). When the NBMPR-resistant uptake was subtracted from the total uptake, leaving only that mediated (theoretically) by the NBMPR-sensitive (es) transporter, the maximum intracellular concentration of [3H]formycin B was 12 ± 2 μM. This is not significantly different from the initial medium concentration of substrate (10 μM) and is similar to the maximum estransporter-mediated uptake determined in Li+ medium (8 ± 2 μM). These latter results are what would be expected from the operation of a Na+-independent, NBMPR-sensitive equilibrative nucleoside transporter. An unexpected finding, however, was that the initial rate of [3H]formycin B uptake was significantly lower (by ∼50%) in the Na+ medium than in the Li+ medium (fig. 5); this difference was observed for both the es- andei-mediated uptake of [3H]formycin B at all concentrations studied (see below).
Full time-courses were then constructed for a range of concentrations of [3H]formycin B (fig. 4B). The kinetic constants (Km , Vmax) derived from these studies are shown in table 2. In the presence of Na+(normal PBS), [3H]formycin B had similarKm values for the NBMPR-sensitive and NBMPR-resistant transport systems (∼70 μM; see table 2). This is in contrast to that seen in the Li+ medium where [3H]formycin B had a 6-fold higher affinity for the NBMPR-resistant transporter. The maximum rate of influx of [3H]formycin B in the presence of Na+(Vmax = 11 pmol/μl · sec) was also 8-fold lower than that observed in the Li+ medium (Vmax = 93 pmol/μl · sec). Approximately 20% of the total uptake of [3H]formycin B in the presence of Na+ was mediated by the NBMPR-resistant transporters (Vmax = 2 pmol/μl · sec), which is a larger proportion than that measured in the Li+ medium and likely reflects the influence of a Na+-dependent nucleoside transporter.
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; table3). 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).
Gemcitabine was then tested over a range of concentrations for its capacity to inhibit the total transporter-mediated uptake and the NBMPR-resistant uptake of [3H]formycin B in both PBS (normal Na+) and Li+ medium (fig.7). In the Li+ medium, gemcitabine inhibited the total uptake in a monophasic manner with an IC50 of about 400 μM and a pseudo Hill coefficient of 0.99 (table 4). When uptake via thees transporter subtype was blocked with 100 nM NBMPR, gemcitabine had a significantly higher IC50 (543 μM, Student’s t test, P < .05) for blocking uptake via the remaining ei transporter isoform (table 4). However, when the NBMPR-resistant influx of [3H]formycin B was measured in the presence of Na+, gemcitabine had an IC50 for blocking influx of 280 nM (95% confidence interval of 150–523 nM, determined from the curve fit to averaged data shown in fig. 7); this is more than 1,000-fold lower than the value determined using the Li+ medium (fig. 7). The pseudo Hill coefficient for gemcitabine inhibition of NBMPR-resistant uptake in the presence of Na+ was also significantly less than 1 (table4); this likely reflects a differential interaction of gemcitabine with multiple transport sites. Data from individual experiments fit best (F test, P < .05) to two-phase competition curves with IC50 values of 17 ± 5 nM and 415 ± 126 μM (n = 5) for each component (table 4). The component with high affinity for gemcitabine represented 47% of the total NBMPR-resistant uptake of [3H]formycin B, and the low affinity component was comparable to that measured for gemcitabine inhibition of [3H]formycin B uptake by cells equilibrated in the Li+ medium.
Discussion
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 byGriffith 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 thees 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., thees transporter). Both the cif and cibsubtypes 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 theei subtype of equilibrative transporter than for thees 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 theei 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; Griffithet al., 1990).
When assays were conducted in the presence of Na+, the Vmax 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 Vmax) 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 (Vmax) was lower in the Na+ medium than in the Li+ medium, the efficiency of the transporters (defined as Vmax/Km ) did not change significantly (see table 2) and is similar to that reported for [3H]formycin B uptake by human erythrocytes (Vmax/Km ∼ 0.12 sec−1; 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 recombinantes transporters (hENT1) expressed in Xenopusoocytes (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 withes, 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 (Ki ∼ 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.
Footnotes
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Send reprint requests to: Dr. James R. Hammond, Department of Pharmacology and Toxicology, Medical Sciences Building, The University of Western Ontario, London, Ontario, Canada N6A 5C1.
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↵1 This work was supported by a grant to J.R.H. from the Medical Research Council of Canada.
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↵2 es = e quilibrative, inhibitor s ensitive; ei = e quilibrative, inhibitor i nsensitive; cs = c oncentrative, inhibitor s ensitive;cif = c oncentrative, inhibitor i nsensitive, f ormycin B (purine) selective;cit = c oncentrative, inhibitor i nsensitive, t hymidine (pyrimidine) selective;cib = c oncentrative, inhibitor i nsensitive, b road substrate selectivity (nomenclature according to Belt et al., 1993).
- Abbreviations:
- NBMPR
- nitrobenzylthioinosine, nitrobenzylmercaptopurine riboside
- PBS
- phosphate-buffered saline
- ATP
- adenosine triphosphate
- Received August 15, 1997.
- Accepted April 27, 1998.
- The American Society for Pharmacology and Experimental Therapeutics