title: Interaction of 2',2'-difluorodeoxycytidine (gemcitabine) and formycin B with the Na+-dependent and -independent nucleoside transporters of Ehrlich ascites tumor cells.
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 with Ki values of 48 +/- 28 and 277 +/- 25 microM, respectively. Similarly, [3H]formycin B had Km values of 111 +/- 52 and 635 +/- 147 microM 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/microliter . sec) than in the Na+ buffer (Vmax = 8.4 +/- 0.9 pmol/microliter . 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 approximately 400 microM). 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.
authors.affiliation: Department of Pharmacology and Toxicology, University of Western Ontario, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Burke
authors.forename: S
authors.initials: S
authors.lastname: Lee
authors.forename: P J
authors.initials: PJ
authors.lastname: Ferguson
authors.forename: J R
authors.initials: JR
authors.lastname: Hammond
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005573:Formycins / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D033703:Nucleoside Transport Proteins; D012964:Sodium / Q000494:pharmacology*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014529:Uridine / Q000378:metabolism; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D002352:Carrier Proteins; D005573:Formycins; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D003841:Deoxycytidine; C034561:formycin B; D013868:Thioinosine; D012964:Sodium; C001789:4-nitrobenzylthioinosine; D014529:Uridine; D000093542:Gemcitabine