Sex Differences in Photoprotective Responses to 1,25-Dihydroxyvitamin D3 in Mice Are Modulated by the Estrogen Receptor-ß.

Susceptibility to photoimmune suppression and photocarcinogenesis is greater in male than in female humans and mice and is exacerbated in female estrogen receptor-beta knockout (ER-ß-/-) mice. We previously reported that the active vitamin D hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D), applied topically protects against the ultraviolet radiation (UV) induction of cutaneous cyclobutane pyrimidine dimers (CPDs) and the suppression of contact hypersensitivity (CHS) in female mice. Here, we compare these responses in female versus male Skh:hr1 mice, in ER-ß-/-/-- versus wild-type C57BL/6 mice, and in female ER-blockaded Skh:hr1 mice. The induction of CPDs was significantly greater in male than female Skh:hr1 mice and was more effectively reduced by 1,25(OH)2D in female Skh:hr1 and C57BL/6 mice than in male Skh:hr1 or ER-ß-/- mice, respectively. This correlated with the reduced sunburn inflammation due to 1,25(OH)2D in female but not male Skh:hr1 mice. Furthermore, although 1,25(OH)2D alone dose-dependently suppressed basal CHS responses in male Skh:hr1 and ER-ß-/- mice, UV-induced immunosuppression was universally observed. In female Skh:hr1 and C57BL/6 mice, the immunosuppression was decreased by 1,25(OH)2D dose-dependently, but not in male Skh:hr1, ER-ß-/-, or ER-blockaded mice. These results reveal a sex bias in genetic, inflammatory, and immune photoprotection by 1,25(OH)2D favoring female mice that is dependent on the presence of ER-ß.

Nicotinamide for skin cancer chemoprevention: effects of nicotinamide on melanoma in vitro and in vivo.

Nicotinamide (NAM), an amide form of vitamin B3, replenishes cellular energy after ultraviolet radiation (UVR) exposure, thereby enhancing DNA repair and reducing UVR's immunosuppressive effects. NAM reduces actinic keratoses and new keratinocyte cancers in high risk individuals, but its effects on melanoma are unknown. Melanomas arising on NAM or placebo within the ONTRAC skin cancer chemoprevention trial (Oral Nicotinamide To Reduce Actinic Cancer) were examined by immunohistochemistry. The effects of NAM (50 µM, 5 mM and 20 mM) on the viability, proliferation and invasiveness of four human melanoma cell lines and on the viability and proliferation of two human melanocyte lines, with and without UV irradiation were also investigated. 50 µM NAM did not affect viability, proliferation or invasion of melanoma or melanocyte cell lines, whereas concentrations too high to be achievable in vivo reduced viability and proliferation. Nicotinamide did not enhance melanoma viability, proliferation or invasiveness in vitro, providing additional confidence in its safety for use in clinical trials in high risk patients. Peritumoral and tumour infiltrating CD4+ and CD8+ lymphocytes were significantly increased in melanomas arising on NAM compared to those arising on placebo. Given the chemopreventive activity of nicotinamide against keratinocyte cancers, its DNA repair enhancing effects in melanocytes and now its potential enhancement of tumour-infiltrating lymphocytes and lack of adverse effects on melanoma cell growth and proliferation, clinical trials of nicotinamide for melanoma chemoprevention are now indicated.

Nicotinamide for photoprotection and skin cancer chemoprevention: A review of efficacy and safety.

Nicotinamide is a water-soluble vitamin B3 derivative that has many roles in medicine. This review examines the role of nicotinamide in dermatology and its actions in preventing photoageing and skin cancers in humans. Nicotinamide prevents ultraviolet radiation (UV) from reducing ATP levels and inhibiting glycolysis, thus preventing the UV radiation-induced energy crisis. This enhances DNA repair and reduces UV-induced suppression of immunity. Randomised controlled clinical trials have also shown that nicotinamide reduces transepidermal water loss and the development of new non-melanoma skin cancers in high-risk humans. This review also examines nicotinamide's safety profile.

A Phase 3 Randomized Trial of Nicotinamide for Skin-Cancer Chemoprevention.

BACKGROUND: Nonmelanoma skin cancers, such as basal-cell carcinoma and squamous-cell carcinoma, are common cancers that are caused principally by ultraviolet (UV) radiation. Nicotinamide (vitamin B3) has been shown to have protective effects against damage caused by UV radiation and to reduce the rate of new premalignant actinic keratoses. METHODS: In this phase 3, double-blind, randomized, controlled trial, we randomly assigned, in a 1:1 ratio, 386 participants who had had at least two nonmelanoma skin cancers in the previous 5 years to receive 500 mg of nicotinamide twice daily or placebo for 12 months. Participants were evaluated by dermatologists at 3-month intervals for 18 months. The primary end point was the number of new nonmelanoma skin cancers (i.e., basal-cell carcinomas plus squamous-cell carcinomas) during the 12-month intervention period. Secondary end points included the number of new squamous-cell carcinomas and basal-cell carcinomas and the number of actinic keratoses during the 12-month intervention period, the number of nonmelanoma skin cancers in the 6-month postintervention period, and the safety of nicotinamide. RESULTS: At 12 months, the rate of new nonmelanoma skin cancers was lower by 23% (95% confidence interval [CI], 4 to 38) in the nicotinamide group than in the placebo group (P=0.02). Similar differences were found between the nicotinamide group and the placebo group with respect to new basal-cell carcinomas (20% [95% CI, -6 to 39] lower rate with nicotinamide, P=0.12) and new squamous-cell carcinomas (30% [95% CI, 0 to 51] lower rate, P=0.05). The number of actinic keratoses was 11% lower in the nicotinamide group than in the placebo group at 3 months (P=0.01), 14% lower at 6 months (P<0.001), 20% lower at 9 months (P<0.001), and 13% lower at 12 months (P=0.001). No noteworthy between-group differences were found with respect to the number or types of adverse events during the 12-month intervention period, and there was no evidence of benefit after nicotinamide was discontinued. CONCLUSIONS: Oral nicotinamide was safe and effective in reducing the rates of new nonmelanoma skin cancers and actinic keratoses in high-risk patients. (Funded by the National Health and Medical Research Council; ONTRAC Australian New Zealand Clinical Trials Registry number, ACTRN12612000625875.).

Melanoma and nonmelanoma skin cancer chemoprevention: A role for nicotinamide?

Ultraviolet radiation (UVR) causes DNA damage in melanocytes by producing photolesions such as cyclobutane pyrimidine dimers and 8-oxo-7-hydrodeoxyguanosine. The production of reactive oxygen species by UVR also induces inflammatory cytokines that, together with the inherent immunosuppressive properties of UVR, propagate carcinogenesis. Nicotinamide (Vitamin B3 ) enhances DNA repair, modulates the inflammatory environment produced by UVR, and reduces UV-induced immunosuppression. As nicotinamide reduces the incidence of actinic keratoses and nonmelanoma skin cancers in high-risk individuals and enhances repair of DNA damage in melanocytes, it is a promising agent for the chemoprevention of melanoma in high-risk populations.

B cells are required for sunlight protection of mice from a CNS-targeted autoimmune attack.

The ultraviolet (UV) radiation contained in sunlight is a powerful immune suppressant. While exposure to UV is associated with protection from the development of autoimmune diseases, particularly multiple sclerosis, the precise mechanism by which UV achieves this protection is not currently well understood. Regulatory B cells play an important role in preventing autoimmunity and activation of B cells is a major way in which UV suppresses adaptive immune responses. Whether UV-protection from autoimmunity is mediated by the activation of regulatory B cells has never been considered before. When C57BL/6 mice were exposed to low, physiologically relevant doses of UV, a unique population of B cells was activated in the skin draining lymph nodes. As determined by flow cytometry, CD1d(low)CD5(-)MHC-II(hi)B220(hi) UV-activated B cells expressed significantly higher levels of CD19, CD21/35, CD25, CD210 and CD268 as well as the co-stimulatory molecules CD80, CD86, CD274 and CD275. Experimental autoimmune encephalomyelitis (EAE) in mice immunized with MOG/CFA was reduced by exposure to UV. UV significantly inhibited demyelination and infiltration of inflammatory cells into the spinal cord. Consequently, UV-exposed groups showed elevated IL-10 levels in secondary lymphoid organs, delayed EAE onset, reduced peak EAE score and significantly suppressed overall disease incidence and burden. Importantly, protection from EAE could be adoptively transferred using B cells isolated from UV-exposed, but not unirradiated hosts. Indeed, UV-protection from EAE was dependent on UV activation of lymph node B cells because UV could not protect mice from EAE who were pharmacologically depleted of B cells using antibodies. Thus, UV maintenance of a pool of unique regulatory B cells in peripheral lymph nodes appears to be essential to prevent an autoimmune attack on the central nervous system.

The alternative complement component factor B regulates UV-induced oedema, systemic suppression of contact and delayed hypersensitivity, and mast cell infiltration into the skin.

Ultraviolet (UV) wavelengths in sunlight are the prime cause of skin cancer in humans with both the UVA and UVB wavebands making a contribution to photocarcinogenesis. UV has many different biological effects on the skin that contribute to carcinogenesis, including suppression of adaptive immunity, sunburn and altering the migration of mast cells into and away from irradiated skin. Many molecular mechanisms have been identified as contributing to skin responses to UV. Recently, using gene set enrichment analysis of microarray data, we identified the alternative complement pathway with a central role for factor B (fB) in UVA-induced immunosuppression. In the current study we used mice genetically deficient in fB (fB-/- mice) to study the functional role of the alternative complement pathway in skin responses to UV. We found that fB is required for not only UVA but also UVB-induced immunosuppression and solar-simulated UV induction of the oedemal component of sunburn. Factor B-/- mice had a larger number of resident skin mast cells than control mice, but unlike the controls did not respond to UV by increasing mast cell infiltration into the skin. This study provides evidence for a function role for fB in skin responses to UV radiation. Factor B regulates UVA and UVB induced immunosuppression, UV induced oedema and mast cell infiltration into the skin. The alternative complement pathway is therefore an important regulator of skin responses to UV.

Safety and tolerability of an intratumorally injected DNAzyme, Dz13, in patients with nodular basal-cell carcinoma: a phase 1 first-in-human trial (DISCOVER).

BACKGROUND: The nuclear transcription factor c-Jun is preferentially expressed in basal-cell carcinoma. Dz13 is a deoxyribozyme that targets JUN messenger RNA and has inhibited the growth of a range of tumours in mice. We did a phase 1 study to assess safety and tolerability in human beings. METHODS: Adults with nodular basal-cell carcinoma were recruited from Royal Prince Alfred Hospital, Sydney, Australia, between September, 2010, and October, 2011. Patients were assigned to receive one intratumoral injected dose of 10, 30, or 100 µg Dz13, in a 50 µL volume of lipid carrier, and were assessed for adverse effects in the first 24 h then at 7, 14, and 28 days after injection. Treated tumours were surgically excised 14 days after injection and compared with the baseline biopsy samples for expression of c-Jun and tumorigenesis markers. FINDINGS: Nine patients were recruited, of whom three received each dose of Dz13. All patients completed the study with no drug-related serious adverse events. No systemic Dz13 exposure was detected. c-Jun expression was reduced in the excised tumours of all nine (100%) patients, compared with baseline, and histological tumour depth had decreased in five (56%) of nine. Proportions of cells positive for caspases 3, 8, and 9 and P53 were increased, but those of cells positive for Bcl-2 and MMP-9 were decreased. Infiltration by inflammatory and immune cells was stimulated. INTERPRETATION: Dz13 was safe and well tolerated after single intratumoral injections at all doses. FUNDING: Cancer Institute NSW, Cancer Council Australia, and National Health and Medical Research Council.

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.

Oral and systemic photoprotection.

Photoprotection can be provided not only by ultraviolet (UV) blockers but also by oral substances. Epidemiologically identified associations between foods and skin cancer and interventional experiments have discovered mechanisms of UV skin damage. These approaches have identified oral substances that are photoprotective in humans. UV inhibits adenosine triphosphate (ATP) production causing an energy crisis, which prevents optimal skin immunity and DNA repair. Enhancing ATP production with oral nicotinamide protects from UV immunosuppression, enhances DNA repair and reduces skin cancer in humans. Reactive oxygen species also contribute to photodamage. Nontoxic substances consumed in the diet, or available as oral supplements, can protect the skin by multiple potential mechanisms. These substances include polyphenols in fruit, vegetables, wine, tea and caffeine-containing foods. UV-induced prostaglandin E2 (PGE2 ) contributes to photodamage. Nonsteroidal anti-inflammatory drugs and food substances reduce production of this lipid mediator. Fish oils are photoprotective, at least partially by reducing PGE2 . Orally consumed substances, either in the diet or as supplements, can influence cutaneous responses to UV. A current research goal is to develop an oral supplement that could be used in conjunction with other sun protective strategies in order to provide improved protection from sunlight.

Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin.

Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide's potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.

Inhibition of UV-induced uric acid production using allopurinol prevents suppression of the contact hypersensitivity response.

Exposure to solar ultraviolet (UV) radiation suppresses adaptive immune responses. This contributes to skin carcinogenesis but may protect from some autoimmune diseases. However, the molecular changes occurring within UV-exposed skin that precipitate the downstream events leading to immune suppression are not fully understood. Using a combination of in vitro and in vivo mouse models, we have discovered that UV induces significant cutaneous production of immune suppressive uric acid. The ability of UV-induced uric acid to inhibit a contact hypersensitivity response was successfully blocked by the gout-treating drug Allopurinol. Up-regulation of NLRP3 mRNA by UV was also found to be dependent on UV-induced uric acid. This suggested that the target of UV-induced uric acid included proteins involved in the formation and activation of the NLRP3-inflammasome. However, in contrast to NLRP3, the adaptor protein ASC, which is required for formation of the NLRP3-inflammasome, was significantly down-regulated. Furthermore, this down-regulation was not dependent on UV-induced uric acid production because Allopurinol treatment failed to prevent the reduction in ASC. Hence, our results identify uric acid as an important molecule involved in sterile UV-induced inflammation and immune suppression. UV-induced uric acid may therefore offer a unique therapeutic target for preventing and treating skin cancer.

The retinoid signalling molecule, TRIM16, is repressed during squamous cell carcinoma skin carcinogenesis in vivo and reduces skin cancer cell migration in vitro.

Retinoid therapy is used for chemo-prevention in immuno-suppressed patients at high risk of developing skin cancer. The retinoid signalling molecule, tripartite motif protein 16 (TRIM16), is a regulator of keratinocyte differentiation and a tumour suppressor in retinoid-sensitive neuroblastoma. We sought to determine the role of TRIM16 in skin squamous cell carcinoma (SCC) pathogenesis. We have shown that TRIM16 expression was markedly reduced during the histological progression from normal skin to actinic keratosis and SCC. SCC cell lines exhibited lower cytoplasmic and nuclear TRIM16 expression compared with primary human keratinocyte (PHK) cells due to reduced TRIM16 protein stability. Overexpressed TRIM16 translocated to the nucleus, inducing growth arrest and cell differentiation. In SCC cells, TRIM16 bound to and down regulated nuclear E2F1, this is required for cell replication. Retinoid treatment increased nuclear TRIM16 expression in retinoid-sensitive PHK cells, but not in retinoid-resistant SCC cells. Overexpression of TRIM16 reduced SCC cell migration, which required the C-terminal RET finger protein (RFP)-like domain of TRIM16. The mesenchymal intermediate filament protein, vimentin, was directly bound and down-regulated by TRIM16 and was required for TRIM16-reduced cell migration. Taken together, our data suggest that loss of TRIM16 expression plays an important role in the development of cutaneous SCC and is a determinant of retinoid sensitivity.

Systemic low-dose UVB inhibits CD8 T cells and skin inflammation by alternative and novel mechanisms.

Exposure to UVB radiation before antigen delivery at an unirradiated site inhibits functional immunological responses. Mice treated dorsally with suberythemal low-dose UVB and immunized with ova in abdominal skin generated ova-specific CD8 T cells with a significantly decreased activation, expansion, and cytotoxic activity compared with unirradiated mice. UVB also impaired the delayed-type hypersensitivity (DTH) reaction to ova. Transfer of CD4⁺CD25⁺cells from UVB-exposed mice did not suppress the ova-specific CD8 T-cell response or DTH reaction in unexposed mice, confirming that systemic low-dose UVB does not induce long-lived functional regulatory CD4⁺CD25⁺ T cells. Repairing cyclobutane pyrimidine dimer-type DNA damage and blocking aryl hydrocarbon receptor signaling also did not reverse the immunosuppressive effect of UVB on ova-specific CD8 T cells and DTH, suggesting that cyclobutane pyrimidine dimers and the aryl hydrocarbon receptor are not required in systemic low-dose UVB-induced immunosuppression. The known UVB chromophore, cis-urocanic acid, and reactive oxygen species triggered the inhibition of DTH caused by UVB, but they were not involved in the modulation of CD8 T cells. These findings indicate that systemic low-dose UVB impedes the primary response of antigen-specific CD8 T cells by a novel mechanism that is independent of pathways known to be involved in systemic suppression of DTH.

The immune-modulating cytokine and endogenous Alarmin interleukin-33 is upregulated in skin exposed to inflammatory UVB radiation.

The cellular and molecular mechanisms by which UV radiation modulates inflammation and immunity while simultaneously maintaining skin homeostasis is complex and not completely understood. Similar to the effects of UV, IL-33 has potent immune-modulating properties that are mediated by the downstream induction of cytokines and chemokines. We have discovered that exposure of mice in vivo or human skin samples ex vivo to inflammatory doses of UVB induced IL-33 expression within the epidermal and dermal skin layers. Using a combination of murine cell lines and primary human cells, we demonstrate that both UV and the oxidized lipid platelet activating factor induce IL-33 expression in keratinocytes and dermal fibroblasts. Highlighting the significance of these results, we found that administering IL-33 to mice in vivo suppressed the induction of Th1-mediated contact hypersensitivity responses. This may have consequences for skin cancer growth because UV-induced squamous cell carcinomas that evade immunological destruction were found to express significantly higher levels of IL-33. Finally, we demonstrate that dermal mast cells and skin-infiltrating neutrophils closely associate with UV-induced IL-33-expressing fibroblasts. Our results therefore identify and support a role for IL-33 as an important early danger signal produced in response to inflammation-inducing UV radiation.

Human 8-oxoguanine-DNA glycosylase-1 is downregulated in human basal cell carcinoma.

Basal cell carcinoma (BCC) is the most common type of skin cancer and is a major public health problem in many Western countries. It usually occurs as a consequence of exposure to ultraviolet radiation (UV) with sunlight. The DNA photolesion 8-oxo-7,8-dihydroguanine (8-oxo-dG) is caused by reactive oxygen species (ROS) produced in response to UVA, UVB, and oxidative metabolism. If this damaged DNA is not repaired prior to cell division, then gene mutations may persist in daughter cells. Human 8-oxoguanine-DNA glycosylase 1 (hOGG1) is the main enzyme that excises 8-oxo-dG from damaged DNA via the base-excision repair pathway. However, the role of hOGG1 in human skin cancer is unknown. In this study, using immunohistochemical staining, we found low hOGG1 protein expression in human BCC compared to overlying epidermis or normal epidermis. We also found higher levels of 8-oxo-dG within the BCC compared to the basal layers of epidermis overlying the BCC lesions (E-BCC). The results suggest that low expression of hOGG1 within BCC results in accumulation of ROS generated 8-oxo-dG due to low levels of DNA repair, thereby implicating hOGG1 in human BCC carcinogenesis. These ROS are likely to be produced by the cancer cells during metabolism, as the BCC nests are too deep for UV to reach. Our data suggests that procedures that increase expression of hOGG1 within BCC, or protect from ROS may be beneficial for reducing progression of BCC.

Dermal mast cells affect the development of sunlight-induced skin tumours.

Ultraviolet (UV) radiation contained in sunlight is considered a major risk in the induction of skin cancer. While mast cells are best known for their role in allergic responses, they have also been shown to play a crucial role in suppressing the anti-tumour immune response following UV exposure. Evidence is now emerging that UV may also trigger mast cell release of cutaneous tissue remodelling and pro-angiogenic factors. In this review, we will focus on the cellular and molecular mechanisms by which UV recruits and then activates mast cells to initiate and promote skin cancer development.

The absence of Brm exacerbates photocarcinogenesis.

Brm is an ATPase subunit of the SWI/SNF chromatin-remodelling complex. Previously, we identified a novel hotspot mutation in Brm in human skin cancer, which is caused by exposure to ultraviolet radiation (UVR). As SWI/SNF is involved in DNA repair, we investigated whether Brm-/- mice had enhanced photocarcinogenesis. P53+/- and Brm-/-p53+/- mice were also examined as the p53 tumor suppressor gene is mutated early during human skin carcinogenesis. Mice were exposed to a low-dose irradiation protocol that caused few skin tumors in wild-type mice. Brm-/- mice with both p53 alleles intact had an increased incidence of skin and ocular tumors compared to Brm+/+p53+/+ controls. Brm loss in p53+/- mice did not further enhance skin or ocular cancer incidence beyond the increased photocarcinogenesis in p53+/- mice. However, the skin tumors that arose early in Brm-/- p53+/- mice had a higher growth rate. Brm-/- did not prevent UVR-induced apoptotic sunburn cell formation, which is a protective response. Unexpectedly, Brm-/- inhibited UVR-induced immunosuppression, which would be predicted to reduce rather than enhance photocarcinogenesis. In conclusion, the absence of Brm increased skin and ocular photocarcinogenesis. Even when one allele of p53 is lost, Brm has additional tumor suppressing capability.

1α,25 dihydroxyvitamin D3 enhances cellular defences against UV-induced oxidative and other forms of DNA damage in skin.

DNA damage induced by ultraviolet radiation is the key initiator for skin carcinogenesis since mutations may arise from the photoproducts and it also contributes to photoimmune suppression. The active vitamin D hormone, 1α,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) reduces thymine dimers, the major photoproduct found in human skin after UV exposure, and suppresses the accumulation of nitric oxide derivatives that lead to more toxic reactive nitrogen species (RNS). We examined whether other forms of DNA damage are reduced by 1,25(OH)(2)D(3), and hypothesized that photoprotection by 1,25(OH)(2)D(3) is, in part, due to the suppression of various forms of promutagenic DNA damage, including thymine dimers, through a reduction of genotoxic RNS. Different forms of UV-induced DNA damage were investigated in irradiated skin cells treated with or without 1,25(OH)(2)D(3), or inhibitors of metabolism and inducible nitric oxide synthase. Keratinocytes were also treated with nitric oxide donors in the absence of UV light. DNA damage was assessed by comet assay incorporating site specific DNA repair endonucleases, and by immunohistochemistry using antibodies to thymine dimers or 8-oxo-7,8-dihydro-2'-deoxyguanosine, and quantified by image analysis. Strand breaks in T4 endonuclease V, endonuclease IV and human 8-oxoguanine DNA glycosylase digests increased more than 2-fold in UV irradiated human keratinocytes, and were reduced by 1,25(OH)(2)D(3) treatment after UV exposure, and also by low temperature, sodium azide and an inhibitor of inducible nitric oxide synthase. Conversely, nitric oxide donors induced all three types of DNA damage in the absence of UV. We present data to show that 1,25(OH)(2)D(3) protects skin cells from at least three forms of UV-induced DNA damage, and provide further evidence to support the proposal that a reduction in RNS by 1,25(OH)(2)D(3) is a likely mechanism for its photoprotective effect against oxidative and nitrative DNA damage, as well as cyclobutane pyrimidine dimers.

Downregulation of Cockayne syndrome B protein reduces human 8-oxoguanine DNA glycosylase-1 expression and repair of UV radiation-induced 8-oxo-7,8-dihydro-2'-deoxyguanine.

Human 8-oxoguanine DNA glycosylase-1 (hOGG1) is the key DNA repair enzyme responsible for initiating repair of UV radiation-induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Previously we have shown that basal cells in human epidermis are particularly sensitive to UVA-mediated DNA damage probably due to low expression of hOGG1. Here we investigate some aspects of the regulatory role of Cockayne syndrome B (CSB) on hOGG1 expression and function. Cockayne syndrome B and hOGG1 genes were knocked down by miRNA technology in the HaCaT human keratinocyte cell line. Loss of the CSB gene decreased hOGG1 mRNA, and loss of hOGG1 increased CSB, indicating that they influence each other's expression. Protein levels were assessed in cells grown into engineered human skin using immunohistochemistry. This confirmed that CSB knockdown with miRNA reduced hOGG1 protein levels, but hOGG1 knockdown did not influence expression of CSB protein. Using comet assay we found that both hOGG1 and CSB knockdown reduced repair of both UVA- and UVB-induced 8-oxo-dG, consistent with CSB downregulation of hOGG1 mRNA and protein. In contrast, CSB but not hOGG1 knockdown reduced repair of UVB- and UVA-induced cyclobutane pyrimidine dimer photolesions. In engineered human skin, repair of UVA-induced 8-oxo-dG was inhibited by both hOGG1 and CSB knockdown, confirming the functional role of both proteins in cells with 3-D cellular contacts. These findings directly indicate that hOGG1 and CSB influence each other's expression. CSB is required for maintaining hOGG1 enzyme levels and function. Cockayne syndrome B could therefore be required for 8-oxo-dG repair due to its regulatory effect on hOGG1 expression. Cockayne syndrome B but not hOGG1 is also required for efficient repair of cyclobutane pyrimidine dimers. Cockayne syndrome B regulation of DNA repair could contribute to the effect of UVA in causing mutations that lead to skin cancer in humans.