thioinosine and Cell-Transformation--Viral

The initial velocity of thymidine uptake was measured in HeLa S3 cells infected with herpes simplex virus type 1 (HSV-1). The rate of nucleoside influx into the cells was shown to increase from as early as 1 h post-infection (p.i.) up to 8 h p.i. This increased uptake was shown to be attributable to a progressively increasing contribution from passive diffusion superimposed upon normal transport. Thus, the specific nucleoside transport system was still operating with unaltered kinetic parameters 8 h after infection. Despite the inhibition of host cell protein synthesis and its replacement by the synthesis of virus-specified proteins, the numbers and affinity of the nucleoside transporters in cells 8 h after infection were virtually unchanged. The increased transport of thymidine in infected cultures was resistant to the nucleoside transport inhibitor dipyridamole, and was correlated with entry of a normally impermeant solute (sucrose) into infected cells. These data suggest that the system for the carrier-mediated facilitated diffusion of nucleosides remains intact in HSV-infected cells, but that progressively increasing passive diffusion takes place. Passive diffusion is the major process operating late after virus infection.

Topics: Affinity Labels; Biological Transport; Cell Transformation, Viral; Dipyridamole; HeLa Cells; Humans; Kinetics; Methionine; Simplexvirus; Sucrose; Thioinosine; Thymidine

Nucleoside transport in various types of animal cells is inhibited by the binding of nitrobenzylthioinosine (NBMPR) to a set of high-affinity sites on the plasma membrane. This work examined the binding of [3H]NBMPR to the nucleoside transporters of cultured Nil 8 hamster fibroblasts and of cells of a virus-transformed clone (Nil SV) derived from Nil 8. Experiments conducted with intact Nil 8 and Nil SV cells and with membrane preparations indicated that the two lines differed significantly in the cellular content of binding sites and only slightly in the affinities of these sites for NBMPR. Nil 8 and Nil SV cells possessed (4.2-8.0) X 10(5) and (2.0-4.0) X 10(6) sites per cell respectively, whereas the dissociation constants of site-bound NBMPR obtained with intact cells and with membrane preparations were similar, ranging from 0.29 to 1.5 nM. Dilazep, a potent inhibitor of nucleoside transport that is structurally unrelated to NBMPR, appeared to compete with NBMPR for binding to the high-affinity sites when tested under equilibrium conditions with Ki values for inhibition of NBMPR binding to Nil 8 and Nil SV cells respectively of 15 +/- 4 and 32 +/- 4 nM. The dissociation of NBMPR from the binding site--NBMPR complex of Nil SV membrane preparations was a first-order decay process with a rate constant of 0.68 +/- 0.26 min-1. The rate of dissociation of NBMPR from the binding-site complex of membrane preparations and intact cells was decreased significantly in the presence of dilazep and increased in the presence of the permeant uridine. These results suggest that the apparent competitive-inhibition kinetics obtained for dilazep under equilibrium conditions should not be interpreted as binding of dilazep to the same site as NBMPR but rather as binding of the two inhibitors to closely associated sites on the nucleoside transporter. Similarly, uridine also appears to bind to a site separate from the NBMPR-binding site.

Topics: Animals; Binding Sites; Biological Transport; Cell Division; Cell Line; Cell Membrane; Cell Transformation, Viral; Clone Cells; Cricetinae; Dilazep; Fibroblasts; Inosine; Kinetics; Mesocricetus; Nucleosides; Thioinosine; Uridine

The uptake of uridine by mammalian cells consists of transport of uridine across the plasma membrane followed by its metabolic conversion, mainly by phosphorylation. S-substituted aromatic derivatives of 6-mercaptopurine ribosides are potent inhibitors of the nucleoside uptake systems in human erythrocytes and in mammalian cells in culture and have been studied extensively. We present here a theoretical analysis which enables one to decide whether transport of metabolites, their metabolic trapping within the cell, or both, are susceptible to inhibition. This analysis was applied in the study of the effect of some inhibitors on uridine and cytosine-beta-D-arabinoside uptake by transformed Nil-8 cells. It was found that in Nil-SV cells, both transport and metabolic conversion are susceptible to inhibition by nitrobenzylmercaptoinosine and by dansylaminoethylmercaptoguanosine. Nitrobenzylmercaptoinosine displays inhibition constants of 20 and 7 nM for transport and phosphorylation, respectively, while for dansylaminoethylmercaptoguanosine the inhibition constants are 1.8 and 0.6 microM, respectively, for the same processes. Cytosine-beta-D-arabinoside is a synthetic nucleoside which is not metabolizable in Nil cells. Its uptake properties are determined by the transport mechanism alone. The transport of this nucleoside into Nil-SV cells in inhibited by nitrobenzylmercaptoinosine and the inhibition constant found is approx. 5 times greater than that for uridine.

Topics: Animals; Cell Line; Cell Transformation, Viral; Cricetinae; Cytarabine; Deoxyglucose; Inosine; Kinetics; Mesocricetus; Phosphorylation; Sarcoma Viruses, Murine; Thioinosine; Uridine