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

Biochem Pharmacol. 1997 Sep 1;54(5):635-8. doi: 10.1016/s0006-2952(97)00259-1.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenocarcinoma
  • Antimetabolites, Antineoplastic / pharmacology*
  • Colonic Neoplasms
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyuracil Nucleotides / analysis
  • Deoxyuracil Nucleotides / metabolism*
  • Dose-Response Relationship, Drug
  • Enzyme Inhibitors / pharmacology
  • Humans
  • Kinetics
  • Pyrophosphatases / metabolism
  • Quinazolines / pharmacology*
  • Templates, Genetic
  • Thiophenes / pharmacology*
  • Thymidylate Synthase / antagonists & inhibitors*
  • Thymine Nucleotides / analysis
  • Thymine Nucleotides / metabolism*
  • Tumor Cells, Cultured


  • Antimetabolites, Antineoplastic
  • Deoxyuracil Nucleotides
  • Enzyme Inhibitors
  • Quinazolines
  • Thiophenes
  • Thymine Nucleotides
  • deoxyuridine triphosphate
  • Thymidylate Synthase
  • DNA-Directed DNA Polymerase
  • Pyrophosphatases
  • dUTP pyrophosphatase
  • raltitrexed
  • thymidine 5'-triphosphate