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[Cancer Research 59, 4997-5001, October 1, 1999]
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[Cancer Research 59, 4997-5001, October 1, 1999]
© 1999 American Association for Cancer Research

Molecular Biology and Genetics

Genomic Structure, Chromosomal Mapping, and Promoter Region Analysis of Murine Uridine Phosphorylase Gene1

Deliang Cao, Manjunath A. Nimmakayalu, Feiya Wang, Dekai Zhang, Robert E. Handschumacher, Patricia Bray-Ward and Giuseppe Pizzorno2

Departments of Internal Medicine (Oncology) and Pharmacology [D. C., F. W., D. Z., R. E. H., G. P.] and Genetics [M. A. N., P. B-W.], Yale University School of Medicine, New Haven, Connecticut 06520


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uridine phosphorylase (UPase) plays an important role in the activation of 5-fluorouracil and in the regulation of tissue and plasma concentration of uridine, a potential biochemical modulator of 5-fluorouracil therapy. UPase expression is affected by the c-H-rasoncogene and various cytokines through unknown mechanisms. To understand its expression and regulation, we cloned the murine UPase gene, defined its genomic organization, determined its 5'- and 3'-end flanking sequences, and evaluated the promoter activity. The UPase gene contains nine exons and eight introns, spanning a total of approximately 18.0 kb. Its promoter lacks canonical TATA and CCAAT boxes, although a CAATAAAAA TATA-like box is seen from -41 to -49. Furthermore, IFN regulatory factor 1, c/v-Myb, and p53 binding sites are present in the promoter region, indicating that UPase expression may be directly regulated by cytokines and oncogene products. The 1.2-kb flanking fragment showed promoter activity driving the expression of the luciferase gene in various mammalian cells. A TGGGG repeat sequence is seen in the 3'-end flanking region. This element is considered to be a potential recombination consensus hot spot that may contribute to the encoding of different UPase isoforms present in different tissues, both normal and neoplastic.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UPase3 (EC 2.4.2.3) reversibly catalyzes the phosphorolysis of uridine and, to a lesser extent, thymidine, with the formation of the corresponding bases. We have shown that UPase is present in most normal tissues and tumor specimens, where its activity is generally elevated compared to the adjacent normal tissue (1) . UPase has a critical role in the homeostatic regulation of the uridine concentration in plasma and tissues (2, 3, 4, 5) and affects the activation and catabolism of fluoropyrimidines influencing the therapeutic properties of 5-FU, an anticancer drug used extensively for the treatment of gastrointestinal and breast malignancies (6, 7, 8, 9) . The plasma uridine level is rigidly maintained at a concentration of 2–4 µM throughout different species (10) , and UPase exerts a regulatory function mainly through its catabolic activity in the liver (11) . A number of preclinical studies have demonstrated the capacity of this pyrimidine nucleoside to reduce 5-FU toxicity without affecting its antitumor activity, if it is properly administered 18–24 h after the cytotoxic agent (3, 4) . Clinical investigators have also demonstrated that the administration of exogenous uridine can modulate the activity of 5-FU-containing regimens by reducing the host toxicity (12) . By selectively protecting normal tissues from the toxic effects of 5-FU, uridine permits the use of a higher dose of the fluoropyrimidine. However, because of its short half-life, the administration of large doses of uridine results in moderate to severe toxicity (13) . This problem could be overcome by using inhibitors of UPase (14) , such as BAU, to conserve endogenous uridine, with a consequent elevation of its concentration in plasma and tissues. In animal models, this approach has also resulted in the reduction of host toxicity while maintaining the antineoplastic effect of 5-FU (3) . A Phase I clinical trial of oral BAU administered as a single agent to cancer patients has shown the ability of this inhibitor to elevate the plasma uridine concentration 2–3-fold without significant host toxicity (10) . A Phase I clinical trial that combines escalating doses of 5-FU followed by BAU administration is currently in progress.

Despite the critical role of UPase in the homeostatic regulation of uridine concentration in plasma and in the activation of 5-FU, little is known about the structure and expression regulation of the UPase gene. It has recently been reported that UPase expression could be induced by c-H-ras in transformed NIH 3T3 cells (15) and by various cytokines such as IFN-{alpha} and -{gamma}, tumor necrosis factor {alpha}, and interleukin 1{alpha} (16, 17, 18) . Combination therapy of cytokines such as IFN- {alpha} with 5-FU or its derivatives such as 5-fluorodeoxyuridine has resulted in a distinctly improved response rate compared to treatment with single agents (19) . However, the mechanism remains to be clearly elucidated (20) .

Our study reports the genomic organization and chromosomal localization of the murine UPase gene. We have also sequenced its 5'-flanking region and determined that the 1.2-kb flanking fragment can promote the expression of the luciferase reporter gene. This information will facilitate the complete elucidation of the biological function of UPase, the mechanisms of induction by cytokines, which may contribute to the improved therapeutic activity of 5-FU in tumors, and genetic experiments in mice.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
BAC Clone.
To isolate the murine UPase gene, we used a UPase cDNA fragment synthesized using primers based on the murine UPase cDNA sequence reported previously (17) . Sense primer P1 was located at the ATG codon region, and three antisense primers, P2, P3, and P4, were located at 860, 920, and 980 bp downstream, respectively. The first-strand murine UPase cDNA was obtained from the total RNA of murine liver using reverse transcription-PCR. The murine UPase cDNA fragment was amplified from the first-strand cDNA and purified using Qiagen columns (Qiagen, Santa Clarita, CA). To confirm the identity with the reported cDNA (17) , the entire sequence of the cDNA fragment was determined by the Protein and Nucleic Acid Chemistry Facility of the Yale Cancer Center. The murine UPase gene was then isolated using the confirmed murine UPase cDNA fragment from a murine ES-129/SvJ BAC library (Genome Systems, St. Louis, MO).

Restriction Mapping.
To identify the insert size of this BAC clone and to determine the presence of the whole murine UPase gene, a restriction mapping analysis was conducted. Briefly, 3-µg aliquots of BAC DNA were digested by 20 commonly used restriction enzymes, including ApaI, AvaI, BamHI, BglI, BglII, EcoRI, EcoRV, KpnI, NheI, NotI, PstI, SacI, SacII, SalI, ScaI, SmaI, SpeI, StyI, XbaI, and XhoI, separated on a 0.8% agarose gel, and blotted onto Nylon membranes (Amersham Life Science, Arlington Heights, IL). The oligonucleotides located at different positions of the murine UPase cDNA were end-labeled in a 20-µl mixture containing 10 pmol of oligonucleotides, 20 µCi of [{gamma}-32P]ATP (Amersham Life Science), 5 units of T4 DNA polynucleotide kinase (New England Biolabs, Beverly, MA), and reaction buffer. The hybridizations were conducted at 42°C using standard procedures, and the membranes were exposed to X-ray film (Amersham Life Science).

Determination of Mouse UPase Gene Exon-Intron Organization.
Using the oligonucleotides mentioned above (P1–P4), the sequences surrounding the exon-intron junction sites were determined by the Protein and Nucleic Acid Chemistry Facility of the Yale Cancer Center. The purified BAC DNA was used directly as a template. The obtained genomic DNA sequences were compared with the UPase cDNA sequence to determine the exon-intron junction sites. New sense and antisense primers in consecutive exons were synthesized based on the cDNA sequence to determine additional boundary sites until all of the junction sites were determined.

Exon-to-Exon PCR.
To determine the sizes of the introns, amplification of intronic DNA located between two consecutive exons of the murine UPase gene was conducted in a volume of 25 µl containing approximately 50 ng of BAC DNA and 10 pmol each of sense and antisense primers with Ready-To-Go PCR beads (Pharmacia Biotech, Piscataway, NJ). The PCR mixtures were then denatured at 94°C for 5 min, and 35 cycles were run at 94°C for 30 s, at 58°C for 30 s, and at 72°C for 3 min, followed by additional 7 min at 72°C in last cycle. It was not possible to amplify intron 2 by this protocol because of its large size (~6.0 kb). Thus, a TaqExtender PCR additive was used according to the manufacturer’s recommendation (Stratagene, La Jolla, CA). The amplified introns were separated on a 0.8% agarose gel. The sizes of the bands were determined using the EaglEye II gel documentation system (Stratagene).

Cloning of the 5'-Flanking Region.
According to the restriction mapping information of the BAC clone, BAC DNA was initially digested by NcoI. After filling in with Klenow DNA polymerase in the presence of 33 µM deoxynucleotide triphosphate mixture (New England Biolabs), the BAC DNA was further digested by XbaI. A 1.2-kb fragment was obtained, identified by an oligonucleotide located at the 5'-end of the murine UPase cDNA (Fig. 3)Citation , and inserted into the SmaI and XbaI sites of pBluescript KS II cloning vector (Stratagene). This construct was named pDCUP/Blue.



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  		Fig. 3. DNA sequence of the 5'-flanking region of the murine UPase gene. The potential promoter elements or transcription factor-binding motifs are underlined, and the corresponding elements are indicated. MUP1 (dashed arrow) indicates the primer used to detect the 5'-flanking portion of UPase; the translation start codon is boxed, and the putative transcription start point is marked by the arrowhead.

 
Transient Expression Analysis of the 5'- Flanking Fragment.
To evaluate the 1.2-kb UPase promoter fragment for basal promoter activity, its DNA sequence was subcloned from the pDCUP/Blue plasmid into the KpnI/SacI sites of the pGL3 luciferase reporter gene vector (Promega, Madison, WI). Furthermore, we made two more luciferase expression constructs that contained -445 (BglII-XbaI) and -84 bp (EcoRV-XbaI) of the 5'-upstream sequence of the UPase gene. The constructs mentioned above and an internal control plasmid DNA, pRL-TK (Promega), were cotransfected into EMT6 murine breast cancer cells and the murine NIH 3T3 fibroblasts using LipofectAMINE PLUS Reagent (Life Technologies, Inc., Grand Island, NY). Twenty-four h before transfection, approximately 1 x 105 cells were seeded to 40–50% confluence in 35-mm plates. On the day of transfection, cells were washed with serum-free medium and then incubated with 1 ml of transfection mixture containing 20 µg of Lipofectin reagent, 1 µg of promoter construct DNA, and 0.05 µg of internal control vector. Three h later, the cells were placed in the presence of their own maintaining media. Forty-eight h later, the cell lysates were prepared to detect luciferase activity by the Dual Luciferase Assay System (Promega).

Chromosomal Mapping of the Murine UPase Gene.
Metaphases from murine spleenocytes were prepared according to a method described previously (21) , with minor modifications. The UPase BAC clone was labeled with digoxigenin-11-dUTP by nick translation. Hybridization conditions, posthybridization washes, and probe detection were performed as described previously (22) . The digoxigenin-labeled clone was cohybridized with biotin-labeled clone pI, a marker for mouse chromosome 11 (23) . Probes were detected with FITC-avidin and rhodamine-antidigoxigenin antibodies as described previously (22) .

The images were captured using a computer-controlled Zeiss Axioskop epifluorescence microscope coupled to a charge-coupled device camera. FITC, rhodamine, and 4',6-diamidino-2-phenylindole fluorescence signals were recorded separately as gray scale images, enhanced, pseudocolored, and merged (Adobe Photoshop; BDS Registration).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolation and Structure of the Murine UPase Gene.
Using a mouse UPase cDNA fragment amplified with reverse transcription-PCR as a probe, a positive clone was isolated from a murine ES-129/SvJ BAC library. The restriction mapping analysis indicated that this clone contained a murine genomic DNA fragment of approximately 50 kb, which included the entire murine UPase gene.

To elucidate the intron-exon boundaries, a series of oligonucleotides were designed based on the cDNA sequence of murine UPase. Our results indicate that the murine UPase gene consists of nine exons (Fig. 1)Citation ranging in length from 66–210 bp and eight introns varying in size from 240 bp to 6.0 kb, with typical donor and acceptor sites (GT-AG rule; Fig. 1Citation ). Exon 1, exon 2, and the 5' part of exon 3 do not encode amino acids; the first in-frame ATG codon is located in exon 3. Exon 8 encodes the COOH terminus of murine UPase protein and contains translation stop codon TGA and the first 70 bp of the 3'-untranslated region. A polyadenylation signal, AATAAA, is present 45 bp downstream of the TGA codon. We estimate the length of the entire murine UPase gene to be approximately 18.0 kb.



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  		Fig. 1. Intron-exon organization of the murine UPase gene. A, scaled representation of the UPase gene. {blacksquare}, exons of the gene. Numbers above the right corner of each box indicate the last nucleotide of the exon, and the numbers below the line represent the intron size (expressed in kb). B, nucleotide sequences around the intron-exon borders of the UPase gene. The gene contains nine exons and eight introns with typical GT/AG sequences at the donor and acceptor sites.

 
After the determination of the splice sites of the murine UPase gene, appropriate primers located at both the 5'- and 3'-ends of each exon were used to amplify the introns by PCR. Introns 1, 7, and 8 were sequenced in their entirety because of their relatively small sizes, and the results were consistent with those determined by PCR. Fig. 2Citation shows the PCR products of intron 1–8. The actual size of each intron is indicated in Fig. 1Citation .



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  		Fig. 2. Gel electrophoresis analysis of PCR-amplified introns of the murine UPase gene. Lane M, molecular size markers; Lanes 1–8, corresponding introns 1–8.

 
Characterization of the 5'-Flanking Region.
A 1201-bp NcoI/XbaI fragment immediately upstream of the murine UPase gene containing 84 bp of the 5'-untranslated region of cDNA was subcloned into a pBluescript KS II cloning vector. The analysis of its sequence (Fig. 3)Citation revealed a TATA-like box, CAATAAAAA, present from -41 to -49 bp upstream of the putative transcription start site (17) . Two GC boxes are located from -197 to -208 bp and from -325 to -337 bp, respectively. A putative SP1 binding site overlaps the first GC box, i.e., at -198 to ~207 bp. Other potential promoter elements include an IFN regulatory factor 1 binding site from -21 to -33 bp; a cAMP-response element from -1096 to -1108 bp; a NF-E2 site from -629 to -640 bp; a C-Rel site from -61 to -70 bp; three overlapping GATA-1 and -2 sites from -8 to -17, -154 to -163, and -211 to -220 bp; a GATA-2 site from -77 to -86 bp; two AP1 sites from -131 to -141 and -143 to -153 bp; and four AP4 sites from -206 to -215 bp, -584 to -593 bp, -831 to -840 bp, and -868 to -877 bp, respectively. More importantly, two proto-oncogenes and tumor suppressor gene product binding sites are also seen in this promoter region, including a p53 binding site from -294 to -303 bp; three C-Myb sites from -19 to -28, -404 to -413, and -807 to -816 bp, respectively; and a V-Myb from -826 to -835 bp.

Characterization of the 3'-Flanking Region.
The sequence of the 3'-untranslated region of the murine UPase gene is identical to the cDNA sequence reported previously (17) . The 218-bp 3'-flanking region (Fig. 4)Citation shows a GT-rich region (24) present 22 bp downstream of the AATAAA polyadenylation signal. Interestingly, a TGGGG tandem repeat, TGGGGG(TGGGG)4, is present 154 bp downstream of the AATAAA polyadenylation signal, which represents a putative recombination consensus sequence found in the immunoglobulin switch region (S region), in the {alpha}-globin gene cluster, in the putative arrest sites for polymerase {alpha}, and in the deletion hot spot (exon 8) of the survival motor neuron gene (25) . This consensus element may be responsible for the presence of different isoforms of UPase.4



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  		Fig. 4. The 3'-flanking nucleotide sequence of murine UPase. cDNA sequence, lowercase letters; 3'-flanking portion, uppercase letters. The putative polyadenylation signal is boxed, and the putative recombination hot spot is underlined.

 
Promoter Activity of the 5'-Flanking Fragment.
To examine the promoter activity of the 5'-flanking fragment of the murine UPase gene, the luciferase activity driven by the whole 1.2-kb region and by the shorter fragments with 5' to 3' deletions was measured from the lysates of transiently transfected EMT6 and NIH 3T3 cells. pRL-TK cDNA expressing Renilla luciferase was used as an internal control to normalize the transfection efficiency. As shown in Fig. 5Citation , the plasmid containing the whole 1.2-kb length of the 5'-flanking region promoted a relatively higher level of luciferase expression. The expression level of the luciferase gene was progressively reduced in the constructs containing the 5'-end deletion fragments.



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  		Fig. 5. Transient expression analysis of the murine UPase gene promoter. EMT6 cells were cotransfected with various 5'-flanking sequences plus 84-bp exon 1 cDNA linked to the luciferase reporter gene and pRL-TK internal control DNA. Luciferase activity was evaluated using the Promega Dual Luciferase Assay System. The lines to the left indicate fragments of various size upstream of the murine UPase gene linked to the luciferase 5'-end in expression vector pGL3. Solid bars on the right indicate the levels of luciferase activity relative to the whole length fragment (set at 100%).

 
Chromosomal Localization of the the Murine UPase Gene.
The murine UPase contained in the BAC clone was mapped by fluorescence in situ hybridization to murine chromosome 11A1-2 (Fig. 6)Citation . More than 25 metaphase spreads from mouse spleenocytes were analyzed for the localization of the probe. Signals on both homologues were observed close to the centromere in >90% of the metaphases.



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  		Fig. 6. Right, fluorescence in situ hybridization mapping of UPase to murine spleenocyte chromosomes. Green signal, UPase; red signal, Pro({alpha})1 collagen. Left, ideogram of murine chromosome 11 indicating the location of the murine UPase gene within 11A1-2.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Based on the hypothesis that 5-FU inhibition of thymidylate synthase is responsible for its antitumor activity and that host toxicity is primarily related to its incorporation into RNA (26) , uridine has been used as a biochemical modulator of 5-FU therapy in a number of clinical trials to reduce host toxicity. UPase is the key enzyme that regulates both 5-FU activation and plasma and intracellular levels of uridine. We have demonstrated in preclinical and clinical studies that UPase inhibitors such as BAU could achieve a uridine rescue similar to that seen with exogenous administration of uridine by conservation of endogenous uridine (3 , 10) . Because of the higher UPase activity observed in neoplastic tissues, a selective depletion of uridine pools will occur in tumors, resulting in the protection of normal tissues from 5-FU toxicity and in the overall improvement of the therapeutic index of the fluoropyrimidine (1) .

The sequence of UPase does not appear to have any features in common with other phosphorylases, including TPase, that share some substrate specificity. The 5'-flanking region of the murine UPase gene, which showed promoter activity in our studies, is no exception; unlike the human purine nucleoside phosphorylase promoter (27) , it does not contain a canonical CAAT box, although a TATA-like sequence, CAATAAAA, is present from -41 to -49 bp upstream of the transcription start point. The lack of both canonical TATA and CAAT consensus sequences is a feature present in a group of genes, many of which have a housekeeping function, such as N-ras and transforming growth factor {alpha} (28) . In addition, some unusual promoter elements are seen in the murine UPase promoter region including: (a) consensus motifs for GATA-1 and -2 transcription factors, which function mainly as regulatory elements in the control of cellular differentiation of hematopoietic cells (29 , 30) ; (b) an IFN regulatory factor 1-like consensus element, which is present just upstream (from -21 to -33) of the putative transcription start site of the UPase gene and represents an important transcription factor in the regulation of the IFN response system for infection, cell growth, and apoptosis (31 , 32) ; and (c) two potential proto-oncogenes, C-Myb and V-Myb (33, 34, 35) , and a tumor suppressor gene p53 product binding site (36, 37, 38) . Considering that UPase is induced in various tumor tissues, one might postulate that the expression of UPase is regulated directly by these proto-oncogenes and tumor suppressor gene products. Preliminary data indicate that the binding of wild-type p53 to one of these motifs could inhibit the expression of the UPase gene.5

Another interesting point in the 3'-flanking region of the murine UPase gene is the presence of pentameric repeat TGGGG(TGGGG)4 154 bp downstream of the poly(A) signal, which represents a putative recombination hot spot homologous to the human deletion hot spot consensus sequence present in the immunoglobulin switch region (25 , 39) and the {alpha}-globin cluster (40) , indicating the possibility of a deletional rearrangement of the UPase gene leading to the encoding of different isoforms that we have recently isolated.4

Although UPase has some overlapping substrate specificity with TPase, the gene structures show distinct differences, as exhibited in the cDNA sequences (28) . TPase is encoded by the same gene as platelet-derived endothelial cell growth factor (41) . This gene contains 10 exons, but the whole gene spans only 4.3 kb.

In summary, we have cloned and characterized the murine UPase gene and uncovered a number of interesting sequences both in the promoter region and in the 3'-flanking region that warrant further evaluation to elucidate the regulation and expression of this gene. Although it belongs to the same phosphorylase family as TPase, no similarities in cDNA or genomic organization were demonstrated between the two genes, suggesting that these two genes have different evolutionary origins. Further characterization of other phosphorylase gene family members will facilitate the understanding of the evolution of the phosphorylase genes.


   ACKNOWLEDGMENTS
 
We thank Dr. Michael Reiss and Vincent Vellucci of the Yale University School of Medicine for helpful discussions and suggestions.


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

1 Supported by National Cancer Institute Grant CA 67035 from the NIH. Back

2 To whom requests for reprints should be addressed, at Department of Internal Medicine (Oncology), Yale University School of Medicine, New Haven, CT 06520. Phone: (203) 785-4549; Fax: (203) 785-7670; E-mail Giuseppe.Pizzorno{at}yale.edu Back

3 The abbreviations used are: UPase, uridine phosphorylase; 5-FU, 5-fluorouracil; BAU, 5-benzylacyclouridine; TPase, thymidine phosphorylase. Back

4 D. Cao and G. Pizzorno, unpublished data. Back

5 D. K. Zhang and G. Pizzorno, unpublished data. Back

Received 3/25/99. Accepted 8/ 5/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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