Absence of p53-dependent apoptosis leads to UV radiation hypersensitivity, enhanced immunosuppression and cellular senescence.

Genotoxic stress triggers the p53 tumor suppressor network to activate cellular responses that lead to cell cycle arrest, DNA repair, apoptosis or senescence. This network functions mainly through transactivation of different downstream targets, including cell cycle inhibitor p21, which is required for short-term cell cycle arrest or long-term cellular senescence, or proapoptotic genes such as p53 upregulated modulator of apoptosis (PUMA) and Noxa. However, the mechanism that switches from cell cycle arrest to apoptosis is still unknown. In this study, we found that mice harboring a hypomorphic mutant p53, R172P, a mutation that abrogates p53-mediated apoptosis while keeping cell cycle control mostly intact, are more susceptible to ultraviolet-B (UVB)-induced skin damage, inflammation and immunosuppression than wild-type mice. p53(R172P) embryonic fibroblasts (MEFs) are hypersensitive to UVB and prematurely senesce after UVB exposure, in stark contrast to wild-type MEFs, which undergo apoptosis. However, these mutant cells are able to repair UV-induced DNA lesions, indicating that the UV hypersensitive phenotype results from the subsequent damage response. Mutant MEFs show an induction of p53 and p21 after UVR, while wild-type MEFs additionally induce PUMA and Noxa. Importantly, p53(R172P) MEFs failed to downregulate anti-apoptotic protein Bcl-2, which has been shown to play an important role in p53-dependent apoptosis. Taken together, these data demonstrate that in the absence of p53-mediated apoptosis, cells undergo cellular senescence to prevent genomic instability. Our results also indicate that p53-dependent apoptosis may play an active role in balancing cellular growth.

P53 protein and pathogenesis of melanoma and nonmelanoma skin cancer.

The p53 tumor suppressor gene and gene product are among the most diverse and complex been shown to have a direct correlation with cancer development and have been shown to occur in nearly 50% of all cancers. p53 mutations are particularly common in skin cancers and UV irradiation has been shown to be a primary cause of specific 'signature' mutations that can result in oncogenic transformation. There are certain 'hot-spots' in the p53 gene where mutations are commonly found that result in a mutated dipyrimidine site. This review discusses the role of p53 from normal function and its dysfunction in precancerous lesions, nonmelanoma and melanoma skin cancers. Additionally, molecules that associate with p53 and alter its function to produce neoplastic conditions are also explored in this chapter.

Oncogenic potential of BRAF versus RAS.

Mutations in the ERK pathway occur in approximately one-third of all human cancers and most often involve production of mutant RAS or BRAF. Several studies, including our own, have shown that mutations in the BRAF and RAS genes are generally mutually exclusive. This study was performed to determine the relative oncogenic potential of the BRAF and RAS oncogenes. BRAF(V600E)-, H-RAS(G12V)-, and N-RAS(Q61R)-transfected mouse embryonic fibroblasts (MEFs) that lack p53 (p53(-/-)) or contain mutations at codon 172 (p53(R172H) and p53(R172P)) were able to induce morphologically transformed foci in p53(-/-) and p53(R172H) MEFs but not in p53(R172P) MEFs. Interestingly, BRAF(V600E) was less potent than mutant H-RAS(G12V) or N-RAS(Q61R) was in cooperating with mutant p53 as the numbers and sizes of foci induced by BRAF(V600E) were significantly lower and smaller. In vitro growth characteristics and anchorage-independent growth of transfected MEFs corroborated the transformed phenotype, and in vivo tumorigenesis confirmed the results. These results indicate that mutant BRAF(V600E) is weakly oncogenic compared with mutant RAS and that they both cooperate with p53(-/-) and p53(R172H) but not with p53(R172P) in oncogenic transformation.

p53 tumor suppressor gene: a critical molecular target for UV induction and prevention of skin cancer.

The relationship between exposure to UV radiation and development of skin cancer has been well established. Several studies have shown that UVB induces unique mutations (C-->T and CC-->TT transitions) in the p53 tumor suppressor gene that are not commonly induced by other carcinogens. Our studies have demonstrated that UV-induced mouse skin cancers contain p53 mutations at a high frequency and that these mutations can be detected in UV-irradiated mouse skin well before the appearance of skin tumors. This observation suggested that it might be possible to use p53 mutations as a biologic endpoint for testing the efficacy of sunscreens in photoprotection studies. Indeed, application of SPF 15 sunscreens to mouse skin before each UVB irradiation resulted in reduction in the number of p53 mutations. Because p53 mutations represent an early essential step in photocarcinogenesis, these results imply that inhibition of this event may protect against skin cancer development. This hypothesis was confirmed by our finding that sunscreens used in p53 mutation inhibition experiments also protected mice against UVB-induced skin cancer.

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

Quantitative proteomes and in vivo secretomes of progressive and regressive UV-induced fibrosarcoma tumor cells: mimicking tumor microenvironment using a dermis-based cell-trapped system linked to tissue chamber.

The alterations of tumor proteome and/or in vivo secretome created by host-tumor cell interaction may be crucial factors for tumors to undergo progression or regression in a host system. Two UV-induced fibrosarcoma tumor cell lines (UV-2237 progressive cells and UV-2240 regressive cells) were used as models to address this issue. Hundreds of proteins including in vivo secretome have been identified and quantified via an isotope-coded protein label (ICPL) in conjunction with high-throughput NanoLC-LTQ MS analysis. A newly designed technology using a dermis-based cell-trapped system was employed to encapsulate and grow 3-D tumor cells. A tissue chamber inserted with a tumor cell-trapped dermis was implanted into mice to mimic the tumor microenvironment. The in vivo secretome created by host-tumor interaction was characterized from samples collected from tissue chamber fluids via ICPL labeling mass spectrometric analysis. Twenty-five proteins including 14-3-3 proteins, heat shock proteins, profilin-1, and a fragment of complement C3 with differential expression in proteomes of UV-2237 and UV-2240 cells were revealed. Three secreted proteins including myeloperoxidase, alpha-2-macroglobulin, and a vitamin D-binding protein have different abundances in the in vivo secretome in response to UV-2237 and UV-2240 cells. Differential tumor proteomes and in vivo secretome were thus accentuated as potential therapeutic targets to control tumor growth.

Models and mechanisms in malignant melanoma.

Human melanoma represents the fastest growing malignancy in the US. The etiology of melanoma is highly debated as is the role of ultraviolet (UV) radiation in the initiation and progression of melanoma. This article discusses data from UV exposure and its relationship to the development of melanoma from various models of melanoma as well as various genetic alterations seen in oncogenic transformation of melanocytes. Genetic alterations such as the p16(INK4a) deletion, melanocortin 1 receptor (MC1R), RAS, and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) may be indicative of a predisposition to melanoma development. Historical research as well as current data on the significance of the hot spot mutation in BRAF is discussed in its relative potential to the activating mutation in RAS.

p53 and the pathogenesis of skin cancer.

The p53 tumor suppressor gene and gene product are among the most diverse and complex molecules involved in cellular functions. Genetic alterations within the p53 gene have been shown to have a direct correlation with cancer development and have been shown to occur in nearly 50% of all cancers. p53 mutations are particularly common in skin cancers and UV irradiation has been shown to be a primary cause of specific 'signature' mutations that can result in oncogenic transformation. There are certain 'hot-spots' in the p53 gene where mutations are commonly found that result in a mutated dipyrimidine site. This review discusses the role of p53 from normal function and its dysfunction in pre-cancerous lesions and non-melanoma skin cancers. Additionally, special situations are explored, such as Li-Fraumeni syndrome in which there is an inherited p53 mutation, and the consequences of immune suppression on p53 mutations and the resulting increase in non-melanoma skin cancer in these patients.

Mass spectrometric proteomics profiles of in vivo tumor secretomes: capillary ultrafiltration sampling of regressive tumor masses.

Identification of in vivo secreted peptides/proteins (secretomes) in tumor masses has the potential to provide important biomarkers and therapeutic targets for cancer therapy. However, limitations of existing technologies have made obtaining these secretomes for analysis extremely difficult. Here we employed an in vivo sampling technique using capillary ultrafiltration (CUF) probes to collect secretomes directly from tumor masses. Mass spectrometric proteomics approaches were then used to identify the tumor secretomes. A UV-induced skin fibrosarcoma cell line (UV-2240) was subcutaneously injected into C3H/NeH mice, resulting in tumor masses that initially progressed, then regressed and eventually eradicated. We then implanted CUF probes into tumor masses at the progressive and regressive stage. Five secreted proteins (cyclophilin-A, S100A4, profilin-1, thymosin beta 4 and 10), previously associated with tumor progression, were identified from tumor masses at the progressive stage. Five secreted proteins including three protease inhibitors (fetuin-A, alpha-1 antitrypsin 1-6, and contrapsin) were identified from tumor masses at the regressive stage. The technique involving CUF probes linked to mass spectrometric proteomics reinforces systems biology studies of cell-cell interactions and is potentially applicable to the discovery of in vivo biomarkers in human disease.

Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis.

Psoralen plus UVA (PUVA) is used as a very effective treatment modality for various diseases, including psoriasis and cutaneous T-cell lymphoma. PUVA-induced immune suppression and/or apoptosis are thought to be responsible for the therapeutic action. However, the molecular mechanisms by which PUVA acts are not well understood. We have previously identified platelet-activating factor (PAF), a potent phospholipid mediator, as a crucial substance triggering ultraviolet B radiation-induced immune suppression. In this study, we used PAF receptor knockout mice, a selective PAF receptor antagonist, a COX-2 inhibitor (presumably blocking downstream effects of PAF), and PAF-like molecules to test the role of PAF receptor binding in PUVA treatment. We found that activation of the PAF pathway is crucial for PUVA-induced immune suppression (as measured by suppression of delayed type hypersensitivity to Candida albicans) and that it plays a role in skin inflammation and apoptosis. Downstream of PAF, interleukin-10 was involved in PUVA-induced immune suppression but not inflammation. Better understanding of PUVA's mechanisms may offer the opportunity to dissect the therapeutic from the detrimental (ie, carcinogenic) effects and/or to develop new drugs (eg, using the PAF pathway) that act like PUVA but have fewer side effects.

Fate of UVB-induced p53 mutations in SKH-hr1 mouse skin after discontinuation of irradiation: relationship to skin cancer development.

Chronic exposure to ultraviolet (UV) radiation causes skin cancer in humans and mice. We have previously shown that in hairless SKH-hr1 mice, UVB-induced p53 mutations arise very early, well before tumor development. In this study, we investigated whether discontinuation of UVB exposure before the onset of skin tumors results in the disappearance of p53 mutations in the skin of hairless SKH-hr1 mice. Irradiation of mice at a dose of 2.5 kJ/m2 three times a week for 8 weeks induced p53 mutations in the epidermal keratinocytes of 100% of the mice. UVB irradiation was discontinued after 8 weeks, but p53 mutations at most hotspot codons were still present even 22 weeks later. During that period, the percent of mice carrying p53(V154A/R155C), p53(H175H/H176Y), and p53R275C mutant alleles remained at or near 100%, whereas the percentage of mice with p53R270C mutation decreased by 45%. As expected, discontinuation of UVB after 8 weeks resulted in a delay in tumor development. A 100% of tumors carried p53(V154A/R155C) mutant alleles, 76% carried p53(H175H/H176Y) mutants, and 24 and 19% carried p53R270C and p53R275C mutants, respectively. These results suggest that different UVB-induced p53 mutants may provide different survival advantages to keratinocytes in the absence of further UVB exposure and that skin cancer development can be delayed but not prevented by avoidance of further exposure to UVB radiation.

Cellular and molecular events leading to the development of skin cancer.

The transition from a normal cell to a neoplastic cell is a complex process and involves both genetic and epigenetic changes. The process of carcinogenesis begins when the DNA is damaged, which then leads to a cascade of events leading to the development of a tumor. Ultraviolet (UV) radiation causes DNA damage, inflammation, erythema, sunburn, immunosuppression, photoaging, gene mutations, and skin cancer. Upon DNA damage, the p53 tumor suppressor protein undergoes phosphorylation and translocation to the nucleus and aids in DNA repair or causes apoptosis. Excessive UV exposure overwhelms DNA repair mechanisms leading to induction of p53 mutations and loss of Fas-FasL interaction. Keratinocytes carrying p53 mutations acquire a growth advantage by virtue of their increased resistance to apoptosis. Thus, resistance to cell death is a key event in photocarcinogenesis and conversely, elimination of cells containing excessive UV-induced DNA damage is a key step in protecting against skin cancer development. Apoptosis-resistant keratinocytes undergo clonal expansion that eventually leads to formation of actinic keratoses and squamous cell carcinomas. In this article, we will review some of the cellular and molecular mechanisms involved in initiation and progression of UV-induced skin cancer.

UV-A fingerprint mutations in human skin cancer.

This review of our work, presented at the Photocarcinogenesis Symposium of the 14th International Congress on Photobiology, shows that UV-A causes a similar number of gene mutations as UV-B in human skin cancer. Areas of about 20 keratinocytes from solar keratoses and squamous cell carcinomas, which are benign and malignant skin cancers, respectively, were sampled by laser capture microdissection. Automated sequencing of the p53 gene was used to detect mutations in these tumor areas, and the cause of the mutations was attributed on the basis of previously published studies. UV-A and UV-B caused similar numbers of p53 gene mutations in both benign and malignant human skin tumors, with UV-B-induced mutations being restricted to the upper areas of the tumors and UV-A-induced mutations predominating at the basal layer. Furthermore, each microdissected region within a tumor had distinct mutations showing that the skin tumors consisted of different clones of cells. This is not consistent with how human skin carcinogenesis is currently understood, and hypotheses to explain our data are presented. We propose that the UV-A waveband of sunlight is as important as UV-B in causing skin cancer in humans.

Inverse relationship between increased apoptosis and decreased skin cancer in UV-irradiated CD1d-/- mice.

We previously demonstrated that CD1d knockout mice were resistant to ultraviolet (UV)-induced immunosuppression. Because immune suppression is a critical factor in the development of UV-induced skin cancers, we investigated the response of wild type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. To investigate the mechanisms involved in the resistance of CD1d-/- mice to UV-induced carcinogenesis, we determined the time course and kinetics of keratinocyte cell death after UV irradiation. After acute UV exposure, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive keratinocytes were eliminated from the skin of WT mice by 72 h post-UV, but they still persisted until 96 h in CD1d-/- mice. The kinetics of p53 protein expression closely followed the kinetics of apoptotic cell death. Chronic UV irradiation resulted in induction of a significantly higher number of apoptotic keratinocytes in CD1d-/- than WT mice. In addition, epidermis and dermis from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. These results indicate that the resistance of CD1d-/- mice to UV carcinogenesis may be due to increased cell death and elimination of keratinocytes and fibroblasts containing DNA damage and p53 mutations.

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.

Resistance of CD1d-/- mice to ultraviolet-induced skin cancer is associated with increased apoptosis.

Inhibition of p53-induced epidermal apoptosis, generation of p53 mutations, and suppressor T cells are the critical events responsible for the induction and development of UV-induced skin cancers. Recently, we demonstrated that CD1d knockout mice were resistant to UV-induced immunosuppression, prompting us to further address the role of CD1d in regulating UV carcinogenesis. We, therefore, investigated the response of wild-type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. Surprisingly, keratinocytes and fibroblasts from CD1d-/- mice were more sensitive to UV-induced apoptosis and persisted longer than cells derived from WT mice. In addition, epidermis and dermis taken from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. Our findings identify an unexpected and novel function for CD1d as a critical molecule regulating UV carcinogenesis, by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts.

Exposure of melanoma cells to dacarbazine results in enhanced tumor growth and metastasis in vivo.

PURPOSE: In recent years, the incidence of cutaneous melanoma has increased more than that of any other cancer. Dacarbazine is considered the gold standard for treatment, having a response rate of 15% to 20%, but most responses are not sustained. Previously, we have shown that short exposure of primary cutaneous melanoma cells to dacarbazine resulted in the upregulation of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). The purpose of the present study was to determine how long-term exposure of melanoma cells to dacarbazine would affect their tumorigenic and metastatic potential in vivo. MATERIALS AND METHODS: The primary cutaneous melanoma cell lines SB2 and MeWo were repeatedly exposed in vitro to increasing concentrations of dacarbazine, and dacarbazine-resistant cell lines SB2-D and MeWo-D were selected and examined for their ability to grow and metastasize in nude mice. RESULTS: The dacarbazine-resistant cell lines SB2-D and MeWo-D exhibited increased tumor growth and metastatic behavior in vivo. This increase could be explained by the activation of RAF, MEK, and ERK, which led to the upregulation of IL-8 and VEGF. More IL-8, VEGF, matrix metalloproteinase-2 (MMP-2), and microvessel density (CD-31) were found in tumors produced by SB2-D and MeWo-D in vivo than in those produced by their parental counterparts. No mutations were observed in BRAF. CONCLUSION: Our results have significant clinical implications. Treatment of melanoma patients with dacarbazine could select for a more aggressive melanoma phenotype. We propose that combination treatment with anti-VEGF/IL-8 or MEK inhibitors may potentiate the therapeutic effects of dacarbazine.

The basal layer in human squamous tumors harbors more UVA than UVB fingerprint mutations: a role for UVA in human skin carcinogenesis.

We hypothesized that a substantial portion of the mutagenic alterations produced in the basal layer of human skin by sunlight are induced by wavelengths in the UVA range. Using laser capture microdissection we examined separately basal and suprabasal keratinocytes from human skin squamous cell carcinomas and premalignant solar keratosis for both UVA- and UVB-induced adduct formation and signature mutations. We found that UVA fingerprint mutations were detectable in human skin squamous cell carcinomas and solar keratosis, mostly in the basal germinative layer, which contrasted with a predominantly suprabasal localization of UVB fingerprint mutations in these lesions. The epidermal layer bias was confirmed by immunohistochemical analyses with a superficial localization of cyclobutane thymine dimers contrasting with the localization of 8-hydroxy-2'-deoxyguanine adducts to the basal epithelial layers. If unrepaired, these adducts may lead to fixed genomic mutations. The basal location of UVA-rather than UVB-induced DNA damage suggests that longer-wavelength UVR is an important carcinogen in the stem cell compartment of the skin. Given the traditional emphasis on UVB, these results may have profound implications for future public health initiatives for skin cancer prevention.

Toxic effects of ultraviolet radiation on the skin.

Ultraviolet (UV) irradiation present in sunlight is an environmental human carcinogen. The toxic effects of UV from natural sunlight and therapeutic artificial lamps are a major concern for human health. The major acute effects of UV irradiation on normal human skin comprise sunburn inflammation (erythema), tanning, and local or systemic immunosuppression. At the molecular level, UV irradiation causes DNA damage such as cyclobutane pyrimidine dimers and (6-4) photoproducts, which are usually repaired by nucleotide excision repair (NER). Chronic exposure to UV irradiation leads to photoaging, immunosuppression, and ultimately photocarcinogenesis. Photocarcinogenesis involves the accumulation of genetic changes, as well as immune system modulation, and ultimately leads to the development of skin cancers. In the clinic, artificial lamps emitting UVB (280-320 nm) and UVA (320-400 nm) radiation in combination with chemical drugs are used in the therapy of many skin diseases including psoriasis and vitiligo. Although such therapy is beneficial, it is accompanied with undesirable side effects. Thus, UV radiation is like two sides of the same coin--on one side, it has detrimental effects, and on the other side, it has beneficial effects.