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Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA isolated from the parental and 5-fluoro-dUrd-resistant cells, proved both nucleotide sequences to be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein database access no. NP_067263). This suggests that the altered properties of TSr are caused by a factor different than protein mutation, presumably posttranslational modification. As a possibility of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsonoff et al. (1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing several potential phosphorylation sites. In order to test possible influence of the protein phosphorylation state on enzymatic properties, endogenous TSp and TSr were purified in the presence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown by electrophoretical separation followed by phosphoprotein detection, the extent of phosphorylation was apparently similar. However, the same two purified enzyme preparations, compared to the corresponding preparations purified in the absence of phosphatase inhibitors, showed certain properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus properties dependence on phosphorylation was indicated.
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Regular paper
Altered mouse leukemia L1210 thymidylate synthase, associated with
cell resistance to 5-uoro-dUrd, is not mutated but rather reects
posranslational modication
Joanna Cieśla
1
, Tomasz Frączyk
1
, Zbigniew Zieliński
1
, Jacek Sikora
2
and
Wojciech Rode
1½
1
Nencki Institute of Experimental Biology, Warszawa, and
2
Institute of Biochemistry and Biophysics, Polish
Academy of Sciences, Warszawa, Poland;
½
e-mail address: rode@nencki.gov.pl
Received: 19 December, 2005; revised: 31 January, 2006; accepted: 10 February, 2006
available on-line: 27 February, 2006
Thymidylate synthase puried from 5-uoro-dUrd-resistant mouse leukemia L1210 cells (TSr)
was less sensitive to slow-binding inhibition by 5-uoro-dUMP than the enzyme from the paren-
tal cells (TSp), both enzyme forms diering also in sensitivity to several other dUMP analogues,
apparent molecular weights of monomer and dimer, and temperature dependence of the cata-
lyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA
isolated from the parental and 5-uoro-dUrd-resistant cells, proved both nucleotide sequences to
be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein
database access no. NP_067263). This suggests that the altered properties of TSr are caused by a
factor dierent than protein mutation, presumably posranslational modication. As a possibility
of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsono et al.
(1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing
several potential phosphorylation sites. In order to test possible inuence of the protein phos-
phorylation state on enzymatic properties, endogenous TSp and TSr were puried in the pres-
ence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown
by electrophoretical separation followed by phosphoprotein detection, the extent of phosphoryla-
tion was apparently similar. However, the same two puried enzyme preparations, compared to
the corresponding preparations puried in the absence of phosphatase inhibitors, showed certain
properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus proper-
ties dependence on phosphorylation was indicated.
Keywords: thymidylate synthase, FdUrd resistance, L1210, posranslational modication, protein phosphorylation
Vol. 53 No. 1/2006, 189–198
on-line at: www.actabp.pl
Abbreviations: FdUrd, 5-uoro-dUrd; FdUMP, 5-uoro-dUMP; meTHF, N
5,10
-methylenetetrahydrofolate; PBS, phosphate-
buered saline; TS, thymidylate synthase; TSp, TS from L1210 parental cells; TSr, TS from 5-uoro-dUrd-resistant cells.
Thymidylate synthase (TS; EC 2.1.1.45) cata-
lyzes the C
(5)
methylation of 2’-deoxyuridine-5’-
monophosphate (dUMP), resulting in the synthesis
of 2’-deoxythymidine-5’-monophosphate (dTMP).
The donor of both the one-carbon group and reduc-
tive equivalents is N
5,10
-methylenetetrahydrofolate
(meTHF), transformed in the reaction to dihydro-
folate (Carreras & Santi, 1995). As the reaction is the
only source of de novo synthesis of thymidylate, the
enzyme is a prominent target for uoropyrimidines
and antifolates in cancer chemotherapy (Jackman et
al., 1985; Douglas, 1987; Danenberg et al., 1999; Na-
pier & Ledermann, 2000; Lehman, 2002).
The anticancer activity of 5-uoro-dUrd
(FdUrd) is based on intracellular conversion of
FdUrd to 5-uoro-dUMP (FdUMP), a potent, slow-
binding TS inhibitor. FdUMP forms a ternary com-
plex with the enzyme and meTHF that results in a
slowly reversible enzyme inactivation. Clinical ef-
fectiveness of antineoplastic drugs is hampered by
development of various types of drug resistance. Re-
sistance of tumors to FdUrd was found to be accom-
panied in most cases by impaired phosphorylation
of FdUrd in cells (Kessel & Wodinsky, 1970; Mulkins
& Heidelberger, 1982), enhanced phosphatase activ-
ity (Fernandes & Cranford, 1985), and increased TS
190 2006
J. Cieśla and others
mRNA and protein levels (Berger et al. 1985; Cieśla
et al., 1995; Aschele et al., 2002; Libra et al., 2004; Ma
et al., 2004), with the laer parameter dependent
also on cellular TS stability (Kitchens et al., 1999).
Several reports appeared on another mechanism of
resistance to FdUrd, an alteration of the target en-
zyme (Heidelberger et al., 1960; Jastrebo et al., 1983;
Bapat et al., 1983; Barbour et al., 1992; Hughey et al.,
1993; Kawate et al., 2002), four of those reports (Ba-
pat et al., 1983; Barbour et al., 1992; Hughey et al.,
1993; Kawate et al., 2002) documenting the role of
the enzyme’s alteration resulting from mutation(s).
The present studies were aimed at explana-
tion of the mechanism of diering properties, in-
cluding sensitivity to inactivation by FdUMP and its
analogues, of TSs from parental and FdUrd-resistant
mouse leukemia L1210 cells. Surprisingly, the results
indicate that the alteration of the enzyme expressed
by the resistant cells does not result from mutation,
but rather is due to posranslational modication(s),
with phosphorylation presumably involved.
MATERIALS AND METHODS
Reagents. Multiplexed Proteomics® Phospho-
protein Gel Stain Kit and SYPRO Ruby Protein Gel
Stain were from Molecular Probes. Methanol, glacial
acetic acid and acetonitrile were from Roth and so-
dium acetate was from Sigma.
L1210 cells. Mouse leukemia L1210 cells were
maintained, harvested, and stored as previously de-
scribed (Rode et al., 1984). An FdUrd-resistant L1210
cell line, developed as described earlier (Rode et al.,
1990), was maintained as for parental L1210 cells,
except that the cell-bearing mice were treated with
FdUrd (75 mg/kg injected ip as 0.5 ml of PBS solu-
tion) on the fourth day following transplantation.
Enzyme assay. The [5-
3
H]dUMP tritium re-
lease assay was performed as previously described
(Rode et al., 1984), all measurements being done in
triplicate. TS activity unit is dened as µmoles of re-
leased tritium per min at 37
o
C.
Thymidylate synthases. TSs from parental
and FdUrd-resistant L1210 cells were puried by
means of anity chromatography on N
10
-formyl-5,8-
dideazafolate as described earlier (Rode et al., 1979).
Both enzymes were also puried by essentially the
same method but with inhibitors of protein phos-
phatases included in buers. All steps were carried
out at 4
o
C. The cell pellets were thawed with 3 vol.
of 50 mM phosphate buer, pH 7.5, containing 0.1 M
KCl, 10 mM 2-mercaptoethanol, 50 mM NaF, 5 mM
Na-pyrophosphate, 0.2 mM EGTA, 0.2 mM EDTA
and 2 mM Na
3
VO
4
, sonicated and centrifuged for
20 min at 20 000 ×
g. Nucleic acids were precipitat-
ed from the supernatant with 2% streptomycin sul-
fate and spun down as above. The supernatant was
fractionated with ammonium sulfate as previously
described (Rode et al., 1979), the pellet precipitated
with 70% ammonium sulfate was then dissolved in
10 mM phosphate buer, pH 7.5, containing 0.1%
Triton X-100, 20 µM dUMP, 10 mM 2-mercaptoetha-
nol, 50 mM NaF, 5 mM Na-pyrophosphate, 0.2 mM
EGTA, 0.2 mM EDTA and 2 mM Na
3
VO
4
(buer A),
and passed through the anity column equilibrated
with the same buer. The column was washed with
5 mM phosphate buer, pH 7.5, containing 0.1%
Triton X-100, 20 µM dUMP, 10 mM 2-mercaptoetha-
nol, 10 mM NaF, 2 mM Na-pyrophosphate, 0.2 mM
EGTA, 0.2 mM EDTA and 2 mM Na
3
VO
4
(buer W),
and the enzyme eluted with buer W lacking dUMP
into a small DEAE-cellulose column, connected to
the anity column in series. TS was eluted from the
DEAE-cellulose column with 0.2 M phosphate buer,
pH 7.5, containing 0.1% Triton X-100, 20% sucrose,
10 mM 2-mercaptoethanol, 10 mM NaF, 2 mM Na
pyrophosphate, 0.2 mM EGTA, 0.2 mM EDTA and
2 mM Na
3
VO
4
. Pooled active fractions were diluted
20-fold with a solution of 0.1% Triton X-100, con-
taining 20 µM dUMP, 10 mM 2-mercaptoethanol, 10
mM NaF, 2 mM Na-pyrophosphate, 0.2 mM EGTA,
0.2 mM EDTA and 2 mM Na
3
VO
4
, passed through
the anity column equilibrated with buer A and
the purication-concentration procedure was repeat-
ed as described above. Active TS fractions eluted
from DEAE-cellulose were stored at –20
o
C.
Kinetic studies. Quantitative analyses of TS
interaction with substrate dUMP and its analogues
were performed as reported earlier (Dąbrowska et
al., 1996). To identify the type of inhibition involved,
the eects of an inhibitor on the dependence of the
reaction rate on either dUMP or meTHF concentra-
tion were examined and analyzed as previously de-
scribed (Dąbrowska et al., 1996). Quantitative analy-
ses of TS inhibition by dUMP analogues, leading to
time-dependent inactivation of the enzyme, were
performed as described earlier (Rode et al., 1990).
Electrophoretic analysis. TS preparations pu-
ried in the absence of phosphatase inhibitors were
analyzed by SDS/polyacrylamide gel electrophore-
sis according to Weber and Osborn (1969). Samples
were prepared as described previously (Rode et al.,
1979).
In order to test the presence of phosphate
groups on TS molecule, enzyme preparations puri-
ed in the presence of phosphatase inhibitors were
analyzed by SDS/polyacrylamide gel (12.5%) electro-
phoresis according to Laemmli (1970).
Analysis of TS phosphorylation. The assays
for the TS phosphorylation and protein detection
were performed using the Multiplexed Proteomics®
Phosphoprotein Gel Stain Kit according to the manu-
facturer’s protocol. Preparations of puried TSs from
Vol. 53 191
Altered thymidylate synthase in Fdurd-resistant cells is not mutated
parental and FdUrd-resistant L1210 cells were sepa-
rated on SDS/polyacrylamide gel (Laemmli, 1970),
that was subsequently treated with Pro-Q Diamond
Phosphoprotein Gel Stain. Phosphorylated species
were visualized on a 300 nm UV transilluminator.
The gel was subsequently stained with SYPRO Ruby
Protein Gel Stain and the protein was again visual-
ized on a 300 nm UV transilluminator. A mixture
of the PeppermintStick™ phosphoprotein molecular
mass standards, containing phosphorylated (ovalbu-
min, 45 kDa; β-casein 23.6 kDa) and non-phosphor-
ylated (β-galactosidase 116.25 kDa; bovine serum al-
bumin, 66.2 kDa; avidin 18 kDa) proteins, provided
controls (both positive and negative).
Gel ltration. The molecular mass of TS
dimer was assessed by Sephadex G-100 ltration.
Before the run samples of TSp or TSr were incubat-
ed for 15 min at 37
o
C with 1 µM [6-
3
H]FdUMP and
1 mM meTHF in 0.2 M Tris/HCl, pH 7.5, contain-
ing 0.01 M 2-mercaptoethanol, in a total volume
of 1 ml. The column was developed at 4
o
C under
conditions preventing dissociation of the ternary
complex TS-[6-
3
H]FdUMP–meTHF, using 0.05 M
Tris/HCl, pH 7.5, buer containing 0.01 M 2-mer-
captoethanol, 0.1 M KCl and 0.1 mM meTHF, and
collected fractions were monitored for radioactiv-
ity.
RT-PCR and sequencing of TS cDNA. Total
RNA was isolated from L1210 cells parental and
resistant to FdUrd with TRIZOL reagent (Gibco)
according to the manufacturer’s protocol. RT-PCR
was performed with standard methods (Sambrook
et al., 1989), using Moloney Murine Leukemia Vi-
rus Reverse Transcriptase (Promega) and oligo(dT)
primer. In Taq polymerase-catalyzed reaction the
following primers were used: forward primer I
(5-GGGAATTCATATGCTGGTGGTTGGCTCCGAG-3)
having sequence identical to the rst 21 nucle-
otides of the coding region plus EcoRI and NdeI
restriction sites (bold), and the reverse primer II
(5-AAAAAGCTTTTAAACAGCCATTTCCATTT-
TAAT-3’) complementary to the last 24 nucleotides
of the coding region plus HindIII restriction site
(bold). The resulting TS cDNAs from L1210 paren-
tal and FdUrd-resistant cells were puried from
agarose gel and sequenced directly (both strands)
with Reader DNA sequencing kit (Fermentas) rea-
gents, but according to our own protocol. Prim-
ers were labelled with [γ-
32
P]ATP by T4 polynu-
cleotide kinase. The matrix (TS cDNA, 0.1 pmol),
labelled primer (1 pmol), 10 × polymerase buer
(6 µl),
Taq polymerase (5 U) and water were
mixed in a nal volume of 15 µl and 3.5 µl aliq-
uots were added to each of four tubes (A,C,T,G)
containing 2 µl of water and 1.5 µl of respective
d/ddNTP. The samples were subjected to cycle
sequencing with 30 cycles of denaturing (94
o
C,
1 min), annealing (primer
t
m
-dependent tempera-
ture, 0.5 min) and extension (72
o
C, 0.5 min), fol-
lowed by addition of 4 µl of Stop Solution. The
samples were separated on polyacrylamide gel and
autoradiographed according to standard methods
(Sambrook et al., 1989).
The EcoRI/HindIII sites were used to clone the
TS coding region into pBluescript and the NdeI/Hin-
dIII restriction sites were used for subcloning of the
TS coding region into an expression vector (Cieśla
et al., 2002). Restriction, ligation, transformation of
Escherichia coli XL-1 cells with recombinant plasmids
and plasmid purication were performed using
standard methods (Sambrook et al., 1989). Recom-
binant plasmids with 1000 bp inserts (pBluescript/
TS) were sequenced with the Sanger’s method using
Sequenase Version 2.0 DNA Sequencing Kit (Amer-
sham).
TS mRNA level assay. mRNA was isolated
from total RNA from L1210 parental and FdUrd-
resistant cells with the use of PolyATtract mRNA
Isolation System (Promega) according to the manu-
facturer’s protocol. Various amounts of both mRNA
preparations were separated on agarose/formalde-
hyde gel, transferred to nylon membrane, hybrid-
ized with
33
P-labelled TS probe (entire coding re-
gion) and subjected to autoradiography. Then the
TS probe was stripped, the membrane rehybridized
with
33
P-labelled β-actin probe (entire coding region)
and autoradiography performed again. The densities
of TSp and TSr mRNA bands in relation to those
of actin mRNA were compared with the use of the
computer program Scion Image (Scion Corporation,
Frederick, MD, USA).
Mass spectrometry analysis. Prior to analy-
sis, bands containing TS were cut out from a 12.5%
SDS/polyacrylamide gel and subjected to standard
“in-gel digestion” procedure during which proteins
were reduced, alkylated and digested with trypsin.
The resulting peptides were eluted from the gel
with 0.1% TFA. Peptide mixture was applied to RP-
18 precolumn (LC Packings), using water containing
0.1% TFA as a mobile phase, and then transferred
to a nano-HPLC RP-18 column (LC Packings, 75 µm
i.d.) and developed with an acetonitrile gradient
(0–50% AcN in 30 min) in the presence of 0.05% for-
mic acid at a ow rate of 150 nl/min. Column out-
let was directly coupled to a Finningan Nanospray
ion source of an LTQ-FT (Thermo) mass spectrom-
eter working in the regime of data dependent MS to
MS/MS switch. A blank run, ensuring lack of cross
contamination from previous samples, preceded
each analysis. The output list of precursor and prod-
uct ions was compared to the NCBI database with
a MASCOT local server (www.matrixscience.com).
Spectra of peptides were analyzed with the use of
the MassLynx v. 3.5 program.
192 2006
J. Cieśla and others
Statistically evaluated results. These are pre-
sented as means ± S.E.M. or means ± percentage dif-
ference between the mean and either two results,
followed by the number of experiments (N) in pa-
rentheses.
RESULTS AND DISCUSSION
The FdUrd-resistant L1210 cells were devel-
oped from L1210 cells grown intraperitoneally in
mice (Rode et al., 1984). Compared to the parental
cells, the resistant ones were found to express TS less
sensitive to the slow-binding inhibition by FdUMP.
This concerned both the enzyme activity studied in
crude cell extracts (not shown) and highly puried
TS preparations (Table 1). The puried preparations
of the enzyme from the parental (TSp) and resistant
(TSr) L1210 cells showed also dierent sensitivities
to time-dependent (slow-binding) inhibition by other
dUMP analogues (Table 1; discussed in Rode et al.,
1990; Dzik et al., 1993) and classical competitive inhi-
bition (reected by the intersection at the ordinate of
the Lineweaver-Burk plot; not shown) by the same
analogues, as well as by the reaction product dTMP
(Table 2). Interestingly, while the basic kinetic prop-
erties (specic activity, molecular activity and the
K
m
values describing interaction with the substrates)
of both preparations appeared the same (Table 3; cf.
Jastrebo et al., 1983; Bapat et al., 1983), the enzyme
monomers separated with the use of SDS/polyacryl-
amide electrophoresis in a continuous buer system
(Weber & Osborn, 1969), as well as dimers analyzed
by Sephadex gel ltration, showed small but repro-
ducible dierences of apparent molecular mass. No-
tably, while the molecular mass of the monomer was
apparently lower for the resistant than parental cell
enzyme, that of the dimer was apparently lower for
Table 1. Parameters for inactivation by FdUMP, N
4
-OH-dCMP, N
4
-OH-5-FdUMP, 2-thio-FdUMP and 4-thio-FdUMP of
thymidylate synthases from L1210 cells parental (TSp) and FdUrd-resistant (TSr) cells.
The plots of log(remaining activity) vs time were usually biphasic (cf. Rode et al., 1990), suggesting dierent interactions
of each inhibitor with the two binding sites on a TS molecule. Consequently, inhibition constants and inactivation rate
constants were calculated with the use of apparent inactivation rate constants during the initial (0.0–1.5 min) and later
(4–10 min) periods of preincubation with a given inhibitor at various concentrations. The corresponding inhibition con-
stants and inactivation rate constants were then K
i
and k
2
and K
i
and k
2
”, respectively.
Enzyme K
i
(nM) K
i
(nM) k
2
(min
-1
) k
2
(min
-1
)
FdUMP
TSp
a
1.8 ± 0.4 (6) 20 ± 5 (4) 0.17 ± 0.02 (6) 0.12 ± 0.04 (5)
TSr
a
12.2 ± 1.4 (6) 14 ± 3 (4) 0.25 ± 0.04 (6) 0.06 ± 0.02 (4)
TSp* 15.3 ± 3.8 (3) 6.5 ± 2.2 (3) 0.22 ± 0.06 (3) 0.10 ± 0.04 (3)
TSr* 47.5 ± 10.7 (3)
c
0.59 ± 0.27 (3)
N
4
-OH-dCMP
TSp
a
63 ± 9 (4) 226 ± 35 (5) 0.17 ± 0.05 (4) 0.02 ± 0.00 (5)
TSr
a
184 ± 61 (6) 1460 ± 330 (5) 0.20 ± 0.04 (6) 0.09 ± 0.02 (5)
N
4
-OH-5-FdUMP
TSp
a
73 ± 13 (4) 71 ± 6 (7) 0.24 ± 0.04 (4) 0.07 ± 0.01 (7)
TSr
a
93 ± 18 (4) 56 ± 9 (7) 0.24 ± 0.03 (4) 0.06 ± 0.00 (7)
2-Thio-FdUMP
TSp
b
41 ± 9 (3) 46 ± 25 (3) 0.12 ± 0.02 (3) 0.02 ± 0.01 (3)
TSr
b
297 ± 93 (3) 93 ± 31 (3) 0.40 ± 0.04 (3) 0.04 ± 0.01 (3)
4-Thio-FdUMP
TSp
b
102 ± 36 (3) 202 ± 36 (3) 0.21 ± 0.04 (3) 0.05 ± 0.00 (3)
TSr
b
14 ± 4 (5) 34 ± 9 (5) 0.11 ± 0.03 (5) 0.03 ± 0.01 (5)
a
Rode et al. (1990);
b
Dzik et al. (1993);
c
The inactivation rate did not change during preincubation of the enzyme with inhibitor; TSp* and
TSr*, enzyme puried in the presence of protein phosphatase inhibitors. Results are presented as means ± S.E.M., followed by the number
of separate experiments in parentheses.
Vol. 53 193
Altered thymidylate synthase in Fdurd-resistant cells is not mutated
the parental than resistant cell enzyme (Table 3 and
Fig. 1), suggesting that the observed dierences may
reect dierent capacity of the monomers to bind
SDS or dierent conformation of dimers, rather than
distinctly dierent molecular mass. The two enzyme
preparations diered also in the dependences of TS
activity on temperature. Although both Arrhenius
plots, reecting those dependences in the 24–42°C
temperature range, were biphasic (Fig 2; cf. Rode et
al., 1986), the resistant cell enzyme showed a lower
activation energy, compared to the parental one, in
the temperature range 37–42°C (6.36 ± 0.14 Kcal/
mol and 8.25 ± 0.23 Kcal/mol for the resistant and
parental cell enzyme, respectively; N = 3), but not at
24–37°C (12.01 ± 0.27 Kcal/mol and 12.46 ± 0.26 Kcal/
mol with the resistant and parental cell enzyme, re-
spectively; N = 3). On the other hand, the two enzyme
preparations did not appear to dier in the mecha-
nism of substrate binding and product release, with
the sequential reaction mechanism suggested by the
kinetics of interaction with the substrates reected
by 1/v vs 1/[dUMP] or 1/[meTHF] plots intersecting
to the le of the ordinate (not shown) and ordered
substrate addition, dUMP prior to meTHF, indicated
by the successful application of anity chromatog-
raphy (cf. Rode et al., 1979).
It should be mentioned that TS mRNA levels,
estimated by Northern blot analysis (Fig. 3), as well
as TS protein levels, judged based on the enzyme
specic activity determined in cell extracts (0.30 ±
0.04 unit/g and 0.42 ± 0.09 unit/g with the parental
and resitant cells, respectively; N = 11) and molecu
-
lar activity of puried preparations (Table 3), were
similar for both parental and resistant cells.
In view of the foregoing, the altered TS pres-
ent in FdUrd-resistant L1210 cells was assumed to
be mutated (cf. Barbour et al., 1992). To verify this
hypothesis, RT-PCR was performed on total RNA
isolated from parental and FdUrd-resistant cells (see
Materials and Methods for details). Surprisingly, the
sequences of the PCR products (TS cDNAs), deter-
mined directly (without cloning), perfectly matched
the mouse TS coding sequence (Deng et al., 1986;
NCBI protein database access number NP_067263),
pointing to identical amino-acid sequences of TSp
and TSr. Interestingly, when cDNA sequencing was
done aer cloning into pBluescript vector of cDNA
originating from either parental or FdUrd-resistant
Table 2. Apparent K
i
values for inhibition of thymidylate synthase from parental (TSp) and FdUrd-resistant (TSr)
L1210 cells by FdUMP, N
4
-OH-dCMP, N
4
-OH-5-FdUMP, 2-thio-FdUMP, 4-thio-FdUMP and dTMP
a
.
Inhibitor TSp TSr
K
i
(µM) K
i
/K
m
K
i
(µM) K
i
/K
m
FdUMP 0.02 0.01 0.03 0.02
N
4
-OH-dCMP 1.70 0.68 9.30 5.47
N
4
-OH-5-FdUMP 0.80 0.32 1.40 0.82
2-Thio-FdUMP 0.07 0.03 0.14 0.08
4-Thio-FdUMP 0.57 0.23 0.44 0.26
dTMP 11.90 4.76 51.50 30.29
a
Each analogue was added to the reaction mixture simultaneously with the substrate and cofactor.
Table 3. Properties of highly puried thymidylate synthase preparations from parental (TSp) and FdUrd-resistant
(TSr) L1210 cells
Enzyme Specic activity
(unit/mg)
Apparent mono-
mer mol. wt (kDa)
Apparent dimer mol.
wt (kDa)
Molecular ac-
tivity
a
(min
–1
)
K
m
(µM)
dUMP meTHF
TSp 0.26 34.0 ± 0.0 (3) 74.2 ± 0.0% (2) 18 2.5 ± 0.3 (3) 21.2 ± 1.5 (3)
TSr 0.35 31.0 ± 0.0 (3) 84.2 ± 6.5 (3) 22 1.7 ± 0.3 (3) 27.6 ± 2.1 (3)
TSp* 4.4 ± 31% (2) 35.2±0.5(3)
b
33.4±0.2(3)
c
ND 310 2.9 (1) ND
TSr* 8.9 ± 69% (2) 35.3±0.4(3)
b
33.3±0.2(3)
c
ND 628 2.5 (1) ND
a
Based on specic activity and dimer molecular mass;
b
Slower band;
c
Faster band; TSp* and TSr*, thymidylate synthases puried from
parental and FdUrd-resistant L1210 cells in the presence of inhibitors of protein phosphatases.
194 2006
J. Cieśla and others
cells, several clones with single, double or triple mu-
tations were always found, beside those encoding
normal mouse TS sequence (not shown), suggesting
certain genetic variation within both parental and
FdUrd-resistant L1210 cell populations. However,
results of the sequencing of the PCR products, indi-
cating non-mutated mouse TS, point to low frequen-
cies of those mutations.
In view of the foregoing, the only apparent
explanation of the diering properties of the parental
and FdUrd-resistant L1210 cell TSs should assume
diering posranslational modication(s). However,
very lile is known about the relation between the
enzyme molecule modication and its properties.
Although a possibility has been demonstrated of rat
TS phosphorylation (Samsono et al., 1997), no data
show the inuence of this modication on the en-
zyme properties. The only other information in this
respect, published by the same laboratory, concerns
modication, by N-acetylation, of the NH
2
termi-
nal methionine in the rat endogenous enzyme. The
modication was speculated to be a possible cause
of a lower, by several-fold, specic activity of elec-
trophoretically homogeneous enzyme preparation,
relative to a similarly pure recombinant enzyme, of
corresponding amino-acid sequence, with a free NH
2
terminal methionine (Cieśla et al., 1995).
Although nothing else is known about the
possible inuence of post-translation modication on
TS properties, several reports should be mentioned
on considerable dierences between certain proper-
ties of dierent enzyme forms of the same specic
origin (Rode & Leś, 1996; Table 1), sometimes also
documented to have the same amino-acid sequence.
In this context, our present and previous (Rode et
al., 1986; 1987; Cieśla et al., 2002; Dąbrowska et al.,
1996; 2004) studies (performed with the use of the
same methods) showed diering catalytic ecien-
cies, and interactions with dUMP and FdUMP, of
several mouse TS preparations (the enzyme forms
from the L1210 parental and 5-FdUrd-resistant cells
and the wild-type recombinant enzyme do not dier
in amino-acid sequence; most probably the same is
true for the enzyme from mouse thymus and Ehr-
lich ascites cells) and of two Trichinella spiralis TS
preparations (the enzyme from muscle larvae also
does not dier in amino-acid sequence from the re-
combinant form). It should also be mentioned that
each of the following pairs of enzyme forms, mouse
L1210 and the corresponding recombinant wild-type
mouse enzyme, and muscle larva T. spiralis and the
corresponding recombinant TS, showed diering
interactions with monoclonal antibodies generated
against recombinant rat TS. While some of those
Figure 2. The dependence
on temperature of the veloc-
ity of the reaction catalyzed
by thymidylate synthase
from parental (le) and
FdUrd resistant (right) L1210
cells, presented as Arrhenius
plots.
Figure 1. SDS/polyacrylamide gel electrophoresis in the continuous buer system according to Weber and Osborn
(1969).
TSp (le) and TSr (right) were pretreated with [6-
3
H]FdUMP and meTHF prior electrophoresis. Arrows indicate molecu-
lar mass standards in kDa.
Vol. 53 195
Altered thymidylate synthase in Fdurd-resistant cells is not mutated
antibodies reacted with the enzyme from T. spiralis,
but not with the corresponding recombinant form,
the same antibodies reacted with the recombinant
wild-type mouse, but not with the L1210, enzyme
(Gołos et al., 2002).
In order to test the capacity of mouse TS to
undergo phosphorylation, its amino-acid sequence
was analysed with three bioinformatics tools (Net-
Phos 2.0 Server [hp://www.cbs.dtu.dk/services/Net-
Phos/], PPsearch [hp://www.ebi.ac.uk/ppsearch/]
and ScanProsite [hp://www.expasy.org/tools/scan-
prosite/]) to reveal nine potential phosphorylation
sites: S60, S96, S118, T69, T70, T161, T245, T281
and T300. NetPhos 2.0 Server and ScanProsite (but
not PPSearch) pointed also to Y147 as a potential
phosphorylation site. Although the structure of the
mouse TS protein is not known, the homology be-
tween mouse, rat and human TSs is ≥90% (Perry-
mann et al., 1986; Cieśla et al., 1995). Therefore we
used the RasMol program for visualization of the
known three-dimensional structures of rat and hu-
man TS proteins to nd out which of those potential
phosphorylation sites are present on the protein sur-
face. The results showed that ve amino-acid resi-
dues corresponding to S60, S96, S118, T161, T300 of
mouse TS are exposed, being potentially available
for protein kinases.
We also tested directly the phosphorylation
state of the two types of endogeneous TSs. In or-
der to prevent potential dephosphorylation, the en-
zyme was puried from both parental and FdUrd-
resistant L1210 cells in the presence of inhibitors
of phosphatases (see Materials and Methods), with
the resulting enzyme preparations referred to as
TSp* and TSr*, respectively. When subjected to
SDS/polyacrylamide gel electrophoresis in a discon-
tinuous buer system (see Materials and Methods),
each of the two preparations showed two closely
located bands of dierent protein content in the re-
gion of the expected TS molecular mass (Fig. 4B).
The two bands resulting from separation of each of
the two TS preparations were cut out from the gel
individually and analyzed by mass spectrometry,
the results identifying both the upper and the low-
er band of TSp* and TSr* as thymidylate synthase.
Notably, the analysis of the spectra indicated also
that the enzyme present in each of the four bands
analyzed has its amino-terminal methionine N-acet-
ylated (cf. Cieśla et al., 1995).
Analysis of TSp* and TSr*, following their
separation by polyacrylamide gel electrophoresis,
with the use of a phosphoprotein detection sys-
tem revealed each TS band to be phosphorylated
(Fig. 4A), apparently to a similar extent, as indicat
-
ed by the phosphoprotein to protein signal ratio (not
shown).
While the presence of phosphatase inhibitors
during enzyme purication did not inuence the K
m
values for dUMP with either enzyme (Table 3), the
slow-binding inhibition of TSp* and TSr* by FdUMP,
reected by time-dependent enzyme inactivation,
was weaker than that of the respective enzymes pu-
ried in the absence of the inhibitors (Table 1). In-
terestingly, compared to TSp*, TSr* was still some
three-fold less sensitive to FdUMP inactivation.
Moreover, unlike with TSp*, TSr* inactivation rate
did not change during preincubation with FdUMP,
pointing to similar interactions of both its active sites
with the inhibitor (cf. Rode et al., 1986; 1987; 1990).
Of note is that both TSp* and TSr* showed much
Figure 3. Thymidylate synthase mRNA level in parental
(L1210p, lanes 1–4) and FdUrd-resistant (L1210r; lanes
5–8) L1210 cells related to β-actin mRNA.
mRNA samples of 0.38 µg (lanes 1 and 5), 0.76 µg (lanes
2 and 6), 1.14 µg (lanes 3 and 7) or 1.52 µg (lanes 4 and
8), were separated on agarose/formaldehyde gel and sub-
jected to Northern assay. The membrane was rst probed
with
33
P-labelled TS probe and then with
33
P-labelled β-
actin probe. Four samples containing dierent amounts of
RNA were used for each kind of cells, in order to be able
to control the range of linear dependence of the signal on
RNA content.
Figure 4. SDS/polyacrylamide gel (12.5%) electrophore-
sis (Laemmli, 1970) of thymidylate synthase from paren-
tal (TSp*; A, lane 2 and B, lane 2) and FdUrd-resistant
(TSr*; A, lane 3 and B, lane 3) cells puried in the pres-
ence of inhibitors of protein phosphatases.
The gel was stained with Pro-Q® Diamond Phosphopro-
tein Gel Stain to visualize phosphorylated species (A) and
subsequently with SYPRO® Ruby Gel Stain to detect pro-
tein (B). The PeppermintStick
TM
phosphoprotein molecular
mass markers (MWM; A, lane 1 and B, lane 1) provide a
mixture of phosphorylated and non-phosphorylated pro-
teins (see Materials and Methods for details).
196 2006
J. Cieśla and others
higher molecular activities compared to TSp and TSr
(Table 3), the dierence being clearly too large to be
accounted for by the higher degree of purication of
TSp* and TSr* than TSp and TSr preparations. The
above quantitative and qualitative dierences be-
tween the enzyme preparations puried in the pres-
ence and in the absence of protein phosphatases in-
hibitors suggest that phosphorylation may inuence
TS inactivation by FdUMP, although for a precise
description of this inuence, and its dependence on
the site(s) and/or extent of modication of the en-
zyme molecule, further studies are needed.
Thus, although the present results do not
make it possible to identify precisely the cause of
the dierent properties of TSs from mouse leukemia
L1210 parental and FdUrd-resistant cells, they sug-
gest it to be connected with a posranslational mod-
ication, pointing to phosphorylation as presumably
involved.
Acknowledgements
Supported by the Ministry of Education and
Science (grant number 2 P05A 118 26).
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... [6][7][8] Several reports presented a possible mechanism of resistance to FdUrd based on alteration of the target enzyme causing less potent inhibition by FdUMP. [9][10][11][12][13][14][15] While several of those reports documented the ability of the enzyme's mutation(s), either naturally occurring 12,13 or experimentally induced, 11,14 to influence properties, in the case of parental and FdUrd-resistant mouse leukemia L1210 cells alteration of thymidylate synthase expressed by the resistant cells was found to involve posttranslational modification(s), rather than a mutation. 15 However, although phosphorylation appeared involved, such conclusions could not be documented unequivocally (ref. ...
... [9][10][11][12][13][14][15] While several of those reports documented the ability of the enzyme's mutation(s), either naturally occurring 12,13 or experimentally induced, 11,14 to influence properties, in the case of parental and FdUrd-resistant mouse leukemia L1210 cells alteration of thymidylate synthase expressed by the resistant cells was found to involve posttranslational modification(s), rather than a mutation. 15 However, although phosphorylation appeared involved, such conclusions could not be documented unequivocally (ref. 15; cf. ...
... 38 However, as presented in Table 1, the enzyme isolated from certain sources showed linear dependence of rate of inactivation by FdUMP on time, and both behaviors could be found in enzyme preparations isolated from sources of the same specific origin. Of particular interest is the group of mouse TSs (Table 1), as the coding sequences of both L1210 parental and FdUrd-resistant cell TSs proved to be identical to that of the mouse enzyme, 15 suggesting the observed differences, including those concerning inactivation parameters, result from posttranslational modification. Furthermore, the difference between inhibition profiles observed with L1210r and L1210r* enzyme preparations (Table 1) pointed to possible influence of phosphorylation. ...
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Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependences of the inactivation rate on time and accompanied by differing inhibition parameters. Considering importance for chemotherapeutic drug resistance, a possibility was tested of thymidylate synthase inhibition to be affected by post-translational modification, e.g. phosphorylation, by comparing sensitivities to inhibition by each of two slow-binding inhibitors, 5-fluoro-dUMP and N4-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparation, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered with each inhibitor studied both the pattern of the dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N5,10-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N4-hydroxy -dCMP was found strongly dependent on [Mg2+], the cations demonstrated previously to influence also activity of endogenous mouse TS isolated from tumour cells.
... Możliwość wpływu fosforylacji na właściwości ST pokazała analiza oczyszczonych niemal do homogenności preparatów enzymu, wyizolowanych w obecności inhibitorów fosfataz z komórek białaczki mysiej L1210 opornych na 5-fluorodezoksyurydynę, której ST jest hamowana słabiej, w porównaniu z enzymem z komórek macierzystych, przez 5-fluorodezoksyurydylan (5-FdUMP, aktywna forma 5-FdUrd), co jest elementem mechanizmu oporności. Różna wrażliwość w stosunku do inhibitora okazała się nie wynikać z różnicy w sekwencji aminokwasów [66], natomiast porównanie właściwości preparatów enzymu otrzymanych z każdej z dwu linii komórek L1210, oczyszczonych w obecności i pod nieobecność inhibitorów fosfataz, wskazało na uzależnienie tej wrażliwości od fosforylacji białka [66]. ...
... Możliwość wpływu fosforylacji na właściwości ST pokazała analiza oczyszczonych niemal do homogenności preparatów enzymu, wyizolowanych w obecności inhibitorów fosfataz z komórek białaczki mysiej L1210 opornych na 5-fluorodezoksyurydynę, której ST jest hamowana słabiej, w porównaniu z enzymem z komórek macierzystych, przez 5-fluorodezoksyurydylan (5-FdUMP, aktywna forma 5-FdUrd), co jest elementem mechanizmu oporności. Różna wrażliwość w stosunku do inhibitora okazała się nie wynikać z różnicy w sekwencji aminokwasów [66], natomiast porównanie właściwości preparatów enzymu otrzymanych z każdej z dwu linii komórek L1210, oczyszczonych w obecności i pod nieobecność inhibitorów fosfataz, wskazało na uzależnienie tej wrażliwości od fosforylacji białka [66]. ...
... Wyniki naszych badań sugerują jednak zależność od fosforylacji nie tylko aktywności katalitycznej, ale także wspomnianych już wyżej zdolności białka syntazy tymidylanowej do wiązania mRNA i hamowania translacji. Badania te dotyczyły enzymu rekombinowanego, wyprodukowanego w komórkach bakteryjnych i rozdzielonego na formy nieufosforylowaną i ufosforylowaną, przy czym modyfikacja tej ostatniej okazała się dotyczyć tylko reszty/reszt histydynowej/histy- [28,[30][31][32][33][34][35]48,66,[71][72][73][74][75][76]. K i (S-B) -stała hamowania powolnego wiązania (ang. ...
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Thymidylate synthase ThyA (EC 2.1.1.45; encoded by the Tyms gene), having been for 60 years a molecular target in chemotherapy, catalyses the dUMP pyrimidine ring C(5) methylation reaction, encompassing a transfer of one-carbon group (the methylene one, thus at the formaldehyde oxidation level) from 6R-N5,10-methylenetetrahydrofolate, coupled with a reduction of this group to the methyl one, with concomitant generation of 7,8-dihydrofolate and thymidylate. New facts are presented, concerning (i) molecular mechanism of the catalyzed reaction, including the substrate selectivity mechanism, (ii) mechanism of inhibition by a particular inhibitor, N4-hydroxy-dCMP, (iii) structural properties of the enzyme, (iv) cellular localization, (v) potential posttranslational modifications of the enzyme protein and their influence on the catalytic properties and (vi) non-catalytic activities of the enzyme.
... In view of such a rich repertoire of potential activities, of obvious interest is a report on possible endogenous phosphorylation of mammalian TS, demonstrated by the detection of labeled phosphoserine in the enzyme isolated from rat hepatoma cells incubated with 32 P i [6]. That TS modification could be physiologically meaningful as phosphorylation, rather than mutation, was suggested to be a potential cause of differing properties, including sensitivity to inactivation by 5-fluoro-dUMP (FdUMP) and some analogues, of TSs from parental and 5-fluoro-dUrd (FdUrd)-resistant mouse leukemia L1210 cells [7]. ...
... The endogenous enzyme proteins from parental and FdUrdresistant mouse leukemia L1210 cells [7] and calf thymus [10] were purified as previously described, but with the use of phosphatase inhibitors throughout [7]. A similar affinity chromatography approach was applied to purify C. elegans [cf. ...
... The endogenous enzyme proteins from parental and FdUrdresistant mouse leukemia L1210 cells [7] and calf thymus [10] were purified as previously described, but with the use of phosphatase inhibitors throughout [7]. A similar affinity chromatography approach was applied to purify C. elegans [cf. ...
... La phosphorylation de la TS a été initialement observée dans les cellules d'hépatome de rat H35 (Samsonoff et al., 1997) puis dans des cellules leucémiques murines L1210 (Cieśla et al., 2006). Plus ARNm hétérologues (codant la DHFR, la DCD (dCMP Désaminase), la SHMT ou encore la TK). ...
... (2015) (Frączyk et al., 2015). De manière intéressante, la phosphorylation de la TS affecte sa sensibilité à l'inactivation par le FdUMP (Cieśla et al., 2006;Ludwiczak et al., 2016). Ainsi la phosphorylation de certains résidus, dont la Ser 10 et Ser 16 , pourrait augmenter la résistance des cellules au 5-FU en empêchant l'inactivation de la TS par le FdUMP (Figures 22 et 23). ...
Thesis
Full-text available
La O-GlcNAcylation (O-N-acétylglucosaminylation) est une MPT (modification post-traductionnelle) dynamique et réversible catalysée par un unique couple d’enzymes antagonistes : l’OGT (O-GlcNAc transférase) et l’OGA (O GlcNAcase). Elle est considérée comme un véritable senseur nutritionnel et régule un grand nombre de mécanismes cellulaires fondamentaux. En ciblant des oncoprotéines et des suppresseurs de tumeur, sa dérégulation est associée à la cancérogenèse et la progression tumorale. En revanche, son rôle dans la réponse aux thérapies anti-cancéreuses est très peu étudié. Il a été néanmoins montré récemment que l’hyper-O-GlcNAcylation impacte la réponse de certains cancers à des drogues telles que le tamoxifène, le cisplatine, le bortézomib et le 5-FU (5-fluorouracile). Le 5-FU est la chimiothérapie de référence du CCR (cancer colorectal) et la TS (Thymidylate Synthase) sa cible principale. La surexpression de la TS est un biomarqueur de résistance au 5-FU utilisé en clinique. La TS a été montrée comme étant O-GlcNAcylée mais le rôle de cette MPT n’a pas été élucidé. Il nous est donc paru intéressant d’analyser le « cross-talk » entre O-GlcNAcylation et réponse au 5-FU dans le CCR dans l’hypothèse que la O-GlcNAcylation pourrait impacter la sensibilité au 5-FU en régulant sa cible TS. Un modèle murin in vivo de CCR humains et des cellules coliques non cancéreuses et cancéreuses ont été utilisés pour analyser l’effet du 5-FU sur la O-GlcNAcylation globale des protéines et réciproquement l’impact de la O-GlcNAcylation sur le niveau et l’activité de la TS, et la réponse au 5-FU. Nos données in vitro corroborent nos résultats in vivo et soutiennent que le 5-FU diminue la O-GlcNAcylation globale et que, réciproquement, la O-GlcNAcylation augmente le niveau de TS et sensibilise le CCR au 5-FU. Nous avons déchiffré le mécanisme moléculaire sous-jacent mettant en lumière le rôle de la O-GlcNAcylation dans la stabilisation de la TS et sa protection contre la dégradation protéasomale. Deux sites de O-GlcNAcylation de la TS ont été identifiés : la Thr251 à l’interface de dimérisation de l’enzyme et la Thr306 dans la séquence dégron carboxy-terminale connue pour contrôler sa dégradation. Ensemble nos résultats proposent une nouvelle stratégie thérapeutique combinant le 5-FU à un inhibiteur de l’OGA afin d’améliorer la réponse du CCR à la chimiothérapie à base de 5-FU.
... With both T. spiralis and C. elegans enzymes the non-phosphorylated fractions were used for crystallization. Separation was inspected visually using Pro-Q Diamond Phosphoprotein Gel Stain and SYPRO Ruby Protein Gel Stain applied after SDS-PAGE as described earlier [66]. ...
... In view of the foregoing conclusion of obvious interest is potential functional consequence of this mechanism, as well as possibility to find structural features enabling the inactive conformation stabilization (arginine residue in a position homologous to hTS 163) in other TS structures. Considering strong influences of posttranslational modifications on kinetics and inhibition of TS-catalyzed reaction [9,26,27] and resulting wide variability of properties of the enzyme preparations of the same specific origin [2,9,27], a simple comparison of mTS and hTS properties would not answer the former question. However, Gibson et al. [28] obtained a mutant R163K of the human enzyme and found its catalytic activity to be higher than that of the parental hTS. ...
Article
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Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2′-deoxyuridine 5′-monophosphate (dUMP) and (3) 5-fluoro-dUMP (FdUMP) and N5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (“inactive” and “active,” respectively) in the loop 181–197, in each of the three mTS structures, the loop 175–191, homologous to hTS loop 181–197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein’s inability to adopt the inactive conformation. The binary structures of either dUMP- or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS–5-FdUMP–meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41–47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.
... Other enzyme modifications that supported drug-resistant profiles appear to be those that involve post-translational modifications. Ciesla et al. (2006) studied the sensitivity to inactivation by FdUMP and its analogs, of TS from parental and FdUrd-resistant mouse leukemia L1210 cells. Their results indicate that the alteration of the enzyme expressed by the resistant cells does not result from mutation , but rather from putative post-translational modification(s), with phosphorylation presumably involved. ...
Article
Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemoresistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug resistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy.
Article
Crystal structure is presented of the binary complex between potassium phosphoramidate-phosphorylated recombinant C. elegans thymidylate synthase and dUMP. On each monomer a single phosphoserine residue (Ser127) was identified, instead of expected phosphohistidine. As 31P NMR studies of both the phosphorylated protein and of potassium phosphoramidate potential to phosphorylate different amino acids, point to histidine as the only possible site of the modification, thermodynamically favored intermolecular phosphotransfer from histidine to serine is suggested.
Article
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Thymidylate synthase (TS) may undergo phosphorylation as endogenous protein present in mammalian cells and as recombinant protein (corresponding to human, rat, mouse or Trichinella spiralis TS) expressed in bacterial cells. The phosphorylated, compared to non-phosphorylated, recombinant enzyme forms show decreased (at least by 3-fold) molecular activity, bind their cognate mRNA (tested only with the rat enzyme) and repress their own mRNA translation (tested with human, rat and mouse enzyme). Recombinant human, mouse, rat and Trichinella spiralis TSs, expressed in E. coli, undergo phosphorylation on histidine residues; so do probably L1210 and calf thymus endogenous TS proteins. Endogenous calf thymus and L1210 thymidylate synthase proteins undergo nitration in vivo. Chemical nitration of human, mouse and C. elegans recombinant TS proteins distinctly affects catalytic potency, by lowering the V max app value.
Article
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Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.
Chapter
The enzyme thymidylate synthetase (TS) which accomplishes the methylation of deoxyuridine monophosphate to thymidine monophosphate has been of interest ever since its discovery in 1957 [1]. Its crucial role in the synthesis of the only nucleotide required exclusively for DNA synthesis makes it an obvious target for antimetabolite attack. The discovery by Cohen [2] that 5-fluorodeoxyuridine monophosphate (FdUMP), a metabolite of the antipyrimidines 5-fluorouracil and 5-fluorodeoxyuridine, was a potent inhibitor of TS, coupled with the documentation of clinical antitumour activity for this drug ensured the continuing studies both of TS and antipyrimidines. The detailed and painstaking studies of the nature of the tight binding of FdUMP to TS in the presence of the cofactor 5,10-CH2FH4, to produce a stable ternary complex have given us enormous insights into the mechanism of TS catalysis and have allowed the rational design of further TS-inhibitory uracil derivatives. The inhibition of TS by FdUMP was accepted for many years as the main basis for the cytotoxicity of the fluorinated pyrimidines, and only recently have the incorporations of these molecules into nucleic acids been fully considered as alternative or contributory cytotoxic events. The knowledge that FU has cytotoxic actions unrelated to TS inhibition implies that an inhibition of TS uncomplicated by other actions has not been evaluated as an antitumour event.
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N{sup 4}-Hydroxy-dCMP (N{sup 4}-OH-dCMP), N{sup 4}-methoxy-dCMP (N{sup 4}-OMe-dCMP), and their 5-fluoro congeners were all slow-binding inhibitors of Ehrlich carcinoma thymidylate synthase (TS), competitive with respect to dUMP, and had differing kinetic constants describing interactions with the two TS binding sites. N{sup 4}-OH-dCMP was not a substrate and its inactivation of TS was methylenetetrahydrofolate-dependent, hence mechanism-based. K{sub i} values for N{sup 4}-OH-dCMP and its 5-fluoro analogue were in the range 10{sup {minus}7}-10{sup {minus}8} M, 2-3 orders of magnitude higher for the corresponding N{sup 4}-OMe analogues. The 5-methyl analogue of N{sup 4}-OHdCMP was 10{sup 4}-fold less potent, pointing to the anti rotamer of the imino form of exocyclic N{sup 4}-OH, relative to the ring N(3), as the active species. This is consistent with weaker slow-binding inhibition of the altered enzyme from 5-FdUrd-resistant, relative to parent, L1210 cells by both FdUMP and N{sup 4}-OH-dCMP, suggesting interaction of both N{sup 4}-OH and C(5)-F groups with the same region of the active center. Kinetic studies with purified enzyme from five sources, viz., Ehrlich carcinoma, L1210 parental, and 5-FdUrd-resistant cells, regenerating rat liver, and the tapeworm Hymenolepis diminuta, demonstrated that addition of a 5-fluoro substituent to N{sup 4}-OH-dCMP increased its affinity from 2- to 20-fold formore » the enzyme from different sources. With the Ehrlich and tapeworm enzymes, N{sup 4}-OH-FdCMP and FdUMP were almost equally effective inhibitors.« less
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The role of the phosphate moiety of dUMP, and some analogues, in their interaction with mammalian thymidylate synthase, has been investigated. Substrate and inhibitor activities, and the pH-dependence of these activities, of dUMP and 5-FdUMP, as well as analogues with modified phosphate groups, were compared. The methyl ester of dUMP was neither a substrate nor an inhibitor. By contrast, the methyl ester of 5-FdUMP was a slow-binding inhibitor of the enzyme from L1210, Ehrlich ascites carcinoma and CCRF-CEM cells, with Ki values in the micromolar range. Both 5-FdUrd and the newly synthesized 5'-methylphosphonate of 5-FdUrd were also slow-binding inhibitors of the Ehrlich carcinoma enzyme, but with Ki values in the millimolar range. The interaction of dUMP, 5-FdUMP, and the methyl ester of the latter decreased with increase in pH, whereas that of the 5'-methyl-phosphonate of 5-FdUrd remained unchanged. The results are discussed in relation to the role of the phosphate hydroxyls of dUMP in binding to the enzyme. 5-FdUMP and its analogues exhibited differing interactions with two binding sites on the enzyme molecule, consistent with cooperativity of binding. A convenient procedure is described for the synthesis of 5-fluoro-2'-deoxyuridine-5'-methylphosphonate, applicable also to the preparation of other 5'-methylphosphonate analogues.
Article
1.1. Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells.2.2. The enzyme is a dimer composed of 35,000 mol. wt monomers.3.3. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP.4.4. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34† C, reflecting a change of rate-limiting step in dTMP formation.5.5. MgATP at millimolar concentrations inhibits mouse thymus enzyme.
Article
Structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. In previous studies, we identified a variant structural form of thymidylate synthase (TS) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. We now report on the use of DNA transfer techniques to examine directly the effects of each TS form on drug response. TS cDNA constructs, corresponding to the normal or variant TS mRNA, were expressed in Chinese hamster lung cells or in Escherichia coli, and response to 5-fluoro-2'-deoxyuridine was determined. We observed that expression of the variant TS, which differs from the normal form by a tyrosine to histidine substitution at residue 33, confers a 4-fold level of drug resistance in the mammalian cells, as well as in bacteria. The possible role of Tyr-33 in 5-fluoropyrimidine-mediated inhibition of TS is discussed.
Article
Cytotoxicity to 5-fluoro-2'-deoxyuridine (FdUrd) derives from its conversion to 5-fluorodeoxyuridine-5'-monophosphate, which binds to and inhibits thymidylate synthase (TS) in the presence of the cofactor, 5,10-methylenetetrahydrofolate. We have selected FdUrd-resistant variants of the human cell line HEp-2 following adaptation to stepwise increases in drug concentration. In the initial selection, maximal drug resistance was associated with a 26-fold increase in the cellular level of TS. Greater TS overproduction (80-fold) was obtained by selection for FdUrd resistance in the presence of 10 microM folinic acid and 100 microM deoxyinosine. The latter agents were included to expand the folate pool to ensure adequate levels of cofactor during the selection process. Using cDNA plasmid pMTS-4, which is complementary to mouse TS mRNA, we show that TS overproduction in the HEp-2 variants is accompanied by a 100-fold increase in TS mRNA and a 100-fold amplification of the TS structural gene. Thus, TS overproduction and gene amplification is a mechanism of resistance to FdUrd in human cells.
Article
Resistance of human CCRF-CEM leukemic cells in tissue culture to 5-fluoro-2'-deoxyuridine (FdUrd) has been examined following a single drug exposure (FS sublines). In two FS sublines generated by soft agar cloning of FdUrd sensitive cells in the presence of 10 nM FdUrd, the level of drug resistance was maintained at 22- to 30-fold following 1 month growth in the absence of FdUrd. Characteristic of the FS sublines was a decreased accumulation and retention of free intracellular 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) averaging 3% of FdUrd sensitive cells, a more rapid rate of disappearance of free FdUMP and FdUMP-bound thymidylate synthase (EC 2.1.1.45, 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase), and enhanced alkaline and acid phosphatase activities. There was no significant difference in the number of nucleoside transport sites per cell among the FS sublines and FdUrd-sensitive cells, indicating that the decreased accumulation of FdUMP in the resistant sublines was not the result of impaired FdUrd transport across the plasma membrane. The more rapid turnover of FdUMP-bound TMP synthase observed in the FS sublines was neither accompanied by a decreased stability of the TMP synthase-FdUMP-5,10-methylenetetrahydrofolate ternary complex, nor an enhanced rate of degradation of FdUrd to the less potent agent, 5-fluorouracil. In addition, the growth rates of the two FS sublines were similar to that of FdUrd sensitive cells in medium containing hypoxanthine, methotrexate, and thymidine, indicating that there was no depletion of thymidine kinase (EC 2.7.1.21, ATP : thymidine-5'-phosphotransferase) in the FS sublines. Therefore, we propose that enhanced activities of acid and alkaline phosphatases, which influence the intracellular accumulation and retention of FdUMP, are important determinants of stable FdUrd resistance in CCRF-CEM cells.