MODULATION OF THE EQUILIBRATIVE NUCLEOSIDE TRANSPORTER BY INHIBITORS OF DNA-SYNTHESIS

Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMP R)-sensitive nucleoside transporter (es), a component of the nucleosid e salvage pathway, was measured during unperturbed growth and followin g exposure to various antimetabolites at growth-inhibitory concentrati ons. The probe 5-(SAENTA-x(8))-fluorescein is a highly modified form o f adenosine incorporating a fluorescein molecule. It binds with high a ffinity and specificity to the (es) nucleoside transporter at a 1:1 st oichiometry, allowing reliable estimates of es expression by flow cyto metry. Using a dual labelling technique which combined the vital DNA d ye Hoechst-33342 and 5-(SAENTA-x(8))-fluorescein, we found that surfac e expression of es approximately doubled between G(1) and G(2) + M pha ses of the cell cycle. To address the question of whether es expressio n could be modulated in cells exposed to drugs which inhibit de novo s ynthesis of nucleotides, cells were exposed to antimetabolite drugs ha ving different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced incre ases in the expression of es. In contrast, cytosine arabinoside (ara-C ) and aphidicolin, which directly inhibit DNA synthesis, produced no s ignificant increase in es expression. Thymidine (TdR), which is an all osteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effe ct on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensit ive to the supply of deoxynucleotides. Because 5-FU (which specificall y depletes dTTP pools) causes a large increase in expression whereas T dR (which depletes all precursors except dTTP) does not, this mechanis m might be particularly sensitive to dTTP pools.