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SONDRA BERGER
Hughey CT, Barbour KW,
Berger FG, and Berger SH.
Functional effects of a naturally occurring amino acid
substitution in human thymidylate synthase. Mol Pharmacol
1993 44:316-323.
A major mechanism underlying the
cytotoxicity of fluoropyrimidine analogs such as
5-fluorouracil and 5-fluoro-2'-deoxyuridine (FdUrd) occurs
via the formation of 5-fluoro-2'-deoxyuridylate (FdUMP), a
tight- binding inhibitor of thymidylate synthase (TS).
Genetic variation in the structure of the TS molecule is an
important determinant of response to fluoropyrimidines,
because such variation may affect the binding of FdUMP to
the enzyme. Previous studies have shown that the colonic
tumor cell line HCT116 expresses two structurally distinct
TS polypeptides that differ by the presence of tyrosine or
histidine at residue 33. Compared with the Tyr-33 form, the
His-33 form confers a 3- 4-fold level of FdUrd resistance to
cells; this was postulated to be derived from the reduced
affinity of the enzyme for FdUMP and N5,N10-
methylenetetrahydrofolate, ligands required for the
formation of a stable inhibitory complex. In the present
study, the Tyr-33 and His-33 forms have been purified to
homogeneity, and their properties have been compared in
detail. The Km values for dUMP and N5,N10-
methylenetetrahydrofolate in the TS reaction were not
significantly different between the two enzymes. In
contrast, the catalytic efficiency (kcat) was 8-fold lower
for the His-33 form. Kinetic and equilibrium binding
measurements demonstrated that the dissociation constant for
FdUMP binding into the ternary complex was 3-4-fold higher
for the His-33 form; this was shown to be due to both a
decrease in the rate of FdUMP association with the enzyme
and an increase in the rate of FdUMP dissociation from the
ternary complex. A TS form containing phenylalanine at
residue 33 was created by site-directed mutagenesis and was
shown to be very similar to the Tyr-33 enzyme with regard to
kcat, pH/activity profile, and effect on FdUrd response.
Thus, it is the presence of histidine at residue 33, rather
than the absence of tyrosine, that is responsible for the
alterations in catalytic and ligand-binding functions
exhibited by the His-33 form. Possible mechanisms by which
the histidine residue perturbs the structure of the TS
active site are discussed.
Davis ST and Berger SH. Variation in
human thymidylate synthase is associated with resistance to
5-fluoro-2'-deoxyuridine. Mol Pharmacol 1993 43:702-708.
Two human colorectal tumor cell lines are
differentially sensitive to growth inhibition by
5-fluorodeoxyuridine (FdUrd); cell line RCA is less
sensitive to FdUrd than is cell line C. Thymidylate synthase
(TS), a target of FdUrd, has been purified to homogeneity
from both cell lines. Because of differences in the avidity
for a folate ligand affinity matrix, TS forms from the cells
were purified by two different procedures. Relative to the
enzyme from C cells, the enzyme from RCA cells demonstrated
higher Km values for the substrates deoxyuridylate and
5,10-methylene-tetrahydrofolate, a lower rate of association
of the inhibitor 5-fluorodeoxyuridylate (FdUMP), a similar
rate of FdUMP dissociation, and lower enhancement of
covalent FdUMP binding by folate derivatives. The activities
of the enzymes in situ and the catalytic efficiencies of the
purified enzymes were similar. Thus, a cell line that is
naturally resistant to FdUrd has been identified that
expresses a TS with reduced affinity for FdUMP and 5,10-
methylenetetrahydrofolate, relative to the enzyme expressed
in a FdUrd- sensitive cell line.
Berger SH, Barbour KW, and Berger FG. A
naturally occurring variation in thymidylate synthase
structure is associated with a reduced response to
5-fluoro-2'-deoxyuridine in a human colon tumor cell line.
Mol Pharmacol 1988 34:480-484.
Inhibition of thymidylate synthase (TS)
is an important mechanism of action of fluoropyrimidine
antimetabolites. Thus, TS structure and expression are
expected to be determinants of response to these agents. The
role of TS in fluoropyrimidine response has been analyzed in
a panel of human colonic tumor cell lines. Previous work has
demonstrated that there is little correlation between TS
concentration and sensitivity to 5-fluoro-2'-deoxyuridine (FdUrd)
among these cell lines, suggesting that parameters other
than the TS levels are responsible for the variations in
drug response. One such parameter has been identified in
cell line HCT 116. This line, which is relatively resistant
to FdUrd, produces two structural forms of TS, as determined
by mobility of the enzyme in isoelectric focusing
polyacrylamide gels. One form is common to all the cell
lines, whereas a variant form, which is more basic and is
encoded by a separate structural gene, is unique to HCT 116.
Cells expressing one or the other TS form have been isolated
and used to demonstrate that the variant form is associated
with FdUrd resistance. Kinetic experiments indicate that the
variant TS has reduced affinities for
5-fluoro-2'-deoxyuridylate and 5,10-
methylenetetrahydrofolate, which are ligands involved in
formation of a stable inhibitory complex with the enzyme.
Thus, the innate resistance of cell line HCT 116 to FdUrd is
derived, at least in part, from production of an altered
structural form of TS having reduced affinity for ligands.
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