Resistance of CD1d-/- mice to ultraviolet-induced skin cancer is associated with increased apoptosis

Abstract

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.

Figure 1

Induction of skin tumors in chronically UV-irradiated WT and CD1d−/− mice. The mice were irradiated with 10 kJ/m² (UVB component) three times per week. A mouse was scored as having a tumor when a skin lesion reached ∼2 to 3 mm in diameter and persisted or increased in diameter for the duration of the experiment.

Figure 2

Response of WT and CD1d−/− mouse skin to acute (5 kJ/m²) UV irradiation. a: At 72 hours after UV irradiation, more TUNEL-positive keratinocytes were present in CD1d−/− mice than in WT mice. DNA was counterstained with PI to identify the nuclei (left). b: Quantitation of TUNEL-positive cells 12 to 120 hours after acute UV exposure. Four mice were sacrificed at each time point, and four microscopic fields in each mouse were used to calculate average number of TUNEL-positive keratinocytes. ***, P < 0.001.

Figure 3

Representative cell-cycle analysis of UV-irradiated (30 J/m² of UVB) primary keratinocytes (a) and fibroblasts (b) from 2- to 3-day-old WT and CD1d−/− mice. Forty-eight hours after UVB irradiation, there were significantly more apoptotic keratinocytes in CD1d−/− mice (28.0%) than in WT mice (8.0%, P < 0.001), and significantly more apoptotic fibroblasts in CD1d−/− mice (15.7%) than in WT mice (4.2%, P < 0.01). The horizontal lines correspond to the apoptotic cell fractions.

Figure 4

p53 and MDM2 expression in UV-irradiated WT and CD1d−/− mice. a: p53 protein expression was measured in both WT and CD1d−/− skin 24 and 72 hours after UV irradiation. b: MDM2 protein expression was measured both in WT and CD1d−/− skin 24 and 72 hours after UV irradiation.

Figure 5

Increased keratinocyte apoptosis in chronically UV-irradiated CD1d−/− mice compared with WT mice. The mice were exposed to UV radiation three time per week for 1 to 12 weeks and TUNEL-positive cells in the skin were measured. The results are averages of four different microscopic fields from two mice at each time point. ***, P < 0.001; **, P < 0.001; *, P < 0.05.

Figure 6

Induction of p53-positive, abnormal cell clusters in chronically UV-irradiated WT and CD1d−/− mouse skin. a: Clusters of p53-positive atypical keratinocytes (top) and fibroblasts (bottom) in a representative WT mouse after 30 weeks of chronic UV irradiation. b and c: Quantitation of p53-positive atypical clusters in the epidermis (b) and dermis (c) of mice exposed to chronic UV irradiation. ***, P < 0.001.

Figure 7

Representative results of AS-PCR detecting p53 mutations. Genomic DNA from epidermal samples of six WT mice and six CD1d−/− mice after 12 weeks of UV irradiation were analyzed for codon 270 (top), and genomic DNA from dermal samples of the same mice were analyzed for codons 175 to 176 (bottom). Amplification of mutant sequences of the sizes expected for codon 270 (134 bp) and codons 175 to 176 (55 bp) are indicated by arrows. M, molecular marker (phiX174/HaeIII); P, tumor DNA known to contain mutation at the specified codons; N, negative control (genomic DNA from normal WT mouse skin).