Nucleoside and nucleobase transport in animal cells

Ethanol increases extracellular adenosine by inhibiting adenosine uptake via the nucleoside transporter.

Transport of nucleoside drugs in animal cells.

Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line.

Although cytosine arabinoside (araC) can induce a remission in a majority of patients presenting with acute myeloblastic leukemia (AML), a minority fail to respond and moreover the drug has less effect in acute lymphoblastic leukemia (ALL). The carrier-mediated influx of araC into purified blasts from patients with AML, ALL, and acute undifferentiated leukemia (AUL) has been compared to that of normal lymphocytes and polymorphs. Blasts showed a larger mediated influx of araC than mature cells, since mean influxes for myeloblasts and lymphoblasts were 6- and 2.3-fold greater than polymorphs and lymphocytes, respectively. Also, the mean influx for myeloblasts was fourfold greater than the mean for lymphoblasts. The number of nucleoside transport sites was estimated for each cell type by measuring the equilibrium binding of [3H]nitrobenzylthioinosine (NBMPR), which inhibits nucleoside fluxes by binding with high affinity to specific sites on the transport mechanism. The mean binding site numbers for myeloblasts and lymphoblasts were 5- and 2.8-fold greater, respectively, than for the mature cells of the same maturation series. The mean number of NBMPR binding sites for myeloblasts was fourfold greater than for lymphoblasts. Patients with AUL were heterogeneous since blasts from some gave values within the myeloblastic range and others within the lymphoblastic range. The araC influx correlated closely with the number of NBMPR binding sites measured in the same cells on the same day. Transport parameters were measured on blasts from 15 patients with AML or AUL who were then treated with standard induction therapy containing araC. Eight patients entered complete remission, while seven failed therapy, among whom were the three patients with the lowest araC influx ( 10-fold between leukemic blasts and normal leukocytes, but transport capacity related directly to the number of nucleoside transport sites on the cell. Finally, low araC transport rates or few NBMPR binding sites on blasts were observed in a subset of patients with acute leukemia who failed to achieve remission with drug combinations containing araC.

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Cytosine Arabinoside Influx and Nucleoside Transport Sites in Acute Leukemia

Transport of uridine by monolayer cultures of HeLa cells was inhibited by nitrobenzylthioinosine, dipyridamole, and lidoflazine. Biphasic concentration-effect curves were obtained for inhibition of nucleoside transport by nitrobenzylthioinosine, but not for inhibition by dipyridamole. Dipyridamole and lidoflazine interfered with high-affinity binding of [ 3 H]nitrobenzylthioinosine to HeLa cells in an apparently competitive fashion; values of 1, 30, and 300 nM were obtained for dissociation constants, respectively, for nitrobenzylthioinosine, dipyridamole, and lidoflazine. The apparent competition with nitrobenzylthioinosine at the latter’s high-affinity binding sites suggests that dipyridamole and lidoflazine inhibit nucleoside transport by interaction with these sites.

A common basis for inhibition of nucleoside transport by dipyridamole and nitrobenzylthioinosine

The toxic effects of various nucleoside antimetabolites toward RPMI 6410 cells in culture were eliminated or reduced when the growth medium contained 5 µM nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport. When cells were "protected" in this way from the growth inhibitory effects of trifluorothymidine, arabinosylcytosine or 6-azauridine, the cellular content of these agents was much reduced relative to that of cells cultured in the absence of NBMPR, indicating that NBMPR impairment of nucleoside transport was the basis of the protective effect. It is implicit in these results that the toxic nucleosides against which protection is afforded by NBMPR are "substrates" for the nucleoside transporter. The cytotoxic effects of a number of nucleosides of diverse structure were ameliorated by NBMPR and these compounds were judged to be substrates for the transporter. Because compounds as diverse as showdomycin, 5-azacytidine, sangivamycin and nebularine were permeants by this criterion, it was concluded that the specificity of the nucleoside transport mechanism of RPMI 6410 cells is low with respect to the base portion of nucleosides.

Low Specificity of the Nucleoside Transport Mechanism of RPMI 6410 Cells

Binding of the potent nucleoside transport inhibitor 6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine (NBMPR) and rates of uptake of several nucleosides were examined at 4-hr intervals during the replication cycle of HeLa S3 cells. Monolayer cultures of synchronous cells, obtained by mitotic detachment, were assayed for high-affinity binding of NBMPR and for rates of uptake of thymidine, uridine, cytidine, adenosine, inosine, and guanosine. The number of NBMPR binding sites per cell doubled between 4 and 16 hr after detachment (late G1 and S phase); during this interval, V max 'S for uptake of cytidine and adenosine doubled, and for uridine and thymidine uptake increased about 4- and 8-fold, respectively. Rates of inosine and guanosine uptake at extracellular concentrations below saturation increased 2-fold between G1 and S phase of the cell cycle. Km 'S for cellular uptake of thymidine, uridine, cytidine, and adenosine did not change with progress through the cycle. The results presented suggest that changes in nucleoside uptake during the HeLa cell cycle were due, in part, to changes in the activity of NBMPR-sensitive transport elements in the membrane.

Fluctuations in Nucleoside Uptake and Binding of the Inhibitor of Nucleoside Transport, Nitrobenzylthioinosine, during the Replication Cycle of HeLa Cells

Eda Y. Lau

Papers

A common basis for inhibition of nucleoside transport by dipyridamole and nitrobenzylthioinosine

Low Specificity of the Nucleoside Transport Mechanism of RPMI 6410 Cells

Fluctuations in Nucleoside Uptake and Binding of the Inhibitor of Nucleoside Transport, Nitrobenzylthioinosine, during the Replication Cycle of HeLa Cells