The enhanced IL-18 production by UVB irradiation requires ROI and AP-1 signaling in human keratinocyte cell line (HaCaT)

Based on our recent observation that enhanced IL-18 expression positively correlates with malignant skin tumors, such as SCC and melanoma, we examined the possible role of UVB, known to be associated with skin cancer development, in the enhancement of IL-18 production using primary human epidermal keratinocytes and human keratinocyte cell line HaCaT. After cells were exposed to UVB irradiation in vitro, IL-18 production was examined by Northern blot analysis and ELISA, and it was found that IL-18 production is enhanced by UVB irradiation in a dose- and time-dependent manner. In addition, we confirmed that it is functionally active form of IL-18 using the inhibitor of caspase-1. The effect of UVB irradiation was blocked by antioxidant, N-acetyl-L-cysteine (NAC), which suggested the involvement of reactive oxygen intermediates (ROI) in the signal transduction of UVB irradiation-enhanced IL-18 synthesis. We also found that UVB irradiation increased AP-1 binding activity by using EMSA with AP-1-specific oligonucleotide. Furthermore, inhibitors of UVB-induced AP-1 activity, such as PD98059, blocked enhanced IL-18 production, indicating that AP-1 activation is required for UVB-induced IL-18 production. Taken together, our results suggest that UVB irradiation-enhanced IL-18 production is selectively mediated through the generation of ROI and the activation of AP-1.     

Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice

UVB radiation damages keratinocytes, potentially inducing chronic skin damage, cutaneous malignancy, and suppression of the immune system. Naturally occurring agents have been considered for prevention and treatment of various kinds of cancer, including skin cancer. Inositol hexaphosphate (IP6), an antioxidant, is a naturally occurring polyphosphorylated carbohydrate that has shown a strong anticancer activity in several experimental models. We assessed the protective effects of IP6 against UVB irradiationinduced injury and photocarcinogenesis by using HaCaT cells (human immortalized keratinocytes) and SKH1 hairless mice. We found that IP6 counteracts the harmful effects of UVB irradiation and increases the viability and survival of UVB-exposed cells. Treatment with IP6 after UVB irradiation (30 mJ/cm(2)) arrested cells in the G(1) and G(2) M phases while decreasing the S phase of the cell cycle. Treatment with IP6 also decreased UVB-induced apoptosis and caspase 3 activation. Topical application of IP6 followed by exposure to UVB irradiation in SKH1 hairless mice decreased tumor incidence and multiplicity as compared with control mice. Our results suggest that IP6 protects HaCaT cells from UVB-induced apoptosis and mice from UVB-induced tumors.     

Protection against ultraviolet B- and C-induced DNA damage and skin carcinogenesis by the flowers of Prunus persica extract

The ethanol extract of the flowers of Prunus persica (Ku-35) (50-200 microg/ml) was found to inhibit UVB- as well as UVC-induced DNA damage measured by the COMET assay in the skin fibroblast cell (NIH/3T3). In addition, Ku-35 inhibited UVB- or UVC-induced lipid peroxidation, especially against UVB-induced peroxidation at higher than 10 microg/ml. We also evaluated the protective effect of Ku-35 against UVB-induced non-melanoma skin cancer in mice. Ku-35 was applied topically before UVB exposure, and its effects on tumor incidence (% of mice with tumors) and tumor multiplicity (number of tumors per mouse) were evaluated. The application of Ku-35 clearly resulted in a delay of tumor development compared to the control. In tumor incidence, 100% mice in the control group and the low dose treatment of Ku-35 had tumors, whereas 94.1% of the mice had tumors after the high dose treatment of Ku-35 at the end of experiment (28 weeks). In tumor multiplicity, low and high treatments of Ku-35 resulted in 25.9 and 53.9% reduction at the end of the experiment (P<0.05, one-way analysis of variance (ANOVA)). The present data indicate that Ku-35 protects against photogenotoxicity in NIH/3T3 fibroblasts. The possible action mechanism of Ku-35 may be through its anti-oxidant activity without pro-oxidant effect. Ku-35 can also show a delay of tumor development against UVB-induced skin carcinogenesis. These results suggest that Ku-35 extract may be useful for protecting UV-induced DNA damage and carcinogenesis when topically applied.     

Exposure to asphalt fumes activates activator protein-1 through the phosphatidylinositol 3-kinase/Akt signaling pathway in mouse epidermal cells

Occupational exposure to asphalt fumes may pose a health risk. Experimental studies using animal and in vitro models indicate that condensates from asphalt fumes are genotoxic and can promote skin tumorigenesis. Enhanced activity of activator protein-1 (AP-1) is frequently associated with the promotion of skin tumorigenesis. The current study investigated the effect of exposure to asphalt fumes on AP-1 activation in mouse JB6 P+ epidermal cells and the skin of transgenic mice expressing the AP-1 luciferase reporter gene. Asphalt fumes were generated from a dynamic generation system that simulated road-paving conditions. Exposure to asphalt fumes significantly increased AP-1 activity in JB6 P+ cells as well as in cultured keratinocytes isolated from transgenic mice expressing AP-1 reporter. In addition, topical application of asphalt fumes by painting the tail skin of mice increased AP-1 activity by 14-fold. Exposure to asphalt fumes promoted basal as well as epidermal growth factor-stimulated anchorage-independent growth of JB6 P+ cells in soft agar. It activated phosphatidylinositol 3-kinase and induced phosphorylation of Akt at Ser-473/Thr-308, and concurrently activated downstream p70 S6 kinase as well as glycogen synthase kinase-3beta. Asphalt fumes transiently activated c-Jun NH2-terminal kinases without affecting extracellular signal-regulated kinases and p38 mitogen-activated protein kinases. Further study indicated that blockage of phosphatidylinositol 3-kinase activation eliminated asphalt fume-stimulated AP-1 activation and formation of anchorage-independent colonies in soft agar. This is the first report showing that exposure to asphalt fumes can activate AP-1 and intracellular signaling that may promote skin tumorigenesis, thus providing important evidence on the potential involvement of exposure to asphalt fumes in skin carcinogenesis.     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

Increased dermal mast cell prevalence and susceptibility to development of basal cell carcinoma in humans

Exposure to ultraviolet B (UVB) radiation (280-320 nm) is the primary etiologic factor associated with the development of basal cell carcinoma (BCC). The outgrowth of these keratinocyte-derived skin lesions is enhanced by the ability of UVB to impair an immune response that would otherwise eliminate them. Studies in a range of inbred mouse strains as well as mast cell-depleted mice reconstituted with mast cell precursors support a functional link between histamine-staining dermal mast cells and the extent of susceptibility to UVB-induced systemic immunomodulation. Humans, like mouse strains, display variations in dermal mast cell prevalence. In a study of Danish and South Australian BCC patients and control subjects, one 4-mm punch biopsy of non-sun-exposed buttock skin was sampled from each participant. This skin site was investigated to avoid any changes in mast cell prevalence caused by sun exposure. Two sections (4 microm) per biopsy were immunohistochemically stained for detection of histamine-containing dermal mast cells. Computer-generated image analysis evaluated dermal mast cell prevalence in both sections by quantifying the total number of mast cells according to the total dermal area (expressed as mast cells per square millimeter). This technique enabled us to detect heterogeneity of dermal mast cell prevalence in buttock skin between individuals and provided evidence of an association between high dermal mast cell prevalence and BCC development in two diverse populations. We hypothesize that mast cells function in humans, as in mouse strains, by initiating immunosuppression following UV irradiation and, thereby, allowing a permissive environment for the development of BCC. Thus, a high dermal mast cell prevalence as demonstrable in buttock skin is a significant predisposing factor for development of BCC in humans.     

Ultraviolet B-induced matrix metalloproteinase-1 and -3 secretions are mediated via PTEN/Akt pathway in human dermal fibroblasts

Matrix Metalloproteinases (MMPs) are crucial enzymes for ultraviolet irradiation-induced photoaging in human skin. Ultraviolet B (UVB) stimulates dermal fibroblasts to increase MMP-1 and -3 expression and extracellular matrix (ECM) degradation in photoaging. We investigated whether phosphatase and tensin homolog (PTEN)/Akt pathway is involved in secretions of MMP-1 and -3 in human dermal fibroblasts. The increase in MMP-1 and -3 expression and secretion occurred along with the increase in PTEN and Akt phosphorylation by UVB irradiation in a dose- and time-dependent manner. However, treatment with a casein kinase 2 inhibitor, 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole, inhibited their phosphorylations and MMP-1 and -3 secretions. Transfection of wild-type PTEN (Wt-PTEN) decreased basal and UVB-induced MMP-1 and -3 secretions, as well as activator protein-1 (AP-1) activity, while transfection of small interference RNA of PTEN (siRNA-PTEN), phosphatase-inactive PTEN (C124S-PTEN), or lipid phosphatase-inactive PTEN (G129E-PTEN) increased basal or UVB-induced MMP-1 and -3 secretions and AP-1 activity. Transfection of constitutively active Akt (Myr-Akt) also increased basal or UVB-induced MMP-1 and -3 secretions, as well as AP-1 activity. However, transfection of kinase-inactive Akt (K179M-Akt) decreased their secretions, but showed no significant change of AP-1 activity without UVB irradiation, and a significant increase of AP-1 activity with UVB irradiation. Treatment with the phosphatidylinositol 3-kinase inhibitors, LY294002 or wortmannin, downregulated basal and UVB-induced MMP-1 and -3 secretions. In conclusion, UVB irradiation increases PTEN and Akt phosphorylation in human dermal fibroblasts, and these inhibition of PTEN and activation of Akt by phosphorylation are involved in UVB-induced MMP-1 and -3 secretions partly through upregulation of AP-1 activity.     

Ultraviolet B(UVB)-induced cox-2 expression in murine skin: an immunohistochemical study

Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostaglandins from arachidonic acid. This enzyme exists in at least two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed in most tissues and plays various physiological roles. However, COX-2 expression is induced by a variety of agents, which include pro-inflammatory agents and mitogens. Evidence exists to indicate that increased expression of COX-2 occurs in several types of epithelial neoplasms. In this study, we show the effect of chronic exposure of murine skin to carcinogenic UVB on cutaneous COX-2 expression. SKH-1 mice were irradiated with 180 mJ/cm(2) UVB daily for five days a week for periods ranging from 1 to 20 weeks. Nontumor bearing skin areas of irradiated mice, skin of age-matched controls and benign papillomas and malignant tumors were assessed immunohistochemically for COX-2 expression in these mice. No epidermal staining occurred in any of the non-UVB-treated controls throughout the experiment. Epidermal COX-2 expression only occurred in UVB-irradiated mice. After 1 and 5 weeks of irradiation, patchy epidermal staining mostly confined to the granular layer and stratum corneum was observed. At week 9, staining intensity had increased, particularly in the granular layer. At week 13, staining was uniformly seen in all epidermal layers with particular prominence in the basal cell layer underlying areas of visible epidermal hyperplasia. It is of interest that the most intense staining was seen in the perinuclear region of keratinocytes and at the plasma membrane. At week 20, COX-2 staining was predominant in the granular layer, although in some tissue sections, the entire epidermis was positive. In benign papillomas, staining was confined to the superficial layers of the epidermis and in squamous cell carcinomas (SCCs), patchy staining in the granular and spinous layers predominated. In general, COX-2 expression was more intense in well-differentiated SCCs than in papillomas. In summary, our results indicate that COX-2 serves as an early marker of epidermal UVB exposure and its expression increases in benign papillomas and in SCCs. These results suggest that pharmacological intervention using specific COX-2 inhibitors could have anticarcinogenic effects in UVB-induced human skin cancer.     

Dose-dependent effects of UVB-induced skin carcinogenesis in hairless p53 knockout mice

Exposure to (solar) UVB radiation gives rise to mutations in the p53 tumor suppressor gene that appear to contribute to the earliest steps in the molecular cascade towards human and murine skin cancer. To examine in more detail the role of p53, we studied UVB-induced carcinogenesis in hairless p53 knock-out mice. The early onset of lymphomas as well as early wasting of mice interfered with the development of skin tumors in p53 null-mice. The induction of skin tumors in the hairless p53+/- mice was accomplished by daily exposure to two different UV-doses of approximately 450 J/m2 and 900 J/m2 from F40 lamps corresponding to a fraction of about 0.4 and 0.8 of the minimal edemal dose. Marked differences in skin carcinogenesis were observed between the p53+/- mice and their wild type littermates. Firstly, at 900 J/m2, tumors developed significantly faster in the heterozygotes than in wild types, whereas at 450 J/m2 there was hardly any difference, suggesting that only at higher damage levels loss of one functional p53 allele is important. Secondly, a large portion (25%) of skin tumors in the heterozygotes were of a more malignant, poorly differentiated variety of squamous cell carcinomas, i.e. spindle cell carcinomas, a tumor type that was rarely observed in daily UV exposed wild type hairless mice. Thirdly, the p53 mutation spectrum in skin tumors in heterozygotes is quite different from that in wild types. Together these results support the notion that a point mutation in the p53 gene impacts skin carcinogenesis quite differently than allelic loss: the former is generally selected for in early stages of skin tumors in wild type mice, whereas the latter enhances tumor development only at high exposure levels (where apoptosis becomes more prevalent) and appears to increase progression (to a higher grade of malignancy) of skin tumors.     

Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model

Activation of activator protein-1 (AP-1) and increased expression of cyclooxygenase-2 (COX-2) have been clearly shown to play a functional role in UVB-induced skin tumor promotion. In this study, we examined UVB-induced signal transduction pathways in SKH-1 mouse epidermis leading to increases in COX-2 expression and AP-1 activity. We observed rapid increases in p38 mitogen-activated protein kinase (MAPK) signaling through activation of p38 MAPK and its downstream target, MAPK activated protein kinase-2. UVB also increased phosphatidylinositol 3-kinase (PI3K) signaling as observed through increases in AKT and GSK-3beta phosphorylation. Activation of the p38 MAPK and PI3K pathways results in the phosphorylation of cyclic AMP-responsive element binding protein, which was also observed in UVB-irradiated SKH-1 mice. Topical treatment with SB202190 (a specific inhibitor of p38 MAPK) or LY294002 (a specific inhibitor of PI3K) significantly decreased UVB-induced AP-1 activation by 84% and 68%, respectively, as well as COX-2 expression. Our data show that in mouse epidermis, UVB activation of the p38 MAPK and PI3K pathways leads to AP-1 activation and COX-2 expression.     

Expression and Functional Role of Sox9 in Human Epidermal Keratinocytes

In this study, we investigated the expression and putative role of Sox9 in epidermal keratinocyte. Immunohistochemical staining showed that Sox9 is predominantly expressed in the basal layer of normal human skin epidermis, and highly expressed in several skin diseases including psoriasis, basal cell carcinoma, keratoacanthoma and squamous cell carcinoma. In calcium-induced keratinocyte differentiation model, the expression of Sox9 was decreased in a time dependent manner. When Sox9 was overexpressed using a recombinant adenovirus, cell growth was enhanced, while the expression of differentiation-related genes such as loricrin and involucrin was markedly decreased. Similarly, when rat skin was intradermally injected with the adenovirus expressing Sox9, the epidermis was thickened with increase of PCNA positive cells, while the epidermal differentiation was decreased. Finally, UVB irradiation induced Sox9 expression in cultured human epidermal keratinocytes, and keratinocytes are protected from UVB-induced apoptosis by Sox9 overexpression. Together, these results suggest that Sox9 is an important regulator of epidermal keratinocytes with putative pro-proliferation and/or pro-survival functions, and may be related to several cutaneous diseases that are characterized by abnormal differentiation and hyperproliferation.     

IL-4 suppresses UVB-induced apoptosis in skin

In this study, cutaneous role of IL-4 in UVB-induced apoptosis was investigated using transgenic mice with skin-specific expression of IL-4 (IL-4 Tg mice). The transgenic mice did not show any gross clinical abnormalities. However, epidermis was thickened and increased MHC class II positive cells were detected as well as enhanced expression of inflammatory cytokines such as IL-1 and TNF-alpha in skin. In addition, histological analysis revealed increased infiltration of lymphocytes, acanthosis, hyperkeratosis, and parakeratosis in skin of IL-4 Tg mice. The physiological effect of IL-4 overexpression in skin against environmental stimulus such as UVB was investigated by irradiating wild-type and IL-4 Tg mice with UVB followed by evaluation of apoptosis. The result demonstrated suppressed apoptosis in epidermis of IL-4 Tg mice compared with wild-type mice. To further assess anti-apoptotic function of IL-4 in keratinocytes, stable cell clones were made where IL-4 was constitutively overexpressed and examined for UVB-induced apoptosis. The results showed that apoptosis was remarkably decreased in IL-4 over-expressing cell clones compared with that in mock transfected cells. Collectively, data presented here shows that IL-4 has an inhibitory effect against UVB-induced apoptosis in keratinocytes, suggesting that IL-4 may be an important regulator in cutaneous immunity against UVB.     

Obesity increases the risk of UV radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling

Obesity has been implicated in several diseases, including cancer; however, the relationship of obesity and susceptibility to ultraviolet (UV) radiation-caused skin diseases has not been investigated. As UV-induced oxidative stress has been implicated in several skin diseases, we assessed the role of obesity on UVB-induced oxidative stress in genetically obese Lep(ob)/Lep(ob) (leptin-deficient) mice. Here, we report that chronic exposure to UVB (120 mJ/cm(2)) resulted in greater oxidative stress in the skin of obese mice in terms of higher levels of H(2)O(2) and NO production, photo-oxidative damage of lipids and proteins, and greater depletion of antioxidant defense enzymes, like glutathione, glutathione peroxidase, and catalase. As UV-induced oxidative stress mediates activation of MAPK and NF-kappaB signaling pathways, we determined the effects of UVB on these pathways in obese mice. Exposure of obese mice to UVB resulted in phosphorylation of ERK1/2, JNK, and p38 proteins of the MAPK family. Compared to wild-type mice, the obese mice exhibited higher levels of phosphorylation of these proteins, greater activation of NF-kappaB/p65, and higher levels of circulating proinflammatory cytokines, including TNF-alpha, IL-1beta and IL-6, on UVB irradiation. Taking these results together, our study suggests for the first time that obesity in mice is associated with greater susceptibility to UVB-induced oxidative stress and therefore may be a risk factor for skin diseases associated with UVB-induced oxidative stress.     

Protective Effect of Indole-3-Pyruvate against Ultraviolet B-Induced Damage to Cultured HaCaT Keratinocytes and the Skin of Hairless Mice

Previous investigations demonstrated that pyruvate protects human keratinocytes against cell damage stemming from exposure to ultraviolet B (UVB) radiation. This study endeavoured to elucidate the protective capacity of aromatic pyruvates (e.g., phenylpyruvate (PPyr), 4-hydroxyphenylpyruvate (HPPyr), and indole-3-pyruvate (IPyr)) against UVB-induced injury to skin cells, both in vitro and in vivo. Cultured human HaCaT keratinocytes were irradiated with UVB light (60 mJ/cm²) and maintained with or without test compounds (1–25 mM). In addition, the dorsal skin of hairless mice (HR-1) was treated with test compounds (100 µmol) and exposed to UVB light (1 J/cm²) for two times. The ability of the test compounds to ameliorate UVB-induced cytotoxicity and inflammation was then assessed. Aromatic pyruvates reduced cytotoxicity in UVB-irradiated HaCaT keratinocytes, and also diminished the expression of interleukin 1β (IL-1β) and interleukin 6 (IL-6). IPyr was more efficacious than either PPyr or HPPyr. Furthermore, only IPyr inhibited cyclooxygenase-2 (Cox-2) expression at both the mRNA and the protein level in UVB-treated keratinocytes. Topical application of IPyr to the dorsal skin of hairless mice reduced the severity of UVB-induced skin lesions, the augmentation of dermal thickness, and transepithelial water loss. Overproduction of IL-1β and IL-6 in response to UVB radiation was also suppressed in vivo by the topical administration of IPyr. These data strongly suggest that IPyr might find utility as a UVB-blocking reagent in therapeutic strategies to lessen UVB-induced inflammatory skin damage.     

The effect of cilostazol on the expression of matrix metalloproteinase-1 and type I procollagen in ultraviolet-irradiated human dermal fibroblasts

AimCilostazol is a selective inhibitor of type III phosphodiesterase that inhibits platelet aggregation. Cilostazol is a useful vasodilator, antithrombotic, and cardiotonic agent. Ultraviolet B (UVB) irradiation increases the production of matrix metalloproteinase-1 (MMP-1) during skin photoaging. The UVB-induced increase of MMP-1 results in connective tissue damage, and the skin becomes wrinkled and aged. Here, we investigated the capacity of cilostazol to inhibit MMP-1 expression in UVB-irradiated human dermal fibroblasts.Main methodsCultured human dermal fibroblasts were irradiated with UVB, followed by the addition of cilostazol to the culture medium.Key findingsPost-treatment with cilostazol attenuated UVB-induced production of MMP-1 and prevented the reduction of type I procollagen. Cilostazol inhibited UVB irradiation-induced phosphorylation of the mitogen-activated protein kinase (MAPK) signaling molecules Jun-N-terminal kinase (JNK) and p38 kinase, as well as activator protein-1 (AP-1) in dermal fibroblasts.SignificanceOverall, these results demonstrate that cilostazol regulates UVB-induced MMP-1 expression and type I procollagen synthesis by inhibiting MAPK signaling and AP-1 activity. Therefore, we suggest that cilostazol may be useful for the prevention and treatment of skin photodamage caused by UVB-irradiation.     

E6/E7 expression of human papillomavirus type 20 (HPV-20) and HPV-27 influences proliferation and differentiation of the skin in UV-irradiated SKH-hr1 transgenic mice

The functional role of UV irradiation, in combination with the E6 and E7 proteins of the cutaneous human papillomavirus (HPV) types in the malignant conversion of benign papillomatous lesions, has not been elucidated. Transgenic SKH-hr1 hairless mice expressing HPV-20 and HPV-27 E6 and E7 proteins in the suprabasal compartment were generated and exposed to chronic UV irradiation. Histological and immunohistochemical examination of skin samples revealed enhanced proliferation of the epidermal layers and papilloma formation in both transgenic strains in comparison to what was observed with nontransgenic mice. Squamous cell carcinoma developed in the HPV-20 E6/E7 transgenic line as well as in the HPV-27 E6/E7 transgenic line. Several weeks after cessation of UV-B exposure, enhanced proliferation, as measured by BrdU incorporation, was maintained only in HPV-20 transgenic skin. Keratin 6 expression was increased in the transgenic mice throughout all cell layers. Expression of the differentiation markers involucrin and loricrin was reduced and disturbed. p63alpha expression was differentially regulated with high levels of cytoplasmic expression in clusters of cells in the granular layer of the skin in the transgenic lines 20 weeks after cessation of UV-B exposure, in contrast to uninterrupted staining in the nontransgenic lines. p53 was expressed in clusters of cells in nontransgenic and HPV-27 transgenic mice, in contrast to an even distribution in a higher number of cells in HPV-20 transgenic animals.