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[Cancer Research 61, 6899-6905, September 15, 2001]
© 2001 American Association for Cancer Research

Molecular Biology and Genetics

p53-dependent Suppression of Uridine Phosphorylase Gene Expression through Direct Promoter Interaction1

Dekai Zhang, Deliang Cao, Rosalind Russell and Giuseppe Pizzorno2

Departments of Internal Medicine (Oncology) and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uridine phosphorylase (UPase) is a key enzyme in the pyrimidine salvage pathway. It reversibly catalyzes the catabolism of uridine to uracil; controls the homeostatic regulation of uridine concentration in plasma and tissues; and plays a role in the intracellular activation of 5-fluorouracil. We cloned the murine UPase gene promoter, a 1703-bp fragment, and determined the transcription initiation sites located at +1 and +92 bp of the cDNA sequence. Through transient expression analysis of the 5'-flanking region of UPase gene, we have evaluated the promoter activity for a series of fragments with 5'- to 3'-deletion in murine breast cancer EMT-6 cells and immortalized murine fibroblast NIH 3T3 cells. Cotransfection of the UPase promoter constructs (from -1619 to -445) containing p53 binding motif with the wild-type p53 construct resulted in a significant reduction of luciferase activity; however, this effect disappeared with the additional deletion of the -445 to -274 sequence to suggest the existence in this promoter region of a putative p53 recognition element. Similar cotransfection in murine embryo fibroblasts p53-/- confirmed the inhibitory role of p53 on the UPase promoter activity. The specificity of the interaction is demonstrated by nuclear protein-specific binding to the putative p53 recognition sequence using gel mobility shift assay and DNase I footprinting analysis. These data indicate the UPase gene is a novel target of p53, and its expression is down-regulated by p53 at the promoter level.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and biomembranes, has also been shown to be a crucial element in the regulation of normal physiological processes as well as pathological states. The biological effects of uridine have been associated with the regulation of the cardiocirculatory system, at the reproduction level, with both peripheral and central nervous system modulation, and with the functionality of the respiratory system (1) . The concentration of uridine in plasma and tissues is tightly regulated, and the liver has been shown to maintain uridine homeostasis by degrading "old" uridine and resynthesizing new uridine in a single pass (2 , 3) . Pharmacologically, uridine has been used to protect normal tissues from the toxic side effects of pyrimidine-based anticancer chemotherapy, mostly as a "rescue" therapy for myeloid and gastrointestinal toxicity produced by FU (4 , 5) . Uridine in combination with 5-benzylacyclouridine (an inhibitor of UPase)3 has also been shown to protect mice against the neurotoxic side effects of FU-containing drug regimens (6 , 8) . UPase is the key enzyme responsible for the reversible phosphorolysis of uridine to uracil and plays a critical role in the homeostatic regulation of uridine concentration in plasma and tissues.

We have shown recently that UPase is elevated in many solid tumors (9) , and specific mutations have been found in human breast cancer specimens but not in paired normal tissues (10 , 11) . Expression of UPase has been shown to be induced in different tumor cell lines, such as colon 26 and HCT-116, when in the presence of the following cytokines: tumor necrosis factor-{alpha}, interleukin 1{alpha}, IFN-{alpha} and -{gamma}, and vitamin D3 (12) . In the treatment of advanced colorectal carcinoma, IFN-{alpha} in combination with FU has resulted in a significant increase in response rate and patient survival when compared with FU alone (13) . In colon 26 tumor cells, a mixture of tumor necrosis factor-{alpha}, interleukin 1{alpha}, and IFN-{gamma} effectively enhanced FU and 5-fluoro-2'- deoxyuridine (5-dFUrd) cytotoxicity 2.7- and 12.4-fold respectively, because of induction of UPase expression (14) . Induction of UPase expression has also been reported in c-H-ras-transformed NIH 3T3 cells resulting in an increased sensitivity to 5'-dFUrd (15) . We have reported recently that the murine UPase gene contains 9 exons and 8 introns, spanning a total of ~18 kb (16) . We have also cloned and partially characterized the UPase promoter region that appears to contain putative regulatory elements for several oncogenic factors and tumor suppressor genes including p53 (16) . Thus, understanding the regulation of the UPase gene affecting both catalytic activity and expression has become critical to elucidate its potential role in the tumorigenesis and to modulate the selectivity of cancer treatment.

The p53 tumor suppressor gene plays a crucial role in cell growth control, DNA damage repair, and apoptosis (17) . p53 functions as a transcription factor regulating a number of target genes at the transcriptional level. Despite the progress achieved toward understanding p53 functions, the mechanisms by which p53 acts as a key regulator of cell growth and tumorigenesis have not been completely elucidated.

The isolation and functional characterization of transcriptional regulatory elements are prerequisites for understanding gene expression. In this study, we report a more in-depth characterization of the UPase promoter region, the mapping of the transcription initiation sites, and conduct the functional analysis of the murine UPase promoter in different murine cell lines. Our analysis demonstrates that wild-type p53 can regulate and repress the activity of UPase at the gene promoter level, possibly regulating the pyrimidine salvage pathway after perturbation of the ribonucleotide pools.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
NIH 3T3 fibroblast cells used in this study were originally obtained from the American Type Culture Collection. The murine breast cancer cell line EMT6 was kindly made available by Dr. Sarah Rockwell (Yale University, New Haven, CT). Early passages p53 -/- and +/+ MEF cells were generously provided by Dr. Larry Donehower (Baylor College of Medicine, Houston, TX; Ref. 18 ). Colon 26 cell cultures were established in our laboratory from in vivo growing tumors. All of the cell lines were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37°C in a 5% CO2 incubator.

Cloning and Sequencing of the 5'-Flanking Region of UPase Gene.
A genomic DNA clone that contains the immediate full-length 5'-flanking UPase sequence was obtained by screening a ES-129/SvJ bacterial artificial chromosome library with a murine UPase cDNA probe. The 1703-bp XbaI/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 (Stratagene). The complete sequence was determined with autosequencing by the Protein and Nucleic Acid Chemistry Facility of the Yale Cancer Center (Yale University).

Primer Extension Analysis.
A 33-mer antisense primer corresponding to bases +133 to +101 of the murine UPase cDNA sequence was end-labeled with T4 polynucleotide kinase using [{gamma}-32P]ATP. Total cellular RNA (15 µg) from colon 26 tumor, which presents high UPase expression, was hybridized with 105 cpm of the 32P-labeled oligonucleotide by heating at 90°C for 5 min in 20 µl of hybridization buffer [50 mM Tris-Cl (pH 8.3), 150 mM KCl, and 1 mM EDTA] followed by incubation at 42°C overnight. The DNA-RNA hybrid was then collected by ethanol precipitation and dissolved in 20 µl of reverse transcription buffer [50 mM Tris-Cl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, and 0.5 mM deoxynucleotide triphosphate]. The primer was extended by SuperScript II RNaseH- Reverse Transcriptase (Life Technologies, Inc.) at 42°C for 1 h. After completion of the reaction, the samples were extracted with phenol-chloroform, precipitated with ethanol, and analyzed on 6% denaturing polyacrylamide gel. The same primer was used in sequencing reactions with the Thermo Sequence radiolabeled cycle sequencing terminator kit (Amersham Pharmacia Biotech).

Plasmid Constructions.
To create the p-1619/+84 plasmid, the genomic clone including the 5'-flanking region of UPase gene was digested with XbaI (position -1619 and +84). The promoterless pGL3 luciferase reporter gene vector (Promega) was digested by HindIII. The single-strand ends of the released fragment and linear vector were made double-stranded using the Klenow fragment of DNA polymerase I. The fragment was then blunt-end ligated into the HindIII site of the pGL3 vector. A series of luciferase expression constructs, based on the p-1619 plasmid, that contained various lengths of the 5'-upstream sequence of the UPase gene were prepared using different restriction enzymes but maintaining the same 3' end digested by SmaI. These include p-1470 (AccI), p-1081 (BstEII), p-570 (NheI), p-445 (BglII), p-274 (BstXI), p-212 (PvuII), and p-84 (EcoRV). All of the restriction enzymes used for the plasmid construction, except for AccI, present only a single cutting point on the UPase promoter. For the fragment obtained at the AccI cutting site, we conducted a partial digestion for the plasmid construct.

Wild-type p53 plasmid construct was kindly provided by Dr. Albert B. Deisserroth (Yale University; Ref. 19 ).

Transfection and Luciferase Assays.
All of the transfections were done in triplicate in 6-well plates. Approximately 105 cells/well were seeded 24 h before transfection. Plasmids were transfected into cells using LipofectAMINE reagent (Life Technologies, Inc.). The cells were incubated in transfection buffer (serum-free DMEM) for 3 h and then harvested after 45 h in culture. Luciferase assays were performed using the Dual Luciferase Assay System (Promega) that already contains an internal control detectable simultaneously with the luciferase reporter gene. Each experiment was conducted at least in triplicate.

Nuclear Extract Preparation and EMSA.
Nuclear extracts were prepared according to the method of Lassar et al. (20) . To obtain the nuclear extract, cell-containing plates were washed three times with Tris-buffered saline solution and 2.5 ml of lysis buffer [20 mM HEPES (pH 7.6), 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml pepstatin, and 100 mg/ml aproteinin] was added to each 15-cm diameter dish. Cell were removed by centrifugation for 5 min at 2,000 rpm at 4°C. Nuclei were resuspended at 2.5 x 107 nuclei/ml in nuclear extraction buffer (identical to the lysis buffer with the addition of 500 mM NaCl). Nuclei were gently shaken for 1 h at 4°C, centrifuged at 10,000 rpm for 10 min, frozen quickly in liquid nitrogen, and stored at -80°C.

The double-stranded DNA probe used in the gel mobility shift assays was the following: 5'-CACCCCCATTCCCAAGCCTTGTCCTTTCGCCAGA-3' from the position -317 bp to -283 bp (coordinated relative to the primary transcription start site) of the murine UPase promoter, synthesized and purified (using oligonucleotide purification cartridge from Perkin-Elmer) by the Protein and Nucleic Acid Chemistry Facility of the Yale Cancer Center (Yale University). This probe included the p53-binding motif. EMSA was performed according to the manufacturer’s instructions for Gel Shift Assay Systems (Promega). Briefly, 2 µg of nuclear extract were mixed with 1 ng of each labeled probe in binding buffer containing 0.5 mM EDTA, 0.5 mM DTT, 4% glycerol, 1 mM MgCl2, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 0.05 mg/ml poly(dl-dC)poly(dl-dC) and incubated for 20 min at room temperature. To demonstrate the sequence-specific binding, a 100-fold excess of the same unlabeled probe and other unlabeled probes as specific and nonspecific competitors were included in a separate reaction. The reaction mixtures were then separated on a 6% nondenaturing polyacrylamide gel at room temperature in 0.5 x TBE buffer at 100 V for ~3 h. The gel was transferred to Whatman No. 3MM paper, dried, and exposed to X-ray film overnight at -70°C with an intensifying screen.

DNase I Footprinting.
Single end-labeled probe (1 x 10-4 cpm) from -445 to -274 bp of murine UPase promoter was incubated with 30 µg of nuclear extract from EMT6 cells in binding buffer containing 25 mM Tris (pH 8.0), 50 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA, 10% glycerol, 0.5 mM DTT, and 2 µg of poly(dI-dC)poly(dI-dC) on ice for 15 min in a total volume of 50 µl. Then, 50 µl of a solution containing 5 mM CaCl2 and 10 mM MgCl2 was added to the mixture. After incubating the samples at room temperature for 1 min, 0.8 units of DNase I were added to each tube, and the incubation was continued at room temperature for an additional 1 min. The reaction was stopped by adding 90 µl of a solution containing 200 mM NaCl, 30 mM EDTA, 1% SDS, and 100 µg/ml yeast tRNA. The DNA samples were purified by a phenol-chloroform extraction and ethanol precipitation and resuspended in loading buffer (0.1 M NaOH-formamide, 0.1% xylene cyanol, and 0.1% bromphenol blue). After denaturation at 75°C, the samples were separated by electrophoresis on a 6% sequencing gel.

Quantitative RT-PCR.
Total RNA was extracted from MEF p53 +/+ and p53 -/- cells using TriZol (Life Technologies, Inc.). For RT-PCR analysis, DNase I-treated total RNA was reverse transcribed using oligo(dT) and SuperScript II (Life Technologies, Inc.). The cDNAs were amplified using mUPase primers P190 (5'-GAC GAA GTG ATT GAC TGG TGG TC-3') and P720a (5'-CGC CTG AAG TGC CAA TGC G-3') together with the internal control mS16 primers (5'-AGG AGC GAT TTG CTG GTG TGG A-3' and 5'-GCT ACC AGG CCT TTG AGA TGG A-3'). PCR products were separated on a 1.0% agarose gel and stained with ethidium bromide.

Western Blot and Enzymatic Activity Assay.
Primary antibodies against UPase were prepared at Yale University (rabbit anti-UPase polyclonal antibody to human recombinant UPase). The Western blot method is reported in detail (9) . UPase enzymatic activity was measured by uridine conversion to uracil, using TLC chromatographic separation as described (21) . Briefly, cell lysates were prepared using 50 mM Tris-HCl, and the supernatant after the 30,000 x g centrifugation was analyzed for both enzymatic activity and Western blot. Enzymatic activity was measured as the percentage of conversion of [3H]uridine to [3H]uracil after separation on silica TLC plates (Kieselgel 60; Merck), using an 85:15:5 mixture of chloroform, methanol, and acetic acid, respectively.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Sequencing of the Murine UPase Gene 5'-Flanking Fragment.
We have cloned previously a 1.2-kb fragment of the UPase gene promoter (16) . Now, to characterize the promoter region of the murine UPase gene and study the possible regulatory elements that control UPase transcriptional activity, we isolated a genomic DNA clone that contains the immediate full-length 5'-flanking UPase sequence (1703 bp). A murine ES-129/SvJ bacterial artificial chromosome library was screened using the murine UPase cDNA probe. The 1703-bp XbaI/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 (p-1619/+84), and the orientation was verified by sequencing both DNA strands. Analysis of the nucleotide sequence of the 5'-flanking region of the murine UPase revealed the absence of a canonical TATA box. At the 5' end of UPase promoter (from -1619 to -1110) are a series of microsatellite and minisatellite repeat bases (Fig. 1)Citation . A potential p53 binding motif, AGcCTTGTCC, is located in the sequence -303 bp to -294 bp presenting one base difference (the small-case c) from the consensus p53 binding sequence, 5'-PuPuPuC(A/T; A/T)GPyPyPy-3' (22) .



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  		Fig. 1. Restriction map and sequence of the 5'-flanking region of the murine UPase gene. A, schematic restriction map for the subcloned UPase gene promoter region. B, nucleotide sequence of the cloned UPase gene promoter and part of exon 1. The potential p53-binding motif is underlined; bold and * base represents the only difference from the consensus-binding sequence. Larger arrow, major transcription initiation site; smaller arrow, minor transcription initiation site.

 
Mapping of UPase Transcription Start Sites.
The transcription initiation sites were determined to facilitate the preparation of the UPase gene promoter constructs. Mapping of the transcription start site of the murine UPase gene was accomplished by primer extension analysis. For the primer extension assay, we used the primer that is located +133 to +101 bp downstream of cDNA sequence and complementary to the minus DNA strand of the murine UPase cDNA. As shown in Fig. 2Citation , Lane 1, the primer extension reaction yielded 133-bp and 225-bp products using RNA extracted from murine colon 26, a tumor cell line with high UPase expression. Sequencing reactions were performed with the same primer on the noncoding DNA strand from murine colon 26 cells to serve as the sequencing ladder and determine the size and nucleotide position of the start sites (Fig. 2)Citation . These experiments determined that two transcriptional start sites are located +1 and +92 bp of the most 5' end of the reported cDNA sequence.



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  		Fig. 2. Determination of the transcription start site of the murine UPase gene. The transcriptional start site was mapped by primer extension analysis. For the primer extension reaction, an oligonucleotide primer corresponding to complementary to +133 to +101 nucleotides of the UPase cDNA was end-labeled with [{gamma}-32P]ATP and hybridized with 15 µg of total RNA from colon 26 tumor cells. Lane 1, primer extension with murine colon 26 cell RNA. Lanes 2–5 correspond to A, G, T, and C nucleotide sequencing reaction using the same primer. Arrows designate the primer extension products. Two transcriptional start sites are located +1 and +92 bp, respectively, of the most 5' end of the reported cDNA sequence.

 
Functional Characterization of the Murine UPase Promoter.
Several 5' deletions of UPase gene promoter transgene constructs were generated to define the DNA regulatory elements. Two distinct cell lines, EMT6 and NIH 3T3, were cotransfected with the UPase plasmid DNA constructs and the pRL-TK vector as an internal control for transfection efficiency. The full-length promoter construct (-1619/+84) was consistently expressed in EMT6 and NIH 3T3 cells. Progressive 5'-deletion mutations of the full-length promoter revealed a pattern of functional activity in the transfected cells (Fig. 3)Citation . The plasmids containing 5' deletions of various lengths from -1081 to -445 bp produced a very modest decrease in promoter activity. Additional deletions from -445 to -274 bp led to an increase in promoter activity higher than the expression obtained with the full-length promoter. Deletion to -84 bp resulted in an extreme reduction of the activity in EMT6 cells with a more modest effect in NIH 3T3 cells (Fig. 3)Citation .



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  		Fig. 3. Deletion analysis of the murine UPase promoter region in (A) EMT6 and (B) NIH 3T3 cells. Left, schematic representation of the 5'-flanking region of the UPase/reporter gene constructs used in the transient transfection analysis of promoter activity. The restriction enzyme sites used in the preparation of the constructs are indicated in Fig. 1Citation . The UPase luciferase constructs were cotransfected with a control plasmid pRL-TK and assayed 48 h after transfection. The luciferase activity elicited by each deletion mutant is expressed as percentage of the activity obtained by the full-length (1703 bp) promoter activity in EMT6 cells. Bars, SE from three samples in three independent experiments.

 
Suppression of the UPase Gene Promoter Activity by Wild-type p53.
Using transient-expression assays in EMT6 and NIH 3T3 cells, cotransfection of UPase promoter construct p-1619/+84 with the wild-type p53 construct resulted in significantly less luciferase activity compared with a cotransfection experiment using an empty vector, an average of 4- and 3.5-fold reduction in relative luciferase activity in EMT6 and NIH 3T3 cells, respectively. To locate the DNA element(s) in the UPase promoter that mediate the transcriptional regulation by wild-type p53, we tested a series of 5'-deletion mutants of the UPase promoter for p53 sensitivity in cotransfection assays (Fig. 4)Citation . The transcription of 5' deletion constructs between -1619 and -445 bp was suppressed by wild-type p53, whereas the constructs missing the putative p53-binding element were not affected. These data indicate that the region between -445 and -274 bp in the UPase promoter is susceptible to regulation by p53. The nucleotide sequence analysis of the murine UPase gene 5'-flanking region had identified a potential p53 binding site at -303 to -294 bp, AGcCTTGTCC. This binding motif differs in one base (small-case base) from the consensus element of p53 binding. In p53 null MEF cells, unlike EMT6, NIH 3T3, and MEF p53 +/+ cells, the luciferase activity was not altered when the constructs deleted from -445 to -274 bp were cotransfected. However, UPase gene promoter activity was repressed significantly by cotransfection of wild-type p53 in the p53 knockout MEF cells with the p-1619/+84 and p-445/+84 constructs. As expected, no change in luciferase activity was observed for the construct p-274/+84 that does not contain the p53 binding sequence (Fig. 5)Citation .



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  		Fig. 4. Suppression of UPase promoter activity by wild-type p53 in NIH/3T3 cells. Cotransfection of murine UPase promoter-reporter gene constructs with wild-type p53 in NIH 3T3 cells. {blacksquare}, luciferase activity of the UPase constructs cotransfected in the presence of wild-type p53 constructs; {square}, luciferase activity of the constructs cotransfected with empty vectors; bars,± SE.

 


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  		Fig. 5. Suppression of UPase promoter activity by wild-type p53 in MEF p53 -/- cells. Cotransfection of UPase promoter constructs p-1619/+84, p-445/+84, and p-274/+84 with the wild-type p53 construct ({blacksquare}) and a control vector ({square}); bars, ± SE.

 
p53 Suppresses UPase mRNA and Protein Expression.
To additionally confirm our observation of p53 down-regulation of UPase gene expression, we evaluated the UPase mRNA and protein expression in MEF p53 -/- and p53 +/+ cells using quantitative RT-PCR, Western blot, and enzyme activity. The data reported in Fig. 6Citation indicated that the level of UPase mRNA expression in MEF p53 -/- cells was elevated compared with the MEF p53 +/+ control cells. The Western blot showed that UPase protein expression was repressed by p53 in MEF p53 +/+ cells compared with MEF p53 -/- cells. The enzyme activity of UPase in MEF p53 -/- was ~30-fold higher than in the MEF p53 +/+ cells (Fig. 7)Citation .



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  		Fig. 6. Suppression of UPase gene mRNA expression by wild-type p53. UPase mRNA expression in MEF p53 +/+ and p53 -/- cells was detected by quantitative RT-PCR. The mS16 primers were used as internal controls in the same PCR reaction.

 


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  		Fig. 7. Western Blot analysis and enzymatic activity of UPase in p53 -/- and p53 +/+ cells. The cell extracts were analyzed by Western blot after SDS-PAGE separation and the enzymatic activity determined as described in "Materials and Methods."

 
p53-specific Binding to the UPase Promoter.
To complete the elucidation of the p53 regulation of the UPase gene, a gel mobility shift assay (EMSA) was performed. A synthesized 34-bp DNA fragment (-317 to -284 bp) containing only the p53-binding element as a probe and nuclear proteins including the full-length wild type p53 protein (393 amino acids) were used in the EMSA. The mobility of the labeled DNA probe was altered in the presence of the p53 protein (Fig. 8)Citation because of the formation of a binding complex. The amount of the shifted complex was diminished by increasing the concentration of self-competitor (nonradiolabeled p53 probe) but not by the addition of nonspecific competitor (SP1 and AP2). The SP1 and AP2 probes used as nonspecific competitors were supplied by Gel Shift Assay Systems (Promega). DNase I footprinting indicated that the binding site was located from -294 to -303 bp of UPase promoter, the position of the p53 binding motif (Fig. 9)Citation . This p53-binding motif is 90% homologous to the reported consensus-binding element of p53.



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  		Fig. 8. Electrophoretic mobility shift assay. A radiolabeled double-stranded DNA probe (34-bp long) containing the p53 promoter-binding region was incubated with NIH 3T3 cell extract and separated on a 6% polyacrylamide gel. To determine binding specificity, cold p53 and other control probes were added as specific and nonspecific competitors, as indicated above the corresponding lanes.

 


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  		Fig. 9. DNase I footprinting analysis of the murine UPase promoter. 5'-End-labeled fragment, including the sequence from -445 to -274 bp was incubated without extract or with 2 µg of EMT6 nuclear extract proteins and partially digested with DNase I. Right, the putative p53 binding site sequence is indicated by {square}.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To study the UPase gene regulation and to understand the alterations in its expression observed in human tumors, we have isolated and sequenced a 1703-bp fragment corresponding to the promoter region of murine UPase and characterized its functional activity in EMT6 and NIH 3T3 cells. DNA sequencing of UPase 5'-flanking region has revealed that the UPase gene lacks basal elements like the TATA box or an initiator sequence. The absence of such sites and the presence of SP1 sites, including one in the basal promoter of UPase (16) , might have been expected to result in the initiation of transcription at several locations. Two transcription start sites were mapped at +1 and +92 bp of the most 5' end of the reported murine UPase cDNA sequence. Promoter constructs containing the whole 1703 bp of 5'-flanking sequence showed comparable luciferase activity in both EMT6 and NIH 3T3 cells. The promoter activity was altered in EMT6 and NIH 3T3 cells after progressive 5'-deletion mutations of the promoter sequence. We found that deletions from -1619 to -445 bp produced a slight decrease in promoter activity. However, additional deletion to -274 bp resulted in the elevation of the activity to levels higher that the full-length promoter. Cotransfection of the UPase promoter constructs with the wild-type p53 construct resulted in significantly less luciferase activity compared with a cotransfection experiment using an empty vector; however, this phenomenon disappeared with the deletion from -445 to -274 bp. This indicates the existence of a p53-inhibitory element in this promoter sequence. A significant promoter activity was also detected between -274 and -84 bp, suggesting that this region contains information necessary for an active transcription of UPase in these cells. In contrast, an additional deletion to -84 resulted in an extreme reduction of activity in EMT6 cell but not in 3T3 cells. A putative IRF-1 binding site is located in this region and could possibly play a different regulatory role in these two cell lines. Additional elucidation of this regulatory element and its function in UPase expression is currently under investigation to clarify the role of cytokines in FU activation and ultimately in pyrimidine-based cancer therapy.

p53 functions as a transcription factor and regulates a number of target genes at the transcriptional level. The central region of the p53 protein interacts with the promoter of the target gene in a sequence-specific manner, binding to two copies of a consensus element {5'-PuPuPuC(A/T; A/T)GPyPyPy-3'; Ref. 22 }. Wild-type p53 efficiently binds to this sequence and transactivates expression of the target genes (22, 23, 24, 25, 26) . p53 can also repress a wide variety of cellular and viral promoters (27) . There are several possible mechanisms to account for the inhibitory activity of p53 on promoter activity. First, p53 inhibition might directly or indirectly inactivate a critical component of the transcription machinery, leading to general inhibition of transcription. However, we found that the promoter of UPase at p-274 still had a high promoter activity even in the presence of cotransfected p53, indicating that the transcriptional machinery is still active under our experimental conditions.

A second possibility is that p53 may inhibit the activity of the promoter by "squelching" or sequestering general transcription factors (28) . Squelching would be expected to inhibit the activity of promoters lacking p53; in some promoters the p53 regulation occurs through binding to the TATA-binding protein causing suppression of the promoter activity (29 , 30) .

A third case is that p53 might inhibit the promoter by directly or indirectly blocking the activity of other factors important for the promoter activity, like SP1 (31) , CCAAT-binding factor (32) , cyclin AMP response element-binding protein (33) , and glucocorticoid receptors (34) .

A fourth alternative, which appears to be the most likely in the case of the UPase gene, is that the inhibition is attributable to the presence of a specific p53-negative response element that is distinct from the core promoter region, as observed previously for the Rb (35) , bcl-2 (36) , and topoisomerase IIa (37) promoters. To explore this possibility, we first analyzed the effects of p53 on UPase promoter activity. We found that the deletion from -1619 to -445 of the UPase promoter had no effect on the ability of p53 to inhibit gene expression; however, the inhibitory activity was altered when the promoter region between -445 and -274 bp was deleted. Using transient-expression assays in EMT6 and NIH 3T3 cells, cotransfection with the wild-type p53 construct resulted in significantly less luciferase activity in the constructs from -1619 to -445, whereas further deletions of the promoter did not affect the activity. These data indicate that the region between -445 and -274 bp is susceptible to regulation by p53 in the UPase promoter. This phenomenon was additionally confirmed in p53-nullified cells. Sequencing analysis of this region found a putative p53-binding motif AGcCTTGTCC located at -303 to -294. This binding motif differs in one base (small-case base) from the consensus-binding element of p53. The gel mobility shift assay and DNase I footprinting have indicated that this putative regulatory motif exhibited specific binding with the p53 protein.

p53 has been shown to be activated by ribonucleotide depletion caused by antimetabolite drugs such as PALA (N-(phosphonacetyl)-L-aspartic) acid even in the absence of DNA damage (38) . As mentioned previously, the phosphorolytic activity of Upase-regulating intracellular uridine levels reveals the critical role of this enzyme in modulating the pyrimidine salvage pathway. The suppressive regulation of p53 on UPase gene indicates the presence of a negative control of the pyrimidine salvage pathway by p53 through UPase, probably as a cellular self-protection mechanism in case of ribonucleotide depletion. p53 has been shown previously to: (a) activate genes that initiate apoptosis to eliminate damaged cells and protect an organism from more severe damage; and (b) cause cell-cycle arrest after DNA damage to prevent the replication of altered DNA. However, thus far, indication of the contribution of p53 to damage repair is quite limited. A recent report (39) has described a p53-induced gene, p53R2, that encodes for a protein similar to one of the two subunits of ribonucleotide reductase, the rate-limiting step in the conversion of ribonucleotides to deoxyribonucleotides. The p53-regulated R2 subunit is found in the nucleus, and its expression is induced by cellular damage ({gamma}-radiation and Adriamycin treatment), suggesting that when repair is needed the nuclear precursors have to be concentrated near the site of damage.

Somehow the p53-regulated suppression of UPase expression exerts similar functions to the control that p53 has on p53R2. A cellular damage causing loss or imbalance in the ribonucleotide pools could cause activation of p53 leading to suppression of UPase expression and activation of the pyrimidine salvage pathway to replenish the affected pyrimidine nucleotide pools. These two p53-regulated mechanisms provide a new level of control on ribo- and deoxyribonucleotide pools. Under normal replication conditions, the regulating mechanisms that control the appropriate balance of nucleotides are based mostly on the direct feedback regulation of the biosynthetic enzymes by some of the precursors or final products. For example, in the case of the pyrimidine nucleotide biosynthesis CTP or UTP (depending on the organism) inhibit the activity of aspartate transcarbamoylase that catalyzes the first reaction of the de novo synthesis. Similarly in the deoxyribonucleotide biosynthesis, dGTP and dTTP stimulate the reduction of ADP and GDP to the corresponding deoxyribonucleotide forms. The report on p53R2 and our data on UPase possibly indicate that in case of cellular damage a more sophisticated level of regulation is triggered to more rapidly provide precursors for nuclear repair.

The elucidation of the negative control regulation of p53 on the UPase gene promoter and UPase expression could also have considerable implication at the clinical level on the therapeutic outcome in the presence of tumors with specific p53 mutations when undergoing antimetabolite-based cancer therapy.


   ACKNOWLEDGMENTS
 
We thank Dr. Donehower (Baylor College of Medicine, Houston, TX) for kindly providing us with the p53 +/+ and p53 -/- MEF cells.


   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 in part by National Cancer Institute Grant CA67035 (to G. P.), the United States Army Medical Research Breast Cancer Research Program (to D. L. C.), and The Anna Fuller Foundation Fellowship (to D. K. Z.). Back

2 To whom requests for reprints should 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; MEF, murine embryo fibroblast; FU, 5-fluorouracil; EMSA, electrophoretic mobility shift analysis; RT-PCR, reverse transcription-PCR. Back

Received 8/10/00. Accepted 7/13/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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