Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression

Josie Pressacco, Biserka Mitrovski, Charles Erlichman, David W. Hedley

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

The effects of de novo dTMP inhibition by N-{5-[N-(3,4-dihydro-2-methyl- 4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl}-L-glutamic acid (D1694) or N6-[4-(morpholinosulfonyl)benz]-N6-diaminobenz[cd]indole glucuronate (AG-331) on clonogenic survival, thymidylate synthase (TS) and thymidine kinase (TK) activity, and expression of S-(p-nitrobenzyl)-6- thioinosine-sensitive nucleoside transporter (NT) sites were addressed in the human bladder cancer cell line, MGH-U1. These two TS inhibitors are structurally diverse. D1694 is a folate-based TS inhibitor, whereas AG-331 is a novel agent that inhibits the cofactor binding site of the enzyme. They also differ with respect to their cytotoxic effects in this cell line; D1694 cytotoxic 50% inhibitory concentration (IC50) and IC50 were 6.0 and 9.0 nM, respectively, and IC50 and IC50 for TS inhibition were 2.5 and 4.8 nM, respectively. In contrast, AG-331 cytotoxic IC50 could not be achieved even at concentrations of up to 20 μM for 24-h exposures, and IC50 and IC50 for TS inhibition were 0.7 and 3.0 μM, respectively. Similar effects for D1694 and AG-331 were observed in their modulation of TK activity and NT expression. 5-(SAENTA-x8)-Fluorescein, a highly modified form of adenosine incorporating a fluorescein molecule which binds with a 1:1 stoichiometry to S-(p-nitrobenzyl)-6-thioinosine-sensitive NT sites, was used to investigate the expression of NT following exposure of cells to D1694 and AG-331. TK activity was addressed by the metabolism of [3H]thymidine to [3H]TMP by cellular extracted protein and by an alternative flow cytometric method using a modified form of thymidine incorporating a fluorescent molecule, dansyl-5- amino-2-deoxyuridine. Results obtained by both methods were comparable. At concentrations of 5 and 10 nM, D1694 increased TK activity 2.3-4.5-fold and NT expression 34-39-fold. AG-331, at concentrations of 5 and 10 μM, increased TK activity 1.8-2.5-fold and NT expression 22-31-fold, respectively. These data suggest that TK activity and NT expression have a common regulatory mechanism which is sensitive to endogenous dTTP pools and that the salvage pathway is a complex system of kinases coordinated with transport of nucleosides.

Original languageEnglish (US)
Pages (from-to)1505-1508
Number of pages4
JournalCancer Research
Volume55
Issue number7
StatePublished - Apr 1 1995
Externally publishedYes

Fingerprint

Nucleoside Transport Proteins
Thymidylate Synthase
Thymidine Kinase
Inhibitory Concentration 50
dTMP kinase
Thymidine
Thymidine Monophosphate
Cell Line
Glucuronic Acid
raltitrexed
Fluorescein
Nucleosides
Folic Acid
Urinary Bladder Neoplasms
Adenosine
AG 331
Phosphotransferases
Binding Sites
Survival

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Pressacco, J., Mitrovski, B., Erlichman, C., & Hedley, D. W. (1995). Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression. Cancer Research, 55(7), 1505-1508.

Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression. / Pressacco, Josie; Mitrovski, Biserka; Erlichman, Charles; Hedley, David W.

In: Cancer Research, Vol. 55, No. 7, 01.04.1995, p. 1505-1508.

Research output: Contribution to journalArticle

Pressacco, J, Mitrovski, B, Erlichman, C & Hedley, DW 1995, 'Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression', Cancer Research, vol. 55, no. 7, pp. 1505-1508.
Pressacco, Josie ; Mitrovski, Biserka ; Erlichman, Charles ; Hedley, David W. / Effects of thymidylate synthase inhibition on thymidine kinase activity and nucleoside transporter expression. In: Cancer Research. 1995 ; Vol. 55, No. 7. pp. 1505-1508.
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