Tumorigenesis by short-wave ultraviolet A: papillomas versus squamous cell carcinomas

The tumorigenic properties of UBV radiation (wavelengths 280-315 nm) are well established, in contrast to those of UVA radiation (315-380 nm). Very little information is available on the short UVA wavelengths (315-340 nm). To expand our knowledge on UVA tumorigenesis we investigated the development of skin tumours in albino hairless mice (SKH-hr1) exposed to custom-built experimental fluorescent tubes (EFL330) with spectral output centred around 330 nm. Two groups received continued daily exposures: one 56 kJ/m2 and the other 20 kJ/m2 per day. The third group was exposed to yellow fluorescent light devoid of UV, and served as a control. Each group consisted of 24 mice. Most of the mice in the high-dose group developed tumours; after 431 days 50% were tumour bearing. Two main types of tumours were observed: papillomas (Pap) and squamous cell carcinomas (SCC). In the low dose group only three mice attracted one papilloma each. No tumours were seen in the control group. Results show that the short-wave as well as the long-wave UVA contributes to carcinogenicity; the short wavelengths being approximately 5 times more efficient. The kinetics of tumour development under UVA exposure appeared to be different from that under UVB exposure. If, however, we exclude papillomas from our analysis, development is very much the same as with UVB irradiation, which yields predominantly SCC.     

The influence of ventral UVA exposure on subsequent tumorigenesis in mice by UVA or UVB irradiation.

Exposure to ultraviolet-B radiation (UVB: 280-315 nm) can result in a decreased immune response. This immune suppression can be restricted to the exposed skin site (local immune suppression) but may also be systemic. To investigate whether ultraviolet-A radiation (UVA: 315-400 nm) could also exert such a systemic effect, we performed the present investigation. The study consisted of two parts. Experiment I: 24 albino hairless mice (SKH:HRI) were ventrally exposed to UVA radiation for 300 days (glass-filtered Philips TLK09 fluorescent tubes, daily dose: 350 kJ/m2), while 24 control mice were left unexposed. After this period the control animals were still tumour free, but 60% of the exposed animals had developed abdominal tumours. Subsequently ventral exposures were stopped and both groups were dorsally exposed to identical UVB regimens (Westinghouse FS40, daily dose: 900 J/m2). Experiment II: this was virtually the same as experiment I, but here the mice were dorsally exposed to UVA radiation (glass-filtered Philips TLK09, daily dose: 290 kJ/m2) instead of UVB radiation. If we look at all tumours induced dorsally, we find no significant influence of pre-exposures to UVA radiation. This holds for dorsal UVB as well as for dorsal UVA exposures. In contrast to UVB, however, the UVA radiation induced many papillomas. Excluding the papillomas from the analysis we find that the induction of non-papillomas (mainly squamous cell carcinomas) under dorsal UVA exposure, is slightly enhanced in the ventrally pre-exposed group (difference significant at the P < 0.05 level). This suggests that UVA radiation induced only a weak systemic effect. Ventral UVA pre-exposure did not appear to affect dorsal skin irritation as expressed by scratch marks. The induction period for hyperkeratosis, however, was significantly shortened by the ventral UVA pre-exposure; this applied to dorsal UVB as well as dorsal UVA exposures.     

Potentiating effect of dietary vitamin A on photocarcinogenesis in hairless mice

Vitamin A and its derivatives (retinoids) exert modulatory effects on epithelial differentiation and are used therapeutically against skin cancers, but the role of dietary vitamin A in ultraviolet (UV)-induced carcinogenesis is far from clear. To study this process, 220 hairless mice were given diets containing low (0.3-0.6 mg/kg; A-) or high (4-6 mg/kg; A+) amounts of retinol, which resulted after 2 months in an approximately 4-fold difference in liver and skin vitamin A levels as determined by HPLC. Commencing after 1 month of diet, daily irradiations with UVB (280-320 nm) or UVAB (280-380 nm) were given to 176 of the animals for 18 weeks (cumulative doses of UVB and UVA: 26 J/cm2 and 168 J/cm2, respectively). The first skin tumours, known to be squamous cell carcinomas, appeared after 35 weeks in the UVAB- irradiated A+ animals and 5-6 weeks later in the other groups. After one year the frequency of tumour-bearing animals was 49-63% in the A+ groups and 28-39% in the A- groups (P = 0.003). Two months later the corresponding figures were 66-72% and 50-53%, respectively (P = 0.014). Disregarding the effect of dietary vitamin A, there was no difference in the final tumour incidence between UVB- and UVAB-irradiated animals. The epidermal vitamin A content at 72 h post-irradiation was approximately 60% lower in A+ animals and approximately 10% lower in A- animals compared with the non-irradiated controls. Rather than protecting against skin cancer, a diet rich in vitamin A seems to facilitate UV carcinogenesis in hairless mice. A possible explanation is that photodecomposition of excessive vitamin A generates short-lived intermediates that may act as photosensitizers during cutaneous carcinogenesis.      

Role of ultraviolet radiation in the induction of melanocytic tumors in hairless mice following 7,12-dimethylbenz(a)anthracene application and ultraviolet irradiation

We examined the role of UVR (UV radiation) (UVA, 320-400 nm; UVB, 290-320 nm; and the combination of UVA and UVB) as a promoter in the induction of cutaneous melanoma. One hundred and seventy hairless mice (Skh-hr2), 6-8 weeks old, were treated in 8 groups: group I, DMBA [7,12-dimethylbenz(a)anthracene] plus UVA; group II, DMBA plus UVA plus UVB; group III, DMBA plus UVB; group IV, DMBA; group V, UVA; group VI, UVA plus UVB; group VII, UVB; group VIII, control. DMBA (0.5% solution) was applied once to promote the formation of dermal melanocytic nevus-like lesions while UVR treatments were conducted 3 times/week for 30 weeks. The mice were examined periodically for the development of multiple pigmented lesions, papillomas, squamous cell carcinomas, melanomas, and lymphomas. Treatment with DMBA plus UVA, DMBA plus UVB, and DMBA plus UVA plus UVB stimulated the development of multiple pigmented nevus-like lesions (85-100%) in mice of groups I, II, III, and IV. Upon necroscopy, 27-33% of animals in groups I, II, and III receiving UVR treatments developed clinically and histologically characterized melanomas. Treatment with DMBA alone did not produce melanomas. DMBA-treated animals in groups I, II, and III which received UVR treatments also developed lymphomas (21-50%). Animals treated with DMBA alone or those that received UVB or the combination of UVB plus UVA (without DMBA) developed only papillomas and squamous cell carcinomas (25-47%). Skin tumors were analyzed for the presence of point mutations in the ras gene. Polymerase chain reaction amplification of DNA and selective oligonucleotide hybridization revealed mutations in the 61st codon of the N-ras gene in the precursor nevus-like lesions and melanoma samples studied. This study suggests that UVR (both UVA and UVB) plays a role as a promoter in the stimulation of melanoma and lymphoma development in hairless mice.     

Chronic UVA irradiation of human HaCaT keratinocytes induces malignant transformation associated with acquired apoptotic resistance

Ultraviolet A (UVA, 315-400 nm), constituting about 95% of ultraviolet irradiation in natural sunlight, represents a major environmental challenge to the skin and is clearly associated with human skin cancer. It has proven difficult to show direct actions of UVA as a carcinogen in human cells. Here, we demonstrate that chronic UVA exposures at environmentally relevant doses in vitro can induce malignant transformation of human keratinocytes associated with acquired apoptotic resistance. As evidence of carcinogenic transformation, UVA-long-treated (24 J/cm(2) once/week for 18 weeks) HaCaT (ULTH) cells showed increased secretion of matrix metalloproteinase (MMP-9), overexpression of keratin 13, altered morphology and anchorage-independent growth. Malignant transformation was established by the production of aggressive squamous cell carcinomas after inoculation of ULTH cells into nude mice (NC(r)-nu). ULTH cells were resistant to apoptosis induced not only by UVA but also by UVB and arsenite, two other human skin carcinogens. ULTH cells also became resistant to apoptosis induced by etoposide, staurosporine and doxorubicin hydrochloride. Elevated phosphorylation of protein kinase B (PKB, also called AKT) and reduced expression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) were detected in ULTH cells. The resistance of ULTH cells to UVA-induced apoptosis was reversed by either inhibition of phosphatidylinositol 3-kinase (PI-3K) or adenovirus expression of PTEN or dominant negative AKT. These data indicate that UVA has carcinogenic potential in human keratinocytes and that the increased AKT signaling and decreased PTEN expression may contribute to this malignant transformation. Further comparisons between the transformed ULTH and control cells should lead to a better understanding of the mechanism of UVA carcinogenesis and may help identify biomarkers for UVA-induced skin malignancies.     

Longwave ultraviolet radiation and promotion of skin cancer

Exposure to solar ultraviolet (UV) radiation is recognized as an important cause of skin cancer. The carcinogenic effects of UV radiation have been attributed almost entirely to wavelengths in the mid-range (UVB, 290-320 nm). However, the development of potent UVB sunscreens has allowed individuals to increase the length of time that they spend sunbathing and, as a consequence, they may be exposed to massive doses of longwave UV radiation (UVA, 320-400 nm). There is now much evidence to suggest that UVA acts to promote tumors that have been initiated by UVB. This review considers possible mechanisms by which UVA promotes tumorigenesis. Evidence is presented which suggests that UVA acts through modulation of protein kinase C.     

Effects of chronic low-dose ultraviolet B radiation on DNA damage and repair in mouse skin.

Chronic exposure to sunlight causes skin cancer in humans, yet little is known about how habitual exposure to low doses of ultraviolet B radiation (UVB) affects DNA damage in the skin. We treated Skh-1 hairless mice with daily doses of suberythemal UVB for 40 days and analyzed the amount and distribution of DNA photodamage using RIAs and immunofluorescence micrography. We found that DNA damage accumulated in mouse skin as a result of chronic irradiation and that this damage persisted in the dermis and epidermis for several weeks after the chronic treatment was terminated. Although the persistent damage was evenly distributed throughout the dermis, it remained in the epidermis as a small number of heavily damaged cells at the dermal-epidermal boundary. Rates of DNA damage induction and repair were determined at different times over the course of chronic treatment in response to a higher challenge dose of UVB light. The amount of damage induced by the challenge dose increased in response to chronic exposure, and excision repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone dimers was significantly reduced. The sensitization of mouse epidermal DNA to photoproduct induction, the reduction in excision repair, and the accumulation of nonrepairable DNA damage in the dermis and epidermis suggest that chronic low-dose exposure to sunlight may significantly enhance the predisposition of mammalian skin to sunlight-induced carcinogenesis.     

Sunscreen use and intentional exposure to ultraviolet A and B radiation: a double blind randomized trial using personal dosimeters

A previous randomized trial found that sunscreen use could extend intentional sun exposure, thereby possibly increasing the risk of cutaneous melanoma. In a similarly designed trial, we examined the effect of the use of sunscreens having different sun protection factor (SPF) on actual exposure to ultraviolet B (UVB) and ultraviolet A (UVA) radiation. In June 1998, 58 European participants 18-24 years old were randomized to receive a SPF 10 or 30 sunscreens and were asked to complete daily records of their sun exposure during their summer holidays of whom 44 utilized a personal UVA and UVB dosimeter in a standard way during their sunbathing sessions. The median daily sunbathing duration was 2.4 hours in the SPF 10 group and 3.0 hours in the SPF 30 group (P = 0.054). The increase in daily sunbathing duration was paralleled by an increase in daily UVB exposure, but not by changes in UVA or UVB accumulated over all sunbathing sessions, or in daily UVA exposure. Of all participants, those who used the SPF 30 sunscreen and had no sunburn spent the highest number of hours in sunbathing activities. Differences between the two SPF groups in total number of sunbathing hours, daily sunbathing duration, and daily UVB exposure were largest among participants without sunburn during holidays. Among those with sunburn, the differences between the two groups tended to reduce. In conclusion, sunscreens used during sunbathing tended to increase the duration of exposures to doses of ultraviolet radiation below the sunburn threshold.     

Ultraviolet B but not ultraviolet A radiation initiates melanoma

Cutaneous malignant melanoma is one of the fastest increasing cancers with an incidence that has more than doubled in the last 25 years. Sunlight exposure is strongly implicated in the etiology of cutaneous malignant melanoma and the UV portion of the sunlight spectrum is considered responsible. Data are, however, conflicting on the roles of ultraviolet B [UVB; 280-320 nanometers (nm)] and ultraviolet A (UVA; 320-400 nm), which differ in their ability to initiate DNA damage, cell signaling pathways and immune alterations. To address this issue, we have used specialized optical sources, emitting isolated or combined UVB or UVA wavebands or solar simulating radiation, together with our hepatocyte growth factor/scatter factor-transgenic mouse model of UV-induced melanoma that uniquely recapitulates human disease. Only UVB-containing sources initiated melanoma. These were the isolated UVB waveband (>96% 280-320 nm), the unfiltered F40 sunlamp (250-800 nm) and the solar simulator (290-800 nm). Kaplan-Meier survival analysis indicated that the isolated UVB waveband was more effective in initiating melanoma than either the F40 sunlamp or the solar simulator (modified log rank P < 0.02). The latter two sources showed similar melanoma effectiveness (P = 0.38). In contrast, transgenic mice irradiated with either the isolated UVA waveband (>99.9% 320-400 nm, 150 kJ/m2), or an F40 sunlamp filtered to remove > 96% of the UVB, responded like unirradiated control animals. We conclude that, within the constraints of this animal model, UVB is responsible for the induction of mammalian cutaneous malignant melanoma whereas UVA is ineffective even at doses considered physiologically relevant. This finding may have major implications with respect both to risk assessment from exposure to solar and artificial UVB, and to development of effective protection strategies against melanoma induction by UVB. Moreover, these differences in wavelength effectiveness can now be exploited to identify UV pathways relevant to melanomagenesis.     

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

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.     

Inhibition of ultraviolet light-induced oxidative events in the skin and internal organs of hairless mice by isoflavone genistein

We have previously demonstrated that soybean isoflavone genistein inhibits ultraviolet-B (UVB)-induced skin tumorigenesis in hairless mice. In the present study, we further investigated the possible mechanism(s) of action whereby genistein inhibits photocarcinogenesis with focuses on UVB-induced oxidative events, including hydrogen peroxide (H(2)O(2)) production, lipid peroxidation (as represented by malondialdehyde, MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in vivo. We demonstrated that subacute exposure to UVB substantially increased the level of H(2)O(2), lipid peroxides, and 8-OHdG in skin of hairless mice. In addition, chronic exposure to low-dose UVB (0.9-1.2 kJ/m(2) for 20 weeks) substantially increased the levels of 8-OHdG not only in the epidermis, but also in the internal organs such as liver, brain, and spleen of mice with exception of kidney. However, genistein did not affect the level of UVB-induced pyrimidine dimmers in the same UVB exposed mouse skin, indicating selective inhibition of oxidative DNA damage by genistein. Induction of H(2)O(2) was independent of UVB fluences whereas the levels of MDA and 8-OHdG were induced in an UVB fluence-dependent manner. The results suggest that H(2)O(2) be generated as an acute cutaneous response to UVB irradiation, while MDA and 8-OHdG are accumulated with increasing UVB exposure and more closely related to chronic effects of UVB radiation. Pre-treatment of animals with 10 micromol of genistein 1 h prior to UVB exposure significantly inhibited UVB-induced H(2)O(2) and MDA in skin and 8-OHdG in epidermis as well as internal organs. Suppression of 8-OHdG formation by genistein has been corroborated in purified DNA irradiated with UVA and B. In summary, our results suggest that UVB irradiation elicit a series of oxidative events, which can be substantially inhibited by isoflavonoid genistein through either direct quenching of reactive oxygen species or indirect antiinflammatory effects. Thus, the antioxidative properties of genistein may explain for the mechanisms of anti-photocarcinogenic action of genistein.     

Effects of ultraviolet radiation on intercellular communication in V79 Chinese hamster fibroblasts

The effects of ultraviolet (UV) radiation on gap junctionalintercellular communication (GJIC) in V79 Chinese hamster fibroblastswere studied by means of a dye transfer assay. Intercellularcommunication was shown to be altered by UVB (297/302 nm) andUVA (365 nm) radiation, the effect depending on the wavelengthof exposure and time between irradiation and microinjectionof the dye in the dye transfer assay. Exposure to 297/302 nmradiation induced a reduction in intercellular communication6 min after exposure. Incubation of the cells post-irradiationreversed the inhibition of GJIC. From 2 to 24 h after exposurean increase in GJIC over the control cells was seen, with amaximum at 8 h post-irradiation. UVA (365 nm) radiation, onthe other hand, induced an increase in the intercellular communication6 min after irradiation. Incubation of the cells post-irradiationled to a decrease in the number of communicating cells, witha minimum seen 4 h after exposure. The reduction in communicationobserved after exposure to UVB and UVA was not correlated withsimilar modifications in the gap junction protein connexin43as found when exposing the cells to the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate. For the higher fluences of UVA, a decrease in immunorecognizableconnexin43 was seen, concomitant with a markedly increased backgroundof higher mol. wt compounds. This may be due to UVA-inducedcrosslinking of connexin43. No correlation was found betweenchanges in communication induced by UV radiation and levelsof cyclic AMP.     

S phase arrest and induction of multinucleated cells after exposure to ultraviolet radiation

The effects or UVA (365 nm) and UVB (297/302 nm)radiation oncellular proliferation, cell cycle progression, aneuploidy andmultinucleus induction have been studied in two different fibroblastcell lines; V79 Chinese hamster lung fibroblasts and 3T3 Swissalbino mouse fibroblasts. UVA and UVB were found to inhibitproliferation of the cells in a fluence-dependent manner. Thisinhibition was due to a temporary accumulation of cells in theS phase of the cell cycle, as determined by flow cytometry ofUV-irradiated V79 cells. The UVA- and UVB-induced S phase delaywas observed a few hours after irradiation and by 48 h post-irradiationthe cells had recovered from cell cycle arrest. For UVA butnot for UVB, the elongation of S phase was followed by a smallaccumulation of cells in the G₂ phase. After exposure to UVAand a post-irradiation time long enough for the cells to recoverfrom cell cycle arrest, a large proportion of the cells werepolyploid, with two or more nuclei. Multinucleated cells werenot, however, induced by UVB irradiation.     

Inhibition of ultraviolet-B skin carcinogenesis by all-trans-retinoic acid regimens that inhibit ornithine decarboxylase induction

There is a correlation between the ability to induce the polyamine-biosynthetic enzyme ornithine decarboxylase (ODC) and the tumor-promoting ability of various carcinogens in mouse epidermis. Some agents which inhibit skin carcinogenesis also inhibit ODC induction. In this study, all-trans-retinoic acid (RA) regimens that inhibited the induction of epidermal ODC by ultraviolet-B (UVB) were tested for their ability to inhibit UVB skin carcinogenesis. Hairless mice were irradiated once daily with UVB for 20 days, receiving a total dose of UVB (17.1 kJ/sq m). Topical RA was applied immediately (RA, one dose) or applied 0, 1, 2, 3, and 4 hr (RA, five doses) after each irradiance. The mice were maintained for 52 weeks and then sacrificed. Groups treated with RA tended to have fewer mice with tumors, fewer tumors per mouse, smaller tumor diameters, and slower growing tumors than did appropriate irradiated control groups. RA given five times was more effective than was RA given one time at inhibiting UVB skin carcinogenesis. These results show that RA treatments that inhibit epidermal ODC induction may be effective in reducing the carcinogenicity of UVB.     

Protein kinase C epsilon is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas

Chronic exposure to UV radiation (UVR), especially in the UVA (315-400 nm) and UVB (280-315 nm) spectrum of sunlight, is the major risk factor for the development of nonmelanoma skin cancer. UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. We found that protein kinase C epsilon (PKCepsilon), a member of the phospholipid-dependent threonine/serine kinase family, is an endogenous photosensitizer, the overexpression of which in the epidermis increases the susceptibility of mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. The PKCepsilon transgenic mouse (FVB/N) lines 224 and 215 overexpressed 8- and 18-fold PKCepsilon protein, respectively, over endogenous levels in basal epidermal cells. UVR exposure (1 kJ/m(2) three times weekly) induced irreparable skin damage in high PKCepsilon-overexpressing mouse line 215. However, the PKCepsilon transgenic mouse line 224, when exposed to UVR (2 kJ/m(2) three times weekly), exhibited minimum cutaneous damage but increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks. UVR exposure of PKCepsilon transgenic mice compared with wild-type littermates (1) elevated the levels of neither cyclobutane pyrimidine dimer nor pyrimidine (6-4) pyrimidone dimer, (2) reduced the appearance of sunburn cells, (3) induced extensive hyperplasia and increased the levels of mouse skin tumor promoter marker ornithine decarboxylase, and (4) elevated the levels of tumor necrosis factor alpha (TNFalpha) and other growth stimulatory cytokines, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. The role of TNFalpha in UVR-induced cutaneous damage was evaluated using PKCepsilon transgenic mice deficient in TNFalpha. UVR treatment three times weekly for 13 weeks at 2 kJ/m(2) induced severe cutaneous damage in PKCepsilon transgenic mice (line 215), which was partially prevented in PKCepsilon-transgenic TNFalpha-knockout mice. Taken together, the results indicate that PKCepsilon signals UVR-induced TNFalpha release that is linked, at least in part, to the photosensitivity of PKCepsilon transgenic mice.     

Ultraviolet A and B differently induce intracellular protein expression in human skin melanocytes--a speculation of separate pathways in initiation of melanoma

Ultraviolet (UV) irradiation has been involved in both initiation and promotion of carcinogenesis in melanoma. Alterations of cellular proliferation proteins, such as p73, Nup88, Id1 and p27 have been considered to play critical roles in melanoma development. However, the molecular mechanisms behind melanoma carcinogenesis are still poorly understood. In this study, we used human skin melanocytes as an experimental model system to investigate effects of UV irradiation on protein expression concerning cellular proliferation. The melanocytes prepared from human foreskin were separately exposed to various doses of UVA or UVB and post-cultivated for 24 or 48 h. Total proteins were isolated from the melanocytes, and expression of p73, Nup88, Id1, p27, bcl-2 and proliferating cell nuclear antigen (PCNA) proteins was examined by western blotting and immunocytochemistry. Results showed that expression of p73 and Nup88 was enhanced by UVA irradiation in a dose- and time-dependent manner. However, expression of Id1, p27, bcl-2 and PCNA proteins was not changed upon exposure to the UVA. Id1 and p27 proteins were over-expressed by exposure to UVB, but expression of p73, Nup88, bcl-2 and PCNA proteins was not changed by the UVB irradiation. The data suggested that UVA and UVB irradiation might lead to alterations of the different intracellular proteins. UVA enhanced protein expression concerning cell growth (p73 and Nup88) and UVB might over-express proteins concerning cellular proliferation (Id1 and p27). UVA and UVB may induce initiation of melanoma via separate intracellular pathways.     

Protective effect of topically applied olive oil against photocarcinogenesis following UVB exposure of mice

Reactive oxygen species have been shown to play a role in ultraviolet light (UV)-induced skin carcinogenesis. Vitamin E and green tea polyphenols reduce experimental skin cancers in mice mainly because of their antioxidant properties. Since olive oil has also been reported to be a potent antioxidant, we examined its effect on UVB-induced skin carcinogenesis in hairless mice. Extra-virgin olive oil was applied topically before or after repeated exposure of mice to UVB. The onset of UVB-induced skin tumors was delayed in mice painted with olive oil compared with UVB control mice. However, with increasing numbers of UVB exposures, differences in the mean number of tumors between UVB control mice and mice pretreated with olive oil before UVB exposure (pre-UVB group) were lost. In contrast, mice that received olive oil after UVB exposure (post-UVB group) showed significantly lower numbers of tumors per mouse than those in the UVB control group throughout the experimental period. The mean number of tumors per mouse in the UVB control, pre-UVB and post-UVB groups was 7.33, 6.69 and 2.64, respectively, in the first experiment, and 8.53, 9.53 and 3.36 in the second experiment. Camellia oil was also applied, using the same experimental protocol, but did not have a suppressive effect. Immunohistochemical analysis of DNA damage in the form of cyclobutane pyrimidine dimers (CPD), (6-4) photoproducts and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in samples taken 30 min after a single exposure of UVB showed no significant difference between UVB-irradiated control mice and the pre-UVB group. In the post-UVB group, there were lower levels of 8-OHdG in epidermal nuclei, but the formation of CPD and (6-4) photoproducts did not differ. Exposure of olive oil to UVB before application abrogated the protective effect on 8-OHdG formation. These results indicate that olive oil topically applied after UVB exposure can effectively reduce UVB-induced murine skin tumors, possibly via its antioxidant effects in reducing DNA damage by reactive oxygen species, and that the effective component may be labile to UVB.     

Formation of 8-hydroxydeoxyguanosine within DNA of mouse keratinocytes exposed in culture to UVB and H2O2.

Exposure to UV light contributes to the development of skin cancer. The importance of reactive oxygen species in UV-radiation carcinogenesis has been recognized for some time and several associated DNA base modifications have been identified. In particular, 8-hydroxydeoxyguanosine (8-OHdG) has been well studied as an indicator of oxidative damage to calf thymus DNA exposed to a variety of oxygen-generating systems, including UV light. However, to date, few studies of 8-OHdG have been conducted in cell or animal systems and those in vitro investigations that studied UV exposure have used UVC (< 290 nm), not the UVB (290-320 nm) or UVA (320-400 nm) ranges to which organisms are exposed through sunlight. The objective of this study was to measure 8-OHdG formation in the DNA of cultured mouse keratinocytes exposed to UVB. Using HPLC with electrochemical detection, background levels of 8-OHdG were approximately 6 fmol/micrograms DNA in DNA isolated and digested to the nucleoside level. UVB induced 8-OHdG up to 100% above that for mock-treated cells at a dose of 630 mJ/cm2 (dose-response range: 210-630 mJ/cm2). UVB exposure at 630 mJ/cm2 combined with 5 mM H2O2 elevated 8-OHdG formation up to 280% above that in control cells, whereas H2O2 alone had no effect. These results suggest that factors which increase the generation of reactive oxygen species by UV light may be potent cofactors of UV-radiation carcinogenesis.     

Induction of nevi and skin tumors in Ink4a/Arf Xpa knockout mice by neonatal, intermittent, or chronic UVB exposures

Nevi and melanomas correlate to childhood and intermittent solar UV exposure, xeroderma pigmentosum patients run increased risk, and p16(Ink4a) expression is often lost in malignant progression. To ascertain the effect of these risk factors, pigmented hairless Ink4a/Arf-, Xpa- knockout mice were subjected to various combinations of neonatal [7,12-dimethylbenz(a)anthracene (DMBA) or UVB exposure] and adult treatments (12-O-tetradecanoylphorbol-13-acetate or subacute daily UVB exposure or intermittent overexposure). Nevi occurred earliest, grew largest, and were most numerous in mice exposed to DMBA followed by intermittent UVB overexposure [effect of six minimal edemal doses (MED), 1 x /2 weeks > 4 MED 1 x /wk]. Neonatal UV exposure enhanced nevus induction but lost its effect after 200 days. The Xpa(-/-) mice proved exquisitely sensitive to UV-driven nevus induction, indicating the involvement of pyrimidine dimer DNA lesions, but Xpa(+/+) mice developed many more nevi (>40 per mouse) at high UV dosages not tolerated by Xpa(-/-) mice. Ink4a/Arf(-/-) mice developed most skin tumors faster, but surprisingly developed nevi slower than their heterozygous counterparts especially after neonatal UV exposure. Despite raising >1,600 nevi, only six melanomas arose in our experiments with Ink4a/Arf knockout mice (five of which in Xpa(+/+) mice at high UV dosages). In contrast to human nevi, these nevi lacked hotspot mutations in Braf or Ras genes, possibly explaining the lack of malignant progression in the Ink4a/Arf(-/-) mice. Hence, although our experiments did not effectively emulate human melanoma, they provided clear evidence that intermittent UV overexposure strongly stimulates and the Ink4a/Arf(-/-) genotype may actually impair nevus development.