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p53 mutations appear to be early events in skin carcinogenesis induced by chronic UVB irradiation. Clusters of epidermal cells that express p53 in mutant conformation ("p53 positive foci") are easily detected by immunohistochemical staining long before the appearance of skin carcinomas or their precursor lesions. In a hairless mouse model, we determined the dose-time dependency of the induction of these p53+ foci and investigated the relationship with the induction of skin carcinomas. The density of p53+ foci may be a good direct indicator of tumor risk. Hairless SKH1 mice were exposed to either of two regimens of daily UVB (500 or 250 J/m2 broadband UV from Philips TL12 lamps; 54% UVB 280-315 nm). With the high-dose regimen, the average number of p53+ foci in a dorsal skin area (7.2 cm2) increased rapidly from 9.0 +/- 2.1 (SE) at 15 days to 470 +/- 80 (SE) at 40 days. At half that daily dose, the induction of p53+ foci was slower by a factor of 1.49 +/- 0.15, very similar to a previously observed slower induction of squamous cell carcinomas by a factor of 1.54 +/- 0.02. In a double-log plot of the average number of p53 + foci versus time, the curves for the two exposure regimens ran parallel (slope, 3.7 +/- 0.7), similar to the curves for the number of tumors versus time (slope, 6.9 +/- 0.8). The difference in slopes (3.7 versus 6.9) is in line with the contention that more rate-limiting steps are needed to develop a tumor than a p53+ focus. By the time the first tumors appear (around 7-8 weeks with the high daily dose), the dorsal skin contains >100 p53+ foci/cm2. To further validate the density of p53+ foci as a direct measure of tumor risk, we carried out experiments with transgenic mice with an enhanced susceptibility to UV carcinogenesis, homozygous Xpa knockout mice (deficient in nucleotide excision repair) and heterozygousp53 knockout mice (i.a. partially deficient in apoptosis). In both of these cancer-prone strains, the p53+ foci were induced at markedly increased rates, corresponding to increased rates of carcinoma formation. Therefore, the frequency of p53+ foci appears to correlate well with UVB-induced tumor risk.

The nucleotide excision repair (NER) system is comprised of two subpathways, i.e., transcription-coupled repair (TCR) and global genome repair (GGR). To establish the relative importance of TCR and GGR for UV effects on the skin, we have used hairless knockout mouse strain lacking either TCR (CSB -/-) or GGR (XPC -/-). In single exposure experiments, we found that CSB -/- mice have a 7-16 times higher susceptibility to sunburn than XPC -/- mice and than heterozygous (+/-) and wild-type (+/+) controls. Exposure to 80 J/m2 UV radiation (i.e., suberythemogenic in CSB -/-) on 10 consecutive days gives rise to epidermal hyperplasia in CSB -/- and XPC -/-, whereas repair-proficient controls do not show epidermal hyperplasia from these exposures. In addition, CSB -/- mice develop marked parakeratosis, whereas XPC -/- mice and controls do not. Under continued exposure to this daily dose, squamous cell carcinomas appear in CSB -/-, XPC -/-, and in the control groups, whereas only in the CSB -/- animals is a fairly high number of benign papillomas also found. The median latency time of squamous cell carcinomas (diameters > or = 1 mm) is 84 days for the XPC -/- mice, 115 days for the CSB -/- mice, and 234-238 days for the heterozygous and wild-type control groups. These results indicate that GGR is more important than TCR in protection against UV-induced carcinomas of the skin but not against other UV effects such as sunburn, epidermal thickening, scaling of the stratum corneum, and development of papillomas. These results also indicate that GGR capacity may serve as a better predictor for skin cancer susceptibility than sensitivity to sunburn. The relative cancer susceptibilities of GGR- and TCR-deficient skin could well depend on the balance between an increased mutation rate and the presence (in CSB -/-) or lack (in XPC -/-) of a compensatory apoptotic response.

Patients with the nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) are highly predisposed to develop sunlight-induced skin cancer, in remarkable contrast to photosensitive NER-deficient trichothiodystrophy (TTD) patients carrying mutations in the same XPD gene. XPD encodes a helicase subunit of the dually functional DNA repair/basal transcription complex TFIIH. The pleiotropic disease phenotype is hypothesized to be, in part, derived from a repair defect causing UV sensitivity and, in part, from a subtle, viable basal transcription deficiency accounting for the cutaneous, developmental, and the typical brittle hair features of TTD. To understand the relationship between deficient NER and tumor susceptibility, we used a mouse model for TTD that mimics an XPD point mutation of a TTD patient in the mouse germline. Like the fibroblasts from the patient, mouse cells exhibit a partial NER defect, evident from the reduced UV-induced DNA repair synthesis (residual repair capacity approximately 25%), limited recovery of RNA synthesis after UV exposure, and a relatively mild hypersensitivity to cell killing by UV or 7,12-dimethylbenz[a]anthracene. In accordance with the cellular studies, TTD mice exhibit a modestly increased sensitivity to UV-induced inflammation and hyperplasia of the skin. In striking contrast to the human syndrome, TTD mice manifest a dear susceptibility to UV- and 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis, albeit not as pronounced as the totally NER-deficient XPA mice. These findings open up the possibility that TTD is associated with a so far unnoticed cancer predisposition and support the notion that a NER deficiency enhances cancer susceptibility. These findings have important implications for the etiology of the human disorder and for the impact of NER on carcinogenesis.

Mutations with clear "UVB fingerprints" have been observed in the p53 gene of human nonmelanoma skin tumors and of experimentally UVB-induced murine skin tumors. Although UVA (315-400 nm) radiation is also a complete carcinogen, its contribution to sunlight-induced mutagenesis remains poorly characterized. There is experimental evidence that the production of reactive oxygen species plays a more dominant role with long-wave UVA than with UVB radiation. We have induced skin tumors (n = 42) in hairless SKH:HR1 mice (n = 14) by daily exposure to long-wave UVA (365-nm) radiation. The incidence of p53 alterations in these tumors is low compared to UVB-induced tumors; positive staining for the p53 protein was observed in only 50% of the tumors, and less than 15% of the tumors showed a mutation in one of the exons 5, 7, or 8 of the p53 gene. The pattern of p53 staining was more irregular and less dense compared to UVB, and the mutations (all C-->T) were mainly (six of seven) located at codon 267. Besides a general p53 hotspot, this codon is also the main hotspot for UVB-induced skin tumors in these mice. No mutations specific for UVA, ie., mutations specific for reactive oxygen species, could be detected.

Although xeroderma pigmentosum (XP) patients are rare, carriers of XP genes (heterozygotes) are much more common. Whether such carriers have an increased skin cancer risk is unknown. Recently developed mouse models for XP have opened up the possibility of determining the skin cancer risk of heterozygotes relative to wild types. Therefore, the XPA knockout trait has been crossed into hairless mice, and squamous cell carcinomas of the skin have been induced by low daily UVB exposures for 500 days in all three genotypes (-/-, +/-, and +/+). The carcinogenic response of the heterozygotes did not significantly differ from that of their wild-type littermates. Tumors in the XPA -/- animals appeared with a latency time that was decreased by a factor of 4.2. From this, we estimate that a functional XPA gene provides a "protection factor" of 60 (95% confidence interval, 15-250) against UV carcinogenesis, which is greater protection than that against acute UV effects, such as erythema and edema (protection factor between 7 and 16). Deficient nucleotide excision repair appears to have a more dramatic impact on skin cancer susceptibility than on sensitivity to acute UV effects.

The rate of tumor induction by UV-A radiation rises more slowly with time and accumulated dose than that by UV-B radiation. It has recently been shown that this difference disappears when frank papillomas are excluded from the analysis. Thus, the rate of development of "nonpapillomas" (mainly squamous cell carcinomas and precursors) can be fully characterized by a typical tumor induction time, e.g., the time until 50% of the mice bear tumors. This has opened the possibility to investigate how UV-A and UV-B exposures add up in the induction of squamous cell carcinomas, which is an important issue in risk assessments of artificial UV-A sources for cosmetic or medical purposes. We present the results of an experiment in which 6 groups of 24 albino SKH:HR1 mice were treated daily for 600 days with either effective UV-A radiation, effective UV-B radiation, or combinations of both. The observed times it took for 50% of the mice to bear tumors in the combination groups were compared with those calculated on the basis of arithmetical addition of effective UV-A and effective UV-B doses. We did not find a statistically significant (P < 0.05) deviation from additivity.

This study describes the induction and repair of UV-induced cyclobutane pyrimidine dimers (CPD) in transcriptionally active and inactive genes in the epidermis of the hairless mouse. Mice were exposed to a single dose of 2000 J/m2 ultraviolet B and kept in darkness for up to 24 h. The CPD frequency was measured in the transcriptionally active hypoxanthine-guanine phosphoribosyltransferase gene, the adenosine deaminase gene, the inactive c-mos protooncogene, and the haptoglobin gene using the CPD-specific enzyme T4 endonuclease V. Sixty % of the CPD was removed from the active genes during the first 4 h, after which no further repair took place up to 24 h. In contrast, the inactive genes did not show any removal of CPD. Assuming that the rate of repair in the c-mos and haptoglobin genes is representative for the repair rate in the genome overall, these results suggest only marginal repair of UV-induced CPD in the mouse epidermis in vivo. The selective repair of active genes in the epidermis of mice resembles that of rodent cells in culture and shows the biological relevance of repair studies performed with cultured rodent cells in vitro.