FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1970395279> ?p ?o ?g. }

• W1970395279 endingPage "3823" @default.
• W1970395279 startingPage "3817" @default.
• W1970395279 abstract "Although p53-inactivating mutations have been described in the majority of human cancers, their role in prostate cancer is controversial as mutations are uncommon, particularly in early lesions. p53 is activated by hypoxia and other stressors and is primarily regulated by the Mdm2 protein. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the conversion of arachidonic acid to prostaglandins and other eicosanoids, is also induced by hypoxia. COX-2 and resultant prostaglandins increase tumor cell proliferation, resistance to apoptosis, and angiogenesis. Previous reports indicate a complex, reciprocal relationship between p53 and COX-2. To elucidate the effects of COX-2 on p53 in response to hypoxia, we transfected the COX-2 gene into the p53-positive, COX-2-negative MDA-PCa-2b human prostate cancer cell line. The expression of functional p53 and Mdm2 was compared in COX-2+ versus COX-2- cells under normoxic and hypoxic conditions. Our results demonstrated that hypoxia increases both COX-2 protein levels and p53 transcriptional activity in these cells. Forced expression of COX-2 increased tumor cell viability and decreased apoptosis in response to hypoxia. COX-2+ cells had increased Mdm2 phosphorylation in either normoxic or hypoxic conditions. Overexpression of COX-2 abrogated hypoxia-induced p53 phosphorylation and promoted the binding of p53 to Mdm2 protein in hypoxic cells. In addition, COX-2-expressing cells exhibited decreased hypoxia-induced nuclear accumulation of p53 protein. Finally, forced expression of COX-2 suppressed both basal and hypoxia-induced p53 transcriptional activity, and this effect was mimicked by the addition of PGE2 to wild-type cells. These results demonstrated a role for COX-2 in the suppression of hypoxia-induced p53 activity via both direct effects and indirect modulation of Mdm2 activity. These data imply that COX-2-positive prostate cancer cells can have impaired p53 function even in the presence of wild-type p53 and that p53 activity can be restored in these cells via inhibition of COX-2 activity. Although p53-inactivating mutations have been described in the majority of human cancers, their role in prostate cancer is controversial as mutations are uncommon, particularly in early lesions. p53 is activated by hypoxia and other stressors and is primarily regulated by the Mdm2 protein. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the conversion of arachidonic acid to prostaglandins and other eicosanoids, is also induced by hypoxia. COX-2 and resultant prostaglandins increase tumor cell proliferation, resistance to apoptosis, and angiogenesis. Previous reports indicate a complex, reciprocal relationship between p53 and COX-2. To elucidate the effects of COX-2 on p53 in response to hypoxia, we transfected the COX-2 gene into the p53-positive, COX-2-negative MDA-PCa-2b human prostate cancer cell line. The expression of functional p53 and Mdm2 was compared in COX-2+ versus COX-2- cells under normoxic and hypoxic conditions. Our results demonstrated that hypoxia increases both COX-2 protein levels and p53 transcriptional activity in these cells. Forced expression of COX-2 increased tumor cell viability and decreased apoptosis in response to hypoxia. COX-2+ cells had increased Mdm2 phosphorylation in either normoxic or hypoxic conditions. Overexpression of COX-2 abrogated hypoxia-induced p53 phosphorylation and promoted the binding of p53 to Mdm2 protein in hypoxic cells. In addition, COX-2-expressing cells exhibited decreased hypoxia-induced nuclear accumulation of p53 protein. Finally, forced expression of COX-2 suppressed both basal and hypoxia-induced p53 transcriptional activity, and this effect was mimicked by the addition of PGE2 to wild-type cells. These results demonstrated a role for COX-2 in the suppression of hypoxia-induced p53 activity via both direct effects and indirect modulation of Mdm2 activity. These data imply that COX-2-positive prostate cancer cells can have impaired p53 function even in the presence of wild-type p53 and that p53 activity can be restored in these cells via inhibition of COX-2 activity. The p53 tumor suppressor protein plays a critical role in the regulation of cell growth, the protection of normal cells from malignant transformation, and the response of tumor cells to radiation and chemotherapy (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6738) Google Scholar, 2Lane D.P. Fischer P.M. Nature. 2004; 427: 789-799Crossref PubMed Scopus (41) Google Scholar). Although alterations in the p53 gene are found in more than half of all human cancers, the role of p53 mutations in human prostate cancer is controversial, as such mutations are uncommon, particularly in early lesions (3Mirchandani D. Zheng J. Miller G.J. Ghosh A.K. Shibata D.K. Cote R.J. Roy-Burman P. Am. J. Pathol. 1995; 147: 92-101PubMed Google Scholar, 4Effert P.J. McCoy R.H. Walther P.J. Liu E.T. J. Urol. 1993; 150: 257-261Crossref PubMed Scopus (88) Google Scholar). After exposure of cells to a variety of stressors, including genotoxic DNA damage, nucleotide depletion and hypoxia, wild-type p53 protein is activated (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6738) Google Scholar). Recent reports demonstrate alterations in p53 activation and stabilization in human premalignant and malignant lesions that retain the expression of functional wild-type p53 (5Liang S.H. Clarke M.F. Eur. J. Biochem. 2001; 268: 2779-2783Crossref PubMed Scopus (162) Google Scholar, 6Issacs J.S. Hardman R. Carman T.A. Barnet J.C. Weissman B.E. Cell Growth Differ. 1998; 9: 545-555PubMed Google Scholar, 7Moll U.M. Laguaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (390) Google Scholar). This wild-type p53 inactivation may result from either abnormal sequestration in the cytoplasm where it is functionally muted (6Issacs J.S. Hardman R. Carman T.A. Barnet J.C. Weissman B.E. Cell Growth Differ. 1998; 9: 545-555PubMed Google Scholar, 7Moll U.M. Laguaglia M. Benard J. Riou G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4407-4411Crossref PubMed Scopus (390) Google Scholar) or increased binding of the protein to its critical negative regulator, Mdm2, which prevents its activation and promotes its degradation via ubiquitination (8Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). Cyclooxygenase-2 (COX-2), 1The abbreviations used are: COX, cyclooxygenase; PG, prostaglandin; HIF, hypoxia-inducible factor; Mdm2, murine double minute-2; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TUNEL, terminal deoxynucletidyl transferase-mediated UTP-biotin nick end labeling. an inducible enzyme that catalyzes the conversion of arachidonic acid to prostaglandins (PGs) and other eicosanoids, is also induced in response to hypoxia and other stressors (9Herschman H.R. Annu. Rev. Biochem. 1991; 60: 281-319Crossref PubMed Scopus (946) Google Scholar, 10Smith W.L. Garavito R.M. Dewitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1851) Google Scholar, 11Schmedtji Jr., J.F. Ji Y.S. Liu W.L. DuBois R.N. Runge M.S. J. Biol. Chem. 1997; 272: 601-608Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 12Liu X.H. Kirschenbaum A. Yao S. Stearns M.E. Holland J.F. Claffey K. Levine A.C. Clin. Exp. Metastasis. 1999; 17: 687-694Crossref PubMed Scopus (153) Google Scholar). COX-2 and resultant PGs increase tumor cell proliferation, resistance to apoptosis, and angiogenesis in colon, prostate, and other cancers (13Tsujii M. Dubois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2137) Google Scholar, 14Tsujii M. Kawano S. Tsuji S. Tsuji S. Sawaoka H. Hori M. DuBois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2214) Google Scholar, 15Tsujii M. Kawano S. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Crossref PubMed Scopus (1328) Google Scholar, 16Kirschenbaum A. Liu X.H. Yao S. Levine A.C. Urology. 2001; 58: 127-131Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 17Liu X.H. Yao S. Kirschenbaum A. Levine A.C. Cancer Res. 1998; 58: 4245-4249PubMed Google Scholar). Our group demonstrated that COX-2 expression and PGE2 production are increased in prostatic inflammation, prostatic intraepithelial neoplasia, and some primary prostate cancer cells (18Kirschenbaum A. Klausner A.P. Lee R. Unger P. Yao S. Liu X.H. Levine A.C. Urology. 2000; 56: 671-676Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). We also reported that COX-2 and PGE2 are key mediators of cellular responses observed after hypoxia resulting from induction of vascular endothelial cell factor (12Liu X.H. Kirschenbaum A. Yao S. Stearns M.E. Holland J.F. Claffey K. Levine A.C. Clin. Exp. Metastasis. 1999; 17: 687-694Crossref PubMed Scopus (153) Google Scholar, 19Liu X.H. Kirschenbaum A. Yao S. Lee R. Holland J.F. Levine A.C. J. Urol. 2000; 164: 820-825Crossref PubMed Google Scholar) and the master oxygen sensor, hypoxia-inducible factor (HIF)-1α (20Liu X.H. Kirschenbaum A. Lu M. Yao S. Dosoretz A. Holland J.F. Levine A.C. J. Biol. Chem. 2002; 277: 50081-50086Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Previous reports indicate complex, functional, and reciprocal interactions between the COX-2 and p53 systems. An association between COX-2 overexpression and p53 mutations or low p53 protein levels has been reported in human gastric and breast cancers (21Leung W.K. To K.F. Ng Y.P. Lee T.L. Lau J.Y.W. Chan F.K.L. Ng E.K.W. Chung S.C.S. Sung J.J.Y. Br. J. Cancer. 2001; 84: 335-339Crossref PubMed Scopus (113) Google Scholar, 22Ristimaki A. Sivula A. Lundin J. Lundin M. Salminen T. Haglund C. Joensuu H. Isola J. Cancer Res. 2002; 62: 632-635PubMed Google Scholar). Moreover, electrophilic PGs produced by COX-2 were shown to inhibit wild-type p53 activity by covalently binding p53, preventing its nuclear accumulation (23Moos P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (66) Google Scholar). It has been reported that inhibition of COX-2 in a colon cancer cell line by celecoxib, a selective COX-2 inhibitor, increases the nuclear localization of active p53 (24Swamy M.V. Herzog C.R. Rao C.V. Cancer Res. 2003; 63: 5239-5242PubMed Google Scholar). Subbaramaiah et al. (25Subbaramaiah K. Altorki N. Chung W.J. Mestre J.R. Sampat A. Dannenberg A.J. J. Biol. Chem. 1999; 274: 10911-10915Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) demonstrated that p53 inhibits COX-2 expression. Recent evidence also suggests that COX-2 and p53 have reciprocal effects on cellular responses to hypoxia via modulation of HIF-1α activity (20Liu X.H. Kirschenbaum A. Lu M. Yao S. Dosoretz A. Holland J.F. Levine A.C. J. Biol. Chem. 2002; 277: 50081-50086Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 26Ravi R. Mookerjee B. Bhujwalla Z.M. Sutter C.H. Artemov D. Zeng Q. Dillehay L.E. Madan A. Semenza G.L. Bedi A. Genes Dev. 2000; 14: 34-44Crossref PubMed Google Scholar). We hypothesized that COX-2 expression in prostate cancer cells decreases wild-type p53 stability, nuclear accumulation, and activity after hypoxia, via both direct effects on p53 activation and indirectly by activation of its primary negative regulator, Mdm2. In the present study, we utilized a prostate cancer cell line, MDA-PCa-2b, as an in vitro model. This cell line expresses the wild-type p53 gene and does not express COX-2 (27Navone N.M. Olive M. Ozen M. Davis R. Troncoso P. Ty S.-M. Johnson D. Pollack A. Pathak S. von Eschenbach A.C. Logethetis C.J. Clin. Cancer Res. 1997; 3: 2493-2500PubMed Google Scholar). We tested the effects of forced expression of COX-2 on cellular proliferation, apoptosis, and the activity of p53 and Mdm2 proteins, under both normoxic and hypoxic conditions. Cell Line, Cell Culture, and Reagents—The MDA-PCa-2b human prostate cancer cell line was purchased from ATCC (Gaithersburg, MD). Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum under normoxic conditions (20% O2, 5% CO2, 75% N2) in a humidified Napco incubator at 37 °C. Hypoxic stimulation was produced with an ambient oxygen concentration of 0.1% using a controlled incubator with CO2/O2 monitoring and CO2/N2 gas sources. Oxygen levels were determined by an indicator. To reach a low oxygen level (0.1%), the chamber was sealed and subjected to five rounds of evacuation followed by flushing with 95% N2-5% CO2 as described by Graeber et al. (28Graeber T.G. Peterson J.F. Tasi M. Monica K. Fornace Jr., A.J. Giaccia A. Mol. Cell. Biol. 1994; 14: 6264-6277Crossref PubMed Google Scholar). To prevent reoxygenation of hypoxic cells, the medium or lysis buffer was pre-equilibrated to the experimental oxygen conditions overnight and added to cells on ice. Prostaglandins, i.e. PGE2, PGI2, PGF2α, and PGD2, were purchased from Cayman Co., Ann Arbor, MI. Transfection—The expression vector pcDNA-hCOX-2, containing the full-length cDNA encoding the human COX-2 gene, was a generous gift from Dr. T. Hla (Department of Physiology, University of Connecticut). Transient transfection was performed using Lipofectamine™ 2000 reagent according to the manufacturer's instructions (Invitrogen). Cells used as a mock control were transfected with empty pcDNA1 vector (Invitrogen). Typically, 3 μg of plasmid DNA were used per 60-mm dish. All assays were performed after 40 h of transfections. TUNEL Assay—MDA-PCa-2b cells (mock-transfected and COX-2+ transfected) were cultured in 12-well cluster plates. Incubations were continued under either normoxic or hypoxic conditions for 8 h. After washing with ice-cold phosphate-buffered saline, apoptotic cells were assayed using the TUNEL method with the ApopTag in situ apoptotic detection kit (Oncor, Gaithersburg, MD) according to the manufacturer's instructions. The labeled cells were examined using a fluorescent microscope. Preparation of Cell Lysates and Cytosolic and Nuclear Proteins— MDA-Pca-2b cells cultured under the desired conditions were lysed as described previously (17Liu X.H. Yao S. Kirschenbaum A. Levine A.C. Cancer Res. 1998; 58: 4245-4249PubMed Google Scholar). Briefly, cells were rinsed twice with ice-cold phosphate-buffered saline and scraped with 1.5 ml of phosphate-buffered saline containing 4 mm iodoacetate. After centrifugation, the pellets were resuspended in CHAPS extraction solution (10 mm CHAPS, 2 mm EDTA, pH 8.0, and 4 mm iodoacetate in phosphate-buffered saline) with protease inhibitors. The samples were incubated for 30 min on ice and centrifuged at 15,000 × g for 10 min. The supernatants were collected and stored at -70 °C. Proteins from the cytosolic and nuclear fractions were isolated using a commercial kit purchased from Pierce, according to the manufacturer's instructions. Protein content was assayed using a kit from Bio-Rad. Immunoprecipitation and Immunoblotting—For immunoprecipitation, the lysates were precleaned with protein G-Sepharose beads for 2 h at 4 °C. The lysates were then incubated with 1 μg of monoclonal anti-Mdm2 antibody (clone IF2) from Calbiochem, followed by the addition of protein G-Sepharose beads. The immunoprecipitated products were then subjected to Western blot analysis. For immunoblotting, the cell lysates were electrophoresed on SDS-polyacrylamide gel, electro-phoretically transferred to a polyvinylidene difluoride membrane (PerkinElmer Life Sciences), and incubated with targeting antibodies overnight at 4 °C. Secondary horseradish peroxidase-linked donkey anti-mouse IgG (Amersham Biosciences) was used. Filters were developed by the enhanced chemiluminescence system (Amersham Biosciences). Antibodies against p53, phospho-p53, and phospho-Mdm2 (Ser-166) were products from Cell Signaling Technology (Beverly, MA). Endogenous Mdm2 antibodies were purchased from Calbiochem, and anti-COX-2 antibodies were obtained from Transduction Labs, Lexington, KY. β-Actin was used as the internal control in all Western blot analyses. Luciferase Assay—The reporter plasmid pGL3-Luc-E1bTATA, a gift from Dr. J. Manfredi (Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine), was used to assay p53 transcriptional activity. The plasmid contains the p21 promoter sequences and the coding region for firefly luciferase upstream of the minimal adenovirus E1b promoter. MDA-PCa-2b cells were cultured in 12-well cluster plates and co-transfected with 1 μg of the reporter plasmid or empty pGL3-Luc vector and pcDNA-hCOX-2 or empty pcDNA plasmid. Internal normalization was performed by co-transfection of a β-galactosidase expression vector driven by the CMV promoter (Clontech). Total DNA transfected into each plate was 3 μg in each 60-mm dish. After 40 h, the transfected cells were lysed by scraping into reporter buffer (Clontech), total protein concentration was determined, and luciferase and β-galactosidase activities were assayed and quantitated using a TD-20e luminometer. The resulting activities were normalized to protein concentrations and β-galactosidase activity. Hypoxia Induces COX-2 Expression in COX-2-transfected MDA-PCa-2b Cells—It has been reported that COX-2 expression is increased by hypoxia and cobalt chloride (a hypoxia surrogate) in human vascular endothelial cells and a metastatic prostate cancer cell line, respectively (11Schmedtji Jr., J.F. Ji Y.S. Liu W.L. DuBois R.N. Runge M.S. J. Biol. Chem. 1997; 272: 601-608Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, 12Liu X.H. Kirschenbaum A. Yao S. Stearns M.E. Holland J.F. Claffey K. Levine A.C. Clin. Exp. Metastasis. 1999; 17: 687-694Crossref PubMed Scopus (153) Google Scholar). In our initial studies, we examined the basal and hypoxic increase in COX-2 protein expression in mock-transfected (control) and COX-2-transfected MDA-PCa-2b cells. As shown in Fig. 1, mock-transfected MDA-PCa-2b cells do not express COX-2 protein under either normoxic or hypoxic conditions (Fig. 1A). When these cells were transfected with the COX-2 gene, we demonstrated that COX-2 protein expression is increased under hypoxic conditions in a time-dependent fashion (Fig. 1B). As the COX-2 construct utilized does not contain the promoter region of the gene, we have assumed that regulation of protein expression by hypoxia is occurring at the post-transcriptional level, a phenomenon that has previously been reported (29Dixon D.A. Curr. Pharm. Des. 2004; 10: 635-646Crossref PubMed Scopus (77) Google Scholar, 30Simmons D.L. Botting R.M. Hla T. Pharmacol. Rev. 2004; 56: 387-437Crossref PubMed Scopus (1344) Google Scholar). COX-2 Expression Prevents Apoptosis in Hypoxic Prostate Cancer Cells—Hypoxia induces cell cycle arrest at the G0/G1 phases and cellular apoptosis via both p53-independent (28Graeber T.G. Peterson J.F. Tasi M. Monica K. Fornace Jr., A.J. Giaccia A. Mol. Cell. Biol. 1994; 14: 6264-6277Crossref PubMed Google Scholar) and p53-dependent (31Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2171) Google Scholar, 32Kunz M. Ibrahim S.M. Mol. Cancer. 2003; 2: 23-36Crossref PubMed Scopus (162) Google Scholar, 33Suzuki H. Tomida A. Tsuruo T. Oncogene. 2001; 20: 5779-5788Crossref PubMed Scopus (203) Google Scholar) pathways. We examined the effect of COX-2 on cell viability and apoptosis under hypoxic conditions. Hypoxic treatment dramatically decreased cell viability and increased apoptosis in the COX-2-, mock-transfected versus COX-2+ MDA-PCa-2b cells (Fig. 2A). TUNEL assay revealed that the decrease in viable cell numbers resulted, at least in part, from induction of apoptosis by hypoxia in the COX-2- cells. COX-2+ cells were resistant to the apoptotic effects of hypoxia (Fig. 2B). COX-2 Modulates Mdm2 Phosphorylation under Normoxic and Hypoxic Conditions—Mdm2 is a critical negative regulator of p53 protein expression, nuclear localization, and transcriptional activity (8Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). Both Mdm2 and p53 proteins are activated by phosphorylation. Hypoxic induction of p53 accumulation is partially modulated by decreased phosphorylation leading to decreased activation of Mdm2 (34Alarcon R. Koumenis C. Geyer R.K. Maki C.G. Giaccia A.J. Cancer Res. 1999; 59: 6046-6051PubMed Google Scholar, 35Blattner C. Hay T. Meek D.W. Lane D.P. Mol. Cell. Biol. 2002; 22: 6170-6182Crossref PubMed Scopus (126) Google Scholar). Fig. 3 demonstrates the effects of forced COX-2 expression on Mdm2 levels and phosphorylation under both normoxic and hypoxic conditions. Consistent with previous reports (34Alarcon R. Koumenis C. Geyer R.K. Maki C.G. Giaccia A.J. Cancer Res. 1999; 59: 6046-6051PubMed Google Scholar, 35Blattner C. Hay T. Meek D.W. Lane D.P. Mol. Cell. Biol. 2002; 22: 6170-6182Crossref PubMed Scopus (126) Google Scholar), parental, mock-transfected MDA-PCa-2b cells expressed measurable basal levels of phospho-Mdm2 protein. Hypoxia significantly decreased Mdm2 phosphorylation in the COX-2-, mock-transfected cells. In contrast, forced expression of COX-2 led to an increase in Mdm2 phosphorylation under normoxic conditions. The enhanced expression of phospho-Mdm2 protein was sustained in the COX-2+ cells, even under hypoxic conditions (Fig. 3A). These data demonstrated that COX-2 not only promotes Mdm2 phosphorylation under normoxic conditions but, furthermore, prevents hypoxic inhibition of phosphorylated Mdm2 protein. In contrast, hypoxia induced a modest increase in endogenous Mdm2 levels in both control and COX-2+ cells (Fig. 3B). Fig. 3C depicts the quantitative analysis of relative expression levels of Mdm2 and phospho-Mdm2 (inactive and active Mdm2, respectively) and demonstrates that forced expression of COX-2 results in increased active, phosphorylated Mdm2 protein expression. COX-2 Abrogates Hypoxic Induction of Ser-20 Phosphorylation of p53 and Promotes the Binding of p53 to Mdm2 in Response to Hypoxia—Phosphorylation is a critical modulator of p53 activity (36Unger T. Juven-Gershon T. Moallem E. Berger M. Sionov R.V. Lozano G. Oren M. Haupt Y. EMBO J. 1999; 18: 1805-1814Crossref PubMed Scopus (315) Google Scholar). It has also been proposed that phosphorylation of p53 could impede binding between p53 and Mdm2 (36Unger T. Juven-Gershon T. Moallem E. Berger M. Sionov R.V. Lozano G. Oren M. Haupt Y. EMBO J. 1999; 18: 1805-1814Crossref PubMed Scopus (315) Google Scholar, 37Ashcroft M. Vousden K.H. Oncogene. 1999; 18: 7637-7643Crossref PubMed Scopus (371) Google Scholar) leading to stabilization and activation of p53 protein (8Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar, 38Shieh S.Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1754) Google Scholar). We examined the levels of p53 phosphoprotein under normoxic and hypoxic conditions in the mock-transfected versus COX-2-transfected cells. Among several potential phosphorylation sites of the p53 protein, Ser-15, Ser-20, and Ser-392 are reported to be involved in the regulation of p53 protein activation in response to a variety of stress signals (36Unger T. Juven-Gershon T. Moallem E. Berger M. Sionov R.V. Lozano G. Oren M. Haupt Y. EMBO J. 1999; 18: 1805-1814Crossref PubMed Scopus (315) Google Scholar, 39Hao M. Lowy A.M. Kapoor M. Deffle A. Liu G. Lozano G. J. Biol. Chem. 1996; 271: 29380-29385Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 40Jimemez G.S. Khan S.H. Stommel J.M. Wahl G.M. Oncogene. 1999; 18: 7656-7665Crossref PubMed Scopus (167) Google Scholar). Under normoxic conditions, there was no demonstrable p53 phosphoprotein in either cell population. In response to hypoxia, p53 protein was subjected to phosphorylation at Ser-20 only in the COX-2-negative, mock-transfected MDA-PCa-2b cells (Fig. 4A). Of note, there were no detectable levels of p53 phosphoprotein at the other sites (data not shown). In contrast, when these cells were transfected with the COX-2 gene, the induction of p53 phosphorylation at Ser-20 was abolished. Hypoxia also induced endogenous p53 protein levels in mock-transfected prostate cancer cells but had no effect on the cells that were engineered to overexpress COX-2 (Fig. 4A). To determine whether hypoxic induction of p53 phosphorylation influences the binding of the protein to Mdm2 (a critical step of p53 protein degradation), we performed immunoprecipitation followed by immunoblot analysis. As demonstrated in Fig. 4B, there was no difference in the binding of p53 to Mdm2 under aerobic conditions between the mock- and COX-2-transfected cells. In response to hypoxia, Mdm2-p53 binding was decreased in the control cells. In contrast, COX-2 gene transfection led to an increase in Mdm2-p53 binding. These results suggested a negative correlation between the amount of phospho-p53 protein (Ser-20) and the ability of p53 to bind to Mdm2. COX-2 Suppresses Basal and Hypoxic Induction of p53 Nuclear Localization—p53 protein translocates from the cytoplasm to the nucleus upon stress stimulation, and this nuclear targeting is essential for its activity as a transcriptional regulator (5Liang S.H. Clarke M.F. Eur. J. Biochem. 2001; 268: 2779-2783Crossref PubMed Scopus (162) Google Scholar). A recent study reported that celecoxib, a selective COX-2 inhibitor, inhibits active p53 nuclear localization in a human colon cancer cell line (24Swamy M.V. Herzog C.R. Rao C.V. Cancer Res. 2003; 63: 5239-5242PubMed Google Scholar). We next examined the effect of COX-2 expression on the subcellular distribution of p53 protein under both normoxic and hypoxic conditions. As shown in Fig. 5, Western blotting using samples derived from the cytosolic and nuclear fractions revealed that forced expression of COX-2 resulted in cytosolic retention and decreased nuclear accumulation of p53 protein under normoxic conditions. In response to hypoxia, p53 protein translocated to the nucleus in the COX-2-, mock-transfected cells. Forced expression of COX-2 prevented the nuclear accumulation of p53 in response to hypoxia (Fig. 5). These results revealed a novel role for COX-2 in the suppression of hypoxia-induced p53 protein nuclear localization, thereby inhibiting p53 activation in response to hypoxic signals. COX-2 Inhibits Basal and Hypoxia-induced p53 Transcriptional Activities—We next investigated the effect of COX-2 expression on p53 transcriptional activity using a luciferase assay. COX-2- and COX-2+ MDA-Ca-2b cells were co-transfected with a plasmid containing the p21 promoter sequence and the coding region for firefly luciferase upstream of the minimal adenovirus E1b promoter. Fig. 6 demonstrates that COX-2-expressing cells had decreased p53 transcriptional activity as compared with COX-2- cells in normoxic conditions. COX-2 expression in these cells also prevented the hypoxic increase in p53 transcriptional activity seen in the parental, mock-transfected cells (Fig. 6). To determine whether the effects of COX-2 are the direct result of its enzymatic activity, four major prostaglandin products of the COX-catalyzed pathway, i.e. PGE2, PGI2, PGF2α, and PGD2, were tested for their possible effects on p53 activity under normoxic and hypoxic conditions. As shown in Fig. 6, although only a modest effect was seen under normoxic conditions, PGE2 exhibited a significant inhibitory effect on p53 transcriptional activity under hypoxic conditions, accounting for 90% of the effect induced by COX-2 overexpression in the same cell line. We also compared PGE2 production in COX-2-transfected and mock-transfected cells under normoxic and hypoxic conditions. Parental cells that were mock-transfected had undetectable levels of PGE2. Forced expression of COX-2 in these cells increased PGE2 production, and a time-dependent further increase was observed when the cells were cultured under hypoxic conditions (data not shown). In comparison, PGF2α modestly inhibited p53 transcriptional activity in both normoxic and hypoxic conditions. Neither PGI2 nor PGD2 had any effect on these parameters. It is well established that the p53 tumor suppressor protein plays a critical role in cancer development and progression. There are also accumulating data implicating COX-2 overexpression and increased secretion of prostaglandins (notably PGE2) in a variety of neoplasms. Several reports confirm a reciprocal relationship between the p53 and COX-2 systems. Wild-type p53 has been shown to decrease COX-2 expression and activity (22Ristimaki A. Sivula A. Lundin J. Lundin M. Salminen T. Haglund C. Joensuu H. Isola J. Cancer Res. 2002; 62: 632-635PubMed Google Scholar, 25Subbaramaiah K. Altorki N. Chung W.J. Mestre J.R. Sampat A. Dannenberg A.J. J. Biol. Chem. 1999; 274: 10911-10915Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Prostaglandins derived from the COX-2-catalyzed pathway inactivate wild-type p53 in colon cancer cell lines (23Moos P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (66) Google Scholar). Furthermore, inhibitors of COX-2 increase the activity of wild-type p53 by promoting its nuclear localization in colon cancer cells (24Swamy M.V. Herzog C.R. Rao C.V. Cancer Res. 2003; 63: 5239-5242PubMed Google Scholar). Although p53 is commonly mutated in solid tumors, the rate of such mutations in prostate cancer, particularly in early lesions, is not high (3Mirchandani D. Zheng J. Miller G.J. Ghosh A.K. Shibata D.K. Cote R.J. Roy-Burman P. Am. J. Pathol. 1995; 147: 92-101PubMed Google Scholar, 4Effert P.J. McCoy R.H. Walther P.J. Liu E.T. J. Urol. 1993; 150: 257-261Crossref PubMed Scopus (88) Google Scholar). Our group and others, however, have demonstrated increased COX-2 expression in preneoplastic and cancerous prostate lesions (18Kirschenbaum A. Klausner A.P. Lee R. Unger P. Yao S. Liu X.H. Levine A.C. Urology. 2000; 56: 671-676Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). We have further shown that COX-2 inhibitors induce prostate cancer cell apoptosis in vitro and in vivo, decrease tumor angiogenesis, and decrease the levels and activity of the master oxygen sensor, HIF-1α (12Liu X.H. Kirschenbaum A. Yao S. Stearns M.E. Holland J.F. Claffey K. Levine A.C. Clin. Exp. Metastasis. 1999; 17: 687-694Crossref PubMed Scopus (153) Google Scholar, 17Liu X.H. Yao S. Kirschenbaum A. Levine A.C. Cancer Res. 1998; 58: 4245-4249PubMed Google Scholar, 19Liu X.H. Kirschenbaum A. Yao S. Lee R. Holland J.F. Levine A.C. J. Urol. 2000; 164: 820-825Crossref PubMed Google Scholar, 20Liu X.H. Kirschenbaum A. Lu M. Yao S. Dosoretz A. Holland J.F. Levine A.C. J. Biol. Chem. 2002; 277: 50081-50086Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Loss of p53 activity in tumor cells enhances the levels of HIF-1α and augments HIF-1-dependent activation of gene transcription (i.e. vascular endothelial cell factor) in response to hypoxia (26Ravi R. Mookerjee B. Bhujwalla Z.M. Sutter C.H. Artemov D. Zeng Q. Dillehay L.E. Madan A. Semenza G.L. Bedi A. Genes Dev. 2000; 14: 34-44Crossref PubMed Google Scholar). In addition, loss of p53 function is associated with reduced cellular apoptotic potential in response to hypoxia (31Graeber T.G. Osmanian C. Jacks T. Housman D.E. Koch C.J. Lowe S.W. Giaccia A.J. Nature. 1996; 379: 88-91Crossref PubMed Scopus (2171) Google Scholar). Therefore, we hypothesized that COX-2 may inhibit wild-type p53 activity, particularly under hypoxic conditions. In the present study, we demonstrated that hypoxia increases both COX-2 protein levels and p53 transcriptional activity in a human prostate cancer cell line. Forced expression of COX-2 conferred resistance to hypoxia-induced apoptosis in these prostate cancer cells. Forced expression of COX-2 also promoted Mdm2 phosphorylation under normoxic conditions and prevented hypoxia-induced decreased phosphorylation of Mdm2. The Mdm2 protein is an important inhibitor of p53 function (8Prives C. Cell. 1998; 95: 5-8Abstract Full Text Full Text PDF PubMed Scopus (629) Google Scholar). Mdm2 both represses p53 transcriptional activity and promotes the degradation of p53 by the ubiquitin-proteasome system. It has previously been demonstrated that phosphorylation of Mdm2 is essential for its translocation from the cytoplasm to the nucleus, where it binds and inactivates p53 (41Mayo L.D. Donner D.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11598-11603Crossref PubMed Scopus (956) Google Scholar, 42Roth J. Dobbelstein M. Freedman D.A. Shenk T. Levine A.J. EMBO J. 1998; 17: 554-564Crossref PubMed Scopus (532) Google Scholar, 43Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3077-3080Crossref PubMed Scopus (297) Google Scholar). It has also been reported that hypoxia-induced p53 accumulation and stabilization is through hypophosphorylation of Mdm2 and down-regulation of Mdm2 protein expression (34Alarcon R. Koumenis C. Geyer R.K. Maki C.G. Giaccia A.J. Cancer Res. 1999; 59: 6046-6051PubMed Google Scholar, 35Blattner C. Hay T. Meek D.W. Lane D.P. Mol. Cell. Biol. 2002; 22: 6170-6182Crossref PubMed Scopus (126) Google Scholar). In addition, Mdm2 has been linked to the regulation of p53 nucleocytoplasmic trafficking (5Liang S.H. Clarke M.F. Eur. J. Biochem. 2001; 268: 2779-2783Crossref PubMed Scopus (162) Google Scholar, 42Roth J. Dobbelstein M. Freedman D.A. Shenk T. Levine A.J. EMBO J. 1998; 17: 554-564Crossref PubMed Scopus (532) Google Scholar, 43Tao W. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3077-3080Crossref PubMed Scopus (297) Google Scholar). Our findings demonstrated that at least one mechanism whereby COX-2 inhibits hypoxic activation of p53 is via phosphorylation and activation of Mdm2. One of the most obvious ways to protect p53 from Mdm2-mediated degradation is to prevent physical interactions between the two proteins. The structural requirements for p53/Mdm2 interactions indicate that phosphorylation at sites within the N terminus of p53 (including serine 15, 20, and 33) may impede binding of the two proteins (36Unger T. Juven-Gershon T. Moallem E. Berger M. Sionov R.V. Lozano G. Oren M. Haupt Y. EMBO J. 1999; 18: 1805-1814Crossref PubMed Scopus (315) Google Scholar, 37Ashcroft M. Vousden K.H. Oncogene. 1999; 18: 7637-7643Crossref PubMed Scopus (371) Google Scholar). We have reported that forced expression of COX-2 directly modulated p53 activity via effects on p53 protein phosphorylation, nuclear localization, and transcriptional activity. In COX-2-null MDA-PCa-2B cells, hypoxia increased p53 phosphorylation at Ser-20, a critical phosphorylation site involved in its nuclear translocation, activation, and protection from degradation (36Unger T. Juven-Gershon T. Moallem E. Berger M. Sionov R.V. Lozano G. Oren M. Haupt Y. EMBO J. 1999; 18: 1805-1814Crossref PubMed Scopus (315) Google Scholar, 44Dumaz N. Milne D.M. Jardine L.J. Meek D.W. Biochem. J. 2001; 359: 459-464Crossref PubMed Scopus (60) Google Scholar). Overexpression of COX-2 inhibited hypoxia-induced phosphorylation at Ser-20, thereby enhancing the binding between p53 and Mdm2, leading to p53 protein ubiquitination and proteasomal degradation. In addition, we provided evidence that overexpression of COX-2 suppressed basal and hypoxia-induced nuclear translocation of p53 protein. Finally, COX-2-expressing cells had decreased p53 transcriptional activity as compared with COX-2-null cells, under both normoxic and hypoxic conditions. Exogenous addition of PGE2, a major product of the COX-2-catalyzed pathway, mimicked the effect of COX-2 overexpression on the repression of p53 transcriptional activity in response to hypoxia. Both PGE2 and PGF2α addition partially mimicked the effect of COX-2 overexpression on p53 transcriptional activity under normoxic conditions. Although these results implied that effects on p53 activity are primarily mediated by COX-2-dependent pathways, we cannot rule out some COX-2-independent effects in this model system. Our results demonstrated that COX-2 can inhibit wild-type p53 activation, particularly in response to hypoxic signals, via direct modulation of p53 protein as well as through the activation of the p53 inhibitor Mdm2. Somatic mutation of p53 is probably a late event in prostate oncogenesis. However, epigenetic modifications of p53 with resultant inactivation of its function have been reported in other tumors (23Moos P.J. Edes K. Fitzpatrick F.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9215-9220Crossref PubMed Scopus (66) Google Scholar, 24Swamy M.V. Herzog C.R. Rao C.V. Cancer Res. 2003; 63: 5239-5242PubMed Google Scholar). Our results provided a possible link between overexpression of COX-2 and inactivation of wild-type p53 function in prostate cancer cells. These effects were most evident under hypoxic conditions and may help explain the relative chemo- and radiation-resistance of hypoxic prostate cancer cells, even those expressing wild-type p53. These data implied that inhibition of COX-2 can restore normal p53 function in response to intratumoral hypoxia and thereby render the cells more sensitive to therapeutic regimens. We thank Dr. T. Hla for the COX-2 expression vector and Dr. J. Manfredi for the pGL3-Luc-E1bTATA vector." @default.
• W1970395279 created "2016-06-24" @default.
• W1970395279 creator A5014280061 @default.
• W1970395279 creator A5023108032 @default.
• W1970395279 creator A5030531770 @default.
• W1970395279 creator A5075926965 @default.
• W1970395279 creator A5085576609 @default.
• W1970395279 date "2005-02-01" @default.
• W1970395279 modified "2023-10-15" @default.
• W1970395279 title "Cyclooxygenase-2 Suppresses Hypoxia-induced Apoptosis via a Combination of Direct and Indirect Inhibition of p53 Activity in a Human Prostate Cancer Cell Line" @default.
• W1970395279 cites W1484675482 @default.
• W1970395279 cites W1824671129 @default.
• W1970395279 cites W1978518304 @default.
• W1970395279 cites W1991106719 @default.
• W1970395279 cites W1991494214 @default.
• W1970395279 cites W2011698701 @default.
• W1970395279 cites W2014170738 @default.
• W1970395279 cites W2028999918 @default.
• W1970395279 cites W2029090558 @default.
• W1970395279 cites W2038197749 @default.
• W1970395279 cites W2043707891 @default.
• W1970395279 cites W2045955128 @default.
• W1970395279 cites W2060584880 @default.
• W1970395279 cites W2062612693 @default.
• W1970395279 cites W2062813587 @default.
• W1970395279 cites W2068912983 @default.
• W1970395279 cites W2072664456 @default.
• W1970395279 cites W2072940800 @default.
• W1970395279 cites W2073413820 @default.
• W1970395279 cites W2079729042 @default.
• W1970395279 cites W2080527581 @default.
• W1970395279 cites W2093884797 @default.
• W1970395279 cites W2097997344 @default.
• W1970395279 cites W2100448419 @default.
• W1970395279 cites W2107359522 @default.
• W1970395279 cites W2127558096 @default.
• W1970395279 cites W2134507382 @default.
• W1970395279 cites W2144662794 @default.
• W1970395279 cites W2158960911 @default.
• W1970395279 cites W2159973565 @default.
• W1970395279 cites W2166213130 @default.
• W1970395279 cites W2319897304 @default.
• W1970395279 cites W2415758662 @default.
• W1970395279 cites W4234142287 @default.
• W1970395279 cites W4242442904 @default.
• W1970395279 cites W4294718043 @default.
• W1970395279 cites W4366572355 @default.
• W1970395279 doi "https://doi.org/10.1074/jbc.m406577200" @default.
• W1970395279 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15550400" @default.
• W1970395279 hasPublicationYear "2005" @default.
• W1970395279 type Work @default.
• W1970395279 sameAs 1970395279 @default.
• W1970395279 citedByCount "52" @default.
• W1970395279 countsByYear W19703952792012 @default.
• W1970395279 countsByYear W19703952792013 @default.
• W1970395279 countsByYear W19703952792014 @default.
• W1970395279 countsByYear W19703952792015 @default.
• W1970395279 countsByYear W19703952792016 @default.
• W1970395279 countsByYear W19703952792017 @default.
• W1970395279 countsByYear W19703952792018 @default.
• W1970395279 countsByYear W19703952792019 @default.
• W1970395279 countsByYear W19703952792020 @default.
• W1970395279 countsByYear W19703952792021 @default.
• W1970395279 crossrefType "journal-article" @default.
• W1970395279 hasAuthorship W1970395279A5014280061 @default.
• W1970395279 hasAuthorship W1970395279A5023108032 @default.
• W1970395279 hasAuthorship W1970395279A5030531770 @default.
• W1970395279 hasAuthorship W1970395279A5075926965 @default.
• W1970395279 hasAuthorship W1970395279A5085576609 @default.
• W1970395279 hasBestOaLocation W19703952791 @default.
• W1970395279 hasConcept C121608353 @default.
• W1970395279 hasConcept C126322002 @default.
• W1970395279 hasConcept C178790620 @default.
• W1970395279 hasConcept C181199279 @default.
• W1970395279 hasConcept C185592680 @default.
• W1970395279 hasConcept C190283241 @default.
• W1970395279 hasConcept C2779689624 @default.
• W1970395279 hasConcept C2780192828 @default.
• W1970395279 hasConcept C2994372470 @default.
• W1970395279 hasConcept C502942594 @default.
• W1970395279 hasConcept C540031477 @default.
• W1970395279 hasConcept C54355233 @default.
• W1970395279 hasConcept C55493867 @default.
• W1970395279 hasConcept C71924100 @default.
• W1970395279 hasConcept C7836513 @default.
• W1970395279 hasConcept C81885089 @default.
• W1970395279 hasConcept C86803240 @default.
• W1970395279 hasConcept C96232424 @default.
• W1970395279 hasConceptScore W1970395279C121608353 @default.
• W1970395279 hasConceptScore W1970395279C126322002 @default.
• W1970395279 hasConceptScore W1970395279C178790620 @default.
• W1970395279 hasConceptScore W1970395279C181199279 @default.
• W1970395279 hasConceptScore W1970395279C185592680 @default.
• W1970395279 hasConceptScore W1970395279C190283241 @default.
• W1970395279 hasConceptScore W1970395279C2779689624 @default.
• W1970395279 hasConceptScore W1970395279C2780192828 @default.
• W1970395279 hasConceptScore W1970395279C2994372470 @default.
• W1970395279 hasConceptScore W1970395279C502942594 @default.

• next