Infrared A radiation promotes survival of human melanocytes carrying ultraviolet radiation‐induced DNA damage

The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR‐induced non‐epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR‐induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR‐induced DNA damage. IR partly reversed the pro‐apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR‐induced DNA damage and thereby might contribute to melanomagenesis.     

Misregulation of Rad50 expression in melanoma cells

DNA double-strand breaks are increased in human melanoma tissue as detected by histone H2AX phosphorylation.(1-3) We investigated two of the downstream effectors of DNA double-strand breaks, Rad50 and 53BP1 (tumor suppressor p53 binding protein 1), to determine if they are altered in human primary melanoma cells. Melanoma cases showed high Rad50 staining (81.8%; 9/11) significantly more frequently than conventional or atypical melanocytic nevi (0%; 0/18). In contrast, the staining pattern for 53BP1 appears similar between melanoma and nevi. This is the first study that shows activation and misregulation of the DNA repair pathway in human melanoma cells. The staining features of Rad50, a component of an essential DNA double-strand break repair complex, are clearly increased in melanoma cells with regards to both staining intensity and the number of positive melanoma cells. Interestingly, among the melanoma cases with increased Rad50 staining, most demonstrated cytoplasmic rather than nuclear staining (88.9%, 8/9). Further studies are needed to determine the cause of this mislocalization and its affects, if any, on DNA double-strand break repair in melanoma.     

Melanoma cells express elevated levels of phosphorylated histone H2AX foci

When human cells sustain a DNA double-strand break (dsb), histone H2AX in chromatin surrounding the DNA break is phosphorylated, marking repair foci. The number of phosphorylated histone H2AX (gammaH2AX) foci approximates the number of dsb present in the cell's nuclear DNA. We observed 0.4 gammaH2AX foci per nucleus in primary human melanocytes. In contrast, in four melanoma cell lines, we detected 7-17 gammaH2AX foci per nucleus, a 17-42 times increase in the basal level of gammaH2AX foci in melanoma cells relative to melanocytes (MC). Thus, untreated melanoma cells express significantly greater numbers of gammaH2AX foci than do untreated MC. Detection and rejoining of ionizing radiation-induced DNA dsb proceeded as rapidly in melanoma cells as in MC. Melanoma cells, however, reduced the number of radiation-induced gammaH2AX foci down only to pre-irradiation levels. Co-localization of the majority of gammaH2AX foci with ataxia telangiectasia mutated, BRCA1, 53BP1, and Nbs1 foci in untreated melanoma cells indicated that the additional foci in melanoma cells were associated with a DNA change that the cells interpret as DNA dsb. Co-localization of gammaH2AX foci with the telomere replication factor 1 protein in untreated melanoma cells indicates that the additional foci in untreated melanoma cells are associated with dysfunctional telomeres that induce a DNA damage stress response.     

Low‐dose 5‐fluorouracil induces cell cycle G2 arrest and apoptosis in keloid fibroblasts

Background Intralesional injection of low‐dose 5‐fluorouracil (5‐FU) has recently been used as an experimental modality for treating keloid scarring and has shown promising efficacy in improving scar appearance and preventing recurrence of the keloid. Objectives We sought to explore the cellular‐ and molecular‐based evidence for the observed clinical benefits. Methods Primary cell lines of keloid fibroblasts were treated with 5‐FU at a range of lower doses (～10 mg mL^?1) in monolayer culture and subjected to examination for cell viability, proliferative potential, apoptosis, cell cycle and associated proteins involved in cell cycle control. Results 5‐FU significantly inhibited cell proliferation of keloid fibroblasts in the full dose range used in this study. The DNA synthesis was completely inhibited by 5‐FU at 72 h, and significant cell apoptosis was observed at concentrations ≥ 1 mg mL^?1 for a period over 72 h. 5‐FU caused a significant delay in cell cycle progression and the G2/M phase arrest. 5‐FU induced p53 and p21 accumulation together with a decrease in cyclin B1 and Bcl‐2 levels in treated keloid fibroblasts. Conclusions Our data indicate that low‐dose 5‐FU (as low as 1 mg mL^?1) induces significant inhibition of proliferation, G2/M cell cycle arrest and apoptosis but not immediate cell death of keloid fibroblasts. The lack of tissue necrosis is a particular benefit as further scarring is likely to be prevented. These results support the use of low‐dose 5‐FU as a potential modality for treating keloid scars.     

Isoflavones in aglycone solution enhance ultraviolet B‐induced DNA damage repair efficiency

The isoflavones daidzein and genistein are natural compounds which have anti‐inflammatory and photoprotective activities, and may be effective in the repair of ultraviolet (UV)‐induced photodamage. In this study, an alcoholic solution of aglycone isoflavones with a genistein:daidzein ratio of 1:4 [Rottapharm (RPH)‐aglycone] was examined for its effects on the repair of DNA damage induced by a single dose of UVB irradiation (20 mJ/cm²). For this purpose, human skin cells were first UVB‐irradiated and then treated with RPH‐aglycone. Comet assay analysis was used to estimate the UVB‐induced DNA damage at different time points after treatment by measuring the tail moment parameter. We found that treatment with 10 μmol/L RPH‐aglycone solution resulted in a significantly reduced tail moment at 1 h after treatment, and 34–35% enhancement of damage repair at 4 h after treatment. These results suggest that isoflavone aglycones are protective against UVB‐induced DNA damage.     

p53 labeling index in assessing the efficacy of a sunscreen in protection against UV‐induced damage

Background Sunscreen efficacy is currently evaluated by the estimation of the sun protection factor (SPF). Our objective was to determine the role of the p53 labeling index in assessing the effectiveness of a sunscreen in the prevention of UV‐induced DNA damage. Methods A broad‐spectrum sunscreen (SPF15) was applied to two of four test areas on the backs of 20 volunteers. Two test areas (with and without prior sunscreen application) were then irradiated with two minimal erythemal doses (MEDs) of solar‐simulated UV radiation (290–400 nm). At 24 h, suction blisters were raised from all four test areas using a vacuum technique, and immunohistochemical staining for p53 was performed. Results In normal epidermis, nonirradiated without sunscreen, p53 was almost undetectable [0.5% positive cells per field (PC/F)]. In UV‐exposed skin without sunscreen, p53 expression was 10.4% PC/F, whereas, in skin treated with sunscreen before UV irradiation, p53 expression was 0.3% PC/F. The mean positive labeling indices (×10³/µm) for UV‐exposed skin with and without sunscreen were 0.002 and 0.059, respectively (P < 0.001). Conclusions In vivo assessment of the p53 labeling index using the suction blister method can serve as a sensitive endpoint in the evaluation of the effectiveness of a sunscreen in the prevention of UV‐induced DNA damage.     

UVA1 impairs the repair of UVB‐induced DNA damage in normal human melanocytes

The exact correlation between melanoma and sun‐light is still a controversially debated issue. Although natural sunlight contains various ratios of UVA and UVB, most investigators so far focused on the effects of single solar wavebands and neglected possible interactions. Therefore, in this study primary human melanocytes of three donors were simultaneously exposed to physiologic doses of UVA1 and UVB. Effects on apoptosis were analysed using annexin V assays and cell death ELISAs, and effects on DNA damage were investigated using southwestern slot blots. While UVA1 did not influence UVB‐induced apoptosis, UVA1 impaired the repair of UVB‐induced cyclobutane pyrimidine dimers (CPD) as the amount of CPD was 1.8 times higher in UVA1 + UVB than in UVB only exposed melanocytes six hours after irradiation. We conclude that UVA1 might contribute to melanomagenesis as it partially inhibits the repair of UVB‐induced CPD in human melanocytes while it does not affect UVB‐mediated apoptosis.     

Ultraviolet B radiation regulates cysteine‐rich protein 1 in human keratinocytes

Background: Cysteine‐rich protein 1 (CRP1) is a growth‐inhibitory cytoskeletal protein that is induced by ultraviolet (UV) C radiation radiation in fibroblasts. Our aim was to investigate the effects of UV radiation on CRP1 in keratinocytes, the main cell type subjected to UV radiation in the human body. Methods: The effects of physiologically relevant doses of UVB radiation on CRP1 protein levels were studied in cultured primary keratinocytes and transformed cell lines (HaCaT, A‐431) by immunoblotting. UVB‐induced keratinocyte apoptosis was assessed by flow cytometry and monitoring caspase activity. Expression of CRP1 in human skin in vivo was studied by immunohistochemistry in samples of normal skin, actinic keratosis (AK) representing UV‐damaged skin and squamous cell carcinoma (SCC), a UV‐induced skin cancer. Results: CRP1 expression increased by UVB radiation in primary but not in immortalized keratinocytes. Upon high, apoptosis‐inducing doses of UV radiation, CRP1 was cleaved in a caspase‐dependent manner. In normal skin, CRP1 was expressed in smooth muscle cells, vasculature, sweat glands, sebaceous glands and hair root sheath, but very little CRP1 was present in keratinocytes. CRP1 expression was elevated in basal cells in AK but not in SCC. Conclusion: CRP1 expression is regulated by UVB in human keratinocytes, suggesting a role for CRP1 in the phototoxic responses of human skin.     

Ionizing radiation,but not ultraviolet radiation,induces mitotic catastrophe in mouse epidermal keratinocytes with aberrant cell cycle checkpoints

Ultraviolet radiation (UVR) and ionizing radiation (IR) are common genotoxic stresses that damage human skin, although the specific damages to the genomic DNA are different. Here, we show that in the mouse glabrous skin, both UVR and IR induce DNA damage, cell cycle arrest, and condensed cell nuclei. However, only IR induces mitotic catastrophe (MC) in the epidermis. This is because UVR induces a complete blockage of pRB phosphorylation and cell cycle arrest in the G1 phase, whereas pRB phosphorylation remains positive in a significant portion of the epidermal keratinocytes following IR exposure. Furthermore, Cyclin B1 expression is significantly downregulated only by IR but not UVR. Finally, there are more MC cells in the epidermis of p53‐/‐ mice after IR exposure as compared to wild‐type mice. Our results suggest that although both IR and UVR are genotoxic, they show distinct impacts on the cell cycle machinery and thus damage the epidermal keratinocytes via different mechanisms.     

Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin

Abstract: Solar ultraviolet (UV) radiation, particularly its UVB (290–320 nm) component, is the primary cause of many adverse biological effects including photoageing and skin cancer. UVB radiation causes DNA damage, protein oxidation and induces matrix metalloproteinases (MMPs). Photochemoprevention via the use of botanical antioxidants in affording protection to human skin against UVB damage is receiving increasing attention. Pomegranate, from the tree Punica granatum, contains anthocyanins and hydrolysable tannins and possesses strong antioxidant and anti‐tumor‐promoting properties. In this study, we determined the effect of pomegranate‐derived products – POMx juice, POMx extract and pomegranate oil (POMo) – against UVB‐mediated damage using reconstituted human skin (EpiDerm^TM FT‐200). EpiDerm was treated with POMx juice (1–2 μl/0.1 ml/well), POMx extract (5–10 μg/0.1 ml/well) and POMo (1–2 μl/0.1 ml/well) for 1 h prior to UVB (60 mJ/cm²) irradiation and was harvested 12 h post‐UVB to assess protein oxidation, markers of DNA damage and photoageing by Western blot analysis and immunohistochemistry. Pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) cyclobutane pyrimidine dimers (CPD), (ii) 8‐dihydro‐2′‐deoxyguanosine (8‐OHdG), (iii) protein oxidation and (iv) proliferating cell nuclear antigen (PCNA) protein expression. We also found that pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) collagenase (MMP‐1), (ii) gelatinase (MMP‐2, MMP‐9), (iii) stromelysin (MMP‐3), (iv) marilysin (MMP‐7), (v) elastase (MMP‐12) and (vi) tropoelastin. Gelatin zymography revealed that pomegranate‐derived products inhibited UVB‐induced MMP‐2 and MMP‐9 activities. Pomegranate‐derived products also caused a decrease in UVB‐induced protein expression of c‐Fos and phosphorylation of c‐Jun. Collectively, these results suggest that all three pomegranate‐derived products may be useful against UVB‐induced damage to human skin.     

Skin‐derived mesenchymal stem cells (S‐MSCs) induce endothelial cell activation by paracrine mechanisms

Please cite this paper as: Skin‐derived mesenchymal stem cells (S‐MSCs) induce endothelial cell activation by paracrine mechanisms. Experimental Dermatology 2010; 19 : 848–850. Abstract: The mesenchymal stem cells (MSCs) are able to accumulate at the site of tissue damage. For this reason, they must transmigrate across the endothelium. In this study, we focused on skin‐derived MSCs (S‐MSCs), because the skin represents a useful stem cell source, and we analysed the VEGF released by S‐MSCs, because it is known to promote endothelial cell proliferation and vascular permeability. Moreover, we evaluated the influence of S‐MSC‐conditioned medium on human aortic endothelial cell intracellular calcium concentration ([Ca²⁺]_i) and nitric oxide (NO) production, given their important role in endothelial permeability modulation. Our results suggest that human S‐MSCs may interact with the endothelium via paracrine mechanisms, probably leading to an alteration of the endothelial barrier. Consequently, we could hypothesize that a therapeutic approach based on human skin‐derived MSCs may have a positive effect on tissue repair.     

Research in practice: More than skin deep –aging of subcutaneous fat tissue

Mitochondria, responsible for the generation of energy in our cells, contain their own genome, mitochondrial (mt)DNA. It is known that mutations of mtDNA accumulate during normal aging and that this can be accelerated by oxidative stress, i.e. induced by ultraviolet radiation. These mutations are functionally relevant and they play a causative role in normal aging as well as premature aging induced by ultraviolet radiation. While the focus of scientific research was more on epidermis and dermis within the last years, alterations of subcutaneous fat tissue were not investigated thus far. Cockayne syndrome (CS) A and B are two proteins known to repair oxidatively induced DNA damage via nucleotide excision repair (NER) in the nucleus. We could show that these two proteins enrich in mitochondria upon oxidative stress, directly interact with mtDNA and the two repair‐associated proteins mtSSBP‐1 and mtOGG‐1 and protect from deletions of mtDNA. If CSA or CSB are lacking, mtDNA mutations accumulate particularly in the cells of subcutaneous fat tissue which appears to mediate loss of adipocytes via apoptosis. Therefore, the two NER‐associated proteins CSA and CSB appear to play a direct role in protection from mutations which in turn are causative in aging‐associated loss of subcutaneous fat tissue.     

Assessment of ultraviolet‐radiation‐induced DNA damage within melanocytes in skin of different constitutive pigmentation

Background Melanoma incidence and pigmentary disorders are known to be related to the degree of skin pigmentation, but few data exist on the specific impact of ultraviolet radiation (UVR) on melanocytes in skin of different constitutive pigmentation. Objectives To analyse UVR‐induced DNA damage within melanocytes in different skin‐colour types. Methods Skin samples were objectively classified into light, intermediate, tan, brown and dark skin according to their individual typology angle (°ITA), based on colorimetric parameters. Samples were exposed to increasing doses of solar simulated radiation. Detection of DNA damage specifically in melanocytes was achieved by cyclobutane thymine dimer (CPD)–tyrosinase‐related protein 1 double staining. Results For light, intermediate and tan skin, accumulation of CPDs in melanocytes was detected at the lowest dose, with a steep increase with dose. At estimated erythemally equivalent doses, around 80–100% of melanocytes were positive for CPDs in tan, intermediate and light skin types. In contrast, in dark and brown skin types, CPDs were found in only approximately 15% of melanocytes at the highest dose. Conclusions This work demonstrates that melanocytes from constitutively highly pigmented skin types are less impacted in terms of UVR‐induced DNA damage than those from lighter skin types, even those that are moderately pigmented.     

Nicotinamide enhances repair of ultraviolet radiation‐induced DNA damage in primary melanocytes

Cutaneous melanoma is a significant cause of morbidity and mortality. Nicotinamide is a safe, widely available vitamin that reduces the immune suppressive effects of UV, enhances DNA repair in keratinocytes and has shown promise in the chemoprevention of non‐melanoma skin cancer. Here, we report the effect of nicotinamide on DNA damage and repair in primary human melanocytes. Nicotinamide significantly enhanced the repair of oxidative DNA damage (8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine) and cyclobutane pyrimidine dimers induced by UV exposure. It also enhanced the repair of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine induced by the culture conditions in unirradiated melanocytes. A significant increase in the percentage of melanocytes undergoing unscheduled but not scheduled DNA synthesis was observed, confirming that nicotinamide enhances DNA repair in human melanocytes. In summary, nicotinamide, by enhancing DNA repair in melanocytes, is a potential agent for the chemoprevention of cutaneous melanoma.     

E‐cadherin mediates ultraviolet radiation‐ and calcium‐induced melanin transfer in human skin cells

Skin pigmentation is directed by epidermal melanin units, characterized by long‐lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X‐mediated control of MC filopodia. Both MC and KC express Ca²⁺‐dependent E‐cadherins. These homophilic adhesion contacts induce transient increases in intra‐KC Ca²⁺, while ultraviolet radiation (UVR) raises intra‐MC Ca²⁺ via calcium‐selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca²⁺ triggers melanin transfer remains unclear. Here we evaluated the role of E‐cadherin in UVR‐mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia‐associated E‐cadherin, Cdc42, VASP and β‐catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E‐cadherin revealed that this cadherin is essential for UVR‐induced MC filopodia formation and melanin transfer. Moreover, Ca²⁺ induced a dose‐dependent increase in filopodia formation and melanin transfer, as well as increased β‐catenin, Cdc42, Myosin X and E‐cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E‐cadherin, which are upregulated by intracellular (UVR‐stimulated) and extracellular Ca²⁺ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca²⁺ signalling influences human pigmentation.     

Effect of ultraviolet (UV) A, UVB or ionizing radiation on the cell cycle of human melanoma cells

BACKGROUND: One important component of the cellular response to irradiation is the activation of cell cycle checkpoints. It is known that both ultraviolet (UV) radiation and ionizing radiation (IR) can activate checkpoints at transitions from G(1) to S phase, from G(2) phase to mitosis and during DNA replication. OBJECTIVES: To evaluate the effects of irradiation with different wavelengths on cell cycle alterations. METHODS: p53-deficient IPC-298 melanoma cells were irradiated with 10 J cm(-2) UVA, 40 mJ cm(-2) UVB, or with 7.5 Gy IR. Cell cycle effects were then determined by DNA/5-bromodeoxyuridine dual-parameter flow cytometry. RESULTS: IPC-298 cells irradiated in G(1) with UVA were not arrested at the G(1)/S transition, but at the G(2)/M transition. Despite p53 deficiency, the cells showed a G(1) arrest after UVB exposure. Furthermore, IR did not affect G(1) or S phase, but induced G(2) phase arrest. Hence, the effects of UVA, but not of UVB, on the cell cycle in p53-deficient melanoma cells are comparable with those of IR. CONCLUSIONS: UVA and IR induce radical-mediated strand breaks and DNA lesions, and UVB essentially induces thymine dimers that lead to excision repair-related strand breaks. Different cell cycle effects may be a consequence of different types of DNA damage. The results showed that UVB-irradiated p53-deficient cells are arrested in G(1). Irradiation with the solar radiation component UVB can therefore result in a beneficial retardation of tumour promotion in human skin carrying p53-mutated cell clones.     

Human papillomavirus infection in Bowen disease: Negative p53 expression,not p16INK4a overexpression,is correlated with human papillomavirus‐associated Bowen disease

Genital Bowen disease (BD) has been linked to the high‐risk types of human papillomavirus (HPV) infection. Recently, it has been recognized that HPV also can be associated with extragenital BD. HPV oncoproteins E6 and E7 interfere with the function of p53 and pRb, respectively, leading carcinogenesis. p16^INK4a overexpression induced by inactivation of pRb is recognized as a surrogate marker for HPV‐associated cervical cancer. In this study, we examined the presence of HPV DNA in 142 BD lesions by polymerase chain reaction (PCR), and determined the type of HPV by PCR restriction fragment length polymorphism or direct DNA sequencing. HPV DNA was detected in 66.7% of genital BD and 8.3% of extragenital BD. The types of HPV detected were HPV types 6, 16, 33, 52, 56, 58 and 59. We also investigated the expression of p16^INK4a, pRb and p53 by immunohistochemistry. Positive expression was detected in 88.6% for p16^INK4a, 25.2% for pRb, and 63.8% for p53. There was no significant difference in p16^INK4a and pRb expression between HPV‐positive and ‐negative BD. However, a strong correlation of HPV positivity with p53 negativity was found. A total of 66.7% of HPV‐positive BD showed no p53 expression, whereas the corresponding rate was 32.8% of HPV‐negative BD. This study demonstrated that HPV can participate in the development of BD, not only in the genital lesion, but also in extragenital lesion. p16^INK4a overexpression is not a marker for HPV infection in BD. Instead, negative p53 expression is correlated with HPV‐associated BD.     

More epidermal p53 patches adjacent to skin carcinomas in renal transplant recipients than in immunocompetent patients: the role of azathioprine

Abstract:  Immunosuppressive medication in renal transplant recipients (RTR) strongly increases the risk of cancers on sun-exposed skin. This increased risk was considered an inevitable collateral effect of immunosuppression, because UV-induced carcinomas in mice were found to be highly antigenic. Here, we posed the question whether immunosuppression also increases the frequency of p53-mutant foci ('p53 patches'), putative microscopic precursors of squamous cell carcinomas. As the majority of RTR was kept on azathioprine for most of the time, we investigated whether this drug could increase UV-induced p53 patches by immunosuppression. As azathioprine can impair UV-damaged DNA repair under certain conditions, we also investigated whether DNA repair was affected. Archive material of RTR and immunocompetent patients (ICP), as well as azathioprine-administered hairless mice were examined for p53 patches. DNA repair was investigated by ascertaining the effect of azathioprine on unscheduled DNA synthesis (UDS) in UV-irradiated human keratinocytes. P53 patches were more prevalent in RTR than in ICP in normal skin adjacent to carcinomas ( P  = 0.02), in spite of a lower mean age in the RTR (52 vs 63 years, P  = 0.001), but we found no increase in UV-induced p53 patches in mice that were immunosuppressed by azathioprine. We found a significant reduction in DNA repair activity in keratinocytes treated with azathioprine ( P  = 0.011). UV-induced UDS in humans is dominated by repair of cyclobutane pyrimidine dimers, and these DNA lesions can lead to 'UV-signature' mutations in the P53 gene, giving rise to p53 patches.