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 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.
Horowitz
R W
RW
Department of Oncology, Montefiore Medical Center, and the Albert Einstein Cancer Center, Bronx, NY 10467, U.S.A.
Zhang
H
H
Schwartz
E L
EL
Ladner
R D
RD
Wadler
S
S
D000230:Adenocarcinoma; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D003110:Colonic Neoplasms; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000032:analysis / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D011755:Pyrophosphatases / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D013698:Templates, Genetic; D013876:Thiophenes / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D013942:Thymine Nucleotides / Q000032:analysis / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D003856:Deoxyuracil Nucleotides; D004791:Enzyme Inhibitors; D011799:Quinazolines; D013876:Thiophenes; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; D013940:Thymidylate Synthase; D004259:DNA-Directed DNA Polymerase; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; C068874:raltitrexed; C024157:thymidine 5'-triphosphate