MEASUREMENT OF DEOXYURIDINE TRIPHOSPHATE AND THYMIDINE TRIPHOSPHATE IN THE EXTRACTS OF THYMIDYLATE SYNTHASE-INHIBITED CELLS USING A MODIFIED DNA-POLYMERASE ASSAY

New inhibitors of the enzyme thymidylate synthase (TS) are now reachin g clinical application. Alteration oi the dUTP:dTTP ratio may be criti cal to TS inhibition-induced tumor cell death. The DNA polymerase assa y with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts oi HT29 human colon carcin oma cells treated with the specific TS inhibitor ZD1694 n-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 ZD169 4 at concentrations of 0.05 to 0.2 mu M with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhib ition. This assay is highly sensitive and reproducible for levels of d UTP and is less labor intensive than traditional assays. (C) 1997 Else vier Science Inc.