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Molecular Pharmacology, Vol 18, 513-520, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics
in the Metabolism of FUdR
in Human Tumor Cells
1 Department of Pharmacology, The University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27514, and
the Department of Experimental Therapeutics and the Grace Cancer Drug Center, Roswell Park Memorial Institute, Buffalo,
New York 14263
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 
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 DNA polymerase will catalyze the incorporation
of FdUTP into DNA. The Km of FdUTP for 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.
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