Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate synthase-inhibited cells using a modified DNA polymerase assay

R W Horowitz, H Zhang, E L Schwartz, R D Ladner, S Wadler

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

New inhibitors of the enzyme thymidylate synthase (TS) are now reaching clinical application. Alteration of the dUTP: dTTP ratio may be critical to TS inhibition-induced tumor cell death. The DNA polymerase assay with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts of HT29 human colon carcinoma cells treated with the specific TS inhibitor ZD1694 [N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid]. These results revealed an increase in the dUTP:dTTP ratio at 2 hr after a 2-hr exposure to ZD1694 at concentrations of 0.05 to 0.2 microM with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhibition. This assay is highly sensitive and reproducible for levels of dUTP and is less labor intensive than traditional assays.

Original languageEnglish
Pages (from-to)635-8
Number of pages4
JournalBiochemical Pharmacology
Volume54
Issue number5
Publication statusPublished - 01 Sep 1997

Keywords

  • Adenocarcinoma
  • Antimetabolites, Antineoplastic
  • Colonic Neoplasms
  • DNA-Directed DNA Polymerase
  • Deoxyuracil Nucleotides
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors
  • Humans
  • Kinetics
  • Pyrophosphatases
  • Quinazolines
  • Templates, Genetic
  • Thiophenes
  • Thymidylate Synthase
  • Thymine Nucleotides
  • Tumor Cells, Cultured
  • Journal Article
  • Research Support, U.S. Gov't, P.H.S.

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