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

Robert W. Horowitz, Hong yang Zhang, Edward L. Schwartz, Robert D. Ladner, Scott Wadler

Research output: Contribution to journalArticle

22 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 μM 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 (US)
Pages (from-to)635-638
Number of pages4
JournalBiochemical Pharmacology
Volume54
Issue number5
DOIs
StatePublished - Sep 1 1997

Fingerprint

Thymidylate Synthase
DNA-Directed DNA Polymerase
Assays
Enzyme Inhibitors
Cell death
Cell Extracts
Tumors
Colon
Cell Death
Cells
Personnel
Carcinoma
thymidine 5'-triphosphate
deoxyuridine triphosphate
raltitrexed
Neoplasms

Keywords

  • Antifolate
  • Colon cancer
  • Deoxynucleotides
  • DNA polymerase
  • Thymidylate synthase

ASJC Scopus subject areas

  • Pharmacology

Cite this

Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate Synthase-Inhibited cells using a modified DNA polymerase assay. / Horowitz, Robert W.; Zhang, Hong yang; Schwartz, Edward L.; Ladner, Robert D.; Wadler, Scott.

In: Biochemical Pharmacology, Vol. 54, No. 5, 01.09.1997, p. 635-638.

Research output: Contribution to journalArticle

@article{e5913d9443414e00867df2f6e31df541,
title = "Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate Synthase-Inhibited cells using a modified DNA polymerase assay",
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 μM 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.",
keywords = "Antifolate, Colon cancer, Deoxynucleotides, DNA polymerase, Thymidylate synthase",
author = "Horowitz, {Robert W.} and Zhang, {Hong yang} and Schwartz, {Edward L.} and Ladner, {Robert D.} and Scott Wadler",
year = "1997",
month = "9",
day = "1",
doi = "10.1016/S0006-2952(97)00259-1",
language = "English (US)",
volume = "54",
pages = "635--638",
journal = "Biochemical Pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",
number = "5",

}

TY - JOUR

T1 - Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate Synthase-Inhibited cells using a modified DNA polymerase assay

AU - Horowitz, Robert W.

AU - Zhang, Hong yang

AU - Schwartz, Edward L.

AU - Ladner, Robert D.

AU - Wadler, Scott

PY - 1997/9/1

Y1 - 1997/9/1

N2 - 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 μM 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.

AB - 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 μM 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.

KW - Antifolate

KW - Colon cancer

KW - Deoxynucleotides

KW - DNA polymerase

KW - Thymidylate synthase

UR - http://www.scopus.com/inward/record.url?scp=0030804554&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030804554&partnerID=8YFLogxK

U2 - 10.1016/S0006-2952(97)00259-1

DO - 10.1016/S0006-2952(97)00259-1

M3 - Article

C2 - 9337081

AN - SCOPUS:0030804554

VL - 54

SP - 635

EP - 638

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 5

ER -