©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification, Cloning, and Expression of Murine Uridine Phosphorylase (*)

Shin-Ichi Watanabe , Ayako Hino , Kenji Wada (1), James F. Eliason , Takafumi Uchida (§)

From the (1) Departments of Oncology and Molecular Genetics, Nippon Roche Research Center, 200 Kajiwara, Kamakura 247, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-, interleukin 1, and interferon .


INTRODUCTION

Pyrimidine nucleoside phosphorylases (PyNPase),() 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.

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- (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) .

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-, IL-1, and IFN-.


MATERIALS AND METHODS

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.

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 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.

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 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.

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.

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.

Peptide peaks differing from the original protein peak were collected and subjected to NH-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).

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.

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.

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).

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.

The substrate specificity of murine UdRPase was examined in further detail as shown in I. The Kvalues 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.

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-, 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.

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.

  
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



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) D44464.

§
To whom correspondence and reprint requests should be addressed: Dept. of Oncology, Nippon Roche Research Center, 200 Kajiwara, Kamakura 247, Japan. Tel.: 81-467-47-2245; Fax: 81-467-45-1675.

The abbreviations used are: PyNPase, pyrimidine nucleoside phosphorylase; UdRPase, uridine phosphorylase; TdRPase, thymidine phosphorylase; 5`-dFUrd, 5`-deoxy-5-fluorouridine; 2`-dFUrd, 2`-deoxy-5-fluorouridine; 5-FUra, 5-fluorouracil; IFN-, 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.


ACKNOWLEDGEMENTS

We thank H. Terashima for excellent technical assistance and Drs. H. Ishitsuka and Y. Furuichi for helpful advice.


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