Reduction of IKKα Expression Promotes Chronic Ultraviolet B Exposure-Induced Skin Inflammation and Carcinogenesis
2010; Elsevier BV; Volume: 176; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2010.091041
ISSN1525-2191
AutoresXiaojun Xia, Eunmi Park, Bigang Liu, Jami Willette‐Brown, Wanghua Gong, Jiming Wang, David L. Mitchell, Susan M. Fischer, Yinling Hu,
Tópico(s)Immune cells in cancer
ResumoUltraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor ®B kinase-α (IKKα) plays an important role in maintaining skin homeostasis, and expression of IKKα was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKα in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkα+/+ and Ikkα+/− mice. Ikkα+/− mice were found to develop a twofold greater number of skin tumors than Ikkα+/+ mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkα+/− than in Ikkα+/+ mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkα+/− compared with those in Ikkα+/+ skin. Also, reduction of IKKα levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFα, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKα expression orchestrates UVB carcinogen, accelerating tumorigenesis. Ultraviolet B light (UVB) is a common cause of human skin cancer. UVB irradiation induces mutations in the tumor suppressor p53 gene as well as chronic inflammation, which are both essential for UVB carcinogenesis. Inhibitor of nuclear factor ®B kinase-α (IKKα) plays an important role in maintaining skin homeostasis, and expression of IKKα was found to be down-regulated in human and murine skin squamous cell carcinomas. However, the role of IKKα in UVB skin carcinogenesis has not been investigated. Thus, here we performed UVB carcinogenesis experiments on Ikkα+/+ and Ikkα+/− mice. Ikkα+/− mice were found to develop a twofold greater number of skin tumors than Ikkα+/+ mice after chronic UVB irradiation. In addition, tumor latency was significantly shorter and tumors were bigger in Ikkα+/− than in Ikkα+/+ mice. At an early stage of carcinogenesis, an increase in UVB-induced p53 mutations as well as macrophage recruitment and mitogenic activity, and a decrease in UVB-induced apoptosis, were detected in Ikkα+/− compared with those in Ikkα+/+ skin. Also, reduction of IKKα levels in keratinocytes up-regulated the expression of monocyte chemoattractant protein-1 (MCP-1/CCL2), TNFα, IL-1, and IL-6, and elevated macrophage migration, which might promote macrophage recruitment and inflammation. Therefore, these findings suggest that reduction of IKKα expression orchestrates UVB carcinogen, accelerating tumorigenesis. Ultraviolet B (UVB) irradiation induces DNA damage. The tumor suppressor gene p53 is an UVB target, and human cutaneous squamous cell carcinoma (SCC) cells contain p53 mutations.1Ziegler A Jonason AS Leffell DJ Simon JA Sharma HW Kimmelman J Remington L Jacks T Brash DE Sunburn and p53 in the onset of skin cancer.Nature. 1994; 372: 773-776Crossref PubMed Scopus (1348) Google Scholar Skin cells can harbor UVB-induced p53 mutations for decades before the onset of human SCC, however,2Jonason AS Kunala S Price GJ Restifo RJ Spinelli HM Persing JA Leffell DJ Tarone RE Brash DE Frequent clones of p53-mutated keratinocytes in normal human skin.Proc Natl Acad Sci USA. 1996; 93: 14025-14029Crossref PubMed Scopus (554) Google Scholar, 3Berg RJ van Kranen HJ Rebel HG de Vries A van Vloten WA Van Kreijl CF van der Leun JC de Gruijl FR Early p53 alterations in mouse skin carcinogenesis by UVB radiation: immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.Proc Natl Acad Sci USA. 1996; 93: 274-278Crossref PubMed Scopus (200) Google Scholar underscoring the importance of coactivators in skin tumorigenesis. In mice, p53 mutations are an early genetic event in UVB skin carcinogenesis, which recapitulates the process of human SCC development.4Rebel H Mosnier LO Berg RJ Westerman-de Vries A van Steeg H van Kranen HJ de Gruijl FR Early p53-positive foci as indicators of tumor risk in ultraviolet-exposed hairless mice: kinetics of induction, effects of DNA repair deficiency, and p53 heterozygosity.Cancer Res. 2001; 61: 977-983PubMed Google Scholar, 5Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar The p53 mutations have been proposed to be important for UVB carcinogenesis because prevention of these mutations prevents skin tumor development, and the number of p53 mutation-positive keratinocytes correlates with the number of skin tumors in mice.6Ananthaswamy HN Ullrich SE Mascotto RE Fourtanier A Loughlin SM Khaskina P Bucana CD Kripke ML Inhibition of solar simulator-induced p53 mutations and protection against skin cancer development in mice by sunscreens.J Invest Dermatol. 1999; 112: 763-768Crossref PubMed Scopus (43) Google Scholar, 7de Gruijl FR Rebel H Early events in UV carcinogenesis–DNA damage, target cells and mutant p53 foci.Photochem Photobiol. 2008; 84: 382-387Crossref PubMed Scopus (98) Google Scholar, 8Hill LL Ouhtit A Loughlin SM Kripke ML Ananthaswamy HN Owen-Schaub LB Fas ligand: a sensor for DNA damage critical in skin cancer etiology.Science. 1999; 285: 898-900Crossref PubMed Scopus (224) Google Scholar, 9Zhang W Hanks AN Boucher K Florell SR Allen SM Alexander A Brash DE Grossman D UVB-induced apoptosis drives clonal expansion during skin tumor development.Carcinogenesis. 2005; 26: 249-257Crossref PubMed Scopus (66) Google Scholar UVB exposure also induces chronic inflammation, cell proliferation, oxidant stress, and immunosuppression, which essentially facilitate UVB carcinogenesis.10Halliday GM Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis.Mutat Res. 2005; 571: 107-120Crossref PubMed Scopus (360) Google Scholar, 11Melnikova VO Ananthaswamy HN Cellular and molecular events leading to the development of skin cancer.Mutat Res. 2005; 571: 91-106Crossref PubMed Scopus (279) Google Scholar Particularly, chronic inflammation can create a microenvironment that is prone to cell proliferation and DNA damage, thereby promoting tumor development.12Lin WW Karin M A cytokine-mediated link between innate immunity, inflammation, and cancer.J Clin Invest. 2007; 117: 1175-1183Crossref PubMed Scopus (1516) Google Scholar On the other hand, UVB exposure also elicits protective responses, such as cell cycle arrest, DNA repair, and apoptosis, which reduce UVB-induced damage.7de Gruijl FR Rebel H Early events in UV carcinogenesis–DNA damage, target cells and mutant p53 foci.Photochem Photobiol. 2008; 84: 382-387Crossref PubMed Scopus (98) Google Scholar, 10Halliday GM Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis.Mutat Res. 2005; 571: 107-120Crossref PubMed Scopus (360) Google Scholar, 11Melnikova VO Ananthaswamy HN Cellular and molecular events leading to the development of skin cancer.Mutat Res. 2005; 571: 91-106Crossref PubMed Scopus (279) Google Scholar Previously, it was reported that mice lacking p53 were defective in inducing apoptosis after UVB irradiation and thus had more skin tumors than wild-type mice did.13Jiang W Ananthaswamy HN Muller HK Kripke ML p53 protects against skin cancer induction by UV-B radiation.Oncogene. 1999; 18: 4247-4253Crossref PubMed Scopus (149) Google Scholar, 14van Kranen HJ Westerman A Berg RJ Kram N van Kreijl CF Wester PW de Gruijl FR Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice.Mutat Res. 2005; 571: 81-90Crossref PubMed Scopus (32) Google Scholar Mice lacking Fas ligand had defects in apoptosis, which increased numbers of cells containing UVB-induced p53 mutations, and the mutant mice were more susceptible to UVB-induced skin tumors than wild-type mice.5Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 8Hill LL Ouhtit A Loughlin SM Kripke ML Ananthaswamy HN Owen-Schaub LB Fas ligand: a sensor for DNA damage critical in skin cancer etiology.Science. 1999; 285: 898-900Crossref PubMed Scopus (224) Google Scholar Therefore, the different defects in inducing protective responses against UVB-induced damage can amplify the severity of the cancer cause, thereby accelerating carcinogenesis. Inhibitor of nuclear factor ®B kinase-α (IKKα) is required for embryonic skin development in mice.15Hu Y Baud V Delhase M Zhang P Deerinck T Ellisman M Johnson R Karin M Abnormal morphogenesis but intact IKK activation in mice lacking the IKKα subunit of IκB kinase.Science. 1999; 284: 316-320Crossref PubMed Scopus (712) Google Scholar, 16Takeda K Takeuchi O Tsujimura T Itami S Adachi O Kawai T Sanjo H Yoshikawa K Terada N Akira S Limb and skin abnormalities in mice lacking IKKα.Science. 1999; 284: 313-316Crossref PubMed Scopus (539) Google Scholar Several laboratories have reported the down-regulated expression and altered localization of IKKα protein as well as deletions and mutations in the Ikkα gene in human SCCs of the skin, lung, esophagus, and head and neck.17Liu B Park E Zhu F Bustos T Liu J Shen J Fischer SM Hu Y A critical role for I{kappa}B kinase {alpha} in the development of human and mouse squamous cell carcinomas.Proc Natl Acad Sci U S A. 2006; 103: 17202-17207Crossref PubMed Scopus (86) Google Scholar, 18Maeda G Chiba T Kawashiri S Satoh T Imai K Epigenetic inactivation of IkappaB Kinase-alpha in oral carcinomas and tumor progression.Clin Cancer Res. 2007; 13: 5041-5047Crossref PubMed Scopus (53) Google Scholar, 19Marinari B Moretti F Botti E Giustizieri ML Descargues P Giunta A Stolfi C Ballaro C Papoutsaki M Alema S Monteleone G Chimenti S Karin M Costanzo A The tumor suppressor activity of IKKalpha in stratified epithelia is exerted in part via the TGF-beta antiproliferative pathway.Proc Natl Acad Sci U S A. 2008; 105: 17091-17096Crossref PubMed Scopus (55) Google Scholar, 20Marinari B Ballaro C Koster MI Giustizieri ML Moretti F Crosti F Papoutsaki M Karin M Alema S Chimenti S Roop DR Costanzo A IKKalpha is a p63 transcriptional target involved in the pathogenesis of ectodermal dysplasias.J Invest Dermatol. 2009; 129: 60-69Crossref PubMed Scopus (37) Google Scholar, 21Descargues P Sil AK Sano Y Korchynskyi O Han G Owens P Wang XJ Karin M IKKalpha is a critical coregulator of a Smad4-independent TGFbeta-Smad2/3 signaling pathway that controls keratinocyte differentiation.Proc Natl Acad Sci USA. 2008; 105: 2487-2492Crossref PubMed Scopus (132) Google Scholar In particular, IKKα deletion in keratinocytes was found to elevate an autocrine loop activity of epidermal growth factor receptor (EGFR), Ras, extracellular signal-related kinase (ERK), EGFR ligands, and the ligands' activators and induced spontaneous skin SCCs in mice.22Liu B Xia X Zhu F Park E Carbajal S Kiguchi K DiGiovanni J Fischer SM Hu Y IKKalpha is required to maintain skin homeostasis and prevent skin cancer.Cancer Cell. 2008; 14: 212-225Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar Reduction of IKKα expression was found to promote chemical carcinogen-induced skin tumorigenesis.23Park E Zhu F Liu B Xia X Shen J Bustos T Fischer SM Hu Y Reduction in IkappaB kinase alpha expression promotes the development of skin papillomas and carcinomas.Cancer Res. 2007; 67: 9158-9168Crossref PubMed Scopus (61) Google Scholar Also, IKKα is an UVB-induced gene, and the defect in IKKα function was linked to inflammation.24Li N Karin M Ionizing radiation and short wavelength UV activate NF-kappaB through two distinct mechanisms.Proc Natl Acad Sci USA. 1998; 95: 13012-13017Crossref PubMed Scopus (402) Google Scholar, 25Liu B Yang Y Chernishof V Loo RR Jang H Tahk S Yang R Mink S Shultz D Bellone CJ Loo JA Shuai K Proinflammatory stimuli induce IKKalpha-mediated phosphorylation of PIAS1 to restrict inflammation and immunity.Cell. 2007; 129: 903-914Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar UVB is a very common cause of human skin cancer; however, the role of IKKα in skin UVB carcinogenesis is largely unknown. Thus, here we examined the effect of reduced IKKα expression on UVB skin carcinogenesis in Ikkα+/− and Ikkα+/+ mice. Ikkα+/− mice were significantly susceptible to UVB skin carcinogenesis than were Ikkα+/+ mice. Because the tumor latency was significantly shorter and many more tumors were found in Ikkα+/− mice than in Ikkα+/+ mice, we analyzed the early events during UVB skin carcinogenesis. This study provided the first evidence showing the importance of IKKα in UVB skin carcinogenesis. The mice used in this study were cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee of The University of Texas M.D. Anderson Cancer Center (animal protocol 04-01-05732). Ikkα+/− mice with a C57Bl6 background were backcrossed with wild-type SKH-1 hairless mice (Charles River Laboratories) for five generations. Female Ikkα+/− and Ikkα+/+ mice with age of 6 to 8 weeks old that were siblings were UVB-irradiated using Westinghouse FS20 sunlamps in an irradiation chamber (Starch Art).26Berton TR Mitchell DL Fischer SM Locniskar MF Epidermal proliferation but not quantity of DNA photodamage is correlated with UV-induced mouse skin carcinogenesis.J Invest Dermatol. 1997; 109: 340-347Crossref PubMed Scopus (71) Google Scholar The broadband range of the UV lamps was 280 to 400 nm, with 80% of the light in the UVB region and 20% in the UVA region. For UVB carcinogenesis experiments, mice were irradiated three times a week beginning at a UVB dose of 90 mJ/cm2 in the first week. This dose was increased by 10% each week to a maximum of 175 mJ/cm2. For time-course experiments, mice were UVB-irradiated at 100 mJ/cm2. Statistic analyses of tumor incidence and multiplicity were done by the statistic core facility at Science Park Research Division, M.D. Anderson Cancer Center. The sunburn cells (SBCs) in the interfollicle epidermis of paraffin-embedded skin sections of mice were stained with hematoxylin and eosin that was performed in the tissue and histology facility of the Department of Carcinogenesis at M.D. Anderson Cancer Center. To count the apoptotic cells, paraffin-embedded skin sections were stained using terminal transferase dUTP nick end labeling (TUNEL) kit (Roche) with immunofluorescence following the manufacturer's manual. Immunohistochemical staining of Ki-67–positive cells and macrophage maker S100A9 in the epidermis of paraffin-embedded skin sections was performed in the tissue and histology facility of the Department of Carcinogenesis at M.D. Anderson Cancer Center.23Park E Zhu F Liu B Xia X Shen J Bustos T Fischer SM Hu Y Reduction in IkappaB kinase alpha expression promotes the development of skin papillomas and carcinomas.Cancer Res. 2007; 67: 9158-9168Crossref PubMed Scopus (61) Google Scholar The number of photoproducts in epidermal DNA of experimental mice was determined using a radioimmunoassay as described previously.26Berton TR Mitchell DL Fischer SM Locniskar MF Epidermal proliferation but not quantity of DNA photodamage is correlated with UV-induced mouse skin carcinogenesis.J Invest Dermatol. 1997; 109: 340-347Crossref PubMed Scopus (71) Google Scholar Briefly, DNA (5 μg) isolated from mice UVB-irradiated at 100 mJ/cm2 to induce (6-4)pyrimidine-pyrimidone photoproducts ([6-4]PPs) and cyclobu-pyrimidine dimers was incubated with 5 to 10 pg of poly(deoxyadenylate-deoxythymidylic acid) (labeled to more than 5 times greater 108 cpm/mg using nick translation with [α-32P]d-TTP) in a total volume of 1 ml of 10 mmol/L Tris (pH 7.8), 150 mmol/L NaCl, 1 mmol/L EDTA, and 0.15% gelatin. Antisera were added to the reaction at a dilution yielding 30% to 60% binding to a labeled ligand, and incubated at 4°C overnight; the immune complex was then precipitated with goat anti-rabbit IgG (CalBiochem) and carrier serum obtained from nonimmunized rabbits. The immune complex was pelletted at 3500 rpm at 4°C for 45 minutes and then solubilized for 15 minutes on a rotator at 37°C. Sample inhibition was extrapolated by a standard curve to determine the number of photoproducts in 106 bases ([6-4]PPs/Mb or CPDs/Mb). For the standard, double-stranded salmon testis DNA was UVB-irradiated at increasing doses of UV radiation and heat-denatured. The percent inhibition of bound 32P from sample DNA was compared with control UV-irradiated salmon testis DNA to determine the quantity of the photoproducts. Rabbit polyclonal (6-4)PPs and CPDs primary antisera were prepared at MD Anderson Cancer Center. Briefly, epidermal or skin tumor DNA was isolated and analyzed using allele-specific PCR for CC→TT mutations at codons 154/155 in exon 5 of the P53 gene and for C to T mutations at codons 270 and 275 in exon 8 of the P53 gene as described previously.5Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 27Ananthaswamy HN Loughlin SM Cox P Evans RL Ullrich SE Kripke ML Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens.Nat Med. 1997; 3: 510-514Crossref PubMed Scopus (193) Google Scholar, 28Guttridge DC Albanese C Reuther JY Pestell RG Baldwin Jr, AS NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1.Mol Cell Biol. 1999; 19: 5785-5799Crossref PubMed Google Scholar, 29Melnikova VO Pacifico A Chimenti S Peris K Ananthaswamy HN Fate of UVB-induced p53 mutations in SKH-hr1 mouse skin after discontinuation of irradiation: relationship to skin cancer development.Oncogene. 2005; 24: 7055-7063Crossref PubMed Scopus (36) Google Scholar The PCR primers used included the codons 154/155: 5′-CCTCCAGCTGGGAGCCGTGCTT-3′ (with mutant-specific sequences) and 5′-GAGGGCTTACCATCACCATC-3′; C to T mutations at codons 270 and 275: the forward mutant-specific sequences 5′-GGACGGGACAGCTTTGAGGTTT-3′ and 5′-GTGTTTGTGCCTGCCT-3′, respectively, and the reverse sequences 5′-GCCTGCGTACCTCTCTTTGC-3′. The PCR primers for exon 5 of the p53 gene were 5′-TCTCTTCCAGTACTCTCCTC-3′ and 5′-GAGGGCTTACCATCACCATC-3′. PCR was performed using 360 ng of genomic DNA, 2 μmol/L each primer, 2.5 μmol/L dNTPs, and 0.2 U of TaqDNA polymerase (Promega) in a buffer containing 1 mmol/L MgCl2 in a final volume of 25 μl. Following an initial denature step (4 minutes at 94°C), 35 cycles (1 minute at 94°C; 1 minute each at 58°C [codon 154 to 155 mutations], 69°C [codon 270] or 65°C [codon 275]; and 1 minute at 72°C) were performed using a DNA thermal cycler (BioRad). An aliquot (15 μl) of the PCR product was mixed with a DNA gel loading solution (3 μl) and electrophoresed on 8% (codons 154) or 6% (codons 270 and 275) polyacrylamide gels at 150 V for 30 minutes and then visualized using SYBR Green II staining (Invitrogen, Carlsbad, CA). Electrophoretic mobility shift assays of NF-®B DNA binding activity were performed using a kit (E3300; Promega). Reaction containing 7.5 μl of 2× electrophoretic mobility shift assay buffer, 1.5 μg of poly dI-dC, 10 μg of protein lysates, 5 × 103 cpm NF-®B probe, and up to 15 μl of water, was incubated at room temperature for 30 minutes. A gel 5% nondenatured polyacrylamide gel was pre-run in 0.5× TBE for 1.5 hours at 100 V, and then the reactions were subjected into the gel. The gel was dried and exposed to film.30Hu Y Baud V Oga T Kim KI Yoshida K Karin M IKKα controls formation of the epidermis independently of NF-κB.Nature. 2001; 410: 710-714Crossref PubMed Scopus (301) Google Scholar Total RNA was isolated from the epidermis or keratinocytes using TRI Reagent (Molecular Research Center).23Park E Zhu F Liu B Xia X Shen J Bustos T Fischer SM Hu Y Reduction in IkappaB kinase alpha expression promotes the development of skin papillomas and carcinomas.Cancer Res. 2007; 67: 9158-9168Crossref PubMed Scopus (61) Google Scholar cDNA was synthesized by a RETROscript kit (Ambion, Inc). PCR primers used included tumor necrosis factor (TNF)α (5′-GATCTCAAAGACAACCAACATGTG-3′ and 5′-CTCCAGCTGGAAGACTCCTCCCAG-3′), interleukin (IL)-6 (5′-CATAGCTACCTGGAGTACATGA-3′ and 5′-CATTCATATTGTCAGTTCTTCG-3′), IL-1 (5′-CCAGGATGAGGACATGAGCACC-3′ and 5′-TTCTCTGCAGACTCAAACTCCAC-3′), and GAPDH.17Liu B Park E Zhu F Bustos T Liu J Shen J Fischer SM Hu Y A critical role for I{kappa}B kinase {alpha} in the development of human and mouse squamous cell carcinomas.Proc Natl Acad Sci U S A. 2006; 103: 17202-17207Crossref PubMed Scopus (86) Google Scholar Antibodies used in this study included those against IKKα (IMG 136; Imgenex), p-ERK (9109; Cell Signaling Technology), ERK1 (SC-93; Santa Cruz Biotechnology), ERK2 (SC-1647; Santa Cruz), Akt (9272; Cell Signaling Technology), p-Akt (9271S; Cell Signaling Technology), MCP-1/CCL2 (554590; BD, NJ), and β-Actin (A-5441; Sigma). Mouse primary keratinocytes were isolated and cultured as described previously.23Park E Zhu F Liu B Xia X Shen J Bustos T Fischer SM Hu Y Reduction in IkappaB kinase alpha expression promotes the development of skin papillomas and carcinomas.Cancer Res. 2007; 67: 9158-9168Crossref PubMed Scopus (61) Google Scholar Briefly, skin specimens isolated from newborn mice were treated with 0.25% trypsin (15050-065, Invitrogen) for 8 to 10 hours at 4°C; the epidermis was separated from the dermis. Isolated keratinocytes were plated on 60-cm cell dishes containing keratinocyte serum-free medium (10785, GIBCO, Rockville, MD). Five days later, cultured keratinocytes were irradiated with 100 mJ/cm2. Cells and supernatants were collected at 24 hours after irradiation. MCP-1 levels in the supernatant of primary cultured keratinoytes were measured by MCP-1 ELISA kit (MJE00, R&D Systems, Minneapolis, MN) in clinical services laboratories, Basic Research Program, SAIC Frederick. Chemotaxis of mouse macrophages was measured with 48-well microchambers and polycarbonate filters (5-μm pore size; NeuroProbe, Cabin John, MD) as previously described.31Chen K Iribarren P Hu J Chen J Gong W Cho EH Lockett S Dunlop NM Wang JM Activation of Toll-like receptor 2 on microglia promotes cell uptake of Alzheimer disease-associated amyloid beta peptide.J Biol Chem. 2006; 281: 3651-3659Crossref PubMed Scopus (184) Google Scholar Briefly, a 27-μl aliquot of testing materials was placed in the wells of the lower compartment of the chamber, and 50 μl of mouse peritoneal macrophages (at 2 × 106/ml) were placed in the wells of the upper compartment. Two compartments of the chemotaxis chamber were separated by an 8-μm pore-sized polycarbonate filter (GE Osmonics Labstore, Minnetonka, MN). After incubation at 37°C for 90 minutes, the filters were removed and stained, and cells that migrated across the filters were counted under light microscopy. The mean number (± SE) of migrated cells in three high-powered fields (×400 magnification) in triplicate samples was obtained, and the results were expressed as chemotaxis index (CI) representing the fold increase in cell migration in response to stimulants over medium control. Student t test was used to examine the statistical significance in the differences of chemotaxis shown by various groups. Recombinant mouse MCP-1 (554590; BD) was used for control of MCP-1. To determine the effect of reduced IKKα expression on UVB skin carcinogenesis, we treated Ikkα+/+ and Ikkα+/− mice with UVB three times a week for 28 weeks. The Ikkα+/− mice appeared to be normal although Ikkα−/− mice die soon after birth because of severe defects in the skin development.15Hu Y Baud V Delhase M Zhang P Deerinck T Ellisman M Johnson R Karin M Abnormal morphogenesis but intact IKK activation in mice lacking the IKKα subunit of IκB kinase.Science. 1999; 284: 316-320Crossref PubMed Scopus (712) Google Scholar, 16Takeda K Takeuchi O Tsujimura T Itami S Adachi O Kawai T Sanjo H Yoshikawa K Terada N Akira S Limb and skin abnormalities in mice lacking IKKα.Science. 1999; 284: 313-316Crossref PubMed Scopus (539) Google Scholar We observed tumors on the dorsal skin of Ikkα+/− mice at week 11 after UVB irradiation, and almost all of these Ikkα+/− mice (95%) rapidly developed tumors over the next 5 weeks (Figure 1A). We observed skin tumors in Ikkα+/+ mice at week 14 after UVB irradiation, and all of these Ikkα+/+ mice had tumors by 23 weeks. These results showed that reduction of IKKα expression decreased tumor latency (P < 0.001, COX proportional hazards model; Figure 1A). Tumor multiplicity analysis showed that Ikkα+/− mice developed significantly more tumors than did Ikkα+/+ mice (P < 0.001, Poisson regression analysis; Figure 1B). At week 28 of the experiment, the Ikka+/− mice had twice as many tumors as the Ikkα+/+ mice (Figure 1C). Histological examination showed that, regardless of genotypes of the Ikkα gene in mice, most skin tumors were SCCs (supplemental Figure S1, see http://ajp.amjpathol.org) and some of small tumors remained papillomas. A comparison of tumor weights found more large tumors in Ikkα+/− mice than in Ikkα+/+ mice (P < 0.005, t test; Figure 1D). Collectively, these results showed that Ikkα+/− mice were more prone to chronic UVB-induced skin carcinogenesis than were Ikkα+/+ mice; in particular, the tumor latency was significantly shorter in Ikkα+/− than in Ikkα+/+ mice. UVB-induced p53 mutations have been shown to be an important early genetic event for carcinogenesis.27Ananthaswamy HN Loughlin SM Cox P Evans RL Ullrich SE Kripke ML Sunlight and skin cancer: inhibition of p53 mutations in UV-irradiated mouse skin by sunscreens.Nat Med. 1997; 3: 510-514Crossref PubMed Scopus (193) Google Scholar UVB-signature CC-TT and C-T mutations in p53 codons 154/155, 270, and 275 were detected in the epidermis as early as after 1 week of chronic UV irradiation.5Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar To identify the early events that may contribute to the tumor development promoted by reduction of IKKα expression, we examined UVB-induced p53V154A/R155C, p53R270C, and p53R275C signature mutations in the skin of mice irradiated for 2 weeks using PCR with mutation-specific primers.8Hill LL Ouhtit A Loughlin SM Kripke ML Ananthaswamy HN Owen-Schaub LB Fas ligand: a sensor for DNA damage critical in skin cancer etiology.Science. 1999; 285: 898-900Crossref PubMed Scopus (224) Google Scholar, 29Melnikova VO Pacifico A Chimenti S Peris K Ananthaswamy HN Fate of UVB-induced p53 mutations in SKH-hr1 mouse skin after discontinuation of irradiation: relationship to skin cancer development.Oncogene. 2005; 24: 7055-7063Crossref PubMed Scopus (36) Google Scholar The result showed that these p53 mutations occurred more frequently in Ikkα+/− than in Ikkα+/+ skin specimens (Figure 2A). However, most of the skin tumors, regardless of the genotype, were positive for these p53 mutations (Figure 2B). Because Ikkα+/− had twice as many tumors as Ikkα+/+ mice and most of the tumors in Ikkα+/+ and Ikkα+/− mice were positive for p53 mutations, the enhanced early p53 damage in the skin of Ikkα+/− mice may contribute to the development of UVB-induced skin tumors. Sites of neighboring pyrimidine bases in DNA strands are preferentially damaged by UVB radiation, forming primarily CPDs and (6-4)PPs.32Mitchell DL Nairn RS The biology of the (6-4) photoproduct.Photochem Photobiol. 1989; 49: 805-819Crossref PubMed Scopus (500) Google Scholar These DNA lesions give rise to unique mutations in the genome. The predominant DNA repair pathway for pyrimidine dimers in mammals is the nucleotide excision repair pathway.33Leibeling D Laspe P Emmert S Nucleotide excision repair and cancer.J Mol Histol. 2006; 37: 225-238Crossref PubMed Scopus (106) Google Scholar To determine whether DNA repair defects increase the number of p53 mutations in Ikkα+/− skin cells, we examined the levels of CPDs and (6-4)PPs in the epidermis of mice at different time points after UVB irradiation using a specific radioimmunoassay.26Berton TR Mitchell DL Fischer SM Locniskar MF Epidermal proliferation but not quantity of DNA photodamage is correlated with UV-induced mouse skin carcinogenesis.J Invest Dermatol. 1997; 109: 340-347Crossref PubMed Scopus (71) Google Scholar, 34Wheeler DL Martin KE Ness KJ Li Y Dreckschmidt NE Wartman M Ananthaswamy HN Mitchell DL Verma AK Protein kinase C epsilon is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas.Cancer Res. 2004; 64: 7756-7765Crossref PubMed Scopus (47) Google Scholar We found similar levels of CPDs and (6-4)PPs in the epidermal DNA of Ikka+/+ and Ikkα+/− mice immediately after UVB irradiation but no detectable CPDs or (6-4)PPs in untreated murine epidermal specimens (supplemental Figure S2, A and B, see http://ajp.amjpathol.org). At 24 hours after irradiation, most of the (6-4)PPs were repaired, and the level of (6-4)PPs dropped to a basal level comparable with that in untreated skin specimens (supplemental Figure S2C, see http://ajp.amjpathol.org); this repair in the epidermal cells in Ikkα+/− and Ikkα+/+ mice was not statistically
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