p53 mutations in basal cell carcinomas arising in routine users of sunscreens

Sun exposure histories were obtained from a series of patients age 35 or younger following diagnosis and removal of a basal cell carcinoma (BCC). The DNA was extracted from tumor biopsy samples derived from BCC of 10 patients who reported that they did not use sunscreens during youth (age 18 or younger) and 10 patients who routinely employed sunscreens during this age period. Exons 5-9 of the p53 gene were then amplified in three fragments from these samples using a nested polymerase chain reaction (PCR) approach and screened for mutations using an RNA heteroduplex assay. All PCR products displaying evidence of a mutation were sequenced. It was found that 6 of the 10 patients who were not routine sunscreen users displayed mutations in these p53 exons. All of the mutations were located at dipyrimidine sites, five of the six were C-->T transitions and one mutation was a tandem double mutation, consistent with a role for solar UVB in BCC formation. In contrast, only one p53 mutation was detected in the group of 10 patients who routinely employed sunscreens during childhood and adolescence. Hence, a significantly (P = 0.029) lower level of p53 mutations was detected in the BCC obtained from sunscreen users compared with tumors derived from nonusers. These findings suggest that the mechanisms involved in the etiology of skin carcinogenesis differ in sunscreen users compared with people who did not routinely employ sunscreens. These data are also indicative of a protective effect associated with sunscreen use against the formation of p53 mutations. It is possible that the patients who were diagnosed with BCC despite their use of sunscreens possessed a genetic susceptibility for skin cancer formation and developed BCC through a p53-independent pathway. Alternatively, solar UVA wavelengths, that were generally not blocked by the suncare products employed by the sunscreen users, may have played a significant role in BCC development through induction of a mutation(s) in an oncogene and/or a tumor suppressor gene, other than p53, for these patients.     

Effects of ultraviolet B exposure on the expression of proliferating cell nuclear antigen in murine skin

Proliferating cell nuclear antigen (PCNA) is an active nuclear protein involved in DNA replication, recombination and repair. PCNA is found throughout the basal layer in normal skin and in all layers of the epidermis in malignancy. This study evaluates PCNA's expression after acute and chronic UV-B irradiation. Skh-1 hairless mice exposed to 1.5 and 4.5 kJ/m2 of UV-B were sacrificed at 6, 12, 24, 48, 72 and 168 h. Immunohistochemical analysis revealed PCNA expression throughout the basal layer of untreated skin, with diminished expression at 6 h, indicative of immediate UV damage, and evidenced by the observable upregulation in pyrimidine dimer formation early on. Subsequently, PCNA immunoreactivity progressively increased, demonstrating an aberrant upward epidermal migratory pattern in association with chronic exposure. The 4.5 kJ/m2 group exhibited prolonged recovery in staining and also demonstrated this altered migratory pattern with chronic exposure. Progressive reactivation of PCNA expression occurs with repair. PCNA migration to upper layers of the epidermis indicates proliferation and possibly a subsequent increased malignant potential. We conclude that PCNA can serve as a marker of DNA repair and indirectly as an indicator of UV-B-induced damage, expression being time dependent and dose related. Specific immunoreactivity patterns and the observable atypia in keratinocytes are relevant in elucidating malignant potentiation.     

The combination of benzo[a]pyrene and ultraviolet A causes an in vivo time-related accumulation of DNA damage in mouse skin

Polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are ubiquitous environmental carcinogens. BaP is metabolized in vivo to reactive intermediates that become covalently bound to DNA and form BaP-DNA adducts, an initial event in carcinogenesis. Ultraviolet A (UVA) synergizes with BaP to significantly enhance genetic damage and accelerate carcinogenic processes. This study was initiated to investigate in vivo cellular changes related to carcinogenesis induced by repeated exposures to BaP plus UVA. Simulated chronic exposure to an environmental carcinogen and sunlight was conducted through biweekly topical application of BaP followed 2 h later by UVA exposure over a 10 week period. BaP diol epoxide (BPDE)-DNA adducts were measured in vivo by immunohistochemistry using an anti-BPDE-DNA monoclonal antibody. Oxidative DNA damage was measured by the detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation using high-performance liquid chromatography. Alterations in the cell cycle that were relevant to carcinogenesis were revealed by changes in p53, as identified in vivo using a polyclonal anti-p53 antibody. We found that cells containing BPDE-DNA adducts and nuclear p53 expression significantly increased between 2 and 10 weeks of BaP-UVA treatment, whereas neither BPDE-DNA adducts nor significant changes in p53 were observed in untreated skin. Using regression analysis, oxidative 8-OHdG damage also showed a parallel increase over 2-10 weeks (r = 0.80). These results indicate that genetic damage caused by exposures to BaP plus UVA accumulates with time and increases the potential for inductive events leading to carcinogenesis and tumor formation.     

COLLAGEN ALTERATIONS IN CHRONICALLY SUN-DAMAGED HUMAN SKIN

Abstract The major histological characteristic of sun-damaged skin is the accumulation of an elastotic material that appears to replace collagen. This elastotic material consists primarily of elastin and histological studies suggest a large loss of collagen in the dermis of chronically sun-damaged skin. In this study, we examine the content and distribution of collagen and procollagen in sun-damaged human skin. The total collagen content of sun-damaged skin was 20% less than nonsolar-exposed skin (524 μ g collagen per mg total protein in sun-damaged skin and 667 μg collagen per mg total protein in nonsolar-exposed skin). In addition, there was a 40% decrease in the content of intact amino propeptide moiety of type III procollagen in sun-damaged skin (0.68 U per 50 mg wet weight) as compared to nonsolar-exposed skin (1.12 U per 50 mg wet weight). The data suggest that this change in collagen content is due to increased degradation. The distribution of collagen in sun-damaged skin was examined by indirect immunofluo-rescence. Mild digestion of sun-damaged skin with elastase removed the elastin and revealed the presence of collagen in the elastotic material. Therefore, the elastin appears to mask the presence of collagen fibers in the dermis of sun-damaged skin.     

p53 gene mutations in SKH-1 mouse tumors differentially induced by UVB and combined subcarcinogenic benzo[a]pyrene and UVA

We compared the frequency and spectra of p53 mutations in skin tumors from UVB-irradiated and benzo(a)pyrene-UVA-treated SKH-1 mice. Analysis of p53 mutations using a combination of polymerase chain reaction, denaturing high-performance liquid chromatography, and sequencing shows that the frequency and spectrum of p53 mutations in BaP-UVA-induced tumors are quite different from those in UVB-induced tumors. SKH-1 mice were treated with BaP-UVA or UVB for 30 weeks after which skin tumors were collected for analysis of p53 mutations. Among the 11 BaP-UVA-induced tumors with diameters of 5-10 mm, two displayed mutations in exon 8 yielding a mutation frequency of 18.2%. In contrast, the mutation frequency among BaP-UVA-induced tumors was 10.5%. In UVB-induced tumors, the mutation frequency in exon 8 was highly correlated with tumor size. A total of 77.8% of tumors with diameters larger than 10 mm contained p53 mutations. The overall mutation frequency among UVB-induced tumors was 17.9% in exon 8 and only 3.8% in exon 5. Hotspots for p53 mutation in UVB-induced tumors were found at codons 128 and 149 (exon 5), and at codons 268, 270, 271 and 273-276 (exon 8). In addition to widely recognized C-->T missense mutations, there were also tandem CC-->AG changes coupled with either an insertion of T, a C-->G substitution or G-->C/T mutations. All of the mutations were found at tri- or tetra-pyrimidine sites. Thirty-nine per cent of all p53 mutations occurred at codons 274 and 275; 53% occurred at codons 268-271. Two multiple mutation clusters were located at codons 268-271 and 274-276. Both BaP-UVA and UVB caused C-->T transitions at codon 275 in exon 8. A C-->T mutation at codon 294 was induced only by BaP-UVA treatment. In contrast to UVB treatment, BaP-UVA treatment did not induce any mutations in exon 5. We show that individually subcarcinogenic levels of BaP and UVA synergistically induce a novel p53-mutation fingerprint. This fingerprint could serve as a prognostic indicator for the development of BaP-UVA-induced skin tumors.     

Induction of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Ultraviolet Radiation in Calf Thymus DNA and HeLa Cells

Abstract— The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10⁵ 2'-deoxyguanosine (dGuo) per kJ/m² for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10⁵ dGuo per kJ/m², respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irra-diated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.