T-oligo Treatment Decreases Constitutive and UVB-induced COX-2 Levels through p53- and NFκB-dependent Repression of the COX-2 Promoter
2005; Elsevier BV; Volume: 280; Issue: 37 Linguagem: Inglês
10.1074/jbc.m503245200
ISSN1083-351X
AutoresVaneeta Marwaha, Yahui Chen, Elizabeth Helms, Simin Arad, Hiroyasu Inoue, Evelyn Bord, Raj Kishore, Raffi Der Sarkissian, Barbara A. Gilchrest, David A. Goukassian,
Tópico(s)NF-κB Signaling Pathways
ResumoChronically irradiated murine skin and UV light-induced squamous cell carcinomas overexpress the inducible isoform of cyclooxygenase (COX-2), and COX-2 inhibition reduces photocarcinogenesis in mice. We have reported previously that DNA oligonucleotides substantially homologous to the telomere 3′-overhang (T-oligos) induce DNA repair capacity and multiple other cancer prevention responses, in part through up-regulation and activation of p53. To determine whether T-oligos affect COX-2 expression, human newborn keratinocytes and fibroblasts were pretreated with T-oligos or diluent alone for 24 h, UV-irradiated, and processed for Western blotting. In both cell types, T-oligos transcriptionally down-regulated base-line and UV light-induced COX-2 expression, coincident with p53 activation. In fibroblasts with wild type versus dominant negative p53 (p53WT versus p53DN), T-oligos decreased constitutive expression of a COX-2 reporter plasmid by >50%. We then examined NFκB, a known positive regulator of COX-2 transcription. In p53WT but not in p53DN fibroblasts and in human keratinocytes, T-oligos decreased readout of an NFκB promoter-driven reporter plasmid and decreased NFκB binding to DNA. After T-oligo treatment and subsequent UV irradiation, binding of the transcriptional co-activator protein p300 to NFκB was decreased, whereas binding of p300 to p53 was increased. Human skin explants provided with T-oligos had markedly decreased COX-2 immunostaining both at base-line and post-UV light, coincident with increased p53 immunostaining. We conclude that T-oligos transcriptionally down-regulate COX-2 expression in human skin via activation and up-regulation of p53, at least in part by inhibiting NFκB transcriptional activation. Decreased COX-2 expression may contribute to the observed ability of T-oligos to reduce photocarcinogenesis. Chronically irradiated murine skin and UV light-induced squamous cell carcinomas overexpress the inducible isoform of cyclooxygenase (COX-2), and COX-2 inhibition reduces photocarcinogenesis in mice. We have reported previously that DNA oligonucleotides substantially homologous to the telomere 3′-overhang (T-oligos) induce DNA repair capacity and multiple other cancer prevention responses, in part through up-regulation and activation of p53. To determine whether T-oligos affect COX-2 expression, human newborn keratinocytes and fibroblasts were pretreated with T-oligos or diluent alone for 24 h, UV-irradiated, and processed for Western blotting. In both cell types, T-oligos transcriptionally down-regulated base-line and UV light-induced COX-2 expression, coincident with p53 activation. In fibroblasts with wild type versus dominant negative p53 (p53WT versus p53DN), T-oligos decreased constitutive expression of a COX-2 reporter plasmid by >50%. We then examined NFκB, a known positive regulator of COX-2 transcription. In p53WT but not in p53DN fibroblasts and in human keratinocytes, T-oligos decreased readout of an NFκB promoter-driven reporter plasmid and decreased NFκB binding to DNA. After T-oligo treatment and subsequent UV irradiation, binding of the transcriptional co-activator protein p300 to NFκB was decreased, whereas binding of p300 to p53 was increased. Human skin explants provided with T-oligos had markedly decreased COX-2 immunostaining both at base-line and post-UV light, coincident with increased p53 immunostaining. We conclude that T-oligos transcriptionally down-regulate COX-2 expression in human skin via activation and up-regulation of p53, at least in part by inhibiting NFκB transcriptional activation. Decreased COX-2 expression may contribute to the observed ability of T-oligos to reduce photocarcinogenesis. Nonmelanoma skin cancer accounts for well over 1 million cases of human malignancy annually in the United States, and the incidence continues to rise (1Jemal A. Murray T. Ward E. Samuels A. Tiwari R.C. Ghafoor A. Feuer E.J. Thun M.J. CA–Cancer J. Clin. 2005; 55: 10-30Crossref PubMed Scopus (5557) Google Scholar, 2Albert M.R. Weinstock M.A. CA–Cancer J. Clin. 2003; 53: 292-302Crossref PubMed Scopus (74) Google Scholar, 3Geller A.C. Zhang Z. Sober A.J. Halpern A.C. Weinstock M.A. Daniels S. Miller D.R. Demierre M.F. Brooks D.R. Gilchrest B.A. J. Am. Acad. Dermatol. 2003; 48: 34-41Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The major initiator and promoter of skin cancer is UVB radiation (4Brash D.E. Ponten J. Cancer Surv. 1998; 32: 69-113PubMed Google Scholar, 5Zoumpourlis V. Solakidi S. Papathoma A. Papaevangeliou D. Carcinogenesis. 2003; 24: 1159-1165Crossref PubMed Scopus (69) Google Scholar). 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Cell Res. 2004; 301: 189-200Crossref PubMed Scopus (47) Google Scholar).In the present study, we show that treatment with either of two oligonucleotides with partial telomere homology decreases constitutive and UVB-induced COX-2 levels in cultured human fibroblasts, human skin explants, and intact murine skin. These responses are shown to occur at least in part through up-regulation and activation of p53, leading to transcriptional repression of COX-2 promoter activity. We propose that in addition to the previously reported protective DNA damage responses, treatment with T-oligos may also decrease the cutaneous inflammatory response through inhibition of COX-2 expression, a possible additional means of reducing photocarcinogenesis.EXPERIMENTAL PROCEDURESCell Culture—Primary human neonatal fibroblast and keratinocytes cultures were established as described (49Eller M.S. Maeda T. Magnoni C. Atwal D. Gilchrest B.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12627-12632Crossref PubMed Scopus (195) Google Scholar, 50Goukassian D.A. Bagheri S. el-Keeb L. Eller M.S. Gilchrest B.A. FASEB J. 2002; 16: 754-756Crossref PubMed Scopus (52) Google Scholar). Cells were incubated at 37 °C in 5% CO2. Cell lines permanently (retroviral transfection) expressing WT p53 (R2FWT) or dominant negative p53 (R2FDD) (52Tubo R.A. Rheinwald J.G. Oncogene Res. 1987; 1: 407-421PubMed Google Scholar, 53Shaulian E. Zauberman A. Ginsberg D. Oren M. Mol. Cell. Biol. 1992; 12: 5581-5592Crossref PubMed Scopus (322) Google Scholar, 54Rheinwald J.G. Hahn W.C. Ramsey M.R. Wu J.Y. Guo Z. Tsao H. De Luca M. Catricala C. O'Toole K.M. Mol. Cell. Biol. 2002; 22: 5157-5172Crossref PubMed Scopus (284) Google Scholar) were the generous gift from Dr. Jim Rheinwald (Department of Dermatology, Harvard Skin Disease Research Center, Harvard Medical School, Boston) and were maintained in R2F medium containing 42.5% Dulbecco's modified Eagle's medium, 42.5% F-12, 15% calf serum, and 0.1% epidermal growth factor at 37 °C in 5% CO2.Oligonucleotides—Previous experiments have shown that 100 μm of thymidine dinucleotide (pTT), representing one-third of the telomere repeat, and 40 μm of pGAGTATGAG (p9-mer), a 55% homologous sequence, are roughly bioequivalent concentrations for the elicitation of UV mimetic responses (49Eller M.S. Maeda T. Magnoni C. Atwal D. Gilchrest B.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12627-12632Crossref PubMed Scopus (195) Google Scholar, 50Goukassian D.A. Bagheri S. el-Keeb L. Eller M.S. Gilchrest B.A. FASEB J. 2002; 16: 754-756Crossref PubMed Scopus (52) Google Scholar, 55Goukassian D.A. Eller M.S. Yaar M. Gilchrest B.A. J. Invest. Dermatol. 1999; 112: 25-31Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 56Maeda T. Eller M.S. Hedayati M. Grossman L. Gilchrest B.A. Mutat. Res. 1999; 433: 137-145Crossref PubMed Scopus (32) Google Scholar), including p53 up-regulation and activation. Oligonucleotides (pTT and p9-mer) were synthesized with phosphodiester linkage by Midland Certified Reagent (Midland, TX) and diluted in H2O to form a 2 mm stock. This stock solution was then diluted in the appropriate culture medium to 100 or 40 μm, respectively, and added to culture dishes for use in experiments. Cells and skin explants were provided T-oligos only once at time 0 and then harvested at intervals, according to the design of the specific experiment. All experiments were conducted using both pTT and p9-mer and gave identical results with either T-oligo.UVB Irradiation—After 48 h of incubation in medium containing pTT, p9-mer, or diluent alone, cells or skin explants were placed in phosphate-buffered saline and irradiated through the plastic culture dish cover by using a solar simulator (Spectral Energy Corp., Westwood, NJ). The 1-kilowatt xenon arc lamp (XMN-1000-21; Optical Radiation Corp., Azuza, CA) irradiance was adjusted to 5 × 10-5 watts/cm2, and dishes were exposed to 15 mJ/cm2 as measured with a research radiometer fitted with a UV light probe at 285 ± 5 nm (model IL1700 A; International Light, Newburyport, MA) (56Maeda T. Eller M.S. Hedayati M. Grossman L. Gilchrest B.A. Mutat. Res. 1999; 433: 137-145Crossref PubMed Scopus (32) Google Scholar, 57Gilchrest B.A. Zhai S. Eller M.S. Yarosh D.B. Yaar M. J. Invest. Dermatol. 1993; 101: 666-672Abstract Full Text PDF PubMed Google Scholar), a protocol that exposes cells to a spectrum of light resembling terrestrial sunlight (58Werninghaus K. Handjani R.M. Gilchrest B.A. Photodermatol. Photomed. 1991; 8: 236-242PubMed Google Scholar). Sham-irradiated cultures were handled identically, except that they were shielded with aluminum foil during irradiation. After irradiation, cells were given fresh medium lacking T-oligos.Western Blot Analysis—Total cellular proteins were collected as described previously (46Eller M.S. Ostrom K. Gilchrest B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1087-1092Crossref PubMed Scopus (240) Google Scholar). Concentrations were determined by the BioRad method, and 50 μg of protein were run in each lane on a 10% denaturing SDS-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane. Antibody reactions were performed with the following antibodies: phospho-p53Ser15 (Cell Signaling Technology, Beverly, MA), p53 DO-1, COX-2, NFκB/p65, and actin (all from Santa Cruz Biotechnology, Santa Cruz, CA). Western blot analysis was then performed as described (46Eller M.S. Ostrom K. Gilchrest B.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1087-1092Crossref PubMed Scopus (240) Google Scholar).Electrophoretic Mobility Shift Assays—Electrophoretic mobility shift assays (EMSAs) using consensus p53 and NFκB oligonucleotides (Santa Cruz Biotechnology) and 5 μg of nuclear protein from variously treated cells were carried out as described previously (59Kishore R. Spyridopoulos I. Luedemann C. Losordo D.W. Circ. Res. 2002; 91: 307-314Crossref PubMed Scopus (13) Google Scholar). Reactions were electrophoresed on 5% nondenaturing polyacrylamide gels, dried, and processed for autoradiography. For competition experiments, 50–100-fold excess of unlabeled DNA were added to the reaction 20 min before the addition of radiolabeled probe.Transfection Studies—Constructs containing the full-length phPES2(-1432/+59) human Cox-2 promoter, a deletion construct of phPES2(-327/+59), and an NFκB binding region site-specific mutant of phPES2(-327/+59) attached to a luciferase reporter (60Inoue H. Yokoyama C. Hara S. Tone Y. Tanabe T. J. Biol. Chem. 1995; 270: 24965-24971Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 61Inoue H. Taba Y. Miwa Y. Yokota C. Miyagi M. Sasaguri T. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1415-1420Crossref PubMed Scopus (110) Google Scholar) or an NFκB reporter plasmid (Promega Corp., Madison, WI) were employed. The pGL2 vector used for cloning the reporter construct was obtained from Promega (pGL2-Basic, Promega Corp., Madison, WI) and was used as an empty vector control. A plasmid containing Renilla luciferase (pRL-CMV, Promega Corp., Madison, WI) was co-transfected as a control for transfection efficiency. R2FWT and R2FDD cells were plated in 35-mm tissue culture dishes and incubated in R2F medium overnight to reach 50–60% confluence the next day. Cells were then transfected using the Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Two μg of plasmid DNA were co-transfected with 0.15 μg of the Renilla luciferase plasmid in each dish. After 4–6 h of transfection, cells were supplemented either with R2F medium alone (diluent) or R2F medium with 40 μm p9-mer or 100 μm pTT. Cells were then incubated for 24 and 48 h at 37 °C in 5% CO2 before harvesting for dual-luciferase assay. Promoter activity was then assayed using a dual-luciferase reporter assay system (Promega Corp., Madison, WI) according to the manufacturer's protocol. Firefly luciferase values were corrected for transfection efficiency according to Renilla luciferase values and revealed minimal differences in transfection efficiency among dishes. Luciferase activity was then expressed as percent of diluent value, setting diluent as 100%.Immunoprecipitation and Immunoblotting—Cell lysates were pre-cleaned with protein G-Sepharose beads for 2 h at 4°C. Then 100 μg of total cell proteins were incubated with 2 μg of monoclonal p300 antibodies (GeneTex® Inc., San Antonio, TX), followed by the addition of protein G-Sepharose beads. The immunoprecipitated products were then subjected to SDS-PAGE and Western blot analysis as described (62Kishore R. Luedemann C. Bord E. Goukassian D. Losordo D.W. Circ. Res. 2003; 93: 932-940Crossref PubMed Scopus (24) Google Scholar). After transferring proteins to nitrocellulose membrane antibody reactions were performed with NFκB (p65) antibodies and p53 (DO-1) (both from Santa Cruz Biotechnology).Human Skin Explant Studies—Human skin fragments from healthy donors (aged 56 ± 15 years, mean ± S.D.) were brought to the laboratory within 30 min after excision during plastic or facial reconstructive surgery. After removing subcutaneous fat and deep dermis, skin was cut into 5 × 5-mm squares and placed in 60-mm tissue culture dishes. Paired skin explants were then incubated in either medium alone or medium supplemented with 100 μm pTT or 40 μm p9-mer for 24 h. Medium consisted of Dulbecco's modified Eagle's medium with 10% calf serum plus KBM-2 with growth factors (50/50 v/v). The skin explants were then irradiated with a single dose of 30 mJ/cm2 UVB. One set was sham-irradiated as a negative control. For each treatment, one explant was harvested immediately after UVB irradiation. The dishes were then re-fed with fresh medium lacking T-oligos, and explants were harvested at 6, 18, and 24 h after UVB irradiation. Harvested skin was snap-frozen at -80 °C in OCT medium for later processing.Immunohistochemistry and Immunofluorescence—Snap-frozen human skin explants were processed for staining by cutting 4–6-μm sections and fixing them in acetone for 10 min at -20 °C. COX-2 staining was performed using the Ultravision Detection System (TQ-015-HA, Labvision Corp., Fremont, CA) according to manufacturer's protocol. Primary antibodies used included anti-COX-2 (Santa Cruz Biotechnology), human anti-p53 DO-7 (DakoCytomation, Carpinteria, CA), and anti-phospho-p53Ser15 (Cell Signaling Technology, Beverly, MA). For the p53 DO-7 and p53Ser15 stainings, sections were blocked in 10% goat normal serum in Tris-buffered saline for 15 min at room temperature and then incubated with primary antibody overnight at 4 °C. Sections were then washed in Tris-buffered saline three times for 5 min each before being incubated with the appropriate fluorescein isothiocyanate-labeled secondary antibody at 37 °C for 45 min. Finally, sections were washed as before and mounted with Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole to visualize nuclei and were stored at -20 °C. We delineated ≈10-μm × 1-mm areas using computer-assisted image analysis and counted p53total and p53Ser15 (+) nuclei in the epidermis. For each time point we analyzed an average of three randomly selected visual fields of p53total and p53Ser15-stained epidermis from three to five donors per treatment condition. To avoid bias all counts were done by a single investigator for whom all samples were blinded by another investigator.Statistical Analysis—Difference in protein expression, Cox-2 promoter activity, and p53total and p53Ser15 (+) nuclei in T-oligo versus control-treated samples were analyzed by the analysis of variance post hoc analysis using the StatView statistical program (SAS Institute, Gary, NC). Groups were considered different when p < 0.05 (50Goukassian D.A. Bagheri S. el-Keeb L. Eller M.S. Gilchrest B.A. FASEB J. 2002; 16: 754-756Crossref PubMed Scopus (52) Google Scholar).RESULTST-oligo Pretreatment Down-regulates Base-line a
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