Inhibition of Cyclooxygenase-2 Gene Expression by p53*

Oncogenes enhance the expression of cyclooxygenase (Cox)-2, but interactions between tumor suppressor genes and Cox-2 have not been studied. In the present work, we have compared the levels of Cox-2 and the production of prostaglandin E2 in mouse embryo fibroblasts that do not express any p53 ((10)1) versus the same cell line ((10.1)Val5) engineered to overexpress wild-type (wt) p53 at 32 °C or mutant p53 at 39 °C. Cells expressing wt p53 showed about a 10-fold decrease in synthesis of prostaglandin E2 compared with those expressing mutant p53. Levels of Cox-2 protein and mRNA were markedly suppressed by wt p53 but not by mutant p53. Nuclear run-offs revealed decreased rates of Cox-2 transcription in cells expressing wt p53. The activity of the Cox-2 promoter was reduced by 85% in cells expressing wt p53 but was reduced only by 30% in cells expressing mutant p53 compared with cells null for p53. The effect of p53 on the suppression of Cox-2 promoter activity was localized to the first 40 base pairs 5′ from the transcription start site. Electrophoretic mobility shift assay revealed that p53 competed with TATA-binding protein for binding to mouseCox-2 or human Cox-2 promoter extending from −50 to +52 base pairs. The results of this study suggest that interactions between p53 and Cox-2 could be important for understanding why levels of Cox-2 are undetectable in normal cells and increased in many tumors.

Two different isoforms of cyclooxygenase (Cox) 1 catalyze the synthesis of prostaglandins from arachidonic acid. Cox-1 is a constitutive enzyme, whereas Cox-2 is inducible (1,2). The Cox-2 gene is an early response gene that is rapidly induced by phorbol esters, cytokines, growth factors, and carcinogens (3)(4)(5)(6)(7). The different responses of the genes encoding Cox-1 and Cox-2 reflect, at least in part, differences in the regulatory elements in the 5Ј-flanking regions of the two genes (8).
Numerous studies support the idea that Cox-2 is important in carcinogenesis. It is known, for example, that Cox-2 is upregulated in transformed cells and in various forms of cancer (9 -16). A null mutation for Cox-2 markedly reduces the number and size of intestinal tumors in a murine model of familial adenomatous polyposis, i.e. APC ⌬716 knockout mice (17). Cox-2 deficiency also protects against the formation of extraintestinal tumors. Thus, Cox-2 knockout mice developed about 75% fewer chemically induced skin papillomas than control mice (18), and a selective inhibitor of Cox-2 caused a nearly complete suppression of azoxymethane-induced colon cancer (19). It is also known that the expression of Cox-2 is enhanced by oncogenes (9 -11), but the effects of tumor suppressor genes, e.g. p53, on Cox-2 are unstudied.
p53 is important in the suppression of cellular growth and transformation (20 -24). In addition, induction of wild-type (wt) p53 can cause cells to undergo apoptosis (25,26). By contrast, inactivation of p53 can lead to deregulation of the cell cycle and DNA replication, selective growth advantage, and tumor formation (27,28). Significantly, p53 can either increase or suppress the expression of a number of target genes (29 -32). Gene activation usually involves binding of p53 to specific consensus sequences. Recent studies indicate that repression of transcription by p53 may involve interaction with basal transcription machinery and may be important in mediating programmed cell death (33)(34)(35)(36).
In the present work, we have investigated the effects of p53 on Cox-2 gene expression. Our data show that wt p53 causes a marked decrease in the expression of Cox-2. In contrast, mutant p53 did not cause a significant reduction in levels of Cox-2. Interactions between p53 and Cox-2 could be important for understanding why levels of Cox-2 are essentially undetectable in normal epithelial cells and elevated in many cancers.  1 The abbreviations used are: Cox, cyclooxygenase; bp, base pair(s); f.p.u., foot print unit; GST, glutathione S-transferase; PGE 2 , prostaglandin E 2 ; TBP, TATA-binding protein; wt, wild-type; SSPE, salinesodium phosphate-EDTA buffer; a.u. arbitrary unit. p53, with the temperature-sensitive p53 allele encoding valine at amino acid 135 (37,38). The (10.1)Val5 line contains a temperature-sensitive p53 mutant that is wt at 32°C and mutant at 39°C (38). PGE 2 Production-(10)1 and (10.1)Val5 cell lines were plated in 6-well plates and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37°C until they were approximately 40% confluent. The cell lines were then shifted to 32°C or to 39°C for 18 h. The medium was then replaced with fresh medium containing 10 M sodium arachidonate. 30 min later, the medium was collected to measure the synthesis of PGE 2 . The levels of PGE 2 released by the cells were measured by enzyme immunoassay (9). Levels of PGE 2 production were normalized to protein concentrations.

Materials
Western Blotting-Cell lysates were prepared as described previously (39). SDS-polyacrylamide gel electrophoresis was performed under reducing conditions on 10% polyacrylamide gels as described by Laemmli (40). The resolved proteins were transferred onto nitrocellulose sheets as detailed by Towbin et al. (41). The nitrocellulose membrane was then incubated with a goat polyclonal anti-Cox-2 antiserum or a polyclonal anti-Cox-1 antiserum. A secondary antibody to IgG conjugated to horseradish peroxidase was used. The blots were probed with the ECL Western blot detection system according to the manufacturer's instructions.
Northern Blotting-Total cellular RNA was isolated from cell monolayers using a RNA isolation kit from QIAGEN Inc. For Northern blots, 10 g of total cellular RNA per lane were electrophoresed in formaldehyde-containing 1.2% agarose gels and transferred to nylon-supported membranes. After baking, membranes were prehybridized for 8 h and then hybridized in a solution containing 50% formamide, 5ϫ SSPE, 5ϫ Denhardt's solution, 0.1% SDS, and 100 g/ml single-stranded salmon sperm DNA. Hybridization was carried out for 16 h at 42°C with radiolabeled murine Cox-2 and 18 S rRNA probes. After hybridization, the membrane was washed for 20 min at room temperature in 2ϫ SSPE and 0.1% SDS, washed twice for 20 min in the same solution at 55°C, and washed twice for 20 min in 0.1ϫ SSPE and 0.1% SDS at 55°C. Washed membranes were then subjected to autoradiography. The density of the bands was quantified by densitometry.
Nuclear Run-off-For each cell line, 2.5 ϫ 10 5 cells were plated in four T150 dishes and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum until they were approximately 50% confluent at 37°C. The cell lines were then shifted to 32°C for 18 h. Nuclei were isolated and stored in liquid nitrogen. For the transcription assay, nuclei (1.0 ϫ 10 7 ) were thawed and incubated in reaction buffer (10 mM Tris, pH 8, 5 mM MgCl 2 , and 0.3 M KCl) containing 100 Ci of [␣-32 P]UTP and 1 mM unlabeled nucleotides. After 30 min, labeled nascent RNA transcripts were isolated. The Cox-2 (TIS10) and 18 S rRNA cDNAs were immobilized onto nitrocellulose and prehybridized overnight in hybridization buffer. Hybridization was carried out at 42°C for 24 h using at least 5 ϫ 10 5 cpm of labeled nascent RNA transcripts for each treatment group. The membranes were washed twice with 2ϫ SSC buffer for 1 h at 55°C and then treated with 10 mg/ml RNase A in 2ϫ SSC at 37°C for 30 min, dried, and autoradiographed.
Transient Transfection Assays-Cells were seeded at a density of 5 ϫ 10 4 cells/well in 6-well dishes and grown to 30 -40% confluence at 37°C in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, unless stated otherwise. For each well, 2 g of plasmid DNA were transfected into cells using the calcium phosphate precipitation method according to the manufacturer's instructions. After 16 h, the medium was changed, and cells were then shifted to 32°C or 39°C. At the end of the treatment period, cells were harvested, and luciferase activity was measured. Each well was washed twice with phosphatebuffered saline. 100 l of 1ϫ lysis buffer (Analytical Luminescence Laboratories, San Diego, CA) were added to each well for 15 min. The lysate was centrifuged for 1 min at 4°C. The supernatant was used to assay luciferase activity with a Monolight 2010 Luminometer (Analytical Luminescence Laboratories) according to the manufacturer's instructions. Luciferase activity is expressed per microgram of protein in the cell extract.
Preparation of GST.p53-Purified p53 protein was prepared as a GST fusion protein according to methods described previously (34).
Gel Shift Assays-A fragment of the human COX-2 or mouse Cox-2 promoter extending from Ϫ50 to ϩ52 encompassing the in vivo transcription initiation site was made by polymerase chain reaction amplification. After purification from agarose gels, the fragment was dephosphorylated using calf intestinal phosphatase and end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase. The labeled probe was separated from unincorporated nucleotides using purification columns from QIAGEN Inc.
Gel shift assays were performed by preincubating TBP (1 f.p.u./ reaction), GST.p53 (100 ng), or a combination of both proteins in 9 l of 20 mM Hepes (pH 7.5), 70 mM KCL, 12% glycerol, 0.05% Nonidet P-40, 100 M ZnSO 4 , 0.05 M dithiothreitol, 1 mg/ml bovine serum albumin, and 0.1 mg/ml poly(deoxyinosinic-deoxycytidylic acid) on ice. After 15 min, 100 pg of the labeled fragment were added, and the reaction was incubated for an additional 10 min at 25°C. Changes in mobility were assessed by electrophoresis through a 5% polyacrylamide gel that was run at 250 V for 2 h. After fixation, the gel was autoradiographed.
Statistics-Comparisons between groups were made by the Student's t test. A difference between groups of p Ͻ 0.05 was considered significant. Fig. 1 show that cells containing wt p53 synthesized about 90% less PGE 2 than cells that are null for p53. By contrast, mutant p53 did not inhibit production of PGE 2 . To evaluate whether the inhibition of prostaglandin synthesis could be related to differences in the levels of Cox, Western blotting of the cell lysate protein was carried out. Fig. 2 shows that wt p53 caused a nearly complete suppression of Cox-2 expression, whereas mutant p53 did not affect the levels of Cox-2. In other experiments, the suppressive effects of wt p53 on amounts of COX-2 protein were more modest. Cox-1 was not detectable by Western blotting in any of the cells tested. To further elucidate the mechanism responsible for the changes in the amount of Cox-2 protein, we determined the steady-state levels of Cox-2 mRNA by Northern blotting. Levels of Cox-2 mRNA were reduced in the presence of wt p53 but not mutant p53 (Fig. 3).

Wild-Type p53 Inhibits the Synthesis of Prostaglandins and Expression of Cox-2-The data in
Cox-2 Transcription Is Suppressed by Wild-Type p53-Because differences in the levels of mRNA could reflect altered rates of transcription or changes in the stability of mRNA, nuclear run-offs were performed to distinguish between these possibilities. As shown in Fig. 4, we found lower rates of synthesis of nascent Cox-2 mRNA in cells expressing wt p53.
To further investigate the importance of p53 in modulating p53 Inhibits Cyclooxygenase-2 Expression the expression of Cox-2, transient transfections were performed using a murine Cox-2 promoter luciferase construct. The activity of the Cox-2 promoter was reduced by about 85% in the cell line expressing wt p53 compared with the p53-null cell line (Fig. 5). In contrast, mutant p53 caused about a 30% decrease in Cox-2 promoter activity (Fig. 5). We also investigated whether p53 could inhibit v-src-mediated induction of Cox-2 promoter activity. As shown in Fig. 6, overexpressing v-src caused about a 6-fold increase in Cox-2 promoter activity in the p53-null cell line. The stimulatory effects of v-src were blocked by overexpressing wt p53 in the same cell system. In separate experiments, wt p53 also blocked phorbol ester-mediated induction of Cox-2 promoter activity (data not shown).
Localization of the Promoter Region Responsible for Repression of Transcription-We next attempted to define the region of the Cox-2 promoter that responded to p53. This was accomplished using a series of murine Cox-2 promoter deletion constructs. As shown in Fig. 7, Cox-2 promoter activity decreased as the length of the promoter decreased. However, with all promoter constructs including TIS10 -40, the activity of the Cox-2 promoter was higher in the p53-null cell line compared with the cell line expressing wt p53. The fact that these differences in promoter activity were maintained despite changes in promoter length indicates that the suppression of Cox-2 promoter activity by p53 was localized to the first 40 bp 5Ј of the transcription start site.
TBP is essential for the transcription of TATA-containing promoters such as the Cox-2 promoter. p53 has been reported to inhibit gene expression via interactions with TBP (33-35). To study possible interactions between p53 and TBP in the FIG. 6. Wild-type p53 suppresses v-src-mediated induction of Cox-2 promoter activity. (10)1 cells, which are null for p53, were grown under serum-free conditions. Cells were transfected with 1.0 g of Cox-2 luciferase construct TIS10L (963 bp) alone (Ϫp53, Ⅺ) or with 0.5 g of expression vector for wt p53 (ϩp53, Ⅺ). Cells were also co-transfected with 1.0 g of TIS10L plus 0.5 g of expression vector for v-src (Ϫp53, f) or 1.0 g of TIS10L plus 0.5 g of expression vectors for v-src and wt p53 (ϩp53, f). The total amount of DNA was kept constant at 2 g by using empty vector DNA. Twenty four h later, luciferase activity was measured. Six wells were used for each transfection condition. Columns, means; bars, S.D.
p53 Inhibits Cyclooxygenase-2 Expression regulation of Cox-2 promoter activity, gel retardation assays were performed. In these experiments, we used a series of labeled 100-bp fragments of murine and human Cox-2 promoter spanning from Ϫ400 to ϩ52 bp together with a GST.p53 fusion protein or purified TBP, or both. We did not observe binding of GST.p53 or TBP to regions of the Cox-2 promoter spanning from Ϫ400 to ϩ52 (data not shown). Binding was only observed with a DNA fragment (from Ϫ50 to ϩ52) that included the TATA box (Fig. 8). Incubation of TBP or GST.p53 with the labeled probe led to two different retarded bands. Moreover, the formation of the TBP-DNA complexes was inhibited by the addition of increasing amounts of GST.p53. We did not observe the formation of complexes with purified GST alone (data not shown).

DISCUSSION
The present data show that the expression of Cox-2 is markedly repressed by wt p53 but not by mutant p53. This observation is important for several reasons. For example, suppression of Cox-2 expression by wt p53 can explain why levels of Cox-2 protein are undetectable in normal epithelial cells and, by contrast, why mutations of p53 may contribute to the increased expression of Cox-2 that is observed in malignant tissues (12)(13)(14)(15)(16). Possibly, the induction of Cox-2 by oncogenes (9,11), growth factors (42), and cytokines (6) may be enhanced in cells expressing mutant p53. Moreover, increased expression of Cox-2 after the mutation of p53 could be an important link in the relationship between the function of p53 and cancer. Thus, the products of Cox-2 activity, i.e. prostaglandins, stimulate cell proliferation (43), inhibit immune surveillance (44,45), increase the invasiveness of malignant cells (46), and enhance the production of vascular endothelial growth factor, which promotes angiogenesis (47). It is already known that transduction of cancer cells with wt p53 decreases the synthesis of vascular endothelial growth factor and inhibits tumor cellinduced angiogenesis (48). Therefore, the present data provide a possible mechanistic explanation for the regulation of expres-sion of vascular endothelial growth factor and angiogenesis by p53. It thus becomes important to determine the correlation between the expression of mutant forms of p53 and amounts of Cox-2 in malignant tissue. It will also be important to determine whether inactivation or loss of other tumor suppressor genes alters the expression of Cox-2.
The repression of transcription by p53 is believed to be important for p53-mediated apoptosis, but the mechanism underlying this effect is not understood (36). On the other hand, overexpression of Cox-2 in intestinal epithelial cells inhibits apoptosis (49). Because Cox-2 interacts directly with nucleobindin, which is an apoptosis-associated protein, Cox-2 might inhibit apoptosis by sequestering nucleobindin (50). The effects of Cox-2 on apoptosis might also be indirect via the modulation of levels of intracellular arachidonic acid as suggested by Chan et al. (51). Levels of arachidonic acid, in turn, regulate the production of ceramide (52), which can induce apoptosis. Independent of the exact mechanism, the finding that wt p53 downregulates the transcription of Cox-2 could be important for understanding p53-mediated apoptosis.
p53 suppresses a variety of promoters that contain TATA elements (30,33,35). This suppression is thought to occur through direct interaction with components of the basal transcription machinery, such as TBP. For example, in prior studies, p53 inhibited the binding of TBP to several promoters, most probably through protein-protein interactions (33)(34)(35)53). Consequently, TBP is no longer able to assemble a functional transcription initiation complex. In the current study, p53 inhibited the formation of complexes between TBP and the murine and human Cox-2 promoters in a cell-free system. One possible explanation for this result is a direct competition between p53 and TBP for the TATA binding site. Interactions between TBP and p53 could decrease the rate of Cox-2 transcription, but this needs to be confirmed in intact cells.