Inhibition of Cyclooxygenase-2 Gene Expression by p53*

Kotha SubbaramaiahDagger §, Nasser Altorkiparallel , Wen Jing ChungDagger , Juan R. Mestre**Dagger Dagger , Anu SampatDagger , and Andrew J. DannenbergDagger §§§

From the Departments of Dagger  Medicine, § Surgery, and parallel  Cardiothoracic Surgery, The New York Presbyterian Hospital-Cornell, ** Head and Neck Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center, and  Strang Cancer Prevention Center, New York, New York 10021

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 mouse Cox-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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

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 (3-7). The different responses of the genes encoding Cox-1 and Cox-2 reflect, at least in part, differences in the regulatory elements in the 5'-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 up-regulated 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. APCDelta 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 (33-36).

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.

    EXPERIMENTAL PROCEDURES
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Materials-- Dulbecco's modified Eagle's medium, fetal bovine serum, and calcium phosphate transfection kits were from Life Technologies, Inc. Sodium arachidonate and poly(deoxyinosinic-deoxycytidylic acid) were from Sigma. Enzyme immunoassay reagents for PGE2 assays were from Cayman Co. (Ann Arbor, MI). Random priming kits were from Boehringer Mannheim. Nitrocellulose membranes were from Schleicher & Schuell. [32P]ATP, [32P]CTP, and [32P]UTP were obtained from DuPont New England Nuclear (Boston, MA). Reagents for the luciferase assay were from Analytical Luminescence (San Diego, CA). RNA was prepared using an isolation kit from QIAGEN Inc. (Chatsworth, CA). Plasmid DNA isolation kits and recombinant TATA-binding protein (TBP) were purchased from Promega (Madison, WI). Goat polyclonal anti-Cox-2 and anti-Cox-1 antisera were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Western blotting detection reagents were from Amersham. Oligonucleotides were synthesized by Genosys Biotechnologies Inc. (The Woodlands, TX). GST-agarose was purchased from Pharmacia Biotech Inc. T4 polynucleotide kinase was purchased from New England Biolabs, Inc. (Beverly, MA). Proteinase K, RNase-free DNase, and RNase A for nuclear run-offs and 18 S rRNA cDNA were from Ambion Inc. (Austin, TX).

Cell Lines-- The murine embryo fibroblast-derived cell lines (10)1 and (10.1)Val5 were obtained from Dr. Arnold Levine (Princeton University, Princeton, NJ). The (10.1)Val5 cell line was generated by stable transfection of parental cell line (10)1, which contains no endogenous 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).

PGE2 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 µM sodium arachidonate. 30 min later, the medium was collected to measure the synthesis of PGE2. The levels of PGE2 released by the cells were measured by enzyme immunoassay (9). Levels of PGE2 production were normalized to protein concentrations.

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 µg 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, 5× SSPE, 5× Denhardt's solution, 0.1% SDS, and 100 µg/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 2× SSPE and 0.1% SDS, washed twice for 20 min in the same solution at 55 °C, and washed twice for 20 min in 0.1× 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 × 105 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 × 107) were thawed and incubated in reaction buffer (10 mM Tris, pH 8, 5 mM MgCl2, and 0.3 M KCl) containing 100 µCi of [alpha -32P]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 × 105 cpm of labeled nascent RNA transcripts for each treatment group. The membranes were washed twice with 2× SSC buffer for 1 h at 55 °C and then treated with 10 mg/ml RNase A in 2× SSC at 37 °C for 30 min, dried, and autoradiographed.

Plasmids-- Cox-2 cDNA and Cox-2 promoter deletion constructs (TIS10L, TIS-300, TIS-180, TIS-80, and TIS-40) were generously provided by Dr. Harvey Herschman (University of California, Los Angeles, CA). The wild-type p53 (pC53-SN3) expression vector was kindly provided by Dr. Arnold Levine. The plasmid construct GST.p53 was a generous gift of Dr. Derek LeRoith (National Institutes of Health, Bethesda, MD) (34). The v-src expression vector was a gift from Dr. David Foster (Hunter College, New York, NY).

Transient Transfection Assays-- Cells were seeded at a density of 5 × 104 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 µg 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 phosphate-buffered saline. 100 µl of 1× 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 -50 to +52 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 [gamma -32P]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 µl of 20 mM Hepes (pH 7.5), 70 mM KCL, 12% glycerol, 0.05% Nonidet P-40, 100 µM ZnSO4, 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 < 0.05 was considered significant.

    RESULTS
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Wild-Type p53 Inhibits the Synthesis of Prostaglandins and Expression of Cox-2-- The data in Fig. 1 show that cells containing wt p53 synthesized about 90% less PGE2 than cells that are null for p53. By contrast, mutant p53 did not inhibit production of PGE2. 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).


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Fig. 1.   Wild-type but not mutant p53 down-regulates the production of PGE2 in mouse embryo fibroblasts. (10)1 and (10.1)Val5 cells were grown at 32 °C and 39 °C for 18 h. (10)1 cells contain no p53 protein. (10.1)Val5 cells express predominantly wt p53 at 32 °C but express mutant p53 at 39 °C. The medium was then replaced with fresh medium containing 10 µM sodium arachidonate. 30 min later, the medium was collected to determine the levels of PGE2. Production of PGE2 was determined by enzyme immunoassay. Columns, means; bars, S.D.; n = 6; *, p < 0.01 compared with (10)1 cells.


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Fig. 2.   Levels of Cox-2 protein are decreased in cells expressing wild-type but not mutant p53. (10)1 and (10.1)Val5 cells grown at 37 °C were shifted to 32 °C or 39 °C for 18 h. Cellular lysate protein (25 µg/lane) was loaded onto a 10% SDS-polyacrylamide gel, electrophoresed, and subsequently transferred onto nitrocellulose. Immunoblots were probed with an antibody specific for Cox-2. Lane 1, ovine Cox-2 standard; lane 2, (10)1 cells at 32 °C; lane 3, (10.1)Val5 cells at 32 °C; lane 4, (10)1 cells at 39 °C; lane 5, (10.1)Val5 cells at 39 °C.


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Fig. 3.   Expression of wt p53 decreases the steady-state level of Cox-2 mRNA. (10)1 and (10.1)Val5 cells were grown at 37 °C before being shifted to either 32 °C or 39 °C for 18 h. Total cellular RNA was isolated. Each lane contained 10 µg of RNA. The Northern blot was hybridized with probes that recognized Cox-2 mRNA and 18 S rRNA. Results of densitometry are expressed in a.u. Lane 1, (10.1)Val5 cells at 32 °C, 81 a.u.; lane 2, (10)1 cells at 32 °C, 206 a.u.; lane 3, (10.1)Val5 cells at 39 °C, 218 a.u.; lane 4, (10)1 cells at 39 °C, 216 a.u.

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.


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Fig. 4.   Wild-type p53 suppresses the transcription of Cox-2. (10)1 and (10.1)Val5 cells grown at 37 °C were shifted to 32 °C for 18 h. Nuclei were isolated. Nuclear run-offs were performed as described under "Experimental Procedures." The Cox-2 and 18 S rRNA cDNAs were immobilized on nitrocellulose membranes and hybridized with labeled nascent RNA transcripts. Lane 1, (10)1 cells; lane 2, (10.1)Val5 cells.

To further investigate the importance of p53 in modulating 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).


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Fig. 5.   Endogenous p53 down-regulates Cox-2 promoter activity in mouse embryo fibroblasts. (10)1 and (10.1)Val5 cells were transfected with 2 µg of murine Cox-2 luciferase construct (TIS10L) containing 963 bases 5' of the Cox-2 transcription start site. After transfection, the cells were grown at 37 °C before being shifted to either 32 °C or 39 °C for 18 h. Six wells were used for each transfection condition. Columns, means; bars, S.D. *, p < 0.01 compared with (10)1 cells.


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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 µg of Cox-2 luciferase construct TIS10L (963 bp) alone (-p53, ) or with 0.5 µg of expression vector for wt p53 (+p53, ). Cells were also co-transfected with 1.0 µg of TIS10L plus 0.5 µg of expression vector for v-src (-p53, black-square) or 1.0 µg of TIS10L plus 0.5 µg of expression vectors for v-src and wt p53 (+p53, black-square). The total amount of DNA was kept constant at 2 µg 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.

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 5' of the transcription start site.


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Fig. 7.   Suppression of Cox-2 promoter activity by p53 is localized to the first 40 bases 5' of the transcription start site. (10)1 and (10.1)Val5 cells were transfected with 2 µg of a series of Cox-2 promoter-luciferase deletion constructs: TIS10L (-963), TIS10-300, TIS10-180, TIS10-80, and TIS10-40. After transfection, the cells were shifted from 37 °C to 32 °C for 18 h to induce wt p53 in the (10.1)Val5 cell line. Six wells were used for each transfection condition. Columns, means; bars, S.D. *, p < 0.01 compared with (10)1 cells.

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 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 -400 to +52 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 -400 to +52 (data not shown). Binding was only observed with a DNA fragment (from -50 to +52) 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).


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Fig. 8.   p53 competes with TBP for binding to the Cox-2 promoter. Gel shift analysis was performed using a fragment of (A) murine Cox-2 or (B) human Cox-2 promoter extending from -50 to +52 bp. The promoter fragments were end-labeled with [gamma -32P]ATP and used in binding reactions with GST.p53, TBP (1 f.p.u.), or both proteins. A and B: lane 1, probe; lane 2, GST.p53 (100 ng); lane 3, TBP; lane 4, TBP and GST.p53 (250 ng); lane 5, TBP and GST.p53 (200 ng); lane 6, TBP and GST.p53 (150 ng); lane 7, TBP and GST.p53 (100 ng).


    DISCUSSION
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INTRODUCTION
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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 (12-16). 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 cell-induced angiogenesis (48). Therefore, the present data provide a possible mechanistic explanation for the regulation of expression 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 down-regulates 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 (33-35, 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.

    ACKNOWLEDGEMENTS

We thank Drs. David Zakim and Babette Weksler for reviewing the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA68136 and the James E. Olson Memorial Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger Supported by a Fellowship from the Cancer Research Foundation of America.

§§ To whom correspondence should be addressed: New York Presbyterian Hospital-Cornell, Division of Gastroenterology, Room F-231, 1300 York Ave., New York, NY 10021. Tel.: 212-746-4403; Fax: 212-746-8447; E-mail: ajdannen{at}mail.med.cornell.edu.

    ABBREVIATIONS

The abbreviations used are: Cox, cyclooxygenase; bp, base pair(s); f.p.u., foot print unit; GST, glutathione S-transferase; PGE2, prostaglandin E2; TBP, TATA-binding protein; wt, wild-type; SSPE, saline-sodium phosphate-EDTA buffer; a.u. arbitrary unit..

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
  1. Smith, W. L., Garavito, R. M., and DeWitt, D. L. (1996) J. Biol. Chem. 271, 33157-33160[Free Full Text]
  2. Herschman, H. R. (1996) Biochim. Biophys. Acta 1299, 125-140[Medline] [Order article via Infotrieve]
  3. Kujubu, D. A., Fletcher, B. S., Varnum, B. C., Lim, R. W., and Herschman, H. R. (1991) J. Biol. Chem. 266, 12866-12872[Abstract/Free Full Text]
  4. Jones, D. A., Carlton, D. P., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (1993) J. Biol. Chem. 268, 9049-9054[Abstract/Free Full Text]
  5. DuBois, R. N., Awad, J., Morrow, J., Roberts, L. J., and Bishop, P. R. (1994) J. Clin. Invest. 93, 493-498[Medline] [Order article via Infotrieve]
  6. Rimarachin, J. A., Jacobson, J. A., Szabo, P., Maclouf, J., Creminon, C., and Weksler, B. B. (1994) Arterioscl. Thromb. 14, 1021-1031[Abstract]
  7. Kelley, D. J., Mestre, J. R., Subbaramaiah, K., Sacks, P. G., Schantz, S. P., Tanabe, T., Inoue, H., Ramonetti, J. T., and Dannenberg, A. J. (1997) Carcinogenesis 18, 795-799[Abstract]
  8. Inoue, H., Yokoyama, C., Hara, S., Tone, Y., and Tanabe, T. (1995) J. Biol. Chem. 270, 24965-24971[Abstract/Free Full Text]
  9. Subbaramaiah, K., Telang, N., Ramonetti, J. T., Araki, R., DeVito, B., Weksler, B. B., and Dannenberg, A. J. (1996) Cancer Res. 56, 4424-4429[Abstract]
  10. Kutchera, W., Jones, D. A., Matsunami, N., Groden, J., McIntyre, T. M., Zimmerman, G. A., White, R. L., and Prescott, S. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4816-4820[Abstract/Free Full Text]
  11. Sheng, G. G., Shao, J., Sheng, H., Hooton, E. B., Isakson, P. C., Morrow, J. D., Coffey, R. J., DuBois, R. N., and Beauchamp, R. D. (1997) Gastroenterology 113, 1883-1891[Medline] [Order article via Infotrieve]
  12. Kargman, S. L., O'Neill, G. P., Vickers, P. J., Evans, J. F., Mancini, J. A., and Jothy, S. (1995) Cancer Res. 55, 2556-2559[Abstract]
  13. Sano, H., Kawahito, Y., Wilder, R. L., Hashiramoto, A., Mukai, S., Asai, K., Kimura, S., Kato, H., Kondo, M., and Hla, T. (1995) Cancer Res. 55, 3785-3789[Abstract]
  14. Ristimaki, A., Honkanen, N., Jankala, H., Sipponen, P., and Harkonen, M. (1997) Cancer Res. 57, 1276-1280[Abstract]
  15. Muller-Decker, K., Scholz, K., Marks, R., and Furstenberger, G. (1995) Mol. Carcinogen. 12, 31-41[Medline] [Order article via Infotrieve]
  16. Parett, M. L., Harris, R. E., Joarder, F. S., Ross, M. S., Clausen, K. P., and Robertson, F. M. (1997) Int. J. Oncol. 10, 503-507
  17. Oshima, M., Dinchuk, J. E., Kargman, S. L., Oshima, H., Hancock, B., Kwong, E., Trazaskos, J. M., Evans, J. F., and Taketo, M. M. (1996) Cell 87, 803-809[Medline] [Order article via Infotrieve]
  18. Tiano, H., Chulada, P., Spalding, J., Lee, C., Loftin, C., Mahler, J., Morham, S., and Langenbach, R. (1997) Proc. Am. Assoc. Cancer Res. 38, 1727
  19. Kawamori, T., Rao, C. V., Seibert, K., and Reddy, B. S. (1998) Cancer Res. 58, 409-412[Abstract]
  20. Baker, S. J., Markowitz, S., Fearon, E. R., Willson, J. K., and Vogelstein, B. (1990) Science 249, 912-915[Medline] [Order article via Infotrieve]
  21. Diller, L., Kassel, J., Nelson, C. E., Gryka, M. A., Litwak, G., Gebhardt, M., Bressac, B., Ozturk, M., Baker, S. J., Vogelstein, B., and Friend, S. H. (1990) Mol. Cell. Biol. 10, 5772-5781[Medline] [Order article via Infotrieve]
  22. Eliyahu, D., Michalovitz, D., Eliyahu, S., Pinhasi-Kimhi, O., and Oren, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8763-8767[Abstract]
  23. Finlay, C. A., Hinds, P. W., and Levine, A. J. (1989) Cell 57, 1083-1093[Medline] [Order article via Infotrieve]
  24. Agarwal, M. L., Agarwal, A., Taylor, W. R., and Stark, G. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8493-8497[Abstract]
  25. Clarke, A. R., Purdie, C. A., Harrison, D. J., Morris, R. G., Bird, C. C., Hooper, M. L., and Wyllie, A. H. (1993) Nature 362, 849-852[CrossRef][Medline] [Order article via Infotrieve]
  26. Lowe, S. W., Schmitt, E. M., Smith, S. W., Osborne, B. A., and Jacks, T. (1993) Nature 362, 847-852[CrossRef][Medline] [Order article via Infotrieve]
  27. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, C. A., Butel, J. S., and Bradley, A. (1992) Nature 356, 215-221[CrossRef][Medline] [Order article via Infotrieve]
  28. Donehower, L. A., Godley, L. A., Aldaz, C. M., Pyle, R., Shi, Y-P., Pinkel, D., Gray, J., Bradley, A., Medina, D., and Varmus, H. E. (1995) Genes Dev. 9, 882-895[Abstract]
  29. Barak, Y., Juven, T., Haffner, R., and Oren, M. (1993) EMBO J. 12, 461-468[Abstract]
  30. Ginsberg, D., Mechtor, F., Yaniv, M., and Oren, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7605-7609[Abstract]
  31. Deb, S. P., Munoz, R. M., Brown, D. R., Subler, M. A., and Deb, S. (1994) Oncogene 9, 1341-1349[Medline] [Order article via Infotrieve]
  32. Ko, L. J., and Prives, C. (1996) Genes Dev. 10, 1054-1072[CrossRef][Medline] [Order article via Infotrieve]
  33. Seto, E., Usheva, A., Zambetti, G. P., Momand, J., Horikoshi, N., Weinmann, R., Levine, A. J., and Shenk, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12028-12032[Abstract]
  34. Werner, H., Karnieli, E., Rauscher, F. J., and LeRoith, D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8318-8323[Abstract/Free Full Text]
  35. Mack, D. H., Vartikar, J., Pipas, J. M., and Laimins, L. A. (1993) Nature 363, 281-283[CrossRef][Medline] [Order article via Infotrieve]
  36. Shen, Y., and Shenk, T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8940-8944[Abstract]
  37. Harvey, D., and Levine, A. J. (1991) Genes Dev. 5, 2375-2385[Abstract]
  38. Wu, X., Bayle, J. H., Olson, D., and Levine, A. J. (1993) Genes Dev. 7, 1126-1132[Abstract]
  39. Zhang, F., Subbaramaiah, K., Altorki, N., and Dannenberg, A. J. (1998) J. Biol. Chem. 273, 2424-2428[Abstract/Free Full Text]
  40. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  41. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract]
  42. Mestre, J. R., Subbaramaiah, K., Sacks, P. G., Schantz, S. P., Tanabe, T., Inoue, H., and Dannenberg, A. J. (1997) Cancer Res. 57, 2890-2895[Abstract]
  43. Sheng, H., Shao, J., Morrow, J. D., Beauchamp, R. D., and DuBois, R. N. (1998) Cancer Res. 58, 362-366[Abstract]
  44. Goodwin, J. S., and Ceuppens, J. (1983) J. Clin. Immunol. 3, 295-314[Medline] [Order article via Infotrieve]
  45. Huang, M., Stolina, M., Sharma, S., Mao, J. T., Zhu, L., Miller, P. W., Wollman, J., Herschman, H., and Dubinett, S. M. (1998) Cancer Res. 58, 1208-1216[Abstract]
  46. Tsujii, M., Kawano, S., and DuBois, R. N. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3336-3340[Abstract/Free Full Text]
  47. Tsujii, M., Kawano, S., Tsuji, S., Sawaoka, H., Hori, M., and DuBois, R. N. (1998) Cell 93, 705-716[Medline] [Order article via Infotrieve]
  48. Bouvet, M., Ellis, L. M., Nishizaki, M., Fujiwara, T., Liu, W., Bucana, C. D., Fang, B., Lee, J. J., and Roth, J. A. (1998) Cancer Res. 58, 2288-2292[Abstract]
  49. Tsujii, M., and DuBois, R. N. (1995) Cell 83, 493-501[Medline] [Order article via Infotrieve]
  50. Ballif, B. A., Mincek, N. V., Barratt, J. T., Wilson, M. L., and Simmons, D. L. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5544-5549[Abstract/Free Full Text]
  51. Chan, T. A., Morin, P. J., Vogelstein, B., and Kinzler, K. W. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 681-686[Abstract/Free Full Text]
  52. Hannun, Y. A. (1996) Science 274, 1855-1859[Abstract/Free Full Text]
  53. Ragimov, N., Krauskopf, A., Navot, N., Rotter, V., Oren, M., and Aloni, Y. (1993) Oncogene 8, 1183-1193[Medline] [Order article via Infotrieve]


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