formycins and Carcinoma--Ehrlich-Tumor

We evaluated the contribution of a nucleoside transporter (NT) consisting of an equilibrative NT (ENT) and a concentrative Na(+)/nucleoside cotransporter (CNT) to the uptake of THP and DOX by mouse Ehrlich ascites carcinoma cells. METHODS. Transport experiments were performed using a silicone layer method. The expression of CNT isoforms was confirmed by RT-PCR analysis.. The effects of inhibition of the ENT inhibitors, nitrobenzylthioinosine (NBMPR) and nitrobenzylthioguanosine, on THP and DOX uptake by Ehrlich cells was negligible. THP uptake, but not DOX uptake, partially depended on an inwardly directed Na(+) gradient, and the uptake was inhibited by all the inhibitors of CNT examined. Furthermore, efflux of [(3)H]uridine from Ehrlich cells was stimulated by the addition of THP to the extracellular compartment, which was definitive evidence of CNT-mediated uptake of THP. The mRNA for CNT2, but not that for CNT3, was detected in Ehrlich cells, which is consistent with the characteristics of [(3)H]uridine uptake. In the cells, formycin B, a representative CNT2 ligand, had cis-inhibitory and trans-stimulatory effects on THP uptake.. These results demonstrate that THP, but not DOX, is taken up into Ehrlich cells partially via a uridine-transportable CNT.

Topics: Animals; Antibiotics, Antineoplastic; Carcinoma, Ehrlich Tumor; Doxorubicin; Formycins; Mice; Nucleoside Transport Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium; Tumor Cells, Cultured; Uridine

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.

Topics: Animals; Antimetabolites, Antineoplastic; Carcinoma, Ehrlich Tumor; Carrier Proteins; Deoxycytidine; Formycins; Gemcitabine; Male; Membrane Proteins; Mice; Nucleoside Transport Proteins; Sodium; Thioinosine; Uridine

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Antibiotics, Antineoplastic; Carcinoma, Ehrlich Tumor; Cells, Cultured; Energy Metabolism; Formycins; Humans; Inosine; Mice; Nucleoside Deaminases; Purine Nucleotides; Time Factors

Topics: Aminoglycosides; Animals; Anti-Bacterial Agents; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Formycins; Mice; Mycobacterium; Nocardia; Pharmacology; Rats; Research; Sarcoma; Sarcoma, Yoshida; Streptomyces; Tissue Culture Techniques

Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Bacteriological Techniques; Carcinoma, Ehrlich Tumor; Chromatography; Drug Resistance; Drug Resistance, Microbial; Formycins; Ion Exchange Resins; Mycobacterium; Pharmacology; Rats; Research; Sarcoma; Sarcoma, Experimental; Tissue Culture Techniques