Agnieszka Wolnicka-Glubisz

Melanoma induction by ultraviolet A but not ultraviolet B radiation requires melanin pigment.

Malignant melanoma of the skin (CMM) is associated with ultraviolet radiation exposure, but the mechanisms and even the wavelengths responsible are unclear. Here we use a mammalian model to investigate melanoma formed in response to precise spectrally defined ultraviolet wavelengths and biologically relevant doses. We show that melanoma induction by ultraviolet A (320–400 nm) requires the presence of melanin pigment and is associated with oxidative DNA damage within melanocytes. In contrast, ultraviolet B radiation (280–320 nm) initiates melanoma in a pigment-independent manner associated with direct ultraviolet B DNA damage. Thus, we identified two ultraviolet wavelength-dependent pathways for the induction of CMM and describe an unexpected and significant role for melanin within the melanocyte in melanomagenesis.

Photodynamic activity of platinum(IV) chloride surface-modified TiO2 irradiated with visible light.

The visible light-induced phototoxicity of titanium dioxide modified with platinum(IV) chloride complexes, [TiO2/PtCl4], was tested. In vitro experiments with the mouse melanoma cells (S-91) have demonstrated phototoxicity of the [TiO2/PtCl4] material. Detection of efficiently generated various reactive oxygen species (.OH, O2. -, H2O2, 1O2) and also reactive chlorine species has proven the photodynamic activity of the tested material, induced by visible light (lambda>455 nm). The cellular death (recognized as a necrosis) is a result of the cell membrane peroxidation.

Pheomelanin in the skin of Hymenochirus boettgeri (Amphibia: Anura: Pipidae)

Pheomelanin is supposed to be the first type of melanin found in vertebrates, in contrast to the main type – eumelanin. Our study aimed at detecting pheomelanin in the skin of Hymenochirus boettgerii. We employed electron paramagnetic resonance (EPR) spectroscopy, and transmission electron microscopy (TEM), supplemented with standard histology and immunochemistry. We identified pheomelanin in the dorsal skin of adult frogs (not only in the dermis, but also in the epidermis) and in the dorsal tadpole. Our work identifies Hymenochirus boettgerii as a model in the basic study on the mechanism, evolution and role of melanogenesis in animals, including human.

Photocytotoxicity of platinum(IV)-chloride surface modified TiO2 irradiated with visible light against murine macrophages

Phototoxicity of titanium dioxide modified with platinum(IV)-chloride complexes, [TiO2/PtCl4], irradiated with visible light was tested on murine macrophage cell line (RAW 264.7) in vitro. Presence of antioxidants such as alpha-tocopherol or beta-carotene during photodynamic treatment significantly increased cells viability. Our results indicate that observed cell death induced by [TiO2/PtCl4] was due to photogeneration of reactive species.

HGF/SF Increases Number of Skin Melanocytes but Does Not Alter Quality or Quantity of Follicular Melanogenesis

Melanins are an important factor determining the vulnerability of mammalian skin to UV radiation and thus to UV-induced skin cancers. Transgenic mice overexpressing hepatocyte growth factor/scatter factor (HGF/SF) have extra-follicular dermal melanocytes, notably in the papillary upper dermis, and are susceptible to UV-induced melanoma. Pigmented HGF/SF neonatal mice are more susceptible than albino HGF/SF animals to UVA -induced melanoma, indicating an involvement of melanin in melanoma formation. This raises the question of the effect of transgenic HGF/SF on melanization. We developed a methodology to accurately quantitate both the production of melanin and the efficiency of melanogenesis in normal, and HGF/SF transgenic mice in vivo. Skin and hair shafts of 5 day old and adult (3 week old) C57BL/6-HGF/SF and corresponding C57BL/6 wild type mice were investigated by electron paramagnetic resonance spectroscopy (EPR) to quantitate melanin, by transmission electron microscopy (TEM) for the presence of melanosomes, and by standard histology and by Western blotting and zymography to determine the expression and activity of melanogenesis-related proteins. Eumelanin but no phaeomelanin was detected in transgenic C57BL/6-HGF and C57BL/6 wild type mice. Transgenic HGF/SF overexpression did not change the type of melanin produced in the skin or hair, did not affect the terminal content of melanin production in standard samples of hair and did not influence hair cycle/morphogenesis-related changes in skin thickness. No melanocytes were found in the epidermis and no melanosomes were found in epidermal keratinocytes. HGF/SF transgenic mice thus lack the epidermal melanin UV-protection found in constitutively dark human skin. We conclude that melanocytes in the HGF/SF transgenic mouse, particularly in the papillary dermis, are vulnerable to UVA which interacts with eumelanin but not phaeomelanin to induce melanoma.

UVA activated 8-MOP and chlorpromazine inhibit release of TNF-α by post-transcriptional regulation

There is evidence that regulation of inflammatory cytokines is among the immunomodulatory effects of photochemotherapy with 8-MOP and UVA. We have recently demonstrated that in the monocytoid cell line U937 incubation with 8-MOP and subsequent exposure to UVA is able to efficiently downregulate the release of TNF-alpha into the culture supernatant. Chlorpromazine, a well known photosensitising drug, was even more potent with regard to this effect. Based on these observations, in this study we further investigate the mechanisms of TNF-alpha inhibition by 8-MOP and CPZ photosensitization. For this purpose we determined intracellular protein levels and gene expression of TNF-alpha by western blot and quantitative real-time PCR, respectively. Our results indicate that the observed inhibition of TNF-alpha secretion after photochemotherapy is not due to downregulation of gene transcription but rather to a post-transcriptional mechanism. The observed decrease of intracellular TNF-alpha with CPZ and 8-MOP points to decreased protein synthesis or enhanced degradation. These findings demonstrate that posttranscriptional regulation of cytokine expression is a possible mechanism of action of photochemotherapy.

p38 but not p53 is responsible for UVA-induced MCPIP1 expression

MCPIP1 (Monocyte Chemotactic Protein-1 Induced Protein) is an important regulator of inflammation and cell apoptosis, but its role in UVA-induced stress response in the epidermis has never been studied. We have found that moderate apoptosis-inducing dose of UVA (27J/cm2) increases the level of MCPIP1 expression in HaCaT cells and normal human keratinocytes (NHEK) within 6-9h after the treatment. MCPIP1 upregulation was dependent on the induction of p38, but not p53, as demonstrated by using p38 inhibitor SB203580 and p53 inducer RG7388, respectively. This increase was also blocked by antioxidants (α-tocopherol and ascorbic acid), suggesting the involvement of MCPIP1 in UVA-induced oxidative stress response. Si-RNA-mediated down-regulation of MCPIP1 expression in HaCaT cells resulted in increased sensitivity to UVA-induced DNA damage and apoptosis. This was accompanied by decreased phosphorylation of p53 and p38 in MCPIP1-silenced cells following UVA irradiation. The activation of p38 in response to low doses of ultraviolet radiation was postulated to be protective for p53-inactive cells. Therefore, MCPIP1 may favor the survival of p53-defective HaCaT cells by sustaining the activation of p38. This creates a loop of mutual positive regulation between p38 and MCPIP1 protein in HaCaT cells, providing the protection against the consequences of UVA irradiation.

MCPIP1 contributes to the inflammatory response of UVB-treated keratinocytes

BACKGROUND Monocyte chemoattractant protein-1-induced protein-1 (MCPIP1), also known as regnase-1, negatively regulates many cellular processes including the cellular response to inflammatory agents, differentiation, viability, and proliferation. It possesses a PilT N-terminus (PIN) domain that is directly involved in regulating the stability of transcripts and miRNAs by recognizing stem loop structures and degrading them by endonucleolytic cleavage. OBJECTIVE We investigated the role of MCPIP1 in the response of human primary keratinocytes to UVB stress. METHODS Keratinocytes were treated with UVB, siRNA against MCPIP1, pharmacological inhibitors of signaling pathways, or subjected to control treatments. The mRNA and protein levels of MCPIP1 and MCPIP1-dependent changes gene expression were analyzed by quantitative (Q)-RT-PCRs and Western blots. Secretion of TNFα and IL-8 was determined by ELISA. RESULTS UVB treatment of keratinocytes induced upregulation of MCPIP1 at the mRNA level after 4-8h and at the protein level after 8-16h. MCPIP1 abundance depended on NF-κB activity. Using an siRNA strategy, we found that diminished MCPIP1 resulted in an up-regulation of transcripts coding for IL-8, TNFα, COX-2, and BCL-2, as well as an enhanced release of IL-8. Moreover, decreased phosphorylation of NF-κB and p38 signaling pathways were observed in addition to a slight up-regulation of ERK1/2 directly after UVB treatment. Twenty-four hours later, decreased phosphorylation was observed only for NF-κB and p38. Furthermore, in MCPIP1-suppressed cells, the levels of pro-apoptotic Puma, the phosphorylated form of p53 and the abundance of its target p21 as well as the activity of caspase 3 decreased, while the level of cyclin D1 increased. CONCLUSION MCPIP1 contributes to the UVB response of keratinocytes by altering metabolic and apoptotic processes and the release of inflammatory mediators.

Role of Mc1r in UV-Induced Melanoma in Animal Models

The role of UV and pigmentation are very difficult to control for in human studies, and mechanisms difficult to infer based on statistical association with melanoma. The animal models are not representative of the human situation. But on the other hand, animal studies can be useful for basic studies that will ultimately help in building up a picture of the overall network of in vivo cellular behavior and intra and inter cellular pathways contributing to melanoma progression and the effects (or not) of UV radiation in individuals with MC1R variants. This review describes that, although the Mc1r is a determinant of coat color phenotype as the MC1R is a determinant of hair and skin color in humans, deficiency of the Mc1r in mice is associated with a paradoxical lower incidence of melanoma.

Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.