Vol. 286, Issue 3, 1333-1340, September 1998

Interaction of 2',2'-Difluorodeoxycytidine (Gemcitabine) and Formycin B with the Na⁺-Dependent and -Independent Nucleoside Transporters of Ehrlich Ascites Tumor Cells¹

Trisha Burke, Stephanie Lee, Peter J. Ferguson and James R. Hammond

Department of Pharmacology and Toxicology, University of Western Ontario (T.B., S.L., P.J.F., J.R.H.) and the London Regional Cancer Centre (P.J.F.), London, Ontario, Canada N6A 5C1

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 with K_i values of 48 ± 28 and 277 ± 25 µM, respectively. Similarly, [³H]formycin B had K_m 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 V_max = 65 ± 10 pmol/µl · sec) than in the Na⁺ buffer (V_max = 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.