The role of deoxyuridine triphosphate nucleotidohydrolase, uracil-DNA glycosylase, and DNA polymerase α in the metabolism of FUdR in human tumor cells

S. J. Caradonna, Y. C. Cheng

Research output: Contribution to journalArticlepeer-review

98 Scopus citations

Abstract

The lack of evidence demonstrating FdUMP residues in DNA or FdUTP in cells after exposure to FUdR could be due either to the breakdown of FdUTP by dUTP nucleotidohydrolase (dUTPase) precluding incorporation of FdUTP into DNA via DNA polymerase or to the action of uracil-DNA glycosylase which would remove fluorouracil from DNA. Evidence presented indicates that the human cell has the capacity to form FdUTP and that FdUTP, once formed, will act as a substrate for dUTPase as well as α DNA polymerase. In addition, if FdUTP escapes hydrolysis and is incorporated into DNA, uracil-DNA glycosylase is capable of removing the FU moiety from DNA, creating an apyrimidinic site which conceivably would undergo repair synthesis. Various cultured cell lines and cells obtained from patients with different forms of leukemia were analyzed for dUTPase and glycosylase activity. The specific activity for dUTPase ranged from 0.5 to 14 nmol dUMP formed/min/mg protein. For the glycosylase, the activity ranged from 0.3 to 42 nmol uracil released/min/mg protein. Cytosols prepared from cultured HeLa S3 and KB cells were used to show that FdUTP can be formed, however, only in the presence of high concentrations of dUTP (1 mM), which presumably slows down the hydrolysis of FdUTP by dUTPase. Enzymatic studies utilizing partially purified nucleoside monophosphate kinase and highly purified thymidylate kinase illustrate that both of these enzyme species have the capability of phosphorylating FdUMP to FdUDP. NDP kinase is in turn responsible for the conversion of FdUDP to FdUTP. The Km value of FdUMP for the partially purified HeLa S3 CMP kinase (species having a pI value of 4.8) is 4 mM. The Km value of FdUMP for the highly purified TMP kinase is 225 μM. FdUTP is a substrate for the purified human dUTPase (Km of 1.2 μM) which catalyzes the conversion of FdUTP to FdUMP. Purified HeLa α DNA polymerase will catalyze the incorporation of FdUTP into DNA. The Km of FdUTP for α DNA polymerase is 4.8 μM. In turn the incorporated fluorouracil will act as a substrate for uracil-DNA glycosylase, having a Km value of 1.4 μM.

Original languageEnglish (US)
Pages (from-to)513-520
Number of pages8
JournalMolecular Pharmacology
Volume18
Issue number3
StatePublished - Dec 1 1980
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

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