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Resistance of CD1d−/− Mice to Ultraviolet-Induced Skin Cancer Is Associated with Increased Apoptosis

2004; Elsevier BV; Volume: 165; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)63350-0

1525-2191

Yasuhiro Matsumura, Angus M. Moodycliffe, Dat X. Nghiem, Stephen E. Ullrich, Honnavara N. Ananthaswamy,

Cancer-related Molecular Pathways

Inhibition of p53-induced epidermal apoptosis, generation of p53 mutations, and suppressor T cells are the critical events responsible for the induction and development of UV-induced skin cancers. Recently, we demonstrated that CD1d knockout mice were resistant to UV-induced immunosuppression, prompting us to further address the role of CD1d in regulating UV carcinogenesis. We, therefore, investigated the response of wild-type (WT) and CD1d−/− mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d−/− mice developed skin tumors. Surprisingly, keratinocytes and fibroblasts from CD1d−/− mice were more sensitive to UV-induced apoptosis and persisted longer than cells derived from WT mice. In addition, epidermis and dermis taken from chronically UV-irradiated CD1d−/− mice harbored significantly fewer p53 mutations than WT mice. Our findings identify an unexpected and novel function for CD1d as a critical molecule regulating UV carcinogenesis, by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts. Inhibition of p53-induced epidermal apoptosis, generation of p53 mutations, and suppressor T cells are the critical events responsible for the induction and development of UV-induced skin cancers. Recently, we demonstrated that CD1d knockout mice were resistant to UV-induced immunosuppression, prompting us to further address the role of CD1d in regulating UV carcinogenesis. We, therefore, investigated the response of wild-type (WT) and CD1d−/− mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d−/− mice developed skin tumors. Surprisingly, keratinocytes and fibroblasts from CD1d−/− mice were more sensitive to UV-induced apoptosis and persisted longer than cells derived from WT mice. In addition, epidermis and dermis taken from chronically UV-irradiated CD1d−/− mice harbored significantly fewer p53 mutations than WT mice. Our findings identify an unexpected and novel function for CD1d as a critical molecule regulating UV carcinogenesis, by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts. Skin cancer incidence is increasing at an astonishing rate. More than 1 million new cases of nonmelanoma skin cancer will be diagnosed in the United States this year.1Gloster HM Brodland DG The epidemiology of skin cancer.Dermatol Surg. 1996; 22: 217-226Crossref PubMed Google Scholar Epidemiological, clinical, and biological studies indicated that solar ultraviolet (UV) radiation is the major etiological agent in skin cancer development.2Strom S Epidemiology of basal and squamous cell carcinomas of the skin.in: Weber R Miller M Goepfert H Basal and Squamous Cell Skin Cancers of the Head and Neck. Williams and Wilkins, Baltimore1996: 1-7Google Scholar Wavelengths in the UVB range (280 to 320 nm) of the solar spectrum are absorbed by the skin, producing erythema, burns, immunosuppression, mutations, and nonmelanoma skin cancer. UV radiation targets the p53 tumor suppressor gene, and UV-induced mutations play a critical role in the induction of nonmelanoma skin cancer.3Brash DE Rudolph JA Simon JA Lin A McKenna GJ Baden HP Halperin AJ Ponten J A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1756) Google Scholar, 4Brash DE Ziegler A Jonason AS Simon JA Kunala S Leffell DJ Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.J Invest Dermatol Symp Proc. 1996; 1: 136-142PubMed Google Scholar, 5Kanjilal S Pierceall WE Cummings KK Kripke ML Ananthaswamy HN High frequency of p53 mutations in ultraviolet radiation-induced murine skin tumors: evidence for strand bias and tumor heterogeneity.Cancer Res. 1993; 53: 2961-2964PubMed Google Scholar, 6Matsumura Y Ananthaswamy HN Molecular mechanisms of photocarcinogenesis.Front Biosci. 2002; 7: D765-D783Crossref PubMed Scopus (114) Google Scholar The p53 protein serves as a guardian of the genome by aiding DNA repair and/or causes elimination of cells with excessive DNA damage. Excessive UV exposure overwhelms DNA repair mechanisms, allowing the survival of mutations. Keratinocytes carrying p53 mutations acquire a growth advantage by virtue of their increased resistance to apoptosis, and so begin to undergo carcinogenesis. CD1d is a novel antigen-presenting molecule encoded by nonmajor histocompatibility complex genes.7Porcelli SA Modlin RL The CD1d system: antigen-presenting molecules for T cell recognition of lipids and glycolipids.Annu Rev Immunol. 1999; 17: 297-329Crossref PubMed Scopus (604) Google Scholar CD1d presents glycolipid antigens to a unique subset of T cells, designated natural killer T (NKT) cells.8Moody DB Porcelli SA Intracellular pathways of CD1d antigen presentation.Nat Rev Immunol. 2002; 3: 11-22Crossref Scopus (157) Google Scholar, 9Exley M Garcia J Balk SP Porcelli S Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8+ T cells.J Exp Med. 1997; 186: 109-120Crossref PubMed Scopus (480) Google Scholar, 10Brossay L Chioda M Burdin N Koezuka Y Casorati G Dellabona P Kronenberg M CD1d-mediated recognition of an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution.J Exp Med. 1998; 188: 1521-1528Crossref PubMed Scopus (569) Google Scholar NKT cell function and development is restricted by CD1d.11Chen YH Chiu NM Mandal M Wang N Wang CR Impaired NK1+ T cell development and early IL-4 production in CD1d-deficient mice.Immunity. 1997; 6: 459-467Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 12Mendiratta SK Martin WD Hong S Boesteanu A Joyce S van Kaer L CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4.Immunity. 1997; 6: 469-477Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar NKT cells play a crucial role in UV-induced immune suppression because they suppress tumor immunity and allow for the progressive growth of highly antigenic UV-induced skin cancers.13Moodycliffe AM Nghiem D Clydesdale G Ullrich SE Immune suppression and skin cancer development: regulation by NKT cells.Nat Immunol. 2000; 1: 521-525Crossref PubMed Scopus (287) Google Scholar In addition, CD1d−/− mice, which are deficient in NKT cells, are resistant to the immunosuppressive effects of UV radiation.13Moodycliffe AM Nghiem D Clydesdale G Ullrich SE Immune suppression and skin cancer development: regulation by NKT cells.Nat Immunol. 2000; 1: 521-525Crossref PubMed Scopus (287) Google Scholar Because immune suppression is a risk factor for UV-induced carcinogenesis,14Kripke M Immunological effects of ultraviolet radiation.J Dermatol. 1991; 18: 429-433Crossref PubMed Scopus (41) Google Scholar we hypothesized that CD1d−/− mice may also be resistant to UV carcinogenesis. To test this hypothesis we compared the response of CD1d−/− and wild-type (WT) mice to acute and chronic UV irradiation. Our results indicate that CD1d−/− mice were more resistant to UV skin carcinogenesis than WT mice and that increased cell death and elimination of potentially malignant keratinocytes and fibroblasts accounted for this resistance. Inbred CD1d−/− and CD1d+/+ mice backcrossed onto the C57BL/6 background were obtained from Dr. Luc Van Kaer (Vanderbilt University School of Medicine, Vanderbilt, TN). The genetic identity of the CD1d−/− and CD1d+/+ mice was confirmed by reciprocal skin grafting experiments. The animals were maintained in facilities approved by Association for Assessment and Accreditation of Laboratory Care, in accordance with current regulations and standards of the National Institutes of Health. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee. A 1000 W xenon UV solar simulator equipped with a Schott WG-320 atmospheric attenuation filter (1 mm thick), a visible/infrared band pass blocking filter (Schott UG-11; 1 mm thick), and a dichroic mirror to further reduce visible and infrared energy (Oriel Corp., Stratford, CT) was used to provide solar-simulated UV radiation (UVA + UVB). The UV dose and spectral output of the light source were measured as previously described.15Ouhtit A Muller HK Davis DW Ullrich SE McConkey D Ananthaswamy HN Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin.Am J Pathol. 2000; 156: 201-207Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar For the acute UV experiments, groups of mice were irradiated with a single dose of 5 kJ/m2 UVB (290 to 320 nm), Skin was excised 6 to 168 hours after UV irradiation. For the chronic UV experiments, the mice were irradiated with 5 kJ/m2 of UVB radiation, three times a week for up to 30 weeks. For the carcinogenesis experiments, groups of 20 mice were irradiated with 10 kJ/m2 of UVB radiation, three times a week until skin tumors developed. Sham-irradiated WT and CD1d−/− mice were used as negative controls. Shaved dorsal skin (∼2 × 4 cm) was excised from each mouse and cut into two pieces. One piece was immediately fixed in 4% formaldehyde for paraffin-embedded sectioning. The other piece was floated dermis-side down in buffered 0.5 mol/L ethylenediaminetetraacetic acid, pH 7.4, for 1 hour at 37°C. The skin was separated into epidermis and dermis, and DNA was extracted from each by phenol-chloroform method. In the carcinogenesis experiments, the mice were sacrificed when the skin tumors reached ∼10 mm in diameter. The tumors were cut into two pieces, one of which was used for paraffin-embedded sectioning and the other for DNA extraction. Immunohistochemistry was performed as described.15Ouhtit A Muller HK Davis DW Ullrich SE McConkey D Ananthaswamy HN Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin.Am J Pathol. 2000; 156: 201-207Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 16Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocyte during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Briefly, 5-μm sections were deparaffinized, treated with target retrieval solution (DAKO, Carpinteria, CA), washed with phosphate-buffered saline (PBS), and incubated in 0.3% H2O2 to block endogenous peroxidase activity. After washing, the sections were preincubated for 10 minutes in 10% normal goat serum and then incubated overnight at 4°C with rabbit polyclonal anti-mouse p53 antibody (CM5; NovoCastra, Newcastle on Tyne, UK), rabbit polyclonal anti-mouse MDM2 antibody (H-221; Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit polyclonal anti-mouse p19ARF antibody (NB200-106; Novus Biologicals, Littleton, CO). After three washes with PBS plus 0.5% Tween, the sections were incubated for 15 minutes with biotin-conjugated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA). After three washes with PBS plus 0.5% Tween, the slides were treated with diaminobenzidine (Vectastain Elite ABC kit, Vector Laboratories) as recommended by the manufacturer. Counterstaining was performed with hematoxylin. As a negative control, tissue sections were stained with the secondary antibody only. TUNEL assays were performed by using a commercial kit according to the manufacture's protocol (Promega Corp., Madison, WI) as described previously.15Ouhtit A Muller HK Davis DW Ullrich SE McConkey D Ananthaswamy HN Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin.Am J Pathol. 2000; 156: 201-207Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 16Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocyte during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Genomic DNA was analyzed by AS-PCR for CC to TT tandem mutations at codons 154 to 155 and 175 to 176 and C to T transitions at codons 270 and 275 of the p53 gene, as previously described.16Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocyte during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Ananthaswamy 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 (197) Google Scholar The mutant-specific forward primers used were: 5′-TTGTGGGTCAGCGCCACTT-3′ for mutations at codons 154 to 155, and 5′-TCGTGAGACGCTGCCCCCATT-3′ for mutations at codons 175 to 176. The reverse primer used for amplification of codons 154/155 and 175/176 was 5′-GCCTGCGTACCTCTCTTTGC-3′. C to T hotspot mutations at codons 270 and 275, were detected using the forward mutant-specific primers 5′-GGACGGGACAGCTTTGAGGTTT-3′ and 5′-GTGTTTGTGCCTGCCT-3′, respectively. The reverse primer used to detect both mutations was 5′-GCCTGCGTACCTCTCTTTGC-3′. The reaction mixture containing DNA (200 ng) was amplified by PCR in a 25-μl solution containing 10 mmol/L Tris-HCl (pH 8.3); 50 mmol/L KCl; 1.5 mmol/L MgCl2; 0.001% gelatin; 75 μmol/L each of dATP, dGTP, dCTP, and dTTP; 2.5 μCi of [32P]dCTP; upstream and downstream primers (200 nmol/L each); and 1.5 U of AmpliTaq (PE Xpress) for 25 cycles. An aliquot of the PCR product was separated on 2.0% agarose gels. Primary keratinocyte and fibroblast cultures were prepared according to Hager and colleagues.18Hager B Bickenbach JR Fleckman P Long-term culture of murine epidermal keratinocytes.J Invest Dermatol. 1999; 112: 971-976Crossref PubMed Scopus (107) Google Scholar Briefly, newborn skin (2 to 3 days after birth) was excised and floated, dermis down, on 0.25% trypsin at 4°C for 18 hours. The epidermis was separated from the dermis, minced with scissors, suspended in Eagle's minimal essential medium (MEM) with Earle's balanced salt solution without calcium (EMEM) (Cambrex, NJ), and stirred for 30 minutes at 4°C. The suspension was filtered through sterile gauze, centrifuged, and the cells were resuspended in 10 ml of EMEM. Keratinocytes were plated on BD BioCoat collagen IV 60-mm culture dishes (BD Biosciences, San Jose, CA) in EMEM plus 0.06 mmol/L CaCl2 and 10% chelexed fetal bovine serum. Dermal fibroblasts were plated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Keratinocytes or fibroblasts (1 × 105 cells per dish) were plated on 6-cm dishes and incubated overnight. The cells were washed with PBS and exposed to 30 J/m2 (UVB component) from a Kodacel-filtered FS40 sunlamp (National Biological Corp., Twinsburg, OH). After a 24- to 72-hour incubation in EMEM, the cells were washed twice with ice-cold PBS, trypsinized, and collected. The cells were resuspended in 5 ml of ice-cold 70% ethanol, incubated at 4°C overnight, centrifuged, and resuspended in 500 μl of PBS containing RNase (100 U) and PI (25 μg). After incubation for 15 minutes at 37°C, the apoptotic cells (sub-G1 DNA content) were detected by flow cytometry. Data are presented as the means ± SEM. The data were analyzed by using an unpaired Student's t-test (StatView 4.0; SAS Institute, Cary, NC). Probabilities less than 0.05 were considered significant. Because UV-induced immune suppression is a major risk factor for sunlight-induced carcinogenesis, and because CD1d−/− mice were resistant to UV-induced immune suppression,13Moodycliffe AM Nghiem D Clydesdale G Ullrich SE Immune suppression and skin cancer development: regulation by NKT cells.Nat Immunol. 2000; 1: 521-525Crossref PubMed Scopus (287) Google Scholar we hypothesized that CD1d−/− mice may also be resistant to UV skin carcinogenesis. To test this hypothesis, WT and CD1d−/− mice (n = 20) were irradiated with solar-simulated UV radiation three times a week and tumor development was measured. The first skin tumor developed in a WT mouse 28 weeks after irradiation. Fifty percent of the WT mice developed skin tumors by week 36, and all WT mice had skin tumors at week 45 (Figure 1). In contrast, the first skin tumor in a CD1d−/− mouse developed after 33 weeks of chronic UV irradiation, and 50% of CD1d−/− mice developed skin tumors at week 42. At week 45, when all of the WT mice had skin tumors, only 60% (12 of 20) of CD1d−/− mice had skin tumors. Continued irradiation until week 55 produced skin tumors in only 70% (14 of 20) of CD1d−/− mice. In all, 25 skin tumors developed in 20 WT mice (average, 1.25 tumors per mouse), and 21 skin tumors in 14 CD1d−/− mice (average, 1.50 tumors per mouse). Analysis of UV-induced skin tumors from WT and CD1d−/− mice revealed the presence of p53 mutations in all of the expected hotspots. In addition, the mutation frequency did not differ significantly between WT and CD1d−/− mice (Table 1).Table 1Number of p53 Mutations in Six WT and Six CD1d−/− Mice after Chronic UV ExposureUV treatmentTissueMiceCodons 154 to 155Codons 175 to 176Codon 270Codon 275Total no. of mutationsP value4 weeksEpidermisWT0/60/61/61/62—CD1d−/−0/60/60/60/60DermisWT0/60/60/60/60—CD1d−/−0/60/60/60/6012 weeksEpidermisWT4/64/63/65/616<0.001CD1d−/−2/61/61/61/65DermisWT1/62/60/61/64<0.05CD1d−/−0/60/60/60/6030 to 35 weeksTumorsWT6/65/65/65/621—CD1d−/−4/64/65/65/618 Open table in a new tab Because UV-exposure induces apoptosis, we determined whether CD1d−/− mice showed altered susceptibility to apoptosis by measuring TUNEL-positive cells in UV-irradiated mouse skin. The numbers of TUNEL-positive keratinocytes peaked at 24 hours in both WT and CD1d−/− mice (Figure 2a). By 72 hours, TUNEL-positive cells decreased rapidly in the skin of WT mice but persisted in the CD1d−/− mice. There were significantly more TUNEL-positive keratinocytes in CD1d−/− mouse skin than in WT mouse skin (48.8 ± 4.9 versus 9.3 ± 2.2, per 100 cells; P < 0.001) at 72 hours and at 96 hours (28.3 ± 3.6 and 4.8 ± 1.0, P < 0.001) (Figure 2b). TUNEL-positive cells were also present in the dermis of both WT and CD1d−/− mice and the pattern of expression at each time point correlated epidermal expression (data not shown). The data presented above indicate slower removal of apoptotic keratinocytes and fibroblasts from the skin of CD1d−/− mice. We wanted to determine whether this is an intrinsic property of CD1d-deficient keratinocytes or if it reflects an alteration in systemic immune function in the NKT cell-deficient mice. Keratinocyte and fibroblast cultures, isolated from neonatal WT and CD1d−/− mice, were exposed to 30 J/m2 UV radiation in vitro, and apoptosis measured by flow cytometry (Figure 3). More apoptotic cells were found in irradiated keratinocytes from CD1d−/− mice than from WT mice (8.4% versus 5.4% 24 hours after UV irradiation). The difference was even more pronounced at the later time points (28.0% versus 8.0% at 48 hours, P < 0.001 and 33.9% versus 11.8% at 72 hours, P < 0.001). Primary fibroblasts from CD1d−/− mice were also more sensitive to UV-induced apoptosis than fibroblasts from WT mice (7.5% versus 3.5% at 24 hours, 15.7% versus 4.2% at 48 hours, P < 0.01, and 24.8% versus 10.8%, at 72 hours, P < 0.01). The fact that we show that keratinocytes and fibroblasts are more sensitive in vitro would suggest that the persistence of apoptosis observed in CD1d−/− mouse skin exposed to acute UV likely reflects increased skin apoptosis rather than delayed removal. Because the p53 protein plays an important regulatory role in UV-induced apoptosis, we determined whether the alteration of apoptosis observed in CD1d−/− cells was because of altered p53 expression. In both WT and CD1d−/− mice, intense nuclear immunostaining was observed 24 hours after UV irradiation (Figure 4a). Although the number of p53-positive keratinocytes had decreased dramatically at 72 hours after UV irradiation in WT mouse skin, the p53-positive cells were still present in CD1d−/− mouse skin. To determine whether the persistent expression of p53 protein was because of altered expression of MDM2 protein, which binds and degrades p53, we analyzed MDM2 expression in UV-irradiated WT and CD1d−/− mice. Figure 4b shows intense MDM2 protein expression in the nuclei and weak cytoplasmic expression 24 hours after UV exposure in WT mouse skin. In contrast, MDM2 was not expressed after UV exposure of CD1d−/− mice. These results suggest that persistent expression of p53 in CD1d−/− mouse skin is because of absence of MDM2. p19ARF protein, which could affect MDM2 expression, was expressed at similar levels in both WT and CD1d−/− mouse skin at 24 hours after UV exposure (data not shown). Although acute UV irradiation induces apoptosis, after chronic exposure to UV radiation, mice develop resistance to apoptosis.16Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocyte during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar To determine whether CD1d deficiency alters the apoptotic response in chronic UV-irradiated mice, mice were irradiated with UV, three times per week for 30 weeks, and TUNEL-positive cells were counted at various time points. Figure 5 shows that early during the course of UV irradiation (1 to 2 weeks), CD1d−/− mouse skin contained significantly more TUNEL-positive keratinocytes than WT mice did. After 1 week of chronic UV exposure, there were 7.5 ± 1.1 TUNEL-positive keratinocytes per 100 cells in CD1d−/− mouse skin but only 3.3 ± 0.7 per 100 in WT mice (P < 0.001). Significant differences were also observed after 2 (6.7 ± 1.4 versus 3.8 ± 0.7, P < 0.01) and 3 weeks (4.8 ± 0.7 versus 3.3 ± 1.1, P < 0.05) of chronic UV exposure. However, there were no significant differences in the numbers of TUNEL-positive cells between CD1d−/− and WT mice after 4 weeks of chronic UV irradiation. These results indicate that CD1d−/− keratinocytes are more sensitive to UV-induced apoptosis and are eliminated from the epidermis of UV-irradiated mice. p53-positive clusters of cells, which are thought to undergo further clonal expansion into skin cancer, are present in UV-damaged skin.19Berg RJW 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 (203) Google Scholar, 20Rebel H Mosnier LO Berg RJW 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, 21Jonason 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 (563) Google Scholar Because CD1d−/− mouse skin exhibited increased apoptosis after chronic UV irradiation, we hypothesized that continued elimination of UV-damaged keratinocytes should decrease the number of p53-positive clusters in CD1d−/− mice. To test this hypothesis, we counted p53-positive clusters in WT and CD1d−/− mouse skin after chronic UV irradiation. After 1 to 6 weeks of chronic UV irradiation, p53-positive keratinocytes were scattered mainly in the basal layer of the epidermis in both WT and CD1d−/− mice (data not shown). After 12 and 30 weeks of chronic UV irradiation, clusters of atypical keratinocytes with relatively large and irregular nuclei were present in WT mouse skin (Figure 6a). Such clusters were also present in CD1d−/− mouse epidermis but were of lesser frequency (Figure 6b). The average number of p53-positive clusters at week 12 was significantly higher in the epidermis of WT mice than in CD1d−/− mouse epidermis. At week 30, the number of p53-positive clusters increased in CD1d−/− mice, but only marginally in WT mice. Atypical p53-positive fibroblast clusters were also present in the dermis of WT mice UV-irradiated for 30 weeks (Figure 6c). At this time point, only one CD1d−/− mouse had a cluster of atypical cells in the dermis. After 35 to 38 weeks of irradiation, the numbers of dermal p53-positive clusters in WT and CD1d−/− were not statistically different (Figure 6c). Although fewer p53-positive clusters in the skin of chronically UV-irradiated CD1d−/− mice may account for the fewer skin cancers in CD1d−/− mice, a more direct test of that hypothesis is to measure the frequency of UV-induced p53 signature mutations.16Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocyte during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Ananthaswamy 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 (197) Google Scholar Therefore, we analyzed the genomic DNA from the epidermis and dermis of UV-irradiated WT and CD1d−/− mice by mutant AS-PCR.9Exley M Garcia J Balk SP Porcelli S Requirements for CD1d recognition by human invariant Valpha24+ CD4-CD8+ T cells.J Exp Med. 1997; 186: 109-120Crossref PubMed Scopus (480) Google Scholar, 19Berg RJW 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 (203) Google Scholar, 20Rebel H Mosnier LO Berg RJW 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 The representative gel of AS-PCR (Figure 7) indicates that three of six epidermal samples from WT mice exposed to UV radiation for 12 weeks but only one of six epidermal samples from CD1d−/− mice had codon 270 mutations. Similarly, two of six dermal samples from WT mice had p53 mutations at codons 175 to 176, but none of six dermal samples from CD1d−/− mice did. The p53 mutation data for all of the hotspot codons in mice exposed to UV radiation are shown in Table 1. After 4 weeks of irradiation, two p53 mutations were found in the epidermis of the six WT mice but none in the dermis. After 12 weeks of irradiation, 16 mutations were found in the epidermis of the six WT mice. In contrast, none of six CD1d−/− mice irradiated for 4 weeks had mutations in the epidermis or dermis. Moreover, the total number of p53 mutations in the epidermis of CD1d−/− mice UV irradiated for 12 weeks was significantly lower (five mutations per six mice, P < 0.001) than that detected in the epidermis of WT mice (16 mutations per six mice). The number of p53 mutations in the dermis of CD1d−/− mice exposed to UV for 12 weeks was also significantly different (0 mutations per six mice, P < 0.05) than the number in the dermis of WT mice (four mutations per six mice). Here, we provide evidence that skin tumor incidence is decreased in CD1d−/− mice. Mice that do not express the CD1d protein eliminated UV-damaged, potentially malignant keratinocytes and fibroblasts by prolonging p53 protein expression and, at the same time, down-regulating MDM2, a negative regulator of p53. Repair of UV-induced DNA damage and removal of cells containing DNA damage decrease the probability of tumor development.4Brash DE Ziegler A Jonason AS Simon JA Kunala S Leffell DJ Sunlight and sunburn in human skin cancer: p53, apoptosis, and tumor promotion.J Invest Dermatol Symp Proc. 1996; 1: 136-142PubMed Google Scholar, 20Rebel H Mosnier LO Berg RJW 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, 21Jonason 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 (563) Google Scholar, 22Ziegler 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 (1363) Google Scholar Although there was no difference in the repair of UV-induced pyrimidine dimers in CD1d−/− and WT mouse skin (data not shown), more UV-damaged keratinocytes and fibroblasts were removed from CD1d−/− than WT mouse skin. Even after chronic UV irradiation for 3 weeks, many more apoptotic cells were present in CD1d−/− mouse skin than in WT mouse skin. We previously showed that chronic UV irradiation results in dysregulation of apoptosis, which leads to hyperproliferation of keratinocytes.16Ouhtit A Gorny A Muller HK Hill LL Owen-Schaub L Ananthaswamy HN Loss of Fas-ligand expression in mouse keratinocyte during UV carcinogenesis.Am J Pathol. 2000; 157: 1975-1981Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar Hyperproliferative keratinocytes