 |
INTRODUCTION |
Thymidylate synthase
(TS,1 EC 2.1.1.45) catalyzes
the reductive methylation of dUMP by
CH2H4PteGlu, generating dTMP and dihydrofolate (for a review, see Ref. 1). Because the enzyme is indispensable in the
de novo synthesis of dTMP, it plays an important role in DNA
replication in actively dividing cells and has been an attractive target at which anti-neoplastic agents are directed. Fluoropyrimidines (e.g. 5-fluorouracil and FdUrd) and, more recently,
anti-folates (e.g. AG337, ZD1694, BW1843U89) have been
useful in the clinical management of tumors of the breast, colon,
stomach, and head and neck (2-5). Fluoropyrimidines exert their
effects through formation of the nucleotide analog FdUMP, which
inhibits TS via formation of a covalent complex containing the analog
CH2H4PteGlu and the enzyme (1). This complex,
which is termed the inhibitory ternary complex, is quite stable and
leads to prolonged inhibition of the enzyme, depletion of dTMP pools,
and thymineless death.
A number of studies with cultured cell lines, tumor models, and
clinical specimens have shown that TS inhibitors induce enzyme levels
by about 2-4-fold (6-8). Because response to TS-directed chemotherapy
is dependent upon the enzyme concentration, such induction has been
viewed as a potential barrier to successful therapeutic outcomes. As a
result, there has been a great deal of interest in the mechanism of the
induction and in strategies to ameliorate its effects. The increases in
TS levels do not involve changes in mRNA concentrations, indicating
that induction occurs at the translational or post-translational level.
Recent studies have shown that TS binds to its mRNA in
vitro and inhibits the translational efficiency of the mRNA
(9); TS ligands disrupt this complex and restore translation (9-12,
see Ref. 13 for a review). These findings have provided the basis for
the so-called translational autoregulation model, which postulates that
ligand-mediated induction of TS occurs through destabilization of the
TS protein-mRNA complex, followed by relief of translational
repression (13).
The autoregulatory translation model is based upon an extensive body of
in vitro studies that are both elegant and persuasive. However, several predictions of the model remain untested. For example,
because the binding of ribosomes to mRNA is rate-limiting in
eukaryotic translation (14), the model predicts that the number of
ribosomes/TS mRNA molecule will increase in cells treated with TS
inhibitors. In addition, it is expected that mutant TS mRNAs that
have lost the ability to bind the enzyme (e.g. see Ref. 11)
will be constitutively translated so that enzyme levels will be
resistant to the inductive effects of inhibitors. In the present study,
we have tested these predictions in human colon tumor cell lines. Our
results do not support the autoregulatory translation model; rather, we
find that TS induction can be accounted for by changes in the stability
of the TS polypeptide. Thus, the translation model may not be a
universal explanation for ligand-mediated induction of TS.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines--
Human colon tumor cell line HCT15 was purchased
from the American Type Type Collection. A FdUrd-resistant derivative of
this line, which expresses the P303L mutant of TS, is described
elsewhere.2 Cells were
typically grown at 37 °C in RPMI 1640 medium containing 10% fetal
bovine serum (Life Technologies, Inc.) in a humidified 5%
CO2 atmosphere.
Measurement of TS Levels--
Extracts of logarithmically
growing cells were prepared by sonication and centrifugation at
100,000 × g for 1 h. The concentration of TS in
the supernatants was determined by either the FdUMP binding assay (16)
or by Western blotting (17). A monoclonal antibody against human TS,
which was provided by Dr. S. Berger, served as the probe for the
Western blot analyses. The TS-bound antibody was detected by
chemiluminescence using the ECL-PLUS kit from Amersham Pharmacia
Biotech; signals were quantitated on a Storm PhosphorImager.
Polysome Analysis--
Polysomes were isolated from cells using
the methods of Palacios et al. (18) with minor modifications
(19). Briefly, cycloheximide (90 µg/ml) was added to the culture
medium. At various times, cells were washed and homogenized at 4 °C
in 25 mM Tris-Cl, pH 7.5, containing 25 mM
NaCl, 5 mM MgCl2, 250 mM sucrose, 1 mg/ml heparin, and 90 µg/ml cycloheximide. The homogenate was
centrifuged at 10,000 rpm for 10 min, and the supernatant was layered
onto a 4.2-ml sucrose gradient (0.5-1.5 M) containing a
1-ml 2.5 M sucrose cushion. Fractions of 0.3 ml were
collected, and RNA was extracted from each fraction and analyzed by
Northern blotting. To verify that RNA sedimenting in the polysomal
region of the gradient is ribosome-bound, controls in which
Na2EDTA was added to the extraction buffer prior to
layering onto the sucrose gradient were included.
Site-directed Mutagenesis--
A GCC
AAA substitution was
introduced immediately upstream of the translational initiation codon
of TS mRNA by polymerase chain reaction mutagenesis. The template
for mutagenesis was plasmid pKB169, which contains a cDNA copy of
the TS mRNA between nucleotides 25 and 1354 (20). The mutagenic
primers corresponded to nucleotides 81-110 within the cDNA; the
forward mutagenic primer was
5'-CGCCCGCCGCAAAATGCCTGTGGCCGGCTC-3' (the mutant
nucleotides are underlined), whereas the reverse mutagenic primer was
5'-GAGCCGGCCACAGGCATTTTGCGGCGGGCG-3'. In one polymerase chain reaction, the reverse mutagenic primer and an upstream flanking primer (5'-GTAATACGACTCACTATAGGGC-3'), corresponding to the T7 promoter region of the vector, were used; in a second reaction, the
forward mutagenic primer and a downstream primer
(5'-CTGCATGCCGAATACCGACA-3'), corresponding to nucleotides 260-279 of
the cDNA, were used. Reaction mixtures contained, in a total volume
of 100 µl, 10 mM Tris-HCl, pH 8.3, 50 mM KCl,
2 mM MgCl2, 250 µM dNTPs, 5%
(v/v) formamide, and 5 units of AmpliTaq DNA polymerase. Polymerase
chain reaction was carried out for 30 cycles (94 °C for 1.5 min,
52 °C for 2.5 min, and 72 °C for 3 min) followed by a 7-min
extension at 72 °C. The DNA products from the two reactions were
combined and co-amplified under the same conditions but using the T7
and the downstream primers.
The final product was digested with HindIII and
PstI and cloned in place of the corresponding fragment in
pKB169; the insert of the resulting plasmid, pTF528, was sequenced to
verify the presence of the GCC
AAA substitution. pTF528 was digested
with HindIII and BglII, and the TS
cDNA-containing fragment was cloned in place of the corresponding
fragment in the expression construct pJZ205, which contains the
wild-type TS cDNA under control of the SV40 promoter; the final
construct was denoted pTF530.
DNA Transfection--
Plasmids pJZ205 and pTF530 were stably
transfected into a TS-deficient Chinese hamster lung cell line
(RJK88.13, Ref. 21) by calcium phosphate-DNA co-precipitation in medium
containing 20 µg/ml thymidine; 16-25 µg of plasmid DNA were added
per culture. After 48 h, the cells were placed in selective medium
lacking thymidine, and surviving colonies were pooled, passed several times through selective medium, and stored until further use. Cells
transfected with the wild-type cDNA plasmid (i.e.
pJZ205) were denoted RJK88.13/TS(wt), whereas cells transfected with
the mutant plasmid (i.e. pTF530) were denoted
RJK88.13/TS(GCC
AAA).
Determination of the Half-life of TS--
Cells were plated at
low density, and the protein synthesis inhibitor cycloheximide was
added to a concentration of 90 µg/ml. At various times after the
addition of inhibitor, cells were collected, extracts were prepared,
and TS levels were assayed by Western blotting as above.
 |
RESULTS |
Analysis of Polysomal TS mRNA--
The binding of ribosomes to
mRNA is rate-limiting in eukaryotic translation (14). As a
consequence, changes in translational efficiency are generally
associated with alterations in the number of ribosomes bound to a
particular mRNA. The translational model of TS regulation predicts
that in the presence of ligands, the number of ribosomes/TS mRNA
molecule will increase. To test this prediction, we examined the
polysome distribution profiles for TS mRNA both prior to and
following treatment with FdUrd. Human colon tumor cell line HCT15 was
treated for 24 h with 6 nM FdUrd, which corresponds to
the ID50 for this cell line.2 Under these
conditions, TS levels increase about 2-3-fold with no change in
mRNA concentrations (data not shown). Cellular extracts were
fractionated by sucrose gradient centrifugation to separate various
size classes of polysomes (Fig.
1A), and the amounts of TS-specific mRNA in the gradient fractions were assessed by
Northern blotting (Fig. 1, B and C). Polysome
profiles were virtually identical for both control and FdUrd-treated
cells. In each circumstance, nearly all of the TS mRNA sedimented
in the polysome region of the gradient with little, if any, mRNA in
nonpolysomal fractions; the polysomal TS mRNA contained
approximately 4-7 ribosomes/molecule (Fig. 1). The addition of
Na2EDTA to the extracts prior to centrifugation resulted in
the TS mRNA being exclusively nonpolysomal, indicating that the
mRNA is indeed ribosome-bound (data not shown).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 1.
Polysome profiles for TS
mRNA. Polysomes prepared from HCT15 and HCT15/200
cells were fractionated by sucrose gradient centrifugation and analyzed
for TS mRNA by Northern blotting. Panel A shows the
distribution of total RNA in the gradient as monitored by the
absorbance at 254 nm (A254). Panels B and
C show the distribution of TS mRNA in control and
FdUrd-treated HCT15 cells, respectively. Panels D and
E show the distribution of TS mRNA in control and
FdUrd-treated HCT15/200 cells, respectively. The locations of the 40 S,
60 S, 80 S, and polysomal RNAs are indicated. The numbers of
ribosomes/mRNA molecule are shown beneath the peaks in the polysome
region of the gradient. The size of TS mRNA is shown to the
left of the Northern blots. kb, kilobases.
|
|
It could be argued that the modest extent of enzyme induction
(i.e. 2-3-fold) is too small to be manifested as a
detectable change in the polysome profile. We therefore made use of a
mutant cell line in which the FdUrd-mediated induction of TS is
amplified relative to that in HCT15. Cell line HCT15/200 is a
FdUrd-resistant derivative of HCT15 that expresses high concentrations
of a mutant TS molecule containing a Pro
Leu substitution at
residue 303.2 This amino acid substitution renders the
polypeptide relatively unstable, as compared with the wild-type
enzyme.2 Treatment of HCT15/200 cells for 24 h in 200 nM FdUrd results in a 10-15-fold induction of TS with no
change in mRNA concentrations (data not shown). The polysome
profiles for TS mRNA from this cell line are presented in Fig. 1,
D and E. The high mRNA concentrations in the
gradient fractions reflect the 40-fold overproduction of mRNA in
HCT15/200 as compared with HCT15.2 The vast majority of TS
mRNA was polysomal and sedimented at a region corresponding to
about 4-7 ribosomes/mRNA. Importantly, the polysome distribution
pattern was identical in both control and FdUrd-treated cells (Fig. 1,
D and E). Thus, even with a 10-15-fold induction
of TS, there is no detectable change in the extent of ribosome binding
to TS mRNA.
Identical results were obtained with several other colon tumor cell
lines (data not shown). Thus, the absence of an effect of FdUrd on the
polysomal distribution of TS mRNA is not specific to cell line HCT15.
Effects of Mutations That Abolish TS Binding to mRNA--
RNA/protein binding experiments have shown that the translational
initiation region of TS mRNA, particularly the AUG initiation codon
and the three bases preceding it, are critical determinants of TS
binding to its mRNA (11). It has been suggested that TS recognizes
and binds a stem loop that is 30 bases in length and encompasses the
AUG codon (11). If destabilization of the TS-TS mRNA complex and
subsequent derepression of translation are central to
fluoropyrimidine-mediated enzyme induction, then the translatability of
a mRNA that is incapable of binding the enzyme should be resistant to FdUrd; enzymes encoded by such a mutant mRNA should not undergo induction in response to drugs.
To test this notion, we made use of the findings of Chu et
al. (11), who reported that a mutant oligoribonucleotide
containing AAA in place of GCC immediately upstream of the
translational initiation codon of TS mRNA failed to compete with
full-length mRNA for binding to TS; a wild-type oligoribonucleotide
fully competed (11). We introduced the GCC
AAA substitution into a full-length TS cDNA expression plasmid, and the resulting construct was stably transfected into a TS-deficient Chinese hamster lung cell
line (RJK88.13, Ref. 21), resulting in cell line
RJK88.13/TS(GCC
AAA). TS induction in response to FdUrd was measured
and compared with that in cell line RJK88.13/TS(wt), which is a stably
transfected line containing the wild-type TS cDNA. Fig.
2 shows that TS levels in cells
expressing the mutant mRNA are induced to the same extent (i.e. 2-3-fold) as cells expressing the wild-type mRNA.
Thus, the GCC
AAA mutation had no detectable effect on enzyme
inducibility, suggesting that binding of TS to the initiation region of
the mRNA is not required for the induction.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of a mutant TS mRNA
lacking the ability to bind TS. FdUrd-mediated induction of
TS was measured by Western blot analysis of stably transfected cell
lines RJK88.13/TS(wt) and RJK88.13/TS(GCC AAA), which express
wild-type TS mRNA and the GCC AAA mutant, respectively.
Lane A, control RJK88.13/TS(wt); lane B,
RJK88.13/TS(wt) treated for 24 h in 6 nM FdUrd;
lane C, control RJK88.13/TS(GCC AAA); lane D,
RJK88.13/TS(GCC AAA) treated for 24 h in 6 nM FdUrd.
The size of TS (37 kDa) is indicated. The retarded migration of ternary
complexes in FdUrd-treated cells has been observed previously
(9-11).
|
|
Stabilization of the TS Polypeptide by FdUrd--
The results
presented above do not support the autoregulatory translation model of
TS induction. This leaves enzyme stability as a possible mechanism. We
therefore measured the half-life of the TS protein in HCT15 cells both
prior to and following treatment with FdUrd. Cycloheximide was added to
cells, and the concentrations of TS protein were assayed by Western
blotting at various times thereafter. The half-life of TS was 7.3 ± 0.3 h in control cells and 25 ± 1.0 h in
FdUrd-treated cells (Fig. 3A).
This 3-4-fold stabilization of the TS polypeptide readily accounts for
the approximately 2-3-fold induction of enzymes in response to
fluoropyrimidines.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Measurement of the half-life of the TS
polypeptide. The concentration of TS was determined by Western
blotting at various times after the addition of cycloheximide to the
media of control and FdUrd-treated cells. The fraction of enzyme
remaining was plotted as a function of time after cycloheximide
addition. Results of a typical experiment, which was carried out three
times, are shown. Panel A, cell line HCT15, control ( )
and treated for 24 h with 6 nM FdUrd ( );
panel B, cell line HCT15/200, control ( ) and treated for
24 h with 200 nM FdUrd ( ).
|
|
In HCT15/200 cells, where the FdUrd-mediated induction of TS is
amplified, the half-life of the enzyme was 2.3 ± 1.7 h,
indicating that the mutant P303L polypeptide expressed in these cells
is significantly less stable than the wild-type polypeptide (Fig. 3B). Treatment with FdUrd increased the half-life to 18 ± 1.4 h (Fig. 3B), a degree of enzyme stabilization
that fully accounts for the induction.
Thus, TS induction by FdUrd appears to result from an increase in the
stability of the TS polypeptide. We find no evidence for a
translational level control mediated by TS binding to its mRNA.
 |
DISCUSSION |
A large body of in vitro studies has clearly shown that
free TS has an affinity for its own mRNA and that this affinity is decreased by TS ligands (9-13). The binding of TS to mRNA
represses the translational efficiency of the latter and inhibits
enzyme synthesis (9-13). TS inhibitors, therefore, have an impact on TS production, as well as on enzyme activity. These findings, in total,
have led to the notion that derepression of TS mRNA translation,
which occurs via disruption of the TS-TS mRNA complex, is a central
mechanism underlying ligand-mediated induction of enzyme concentrations
in cells (13). The experiments reported in the current paper are not
consistent with such a model. First, the polysomal distribution
patterns for the mRNA were virtually identical in control and
FdUrd-treated cells. If TS induction occurs via a translational
mechanism, then an increase in the number of ribosomes/mRNA would
be expected in the presence of drugs. Second, a 3-base substitution in
TS mRNA that abolishes its ability to bind the enzyme had no
observable effect on induction. Thus, interactions between TS and its
mRNA do not appear to be required for the drug-mediated increases
in cellular TS concentrations. Finally, direct biochemical measurements
showed that treatment with FdUrd stabilizes the TS molecule to an
extent that fully accounts for the induction. We conclude that
fluoropyrimidine-mediated increases in TS levels occur through an
effect on enzyme stability, with no effect on mRNA translation.
This conclusion is similar to that made by Washtien (22) many years
ago, following the analysis of human gastrointestinal cell lines.
The finding that ligands stabilize TS is entirely consistent with
current views on TS structure and function. It is well documented that
upon formation of either the catalytic or the inhibitory ternary
complex, the enzyme, particularly its C-terminal residues, undergoes a
major conformational shift that is stabilized by a hydrogen bonding
network involving the folate co-substrate (1). Indeed, this shift
alters the susceptibility of TS to proteolytic enzymes in
vitro (15). It is therefore not surprising that the conformational
change stabilizes the enzyme in vivo.
It has been suggested that increases in TS levels cause resistance to
TS-directed agents and may represent a significant barrier to
successful cancer chemotherapy (13). Distinguishing between mRNA
translation and enzyme stabilization as mechanisms for TS induction has
implications with regard to how such resistance occurs. In the
translation model, increases in the TS level are derived from a newly
synthesized enzyme, which confers resistance by enhancing the rate of
dTMP synthesis. In contrast, in the enzyme stabilization model, the
induced enzyme is bound into the inhibitory ternary complex; resistance
in this model is conferred by "titrating out" FdUMP, thereby
lowering the effective concentration of free nucleotide analog
available for inhibition of newly synthesized enzyme. Clearly, further
insights will require sorting out the relative importance of enzyme
stability and mRNA translation in the regulation of cellular TS
levels, particularly in response to TS-directed anti-metabolites.
The present results, coupled with the absence of rigorous experiments
testing the in vivo relevance of TS binding to its mRNA, raise questions concerning the general applicability of the
translational model of TS induction. Of course, it is formally possible
that cell lines other than those focused upon here may exhibit a
translational derepression mechanism. In the least, it seems reasonable
to conclude that the translation model cannot be invoked as a universal
explanation for the induction of TS by its inhibitors.