INTERACTION OF 2',2'-DIFLUORODEOXYCYTIDINE (GEMCITABINE) AND FORMYCIN-B WITH THE NA-DEPENDENT AND NA+-INDEPENDENT NUCLEOSIDE TRANSPORTERS OF EHRLICH ASCITES TUMOR-CELLS()

The uptake of [H-3]formycin B by Ehrlich ascites tumor cells was exami ned in both normal Na+ buffer (physiological) and nominally Na+-free b uffer (iso-osmotic replacement with Li+). These studies were conducted to further characterize the equilibrative nucleoside transporter subt ypes of Ehrlich cells and to assess the contribution of Na+-dependent concentrative transport mechanisms to the cellular accumulation of nuc leoside analogues by these cells; Formycin B is poorly metabolized by mammalian cells and, hence, can be used as a substrate to measure tran sport kinetics in energetically competent cells. Initial studies estab lished that formycin B inhibited [H-3]uridine uptake by the ei (equili brative inhibitor-insensitive) and es (equilibrative inhibitor-sensiti ve) transporters of Ehrlich cells with K-i values of 48 +/- 28 and 277 +/- 25 mu M, respectively. Similarly, [H-3]formycin B had K-m values of 111 +/- 52 and 635 +/- 147 mu M for uptake by the ei and es transpo rters, respectively. When assays were conducted in the presence of Na, plus 100 nM nitrobenzylthioinosine to prevent efflux via the es tran sporters, the intracellular concentration of [H-3]formycin B exceeded the initial medium concentration by more than 3-fold, indicating the a ctivity of a Na+-dependent transporter. Interestingly, the initial rat e of uptake of [H-3]formycin B was significantly higher in the Li+ buf fer (es-mediated V-max = 65 +/- 10 pmol/mu l . sec) than in the Na+ bu ffer (V-max = 8.4 +/- 0.9 pmol/mu l . sec); this may reflect trans-acc eleration of [H-3]formycin B uptake by elevated intracellular adenosin e levels resulting from the low Na+ environment. This model was then u sed to assess the interaction of gemcitabine (2',2'-difluorodeoxycytid ine) 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 [H-3]formycin B u ptake via the equilibrative transporters (IC50 similar to 400 mu M). I n contrast, gemcitabine was a potent inhibitor of the Nac-dependent nu cleoside transporter of Ehrlich cells (IC50 = 17 +/- 5 nM). These resu lts suggest that the cellular expression/activity of Na+-dependent nuc leoside transporters may be an important determinant in gemcitabine cy totoxicity and clinical efficacy.