From the
Uridine phosphorylase was purified 10,300-fold from tumors of
the murine colorectal adenocarcinoma cell line, Colon-26. Degenerate
DNA probes were synthesized corresponding to partial amino acid
sequences and used to screen a Colon-26 cDNA library. A cDNA clone of
1327 base pairs that contains a 5` untranslated region, a coding region
of 933 base pairs, and a 3` nontranslated region with a polyadenylated
tail was identified. The cDNA was confirmed to be uridine phosphorylase
by 1) sequence comparison to uridine phosphorylase of Escherichia
coli, 2) substrate specificity studies with recombinant protein
expressed in COS-7 cells that demonstrated relatively high enzyme
activity with uridine as substrate compared low levels when thymidine
was used, and 3) inhibition of enzyme activity by the competitive
inhibitor 2,2`-anhydro-5-ethyluridine. Northern blot analysis using the
cDNA as a probe, demonstrated high levels of mRNA expression in
Colon-26. Expression was low in NIH3T3 cells, but high in DMBA-3 and
PH-1 cells, which are NIH3T3-derived cells that have been transformed
with mutated murine Ha-ras and viral Ha-ras,
respectively. Expression of uridine phosphorylase mRNA in these cell
lines was further enhanced by treating the cells with the inflammatory
cytokines, tumor necrosis factor-
Pyrimidine nucleoside phosphorylases (PyNPase),
Both of these two PyNPases also convert the
anti-cancer pro-drug 5`-deoxy-5-fluorouridine (5`-dFUrd) to its active
form, 5-fluorouracil (5-FUra), in tumor cells
(1, 2) .
Because PyNPase is more abundant in many tumors than in their normal
counterparts, 5`-dFUrd has better therapeutic indices than 5-FUra in
model tumor systems
(1, 3, 4) . With 5-FUra
itself, PyNPases can add ribose or deoxyribose to form nucleosides that
can be incorporated into RNA or DNA. Studies on the substrate
specificity of PyNPases in murine and human tumors demonstrated that
there is a species selective expression
(5) . In murine tumors,
UdRPase is most abundant
(1) , whereas TdRPase is the primary
enzyme in human tumors
(6) .
Recent studies have shown that
the expression of PyNPase activity is up-regulated by treatment with
various cytokines such as interferon-
To clarify the regulation of
UdRPase expression, we have purified the murine UdRPase protein from a
mouse colorectal tumor cell line, Colon-26, and have cloned the cDNA.
We have examined expression of the UdRPase gene in fibroblasts
transformed by mutated Ha-ras oncogenes as well as in cancer
cells treated with the cytokines TNF-
Aliquots of cell lysates, tumor homogenates, or column
fractions were adjusted to give a final volume of 120 µl in a
reaction mixture containing 183 mM potassium phosphate (pH
7.4) and 10 mM 5`-dFUrd. Reactions were performed at 37 °C
for 60 min and terminated by addition of 360 µl of methanol. After
removal of the precipitates by centrifugation at 3,000
All the purification steps
were performed at 4 °C. Tumors were cut into 2-4-mm pieces
with surgical scissors and then homogenized in a Teflon-glass Potter
homogenizer in 240 ml of buffer A. The homogenate was centrifuged at
160,000
Each
of the enzymatically active fractions from the final step of
purification was examined by SDS-10-20% (w/v) gradient
polyacrylamide gel electrophoresis (PAGE). The proteins were stained
with Coomassie Brilliant Blue for visualization. Four milliliters of
the final preparation were concentrated to 900 µl and dialyzed
against buffer A before using for amino acid sequence analysis.
Peptide peaks differing from
the original protein peak were collected and subjected to
NH
The PCR products were separated on
4% agarose gels, and only a 75-bp band derived from the primers for P-2
was detected. This band was eluted from the gel, digested with
EcoRI, and cloned into pUC19. Plasmid DNAs were isolated from
10 different clones and sequenced
(16) . Based on the results
from sequencing, a 57-bp oligonucleotide named UP5
(5`-AATACCTTCATAAAGTATGTGGCTGCAGAGCTGGGCCTTGACCACCCCGCAAAGAG-3`) was
synthesized as a probe for identification of UdRPase cDNA.
The kinetics of these reactions were examined using
a similar system with various concentrations of uridine (0.02-3
mM), thymidine (0.01-1 mM), or 5`-dFUrd
(0.02-3 mM) and shorter incubation times (0 to 3 min for
uridine and 5`-dFUrd, 0 to 10 min for thymidine).
The substrate specificity of murine UdRPase was examined in
further detail as shown in I. The
K
In Northern blot analysis, the cloned UdRPase cDNA was
used to examine mRNA levels in the mouse fibroblast cell lines, NIH3T3,
DMBA-3, and PH-1, as well as in the colorectal cell line Colon-26.
Expression was much higher in the mutant ras-transformed lines
DMBA-3 and PH-1 than in the parental line NIH3T3. As expected,
expression was also high in Colon-26. The mRNA levels appeared to
correspond with the activity of UdRPase in these cells as measured by
their sensitivities to 5`-dFUrd. The mechanism by which mutated
Ha-ras up-regulates UdRPase is not known. However, mutations
of Ha-ras are frequently found in human colorectal tumors, and
these tumors are frequently treated with 5-FUra and 5`-dFUrd. It will
be interesting to determine if TdRPase is also up-regulated by mutated
Ha-ras in human cells, or if UdRPase levels are increased in
human tumors expressing this oncogene.
We have demonstrated that
UdRPase expression is increased in Colon-26 cells in response to a
mixture of the cytokines TNF-
It has
been shown that TdRPase is identical with platelet-derived endothelial
cell growth factor and has angiogenic
activity
(10, 11, 12) . Recently, it has been
suggested that the angiogenic activity of TdRPase is closely related to
its enzyme activity, because it can be blocked by the competitive
inhibitor 6-amino-5-chlorouracil
(28) . Furthermore,
2-deoxy-D-ribose, a product of the catabolism of thymidine,
appears to have chemotactic activity for bovine aortic endothelial
cells and to induce angiogenesis in chorioallantoic membrane in chicken
eggs
(28) . It possible that, even though there is virtually no
homology between the two enzymes, UdRPase may also have angiogenic
activity through a similar mechanism, because both may give rise to
2-deoxy-D-ribose as a reaction product under the appropriate
circumstances.
In conclusion, we have cloned the murine UdRPase gene
and shown that its expression is regulated at the mRNA level by
Ha-ras and cytokines. It remains to be determined whether this
regulation is transcriptional or post-transcriptional, through mRNA
processing or stability. The cloning of this gene should make it easier
to determine the relative role of UdRPase and TdRPase in animal cancer
models with syngeneic transplantable tumors and human tumor xenografts.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank
We thank H. Terashima for excellent technical
assistance and Drs. H. Ishitsuka and Y. Furuichi for helpful advice.
, interleukin 1
, and
interferon
.
(
)
are key enzymes in the salvage pathway of pyrimidine
nucleoside biosynthesis. There are two kinds of PyNPases, uridine
phosphorylase (UdRPase; EC 2.4.2.3) and thymidine phosphorylase
(TdRPase; EC 2.4.2.4), which, in the presence of orthophosphate,
catalyze the reversible phosphorolysis of uridine and thymidine or
deoxyuridine, respectively, to free bases and ribose 1-phosphate or
deoxyribose 1-phosphate. These degradation products are then utilized
as carbon and energy sources or for rescue of pyrimidine bases for
nucleotide synthesis.
(IFN-
), tumor necrosis
factor-
(TNF-
), interleukin-1
(IL-1
), and
interferon-
(IFN-
) in various human tumor cells (7, 8) and in
mouse tumor cells
(9) . Because of the importance of combination
therapies with cytokines in cancer therapy, it is important to
understand the nature of this up-regulation in more detail. Thymidine
phosphorylase has been purified, cloned, and shown to be identical with
platelet-derived endothelial cell growth factor
(10, 11, 12) and gliostatin
(13) . In human cancer cells,
up-regulation of TdRPase occurs at the mRNA level
(8) . However,
information about up-regulation of UdRPase in murine cancer cells has
been only indirect using an inhibitor of enzyme activity,
2,2`-anhydro-5-ethyluridine
(9) .
, IL-1
, and IFN-
.
Cell Lines and Cell Culture
The Balb/c mouse
colorectal adenocarcinoma cell line, Colon-26, was obtained from Dr. T.
Kataoka (Japanese Foundation for Cancer Research, Tokyo, Japan). Cells
were cultured in RPMI 1640 medium containing 10% fetal calf serum and
were incubated at 37 °C in a humidified atmosphere of 5% CO in air. The normal murine fibroblast line NIH3T3 and its
transformed counterpart DMBA-3 that expresses mutated c-Ha-ras(14) were supplied by Dr. A. Wood (Hoffmann-La Roche). A second
transformed NIH3T3 line expressing v-Ha-ras, PH-1, was
obtained from Dr. T. Sekiya (National Cancer Institute, Tokyo, Japan).
COS-7 cells were purchased from ATCC (Bethesda, MD). The fibroblast
lines were cultured in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 10% fetal calf serum.
Uridine Phosphorylase Assay Using 5`-dFUrd as
Substrate
Cultured NIH3T3, DMBA-3 or PH-1 cells were harvested
with a cell scraper and sonicated in 1.5 ml of 15 mM sodium
chloride, 1.5 mM magnesium chloride, and 50 mM
potassium phosphate (pH 7.4) for 20 s on ice with an ultrasonic
homogenizer (Handy Sonic model UR-201, Tomy Seiko Co., Ltd., Tokyo,
Japan). The homogenate was centrifuged at 105,000 g for 90 min. The supernatants were dialyzed against buffer A (20
mM potassium phosphate (pH 7.4), 5 mM
2-mercaptoethanol, and 1 mM EDTA) overnight with two changes
of buffer.
g for 10 min, 100 µl of the supernatants were supplemented with
20 µM 5-chlorouracil as an internal standard and were then
applied to a high performance liquid chromatography (HPLC) column (6
200 mm; ERC-ODS-1171, ERMA CR., Inc.). The column was eluted
with 50 mM sodium phosphate buffer (pH 6.8) containing 5
mM 1-decanesulfonic acid:methanol (85:15, v/v) at a flow rate
of 1 ml/min. The amount of 5-FUra produced by phosphorolysis from
5`-dFUrd was measured with a UV detector (280 nm). Protein
concentration was determined by the method of Lowry et
al.(15) .
Purification of Murine UdRPase
Subcutaneous tumors
were produced by inoculating 1 10
Colon-26 cells
into each of 100 CDF
mice (SLC, Shizuoka, Japan). The
animals were sacrificed after 20 days, and tumors were removed. A total
of 81.5 g of tumor tissue was obtained.
g for 60 min, and protein in the supernatant
was precipitated by addition of solid
(NH
)
SO
. The precipitate obtained
with (NH
)
SO
concentrations between
30% and 60% (w/v) saturation was suspended in 40 ml of buffer A and
dialyzed against two changes of buffer. After dialysis, the remaining
precipitate was removed by centrifugation at 20,000
g for 60 min, and the supernatant was applied to a DEAE-Toyopearl
(Tosoh, Tokyo, Japan) column (18
110 mm). This column was
eluted with a linear gradient of KCl (0-150 mM) at a
flow rate of 3 ml/min. Fractions having UdRPase activity were collected
(90 ml), dialyzed against buffer A, and concentrated to 10 ml using an
Amicon stirred cell system. After centrifugation at 20,000
g for 20 min to remove any undissolved precipitate, the
solution was then applied to a column (7.5
600 mm) of
TSK-G3000SW (Tosoh) that had been equilibrated with buffer A. This
column was eluted with buffer A at a flow rate of 0.5 ml/min. Fractions
containing UdRPase were collected (5 ml) and then applied to a
TSK-DEAE-5PW (Tosoh) column (6
70 mm) equilibrated with buffer
A. UdRPase was eluted with 45 ml of a linear concentration of KCl
gradient (0-150 mM) at a flow rate of 1 ml/min.
Amino Acid Sequence Analysis
Approximately 50
µg of purified UdRPase were precipitated by adding 10% (w/v)
trichloroacetic acid (final concentration), and the precipitate was
incubated at 37 °C overnight with 2 µg of trypsin (Sigma) in 40
µl of 200 mM Tris-HCl buffer (pH 8.0). The resultant
peptides were purified by HPLC on a CAPCELL-PAK-SG300 (Shiseido, Tokyo,
Japan) column (4.6 250 mm) eluted with 50 ml of a linear
gradient of acetonitrile (0-80%, v/v) in 0.1% (v/v)
trifluoroacetic acid at a flow rate of 1 ml/min. A second batch of 50
µg of UdRPase was precipitated as described above. The precipitate
was incubated for 14 h at room temperature with 70% (v/v) formic acid
containing 1% (w/v) CNBr and evaporated to dryness. The dried protein
was dissolved in 200 µl of 0.1% (v/v) trifluoroacetic acid and
separated by HPLC as described above.
-terminal sequence analysis on a protein sequencer (model
470A, ABI, Foster City, CA). The amino acid sequences determined from
trypsin and CNBr-digested peptides were discrete sequences comprising
16 amino acids (P-1: LQGDQINTPHDVLVEY) and 19 amino acids (P-2:
NTFIKYVAAELGLDHPGKE), respectively.
PCR Amplification of Probe DNA
Four degenerate
primers were synthesized based on the amino acid sequences of P-1 and
P-2. These were named UP1:
5`-cgcgaattc(C/T)T(A/G/C/T)CA(A/G)GG(A/G/C/T)GA(T/C)CA(A/G)AT-3`, UP2:
5`-cgcgaattc(A/G)TA(T/C)TC(A/G/C/T)AC(A/G/C/T)A(A/G)(A/G/C/T)AC(A/G)T-3`,
UP3: 5`-cgcgaattcAA(T/C)AC(A/G/C/T)TT(T/C)AT(T/C/A)AA(A/G)TA-3`, and
UP4: 5`-cgcgaattc(T/C)TC(T/C)TT(A/G/C/T)CC(A/G/C/T)GG(A/G)TG(A/G)T-3`.
In this representation, extraneous nucleotides are given in lowercase
with the synthetic EcoRI site underlined. The primers UP1 and
UP2 correspond to the N and C termini of P-1; UP3 and UP4 correspond to
those of P-2. PCR was performed for 30 cycles at 94 °C (40 s), 52
°C (1 min), and 72 °C (1 min) using 100 ng of single strand
cDNA as template. The cDNA was synthesized from Colon-26 mRNA by
Moloney murine leukemia virus reverse transcriptase (Clontech) primed
with 12 nucleotides of oligo(dT).
Library Construction and Cloning
Total RNA from
Colon-26 cells was purified by guanidinium thiocyanate extraction and
purification by cesium trifluoroacetic acid centrifugation
(17) .
Poly(A) RNA was isolated using Oligotex-dT30 (Takara,
Osaka, Japan) and used to prepare cDNA according to the Stratagene cDNA
kit protocol (Stratagene). The cDNA was then inserted into the
Zap
XR vector and packaged with Gigapack Gold extracts (Stratagene). The
primary library had approximately 1
10
independent
clones with a frequency of nonrecombinant
vectors of 2%. A total
of 6
10
phage plaques were lifted onto nylon
membranes (Colony/Plaque Screen, DuPont NEN) and prehybridized for 3 h
at 42 °C in 6
SSC (1
SSC: 0.15 M NaCl,
0.015 M sodium citrate), 5
Denhardt's solution
(1
: 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% serum
albumin), 0.5% SDS, 50% (v/v) formamide, and 100 µg/ml salmon sperm
DNA. The probe was labeled by [
-
P]dCTP
using the Klenow enzyme, UP5, as template and primed by oligonucleotide
UP6 (5`-AATACCTTCATAAAGTA-3`)
(18) . Hybridization was performed
at 42 °C for 14 h. Membranes were washed twice at room temperature
with a solution containing 2
SSC and 0.1% SDS for 30 min and
twice at 60 °C for 20 min with a solution containing 0.5
SSC and 0.1% SDS. The membranes were exposed to Kodak X-Omat AR film at
-70 °C. The phagemid-harboring insert was excised in vivo according to the vendor's protocol (Stratagene) and was
sequenced. The insert obtained was 946 bp in length and contained 49 bp
of poly(A) tail.
Rapid Amplification of cDNA Ends (5`-RACE) of the UdRPase
Gene
The 5`-AmpliFINDER RACE kit (Clontech) was used for the
RACE reaction as described
(19) except that Pfu DNA polymerase
(Stratagene) was used for the final PCR reaction. Reverse transcription
was performed using 10 units of avian myeloblastosis virus reverse
transcriptase primed with UP7 (5`-TGCAAAAAATAGATTTATTGCCCAG-3`) using 2
µg of Colon-26 mRNA as template. Using the ligation product of the
anchor (5`-cacgaattcACTATCGATTCTGGAACCTTCAGAGG-NH-3`) and
UP7-primed single strand cDNA as template, PCR was performed for 30
cycles at 94 °C (1 min), 58 °C (1 min), and 75 °C (4 min)
with anchor primer (5`-ctggttcggcccaCCTCTGAAGGTTCCAGAATCGATAG-3`) and
nested primer UP8 (5`-cgcgaattcGAGTTGCAGAGGCTTCT-3`) by addition of 5
units of Pfu DNA polymerase. The reaction products from 5`-RACE were
separated on a 1.2% agarose gel, and a band of approximately 1.3
kilobases was identified.
cDNA Expression in COS-7 Cells
The 1.3-kilobase
RACE product was eluted from the agarose gel, digested with
EcoRI, and subcloned into a pSG5 vector
(20) . The
plasmid having the longest insert with sequence identical with the
insert of the cloned phage was selected. This plasmid (pUP-SG5)
was amplified on a large scale and purified by CsCl
ultracentrifugation
(17) . Two million COS-7 cells per
175-cm
bottle were cultured for 24 h before transfection
with 20 µg of pSG5 and pUP-SG5 with 20 ml of Opti-MEM (Life
Technologies, Inc.) containing 200 µl of Lipofect-AMINE reagent
(Life Technologies, Inc.). They were incubated for an additional 6 h,
then the transfection mixture was removed and 40 ml of fresh DMEM
supplemented with 10% of fetal calf serum was added. After a final 24 h
of incubation, the cells were harvested and lysed. The cDNA for human
TdRPase, cloned as described previously
(8) , was also expressed
in COS-7 cells using the same vector system (pTP-SG5).
Assay for Substrate Specificity
The substrate
specificities of PyNPases in lysates of COS-7 cells transfected with
pSG5, pUP-SG5, or pTP-SG5 were determined by assaying the enzyme
activities with four different substrates, uridine, thymidine,
5`-dFUrd, and 2`-deoxy-5-fluorouridine (2`-dFUrd). Aliquots of 120
µl of the reaction mixtures containing 183 mM potassium
phosphate buffer (pH 7.4), and 10 mM concentration of each
substrate were incubated at 37 °C for 30 min. In some tubes, 1
mM 2,2`-anhydro-5-ethyluridine, a competitive
inhibitor
(21) , was added. The reactions were terminated by
adding 360 µl of methanol. After centrifugation at 3000
g for 10 min, the phosphorolysis activity of UdRPase in the
supernatant was assayed by measuring the amount of uracil, thymine, or
5-FUra by HPLC as described above. Under these conditions, the activity
increased linearly with time of incubation and with concentration of
lysate protein.
Northern Blotting and Hybridization
Two million
NIH3T3, DMBA-3, PH-1, and Colon-26 lines were plated per flask (175
cm) and incubated for 24 h. They were then treated with
1000 units/ml of human TNF-
, 100 units/ml mouse IL-1
, and
mouse 10 units/ml IFN-
(Hoffmann-La Roche, Basel, Switzerland) for
24 h. Total RNA was prepared from treated and untreated control cells
by extraction with guanidinium thiocyanate
(22) , and 20 µg
of the RNAs were loaded on 1% agarose/formaldehyde gels for
electrophoresis. The separated RNAs were transferred onto nylon
membranes (Hybond-N; Amersham-Buchler, Braunsschweig, Germany). The
membranes were hybridized with
P-labeled insert cDNA from
pUP-SG5 using conditions as described above. The membranes were boiled
twice in 2 liters of water containing 0.1% SDS and rehybridized with a
P-labeled human
-actin DNA (Clontech) probe to
ascertain that the equal amounts of mRNA were present in all the
samples.
Cell Proliferation Assay
Cell proliferation was
assayed using a colorimetric assay for viable cell numbers essentially
as described
(23) . Cells were cultured in DMEM supplemented with
10% fetal calf serum at a concentration of 1 10
cells/ml. Aliquots of 100 µl were added to 96-well microtiter
plates. The plates were incubated for 24 h at 37 °C in a fully
humidified atmosphere of 5% CO
in air before addition of
drugs (uridine, thymidine, and 5`-dFUrd). Stock solutions of these
drugs were further diluted in DMEM just prior to addition to the
cultures in 100 µl of medium. After a total of 7 days of
incubation, 50 µl of a 3 mg/ml solution of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma)
were added. The cells were incubated for 6 h at 37 °C, after which
time 50 µl of a 25% (w/v) solution of sodium dodecyl sulfate (pH
2.0) were added. The plates were incubated at 37 °C overnight to
dissolve the formazan crystals, and then absorbance at 540 nm was
measured using a microplate reader (Bio-Rad, model 3550).
RESULTS
Purification of UdRPase
Tumors of the Colon-26
cell line were used as the source for protein purification.
Approximately 60% of the activity of the tumor homogenates was
recovered in the precipitate of 30-60% saturated ammonium sulfate
(). This material was further purified by ion exchange
chromatography, followed by gel filtration chromatography and a final
ion exchange column ( Fig. 1and summarized in ). The
material eluted from the TSK-DEAE-5PW column was analyzed by SDS-PAGE
and revealed a strong band at 35 kDa (Fig. 2). The final
purification of UdRPase was 10,300-fold with a recovery of 23.9%.
Figure 1:
Isolation of murine UdRPase.
a, Fraction II, the precipitate from the ammonium sulfate
fractionation (30 and 60% saturation), was purified by ion exchange
column chromatography on DEAE-Toyopearl. The active fractions were
pooled (Fraction III) and further purified by gel filtration column
chromatography on TSK-G3000SW (b). The active fractions from
gel filtration (Fraction IV) were pooled and purified by a second step
of ion exchange chromatography on TSK-DEAE-5PW (c). Column
eluates were assayed for UdRPase activity by 5`-dFUrd conversion to
5-FUra () and by absorbance at 280 nm (--). For ion
exchange column chromatography, elution was performed with a gradient
of KCl (- - -). The lines with double arrows indicate
the fractions pooled for the next step.
Figure 2:
SDS-PAGE analysis of fractions eluted from
the TSK-DEAE-5PW column (Fig. 1c). Electrophoresis of the
proteins in Fractions 25 (lane 1) and 26 (lane 2) was
performed on an SDS-10-20% (w/v) gradient polyacrylamide gel. The
gel was stained with Coomassie Brilliant
Blue.
Amino Acid Sequences of Peptide Fragments
The
amino terminus of UdRPase appeared to be blocked, so peptide fragments
were made by cleavage with trypsin and CNBr. The peptides were isolated
by reverse-phase HPLC. The amino acid sequences of two fragments, P1
and P2, were determined (Fig. 3).
Figure 3:
Nucleic
acid and deduced protein sequences of murine UdRPase. The deduced amino
acid sequence of mouse UdRPase is shown under the nucleotide sequence.
Peptide sequences corresponding to trypsin digestion (P-1) and CNBr
digestion (P-2) are double-underlined. The dotted lines
with arrows (UP-3 and UP-4) show the positions for degenerate
primers. The amplified DNA between UP-3 and UP-4 was used as the probe
for screening. The solid lines with arrows were UP-7, the
primer for synthesizing UdRPase cDNA, and UP-8, one of the primers for
nested PCR 5`-RACE.
Identification and Expression of UdRPase
The amino
acid sequence information of P-1 and P-2 enabled us to obtain a clone
from a Colon-26 cDNA library as described under ``Materials and
Methods.'' The complete sequence of this cDNA is shown in
Fig. 3
. It is 1327-bp in length and includes sequences that
correspond to P-1 and P-2. The deduced amino acid sequence was compared
with proteins in the Swiss Protein Data Bank. The protein with highest
homology was the UdRPase gene of Escherichia coli, whose
predicted amino acid sequence showed 23.9% identity with that of the
mouse protein. The sequences of murine and E. coli UdRPase
proteins are compared in Fig. 4a. Comparison of the
sequence murine UdRPase gene with that of human TdRPase showed no
homology except in a small 28-bp region from amino acids 92-119
having 21% sequence identity to amino acids 434-461 of TdRPase
(Fig. 4b). This small region of TdRPase has some
homology with E. coli UdRPase as well.
Figure 4:
a, sequence comparisons of murine and
E. coli UdRPase. Hyphens represent gaps introduced
for optimal alignment. Asterisks indicate identical amino
acids, and periods designate conservative substitutions in the
homologous regions. b, comparison of homologous regions of the
two UdRPases and TdRPase.
The substrate
specificity of UdRPase was examined by transfecting the cDNA into COS-7
cells and assaying the cell lysates with four different substrates,
uridine, thymidine, 5`-dFUrd, and 2`-dFUrd. For comparison, COS-7 cells
were also transfected with vector alone (pSG5) or with human TdRPase.
As summarized in , the specific activity of UdRPase in
cells transfected with UdRPase-pSG5 was 120-fold higher than in cells
transfected with vector alone when uridine was used as substrate. The
activity measured with thymidine was 112-fold higher. With 5`-dFUrd, it
was 512 times higher, and, with 2-dFUrd, it was 336-fold higher than
that of COS-7 cells transfected with vector. The specific enzyme
activity measured with uridine was 12.6-fold greater in cells
transfected with UdRPase than in cells transfected with human TdRPase,
whereas it was only one-half as active as TdRPase when thymidine was
used.
values for uridine, thymidine, and
5`-dFUrd are similar to those for UdRPase partially purified from mouse
liver, reported to be 65, 105, and 1750 µM,
respectively
(24) . Comparison of
V
/K
, or
efficiencies of catalysis, indicates that the murine UdRPase cleaves
thymidine only 10% as efficiently as it does uridine, comparing well
with 4% for the murine liver enzyme
(24) . The relative
efficiency with 5`-dFUrd was 4% with both the recombinant and native
enzymes. The enzymatic activity of UdRPase was completely inhibited by
1 mM 2,2`-anhydro-5-ethyluridine, a competitive inhibitor for
uridine
(21) , whereas thymidine cleavage was inhibited by less
than 20% (I).
Induction of UdRPase Expression by Mutated Ha-ras and
Cytokines
Northern blot analysis was used to examine the
expression of UdRPase mRNA in normal mouse fibroblasts, NIH3T3, and two
derivative cell lines, DMBA-3 with a chemically induced mutation at
codon 61 of c-Ha-ras(14) and PH-1, which is
transformed by transfection with v-Ha-ras. Message levels in
the mouse colorectal carcinoma cell line Colon-26 were also examined.
All four cell lines were treated with a mixture of cytokines,
TNF-, IL-1
, and IFN-
that has been shown to be highly
effective for inducing UdRPase activity in Colon-26 cells
(9) .
Expression of UdRPase was not detected in parental NIH3T3, even when
treated with cytokines (Fig. 5). However, it was clearly
expressed in the other three lines, DMBA-3, PH-1, and Colon-26. These
cells are also more sensitive to the toxic effects of 5`-dFUrd than are
the parental cells with IC
values that are 12- to 15-fold
lower. Both lines are about 4-fold more sensitive to 5-FUra than NIH3T3
as well (). The expression of UdRPase in these three lines
was markedly increased by treatment with the cytokine mixture
(Fig. 5).
Figure 5:
Northern blot analysis of UdRPase
expression in NIH3T3, DMBA-3, PH-1, and Colon-26. Total RNA was
prepared from control cells (-) or cells treated with the mixture
of human TNF- (1000 units/ml), mouse IL-1
(100 units/ml), and
mouse IFN-
(10 units/ml) (+). After electrophoresis, the gels
were blotted on a Nylon membrane. The RNAs were hybridized with
P-labeled mouse UdRPase cDNA. The filter was rehybridized
with
P-labeled human
-actin
cDNA.
DISCUSSION
To study the regulation of UdRPase with more precision, we
have purified the enzyme from the murine colorectal cell line Colon-26
and, based upon partial amino acid sequences, have cloned the
full-length gene. The identity of the cloned gene as UdRPase was
confirmed in several ways. First, the deduced amino acid sequence has a
low degree of homology to UdRPase from E. coli, but to no
other proteins in the protein sequence bank. Second, the recombinant
protein, expressed in COS-7 cells, had a different substrate
specificity from that of human TdRPase with higher activity for uridine
than for thymidine. The parameters determined for the enzyme kinetics
of the recombinant murine UdRPase were similar to those for the
partially purified enzyme from mouse liver
(24) . These results
indicated that uridine was cleaved about 10 times more efficiently than
was thymidine. Finally, the activity of the enzyme was completely
inhibited by a competitive inhibitor of UdRPase,
2,2`-anhydro-5-ethyluridine, when uridine or 5-dFUrd was used as
substrate, but only partially inhibited when thymidine was the
substrate.
, IL-1
, and IFN-
,
confirming earlier work that examined enzyme activity
(9) .
Increased expression was seen in the DMBA-3 and PH-1 lines as well.
Interestingly, the expression of TdRPase gene is also up-regulated in
human cancer cells by these cytokines
(8) . The increased
expression of these enzymes may explain why combination therapy with
IFN-
, which also enhances PyNPase activity in human tumor
cells
(7, 25) , and 5-FUra gives increased responses
compared to treatment with single agents (26). Treatment of colorectal
carcinoma cells with IFN-
significantly increases the activity of
5`-dFUrd in vitro (7) and in vivo(27) .
Because of the importance of combination therapies in cancer treatment,
the regulation of UdRPase and TdRPase should be examined further to
determine if induction by cytokines is direct or indirect.
Table:
Purification of UdRPase from the mouse
colorectal carcinoma cell line Colon-26
Table:
Specific activities of recombinant murine
UdRPase and human TdRPase in extracts of transfected COS-7 cells
Table:
Kinetic parameters of UdRPase from lysates of
transfected COS-7 cells with different substrates and inhibition by
2,2`-anhydro-5-ethyluridine
Table:
Effect of the Ha-ras oncogene on
expression of UdRPase in murine fibroblast cell lines and their
susceptibilities to 5-FUra and 5`-dFUrd
/EMBL Data Bank with accession number(s)
D44464.
, interferon-
; TNF-
, tumor necrosis
factor-
; IL-1
, interleukin-1
; IFN-
,
interferon-
; PAGE, polyacrylamide gel electrophoresis; HPLC, high
performance liquid chromatography; DMEM, Dulbecco's modified
Eagle's medium; PCR, polymerase chain reaction; bp, base pair(s);
RACE, rapid amplification of cDNA ends.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.