Loss of CDKN2A and p14ARF expression occurs frequently in human nonmelanoma skin cancers.

BACKGROUND: The CDKN2A locus on human chromosome 9p21 encodes two proteins named p16INK4a and p14ARF, known to function as tumour suppressors via the retinoblastoma (Rb) or the p53 pathway. The p53 tumour suppressor gene is the most commonly mutated gene in human and mouse cancers. Disruption of the p53 and Rb pathways is a fundamental trend of most human cancer cells. Recent studies have shown that the CDKN2A gene plays an active role in the p53 and Rb tumour suppressor pathways. Genetic abnormalities in CDKN2A have been well documented in human melanoma, but their involvement in nonmelanoma skin cancer (NMSC) is less clear. OBJECTIVES: To determine whether genetic abnormalities in CDKN2A and p53 genes play a role in the development of NMSC. METHODS: We analysed 40 primary NMSCs in 40 patients (21 squamous cell carcinomas, 17 basal cell carcinomas and two actinic keratoses) for p16INK4a and p14ARF protein expression and for genetic alterations in exons 1alpha, 1beta and 2 of CDKN2A. RESULTS: Immunohistochemical analysis revealed loss of expression of p16INK4a and p14ARF proteins in 38 and 39 of 40 NMSCs, respectively. Amplification of genomic DNA by polymerase chain reaction revealed homozygous deletion of exon 1beta in 20% of tumours and of exon 2 in 82.5% of tumours. Of 22 NMSCs with p53 mutations, 13 (59%) had ultraviolet (UV) signature mutations in the p53 gene; all of them were strongly positive for p53 immunostaining. CONCLUSIONS: In addition to mutations in the p53 gene, loss of expression of CDKN2A via deletion also plays an important role in the pathogenesis of human NMSC. While p53 mutations are induced by UVB, deletions in CDKN2A could arise spontaneously, perhaps during tumour progression.

Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells.

One of the major targets for breast cancer therapy is the epidermal growth factor receptor (EGFR) and related receptors, which signal via different signal transduction pathways including the mitogen-activated protein kinase (MAPK) pathway. This study determined whether there is a correlation between EGFR/HER2 status and MAPK (ERK1/2) phosphorylation in breast cancer cells, and how this affects the response to an inhibitor of the receptors. Expression of EGFR, HER2 and phosphorylated ERK1/2 were measured by immunoblotting in a panel of breast cancer cell lines. Several lines expressed high levels of pERK1/2, with no obvious correlation with the level of EGFR/HER2. The EGFR tyrosine kinase inhibitor PKI166 inhibited growth and induced apoptosis in some cells with high levels of growth factor receptors (MDA-MB-468, SUM149, SKBR3), but was less effective in cells that also had high basal ERK1/2 activity (MDA-MB-231). The combination of an inhibitor of MAPK signalling (U0126) and PKI166 produced significantly more inhibition and apoptosis than either agent alone. This suggests that constitutive activation of the MAPK pathway may bypass inhibition of EGFR/HER2 tyrosine kinases, and lead to insensitivity to agents targeting the receptors. However, inhibiting both EGFR/HER2 and MAPK signalling can result in significant growth inhibition and apoptosis of EGFR-expressing breast cancer cells.

p53 and Fas ligand are required for psoralen and UVA-induced apoptosis in mouse epidermal cells.

A combination of 8-methoxypsoralen (8-MOP) and ultraviolet-A (UVA) radiation (320-400 nm) (PUVA) is widely used in the treatment of psoriasis and other skin diseases. PUVA is highly effective in eliminating hyperproliferative cells in the epidermis, but its mechanism of action has not been fully elucidated. In this study, we used immortalized JB6 mouse epidermal cells, p53(-/-), and Fas ligand deficient (gld) mice to investigate the molecular mechanism by which PUVA induces cell death. The results indicate that PUVA treatment induces apoptosis in JB6 cells. In addition, PUVA treatment of JB6 cells results in p53 stabilization, phosphorylation, and nuclear localization as well as induction of p21(Waf/Cip1) and caspase-3 activity. In vivo studies reveal that PUVA treatment induces significantly less apoptosis in the epidermis of p53(-/-) mice compared to p53(+/+) mice. Furthermore, FasL-deficient (gld) mice are completely resistant to PUVA-induced apoptosis compared to wild-type mice. These results indicate that PUVA treatment induces apoptosis in mouse epidermal cells in vitro and in vivo and that p53 and Fas/Fas ligand interactions are required for this process, at least in vivo. This implies that similar mechanisms may be involved in the elimination of psoriatic keratinocytes from human skin following PUVA therapy.

Ultraviolet a radiation suppresses an established immune response: implications for sunscreen design.

The ultraviolet radiation present in sunlight is the primary cause of nonmelanoma skin cancer and has been implicated in the development of cutaneous malignant melanoma. In addition, ultraviolet is immune suppressive and the suppression induced by ultraviolet radiation has been identified as a risk factor for skin cancer induction. Ultraviolet also suppresses the immune response to infectious agents. In most experimental models, ultraviolet is applied to immunologically naive animals prior to immunization. Of equal concern, however, is the ability of sunlight to suppress established immune reactions, such as the recall reaction in humans, which protects against microbial infections. Here we demonstrate that solar-simulated ultraviolet radiation, applied after immunization, suppresses immunologic memory and the elicitation of delayed-type hypersensitivity. Further, we found that wavelengths in the ultraviolet A region of the solar spectrum were critical for inducing immune suppression. Ultraviolet A (320-400 nm) radiation was as effective as solar-simulated ultraviolet A + B (290-400 nm) in suppressing the elicitation of an established immune response. Irradiation with ultraviolet AI (340-400 nm) had no effect. Supporting a critical role for ultraviolet A in ultraviolet-induced immune suppression was the observation that applying a sunscreen that contained an ultraviolet B only filter had no protective effect, whereas, a sunscreen containing both ultraviolet A and ultraviolet B filters totally blocked ultraviolet-induced immune suppression. These data suggest that sunlight may depress the protective effect of prior vaccination. In addition, the observation that ultraviolet A is immunosuppressive indicates the need for ultraviolet A protection when designing sun protection strategies.

UV irradiation augments lymphoid malignancies in mice with one functional copy of wild-type p53.

Epidemiological studies have suggested an association between exposure to solar UV radiation and the incidence of lymphoid malignancies, which has increased substantially worldwide during the last two decades. Findings from animal studies have raised the question of whether UV radiation might influence the development of lymphoid malignancies by means of its immunosuppressive effect. In this study, we examined the effect of UV irradiation on the development of lymphoid malignancies in mice with no or only one functional copy of p53. Mice that lack both copies of p53 spontaneously develop high frequency of lymphoid malignancies in the thymus and spleen. p53 heterozygous mice with only one copy of the wild-type allele also develop lymphoid malignancies, but with a much lower frequency and a long latent period. In our study using mice of the C57BL/6 background, only one of the unirradiated mice lacking one copy of p53 (p53(+/-)) spontaneously developed a lymphoid tumor (6%), whereas 88% of UV-irradiated p53(+/-) mice developed lymphoid tumors in the spleen or liver. None of the control or UV-irradiated p53 wild-type mice developed lymphoid tumors during the 60-week observation period. Both UV-irradiated and unirradiated mice lacking both copies of p53 (p53(-/-)) rapidly developed thymic lymphomas and/or lymphoid tumors in spleen or liver. All of the lymphoid tumors tested were of T cell type. The immune responses of the mice to contact sensitization were identical and were suppressed to the same extent by UV irradiation regardless of the genotype. These results indicate that differences in immune reactivity do not account for the different effects of UV radiation on lymphoid malignancies and, in addition, that p53 is not required for generation of T cell-mediated immunity. Interestingly, whereas p53 mutations or loss of heterozygosity did not account for the accelerated development of lymphoid tumors in UV-irradiated p53(+/-) mice, deletions in the p16(INK4a) gene were quite common. These data provide the experimental evidence that UV irradiation induces lymphoid neoplasms in genetically susceptible mice and support the hypothesis that extensive sunlight exposure contributes to the induction of lymphoma in humans.

Impact of Bcl-2 and Ha-ras on keratinocytes in organotypic culture.

In this study, the role of specific molecular alterations associated with multistep skin carcinogenesis was assessed using in vitro organotypic cultures of the spontaneously immortalized, nontumorigenic HaCaT keratinocyte cell line. HaCaT vector control clones and clones expressing bcl-2, activated Ha-ras, or both genes were generated. Clones were induced to stratify and differentiate by culturing on dermal equivalents for 2 wk at the air-medium interface. In parental and vector control HaCaT rafts the expression and distribution of cytokeratin K1, K14, involucrin, proliferating cell nuclear antigen, and p21cip1/waf1 were assessed using immunohistochemistry and immunoblotting and were similar to normal epidermis. Apoptosis was also examined using the TUNEL technique. HaCaT-bcl-2 rafts were similar to control rafts but exhibited lower spontaneous rates of apoptosis and a moderate increase in the rate of proliferation. Differentiation was significantly inhibited in HaCaT-ras organotypic cultures and was associated with high rates of proliferation and lower rates of spontaneous apoptosis. Additionally, HaCaT-ras rafts exhibited significantly higher rates of apoptosis following ultraviolet irradiation compared with vector control or HaCaT-bcl-2 rafts. Bcl-2 was able to largely restore normal differentiation, proliferation, and apoptosis in HaCaT-ras/bcl-2 organotypic cultures. Bcl-2 also abrogated apoptosis induction following ultraviolet irradiation in HaCaT-ras/bcl-2 organotypic cultures. Organotypic keratinocyte culture represents a valuable in vitro system to evaluate the impact of individual molecular genetic alterations on the coordinate regulation of cell proliferation, differentiation, and cell death.

Loss of Fas-ligand expression in mouse keratinocytes during UV carcinogenesis.

Skin cells containing excessive ultraviolet (UV) radiation-induced DNA damage are eliminated by apoptosis that involves the p53 pathway and Fas/Fas-Ligand (Fas-L) interactions. To determine whether dysregulation of apoptosis plays a role in skin cancer development through disruption of Fas/Fas-L interactions, hairless SKH-hr1 mice were exposed to chronic UV irradiation from Kodacel-filtered FS40 lamps for 30 weeks. Their skin was analyzed for the presence of sunburn cells (apoptotic keratinocytes) and for Fas and Fas-L expression at various time points. A dramatic decrease in the numbers of morphologically identified sunburn cells and TUNEL-positive cells was detected as early as 1 week after chronic UV exposure began. After 4 weeks of chronic UV exposure, these cells were barely detectable. This defect in apoptosis was paralleled by an initial decrease in Fas-L expression during the first week of chronic UV irradiation and a complete loss of expression after 4 weeks. Fas expression, however, increased during the course of chronic UV exposure. p53 mutations were detected in the UV-irradiated epidermis as early as 1 week after irradiation began and continued to accumulate with further UV exposure. Mice exposed to chronic UV began to develop skin tumors after approximately 8 weeks, and all mice had multiple skin tumors by 24 weeks. Most of the tumors expressed Fas but not Fas-L. We conclude that chronic UV exposure may induce a loss of Fas-L expression and a gain in p53 mutations, leading to dysregulation of apoptosis, expansion of mutated keratinocytes, and initiation of skin cancer.

Temporal events in skin injury and the early adaptive responses in ultraviolet-irradiated mouse skin.

We examined the effects of ultraviolet (UV) radiation on the time course for induction of sunburn (apoptotic) cells and expression of proteins known to be associated with growth arrest and apoptosis in SKH-hr1 mouse skin. Mice were irradiated with a single dose (2.5 kJ/m(2)) of UV from Kodacel-filtered (290-400 nm) FS40 sunlamps and the skin tissues were analyzed at various times after irradiation for the presence of apoptotic cells and expression of p53, p21(Waf-1/Cip1), bcl-2, bax, and proliferating cell nuclear antigen. The results indicated that p53 expression was induced early in the epidermis, reaching maximum levels 12 hours after irradiaton, and p21(Waf-1/Cip1) expression in the epidermis peaked at 24 hours after irradiation. In contrast, UV radiation induced high levels of bax at 24 to 72 hours after irradiation with a concomitant decrease in bcl-2 expression. Coinciding with these changes, apoptotic cells began to appear 6 hours after irradiation and reached a maximum at 24 hours after irradiation. Interestingly, proliferating cell nuclear antigen expression, which was initially confined to the basal layer, became dispersed throughout the basal and suprabasal layers of the skin at 48 hours and paralleled marked hyperplasia. These results suggest that UV irradiation of mouse skin induces apoptosis mediated by the p53/p21/bax/bcl-2 pathway and that the dead cells are replaced by hyperproliferative cells, leading to epidermal hyperplasia. This implies that UV-induced apoptosis and hyperplasia are closely linked and tightly regulated and that dysregulation of these two events may lead to skin cancer development.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis.

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC-/- mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC-/- skin tumor analysed displayed C-->T or CC-->TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC-/- and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC-/- mouse skin after 3 - 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC-/- mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC-/- mice.

Sunscreen effects on UV-induced immune suppression.

In order to protect the public against the adverse effects of sunlight, the scientific, medical, and particularly the dermatologic community has promoted "safe sun exposure." This strategy includes sun avoidance whenever possible, wearing hats and other protective clothing and/or devices, such as sunglasses, and extensive use of sunscreens. Sunscreen efficacy is determined by measuring the ability of the sunscreen to block ultraviolet (UV)-induced erythema (sun protection factor or SPF), and most sunscreen formulations on the market, if used properly, are very good at preventing erythema and sunburn. How well sunscreens protect against the other adverse effects of sunlight, such as immune suppression, is not as clear. The purpose of this paper is to review and discuss the literature in this area, concentrating on some of the complications of determining how well sunscreens protect against UV-induced immune suppression.

Fas ligand: a sensor for DNA damage critical in skin cancer etiology.

DNA-damaged cells can either repair the DNA or be eliminated through a homeostatic control mechanism termed "cellular proofreading." Elimination of DNA-damaged cells after ultraviolet radiation (UVR) through sunburn cell (apoptotic keratinocyte) formation is thought to be pivotal for the removal of precancerous skin cells. Sunburn cell formation was found to be dependent on Fas ligand (FasL), a pro-apoptotic protein induced by DNA damage. Chronic exposure to UVR caused 14 of 20 (70 percent) FasL-deficient mice and 1 of 20 (5 percent) wild-type mice to accumulate p53 mutations in the epidermis. Thus, FasL-mediated apoptosis is important for skin homeostasis, suggesting that the dysregulation of Fas-FasL interactions may be central to the development of skin cancer.

p53 protects against skin cancer induction by UV-B radiation.

To assess the role of the p53 tumor suppressor gene in skin carcinogenesis by UV radiation, mice constitutively lacking one or both copies of the functional p53 gene were compared to wild-type mice for their susceptibility to UV carcinogenesis. Heterozygous mice showed greatly increased susceptibility to skin cancer induction, and homozygous p53 knockout mice were even more susceptible. Accelerated tumor development in the heterozygotes was not associated with loss of the remaining wild-type allele of p53, as reported for tumors induced by other carcinogens, but in many cases was associated with UV-induced mutations in p53. Tumors arose on the ears and dorsal skin of mice of all three genotypes, and homozygous knockout mice also developed ocular tumors, mainly melanomas. Skin tumors in the p53 knockout mice were predominately squamous cell carcinomas and were associated with premalignant lesions resembling actinic keratoses, whereas those in the heterozygous and wild-type mice were mainly sarcomas. These results demonstrate the importance of p53 in protecting against UV-induced cancers, particularly in the eye and epidermis.

Detection of mutations and polymorphisms in the p53 tumor suppressor gene by single-strand conformation polymorphism analysis.

Deciphering the genetic mechanisms in cancer development requires analysis of a large number of tumors for consistent genetic alterations. Single-strand conformational polymorphism (SSCP) analysis is a fast and efficient method for detecting mutations, deletions, insertions and loss of alleles. The primary advantage of this method is speed and ability to screen a large number of samples at one time. Here we report the use of the SSCP technique for rapidly screening tumor and normal tissues for mutations and polymorphisms in the p53 tumor suppressor gene. Because the DNA extracted from specific aberrant bands from different samples always give rise to the same nucleotide sequence upon sequencing analysis, the SSCP technique can be used as a diagnostic tool to identify the presence of such genetic alterations without having to spend time on further sequencing analysis.

Direct correlation between nitric oxide synthase II inducibility and metastatic ability of UV-2237 murine fibrosarcoma cells carrying mutant p53.

The relationship between nitric oxide synthase II (NOS II) inducibility and the metastatic ability of UV-2237 murine fibrosarcoma cells was determined. Highly metastatic cells survived to produce numerous lung metastases after i.v. injection in syngeneic C3H/HeN mice, whereas poorly metastatic cells did not. Highly metastatic clones exhibited higher levels of NOS II than did poorly metastatic clones in response to interleukin 1alpha and IFN-gamma stimulation. Furthermore, both poorly and highly metastatic clones contained an identical p53 mutation. Overexpression of NOS II in a highly metastatic clone by transfection with NOS II gene retarded tumor growth and completely suppressed metastasis. Our data indicate that a low to moderate level of NOS II expression directly correlates with metastatic ability of UV-2237 fibrosarcoma cells carrying mutant p53 and that a high level of nitric oxide production suppresses tumor growth and metastasis.

Direct comparison of DNA damage, isomerization of urocanic acid and edema in the mouse produced by three commonly used artificial UV light sources.

Exposure to sunlight can result in a number of harmful effects, including sunburn, erythema, premature aging of the skin, immune suppression and skin cancer. Studies designed to understand the underlying mechanisms often depend upon the use of artificial sources of UV radiation. Unfortunately, conclusions from different laboratories using different lamps often conflict, and it is entirely possible that the different spectra of sunlights used in each may be a source of conflict. To minimize confounding variables, we employed two of the more commonly used UV light sources, fluorescent sunlamps, such as the FS-40 and Kodacel-filtered FS-40 sunlamps, and a xenon arc solar simulator and compared, in one series of standardized experiments, the effects of each light source on DNA damage, urocanic acid isomerization and edema formation. The dose-response curves, calculated by linear regression or curve fitting were compared. The data indicate that DNA damage and urocanic acid isomerization were more sensitive to shorter wavelengths of UV than longer wavelengths, and the biological endpoint of edema most closely correlated with the induction of DNA damage. The results emphasize the dominance of shorter wavelengths within the UV spectrum in damaging biological tissues, even when the solar simulator, which contains significant amounts of UVA, was used and demonstrate that each light source has a characteristic pattern of induction of biochemical and biological endpoints.

Inhibition of solar simulator-induced p53 mutations and protection against skin cancer development in mice by sunscreens.

We demonstrated previously that p53 mutations can be detected in ultraviolet B-irradiated mouse skin months before the gross appearance of skin tumors and that applying sun protection factor 15 sunscreens to mouse skin before each Kodacel-filtered FS40 sunlamp irradiation resulted in the reduction of such mutations. To determine whether there is an association between reduction of ultraviolet-induced p53 mutations by sunscreens and protection against skin cancer using an environmentally relevant light source, we applied sunscreens (sun protection factors 15-22) on to the shaved dorsal skin of C3H mice 30 min before each exposure to 4.54 kJ ultraviolet B (290-400 nm) radiation per m2 from a solar simulator. Control mice were treated 5 d per wk with ultraviolet only or vehicle plus ultraviolet. p53 mutation analysis indicated that mice exposed to ultraviolet only or vehicle plus ultraviolet for 16 wk (cumulative exposure to 359 kJ ultraviolet B per m2) developed p53 mutations at a frequency of 56%-69%, respectively, but less than 5% of mice treated with sunscreens plus ultraviolet showed evidence of p53 mutations. More importantly, 100% of mice that received a cumulative dose of 1000 kJ ultraviolet B per m2 only, or vehicle plus ultraviolet B developed skin tumors, whereas, the probability of tumor development in all the mice treated with the sunscreens plus 1000 kJ ultraviolet B per m2 was 2% and mice treated with sunscreens plus 1500 kJ ultraviolet B per m2 was 15%. These results demonstrate that the sunscreens used in this study not only protect mice against ultraviolet-induced p53 mutations, but also against skin cancers induced with solar-simulated ultraviolet. Because of this association, we conclude that inhibition of p53 mutations is a useful early biologic endpoint of photoprotection against an important initiating event in ultraviolet carcinogenesis.

Suppression of delayed and contact hypersensitivity responses in mice have different UV dose responses.

Although acute exposure to UV radiation suppresses the induction of delayed-type (DTH) and contact (CHS) hypersensitivity in mice, it is not clear whether the photobiological mechanism(s) involved in suppressing these closely related immune reactions is the same. A careful examination of the UV dose responses and wavelength dependencies involved in suppressing CHS and DTH may provide important insights into the mechanisms involved. We compared the UV dose-response curves for suppressing four closely related immune reactions, local and systemic suppression of CHS to dinitrofluorobenzene, systemic suppression of DTH to Candida albicans and systemic suppression of DTH to alloantigen using three different UV spectra (FS40 sunlamps, Kodacel-filtered FS40 sunlamps and solar-simulated light). For each immune response studied, the amount of UVB radiation required to induce 50% immune suppression was lowest when FS40 sunlamps were used, highest with solar-simulated light and intermediate when Kodacel-filtered FS40 sunlamps were used, but the differences observed were not statistically significant. The UV dose-response curves for immune suppression differed significantly depending on the assay used, the site of antigenic sensitization and the antigen used. These findings suggest that the mechanisms by which UV radiation induces immune suppression differ for the four immunological reactions studied.