Ultraviolet radiation-mediated development of cutaneous melanoma: An update.

Ultraviolet (UV) light is absorbed by nucleic acids, proteins or other endogenous chromophores, such as porphyrins, flavins and melanin, triggering biological processes in skin cells. Both UV-induced mutations in melanocytes and changes in the immune microenvironment are understood to play a role in the development of cutaneous melanoma. The degree of UV-induced stress and the protection against this stress are influenced by both intracellular and intercellular molecular interactions. The present review summarizes the known major molecular biological changes induced by UV light in the skin that play a role in melanoma initiation and promotion. Nevertheless, cutaneous melanoma is not a homogenous disease, and the interaction of variable environmental exposure and different genetic susceptibility and other host factors lead to the formation of melanomas with different biological behavior and clinical characteristics. This review highlights the challenges in the understanding of how UV radiation contributes to the formation of cutaneous melanoma, and reviews the new results of photobiology and their link to tumor genetics and tumor immunology with potential implications on melanoma prevention and therapeutic strategies. The information presented here is expected to add clarity to ongoing research efforts in this field to aid the development of novel strategies to prevent and treat melanoma.

Identification of Cyclobutane Pyrimidine Dimer-Responsive Genes Using UVB-Irradiated Human Keratinocytes Transfected with In Vitro-Synthesized Photolyase mRNA.

Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases.

Effects of non-toxic zinc exposure on human epidermal keratinocytes.

Zinc is an essential microelement; its importance to the skin is highlighted by the severe skin symptoms in hereditary or acquired zinc deficiency, by the improvement of several skin conditions using systemic or topical zinc preparations and by the induced intracellular zinc release upon UVB exposure, which is the main harmful environmental factor to the skin. Understanding the molecular background of the role of zinc in skin may help gain insight into the pathology of skin disorders and provide evidence for the therapeutic usefulness of zinc supplementation. Herein, we studied the effects of zinc chloride (ZnCl2) exposure on the function of HaCaT keratinocytes, and the results showed that a non-toxic elevation in the concentration of extracellular zinc (100 µM) facilitated cell proliferation and induced significant alterations in the mRNA expression of NOTCH1, IL8, and cyclooxygenase-2. In addition, increased heme oxygenase-1 (HMOX1) expression and non-toxic generation of superoxide were detected in the first 4 h. Regarding the effects on the UVB-induced toxicity, although the level of cyclobutane pyrimidine dimers in the keratinocytes pre-treated with zinc for 24 h was reduced 3 h after UVB irradiation, significantly enhanced superoxide generation was observed 10 h after UVB exposure in the zinc pre-exposed cells. The overall survival was unaffected; however, there was a decrease in the percentage of early apoptotic cells and an increase in the percentage of late apoptotic plus necrotic cells. These results suggest that the exposure of human keratinocytes to non-toxic concentrations of ZnCl2 impacts gene expression, cell proliferation and the responses to environmental stress in the skin. It would be important to further examine the role of zinc in skin and further clarify whether this issue can affect our thinking regarding the pathogenesis of skin diseases.

Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential.

UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine.

Reference genes for quantitative real time PCR in UVB irradiated keratinocytes.

Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in keratinocytes, and UVB elicited changes have extensively been studied by qRT-PCR, yet a comparison of commonly used reference genes in UVB treatment is lacking. To find the best genes for compensating slight inter-sample variations in keratinocytes in UVB experiments and to understand the potential effects of improper reference gene (RG) selection we have analyzed the mRNA expression of 10 housekeeping genes in neonatal human epidermal keratinocytes (NHEK) after UVB treatment. The biological effect of the used UVB light source was validated by trypane blue exclusion, MTT and comet assays. 20-40mJ/cm(2) dose was chosen for the experiments. The stability of the 10 RGs was assessed by the GeNorm and Normfinder software tools. Regardless of their slightly different algorithm the programs found succinate dehydrogenase complex subunit A (SDHA) to be the best individual RG and SDHA and phosphoglycerate kinase-1 (PGK1) as the most suitable combination. Analysis of the expression of tumor necrosis factor alpha (TNFalpha) and vascular endothelial growth factor (VEGF) found that while the perception of changes in TNF-alpha, a gene undergoing marked upregulation after UVB irradiation is independent of the used RG, changes seen in the more modestly upregulated VEGF are greatly effected by reference gene selection. These findings highlight the importance of reference gene selection in UVB irradiation experiments, and provide evidence that using SDHA or the combination of SDHA and PGK1 as standards could be a reliable method for normalizing qRT-PCR results in keratinocytes after UVB treatment.

[The role of free radicals in the UV-induced skin damage. Photo-aging]. / Szabad gyökök szerepe az ultraibolya fény okozta borkárosodásokban.

The natural (intrinsic) ageing of the skin is enhanced by environmental factors (extrinsic ageing). One of the most important exogenous factors is the solar UV exposure, which results in photo-aging. Besides this, epidemiological and experimental data show a rapid increase in the incidence of human skin cancers, which is also in relation to the increased sunlight exposure of the skin. In the background of these processes there are cell biological effects, photochemical reactions, membrane receptor changes, lipid- and protein modifications, DNA-damage induced by UV. The qualities and quantities of them are wavelength dependent. The UVB photons are absorbed mostly by the DNA of the epidermal keratinocytes, therefore this spectrum is more relevant for photocarcinogenesis. The effect of UVA-irradiation is mainly manifested in the induction of free radicals, which have not only DNA-damaging, but also immunomodulating effect, which also can influence on tumour development. Furthermore, the free radicals cause dermal connective tissue damage as well via activating transcription factors, inducing matrix metalloproteinases, diminishing the procollagen I and fibrillin-1 synthesis. These processes are augmented by mitochondrial DNA mutations, protein oxidation, apoptosis induction. Therefore the enzymes neutralising free radicals and antioxidant molecules, respectively, have an important role in the defence mechanisms. In the therapy of photo-aging the local retinoids lived up to expectations, but the clinical effectiveness of antioxidant vitamins is lower than expected. The most important factor in the prevention of the photo-aging and photocarcinogenesis is the sun protection at present.

Hemochromatosis (HFE) gene mutations and hepatitis C virus infection as risk factors for porphyria cutanea tarda in Hungarian patients.

AIM: It is not clear whether the mutations in hemochromatosis (HFE) gene and hepatitis C virus (HCV) infection act independently in the pathogenesis of porphyria cutanea tarda (PCT). The prevalence of both risk factors varies greatly in different parts of the world. PCT patients from Hungary were evaluated to assess both factors. METHODS: The prevalence of C282Y and H63D mutations in the HFE gene was determined in 50 PCT patients and compared with the reported control frequencies. Furthermore, the presence of HCV infection was determined and related to the patients' HFE gene status. RESULTS: The C282Y mutation was found in 8/50 cases (three homozygotes and five heterozygotes), with an 11% allele frequency (vs. 3.8% control) (P<0.05). Seventeen patients were heterozygous, one was homozygous for the H63D mutation, allele frequency 19%, which did not differ significantly from the reported control prevalence of 12.3%. Twenty-two patients (44%) were HCV-RNA positive; six out of them were heterozygous for H63D mutation, one only for the C282Y mutation and one was compound heterozygous for both mutations. CONCLUSION: HCV infection and HFE C282Y mutation may probably be independent predisposing factors for development of PCT in Hungarian patients.