Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis

J. Pressacco, J. S. Wiley, G. P. Jamieson, C. Erlichman, D. W. Hedley

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-xg)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-xg)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools.

Original languageEnglish (US)
Pages (from-to)939-942
Number of pages4
JournalBritish Journal of Cancer
Volume72
Issue number4
StatePublished - 1995

Fingerprint

Nucleoside Transport Proteins
Nucleic Acid Synthesis Inhibitors
Fluorescein
Antimetabolites
Cytarabine
Fluorouracil
DNA
Aphidicolin
Ribonucleotide Reductases
Hydroxyurea
G2 Phase
Growth
Nucleosides
Pharmaceutical Preparations
Cell Division
Adenosine
Thymidine
Cell Cycle
Flow Cytometry
Coloring Agents

Keywords

  • 5-(SAENTA-xg)-fluorescein
  • 5-fluorouracil
  • Cytosine arabinoside
  • Flow cytometry
  • Nucleoside transporter

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Pressacco, J., Wiley, J. S., Jamieson, G. P., Erlichman, C., & Hedley, D. W. (1995). Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis. British Journal of Cancer, 72(4), 939-942.

Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis. / Pressacco, J.; Wiley, J. S.; Jamieson, G. P.; Erlichman, C.; Hedley, D. W.

In: British Journal of Cancer, Vol. 72, No. 4, 1995, p. 939-942.

Research output: Contribution to journalArticle

Pressacco, J, Wiley, JS, Jamieson, GP, Erlichman, C & Hedley, DW 1995, 'Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis', British Journal of Cancer, vol. 72, no. 4, pp. 939-942.
Pressacco J, Wiley JS, Jamieson GP, Erlichman C, Hedley DW. Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis. British Journal of Cancer. 1995;72(4):939-942.
Pressacco, J. ; Wiley, J. S. ; Jamieson, G. P. ; Erlichman, C. ; Hedley, D. W. / Modulation of the equilibrative nucleoside transporter by inhibitors of DNA synthesis. In: British Journal of Cancer. 1995 ; Vol. 72, No. 4. pp. 939-942.
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abstract = "Expression of the equilibrative, S-(p-nitrobenzyl)-6-thioinosine (NBMPR)-sensitive nucleoside transporter (es), a component of the nucleoside salvage pathway, was measured during unperturbed growth and following exposure to various antimetabolites at growth-inhibitory concentrations. The probe 5-(SAENTA-xg)-fluorescein is a highly modified form of adenosine incorporating a fluorescein molecule. It binds with high affinity and specificity to the (es) nucleoside transporter at a 1:1 stoichiometry, allowing reliable estimates of es expression by flow cytometry. Using a dual labelling technique which combined the vital DNA dye Hoechst-33342 and 5-(SAENTA-xg)-fluorescein, we found that surface expression of es approximately doubled between G1 and G2 + M phases of the cell cycle. To address the question of whether es expression could be modulated in cells exposed to drugs which inhibit de novo synthesis of nucleotides, cells were exposed to antimetabolite drugs having different modes of action. Hydroxyurea and 5-fluorouracil (5-FU), which inhibit the de novo synthesis of DNA precursors, produced increases in the expression of es. In contrast, cytosine arabinoside (ara-C) and aphidicolin, which directly inhibit DNA synthesis, produced no significant increase in es expression. Thymidine (TdR), which is an allosteric inhibitor of ribonucleotide reductase that depletes dATP, dCTP and dGTP pools while repleting the dTTP pool, had no significant effect on es expression. These data suggest that surface expression of the es nucleoside transporter is regulated by a mechanism which is sensitive to the supply of deoxynucleotides. Because 5-FU (which specifically depletes dTTP pools) causes a large increase in expression whereas TdR (which depletes all precursors except dTTP) does not, this mechanism might be particularly sensitive to dTTP pools.",
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