Repair Of UVB-induced DNA Damage Research Articles

The scaffold protein JLP plays a key role in regulating ultraviolet B‐induced apoptosis in mice

The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB-induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c-Jun NH2 -terminal kinase-associated leucine zipper protein (JLP), a scaffold protein for mitogen-activated protein kinase (MAPK) signaling cascades, in UVB-induced apoptosis. We found that UVB-induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte-specific Jlp KO mice. Analysis of the repair of UVB-induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB-stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte-specific Jlp KO mice. Moreover, topical treatment of UVB-irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild-type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB-induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation.

Dual role of SIRT1 in UVB-induced skin tumorigenesis.

The protein deacetylase SIRT1 regulates various pathways in metabolism, aging and cancer. However, the role of SIRT1 in skin cancer remains unclear. Here, using mice with targeted deletions of SIRT1 in their epidermis in both resistant B6 and sensitive SKH1 hairless backgrounds, we show that the role of SIRT1 in skin cancer development induced by ultraviolet B (UVB) radiation is dependent on its gene dose. Keratinocyte-specific heterozygous deletion of SIRT1 promotes UVB-induced skin tumorigenesis, whereas homozygous deletion of SIRT1 suppresses skin tumor development but sensitizes the B6 mice to chronic solar injury. In mouse skin, SIRT1 is haploinsufficient for UVB-induced DNA damage repair and expression of xeroderma pigmentosum C (XPC), a protein critical for repairing UVB-induced DNA damage. As compared with normal human skin, down-regulation of SIRT1 is in parallel with down-regulation of XPC in human cutaneous squamous cell carcinoma at both the protein and mRNA levels. In contrast, homozygous SIRT1 deletion in mouse skin augments p53 acetylation and expression of its transcriptional target Noxa, and sensitizes the epidermis to UVB-induced apoptosis in vivo, while heterozygous SIRT1 deletion has no such effect. The gene dosage-dependent function of SIRT1 in DNA repair and cell survival is consistent with the dual roles of SIRT1 in UVB-induced skin tumorigenesis. Our results reveal the gene dosage-dependent in vivo functions of SIRT1 in skin tumorigenesis and may shed light on the role of SIRT1 in epithelial cancer induced by DNA damage.

Chafuroside B, an Oolong Tea Polyphenol, Ameliorates UVB-Induced DNA Damage and Generation of Photo-immunosuppression Related Mediators in Human Keratinocytes

Chafuroside B was recently isolated as a new polyphenolic constituent of oolong tea leaves. However, the effects of chafuroside B on skin function have not been examined. In this study, we investigated the protective effects of chafuroside B against UVB-induced DNA damage, apoptosis and generation of photo-immunosuppression related mediators in cultured normal human epidermal keratinocytes (NHEK). Chafuroside B at 1 µM attenuated both UVB-induced apoptosis, evaluated in terms of caspase-3/7 activity, and UVB-induced DNA damage, evaluated in terms of formation of cyclobutane pyrimidine dimers (CPD), in NHEK exposed to UVB (20 mJ/cm2). In addition, chafuroside B at 0.3 or 1 µM suppressed the UVB-induced production of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and prostaglandin E2 (PGE2), as determined by ELISA, and conversely enhanced IL-12 mRNA expression and production, as measured by RT-PCR and ELISA. Further, chafuroside B at 1 µM also suppressed UVB-induced expression of receptor activator of nuclear factor κB ligand (RANKL) mRNA. These results indicate that chafuroside B promotes repair of UVB-induced DNA damage and ameliorates the generation of IL-10, TNF-α, PGE2, and RANKL, all of which are UVB-induced immunosuppression related mediators. These effects of chafuroside B may be mediated at least in part through induction of IL-12 synthesis in human keratinocytes. Because chafuroside B might have practical value as a photoprotective agent, a further study of the in vivo effects of chafuroside B seems warranted.

Abstract 1068: Keratinocyte-specific deletion in mice reveals gene dose-dependent function of SIRT1 in tumorigenesis.

Abstract The protein deacetylase SIRT1, a mammalian counterpart of the yeast silent information regulator 2 (Sir2) and a proto member of the sirtuin family, regulates various pathways in metabolism, DNA repair, and cell survival. However, the role of SIRT1 in cancer is still under debate. Here we show that the role of SIRT1 in skin cancer development induced by the human skin carcinogen UVB radiation is dependent on its gene dose. Keratinocyte-specific heterozygous deletion of SIRT1 increases UVB-induced skin tumorigenesis, whereas homozygous deletion of SIRT1 decreases skin tumorigenesis. In mouse skin, SIRT1 is haploinsufficient for DNA repair and the expression of xeroderma pigmentosum C (XPC), a protein critical for repairing UVB-induced DNA damage. Similar to homozygous SIRT1 deletion, heterozygous SIRT1 deletion reduces the XPC protein levels and UVB-induced DNA damage repair. As compared with normal human skin, down-regulation of SIRT1 is in parallel with down-regulation of XPC in human cutaneous squamous cell carcinoma at both the protein and mRNA levels. In contrast, homozygous SIRT1 deletion in mouse skin augments p53 acetylation, and the expression of its transcriptional target Noxa, and sensitizes epidermis to UVB-induced apoptosis in vivo, while heterozygous SIRT1 deletion has no effect. Although mice with homozygous SIRT1 deletion do not develop tumors, these mice suffer severe sunburn. The gene dose-dependent function of SIRT1 in DNA repair and cell survival is consistent with its role in UVB-induced skin tumorigenesis and injury. Taken together, our results indicate that, depending on the SIRT1 levels, SIRT1 acts as a tumor suppressor and as an oncogene. Citation Format: Mei Ming, Keyoumars Soltani, Christopher R. Shea, Xiaoling Li, Yu-ying He. Keratinocyte-specific deletion in mice reveals gene dose-dependent function of SIRT1 in tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1068. doi:10.1158/1538-7445.AM2013-1068

Bioactive Grape Proanthocyanidins Enhance Immune Reactivity in UV-Irradiated Skin through Functional Activation of Dendritic Cells in Mice

Ultraviolet (UV) radiation-induced immunosuppression has been implicated in skin carcinogenesis. Grape seed proanthocyanidins (GSPs) have anti-skin carcinogenic effects in mice and GSPs-fed mice exhibit a reduction in UV-induced suppression of allergic contact hypersensitivity (CHS), a prototypic T-cell-mediated response. Here, we report that dietary GSPs did not inhibit UVB-induced suppression of CHS in xeroderma pigmentosum complementation group A (XPA)-deficient mice, which lack nucleotide excision repair mechanisms. GSPs enhanced repair of UVB-induced DNA damage (cyclobutane pyrimidine dimers) in wild-type, but not XPA-deficient, dendritic cells (DC). Co-culture of CD4(+) T cells with DCs from UVB-irradiated wild-type mice resulted in suppression of T-cell proliferation and secretion of T-helper (TH) 1-type cytokines that was ameliorated when the DCs were obtained from GSP-fed mice, whereas DCs obtained from GSP-fed XPA-KO mice failed to restore T-cell proliferation. In adoptive transfer experiments, donor DCs were positively selected from the draining lymph nodes of UVB-exposed donor mice that were sensitized to 2,4,-dinitrofluorobenzene were transferred into naïve recipient mice and the CHS response assessed. Naïve recipients that received DCs from UVB-exposed wild-type donors that had been fed GSPs exhibited a full CHS response, whereas no significant CHS was observed in mice that received DCs from XPA-KO mice fed GSPs. These results suggest that GSPs prevent UVB-induced immunosuppression through DNA repair-dependent functional activation of dendritic cells in mice. Cancer Prev Res; 6(3); 242-52. ©2013 AACR.

1SAP-02 植物におけるCPD光回復酵素とUVB抵抗性(1SAP 新学術領域「植物の環境感覚 : 刺激受容から細胞応答まで」共催,カラフルな植物光環境感覚タンパク質,シンポジウム,日本生物物理学会第51回年会(2013年度))

1SAP-02 植物におけるCPD光回復酵素とUVB抵抗性(1SAP 新学術領域「植物の環境感覚 : 刺激受容から細胞応答まで」共催,カラフルな植物光環境感覚タンパク質,シンポジウム,日本生物物理学会第51回年会(2013年度))

Deregulation of XPC and CypA by Cyclosporin A: An Immunosuppression-Independent Mechanism of Skin Carcinogenesis

Skin cancer is the most common malignancy in organ transplant recipients, causing serious morbidity and mortality. Preventing and treating skin cancer in these individuals has been extraordinarily challenging. Following organ transplantation, cyclosporin A (CsA) has been used as an effective immunosuppressive to prevent rejection. Therefore immunosuppression has been widely assumed to be the major cause for increased skin carcinogenesis. However, the mechanism of skin carcinogenesis in organ transplant recipients has not been understood to date; specifically, it remains unknown whether these cancers are immunosuppression dependent or independent. Here, using both immunocompromised nude mice which are defective in mature T lymphocytes as an in vivo model and human keratinocytes as an in vitro model, we showed that CsA impairs genomic integrity in the response of keratinocytes to ultra violet B (UVB). Following UVB radiation, CsA inhibited UVB-induced DNA damage repair by suppressing the transcription of the DNA repair factor xeroderma pigmentosum C (XPC). In addition, CsA compromised the UVB-induced checkpoint function by upregulating the molecular chaperone protein cyclophilin A (CypA). XPC mRNA levels were lower, whereas CypA mRNA and protein levels were higher in human skin cancers than in normal skin. CsA-induced phosphoinositide 3-kinase(PI3K)/AKT activation was required for both XPC suppression and CypA upregulation. Blocking UVB damage or inhibiting the PI3K/AKT pathway prevented CsA-sensitized skin tumorigenesis. Our findings identified deregulation of XPC and CypA as key targets of CsA, and UVB damage and PI3K/AKT activation as two principal drivers for CsA-sensitized skin tumorigenesis, further supporting an immunosuppression-independent mechanism of CsA action on skin tumorigenesis.

Role of AMPK in UVB-induced DNA damage repair and growth control

Skin cancer is the most common cancer in the U.S., while DNA-damaging UVB radiation from the sun remains the major environmental risk factor. Reducing skin cancer incidence is becoming an urgent issue. The energy-sensing enzyme 5’-AMP-activated protein kinase (AMPK) plays a key role in the regulation of cellular lipid and protein metabolism in response to stimuli such as exercise and changes in fuel availability. However, the role AMPK in the response of skin cells to UVB damage and in skin cancer prevention remains unknown. Here we show that AMPK activation is reduced in human and mouse squamous cell carcinoma as compared with normal skin, and by UVB irradiation, suggesting that AMPK is a tumor suppressor. At the molecular level, AMPK deletion reduced the expression of the DNA repair protein xeroderma pigmentosum C (XPC) and UVB-induced DNA repair. AMPK activation by its activators AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) and metformin (N’,N’-dimethylbiguanide), the most widely used anti-diabetic drug, increased the expression of XPC expression and UVB-induced DNA repair in mouse skin, normal human epidermal keratinocytes, and AMPK wild-type cells but not in AMPK deficient cells, indicating an AMPK-dependent mechanism. Topical treatment with AICAR and metformin not only delayed onset of UVB-induced skin tumorigenesis but also reduced tumor multiplicity. Furthermore, AMPK deletion increased ERK activation and cell proliferation, while AICAR and metformin inhibited ERK activation and cell proliferation in keratinocytes, mouse skin, AMPK wild-type and AMPK deficient cells, suggesting an AMPK-independent mechanism. Finally, in UVB-damaged tumor-bearing mice, both topical and systemic metformin prevented the formation of new tumors and suppressed growth of established tumors. Our findings not only suggest that AMPK is a tumor suppressor in the skin by promoting DNA repair and controlling cell proliferation, but also demonstrate previously unknown mechanisms by which the AMPK activators prevent UVB-induced skin tumorigenesis.

Nrf1 CNC-bZIP Protein Promotes Cell Survival and Nucleotide Excision Repair through Maintaining Glutathione Homeostasis

Skin cancer is the most common cancer in the United States. Its major environmental risk factor is UVB radiation in sunlight. In response to UVB damage, epidermal keratinocytes activate a specific repair pathway, i.e. nucleotide excision repair, to remove UVB-induced DNA lesions. However, the regulation of UVB response is not fully understood. Here we show that the long isoform of the nuclear factor erythroid 2-related factor 1 (Nrf1, also called NFE2L1), a cytoprotective transcription factor critical for the expression of multiple antioxidant response element-dependent genes, plays an important role in the response of keratinocytes to UVB. Nrf1 loss sensitized keratinocytes to UVB-induced apoptosis by up-regulating the expression of the proapoptotic Bcl-2 family member Bik through reducing glutathione levels. Knocking down Bik reduced UVB-induced apoptosis in Nrf1-inhibited cells. In UVB-irradiated surviving cells, however, disruption of Nrf1 impaired nucleotide excision repair through suppressing the transcription of xeroderma pigmentosum C (XPC), a factor essential for initiating the global genome nucleotide excision repair by recognizing the DNA lesion and recruiting downstream factors. Nrf1 enhanced XPC expression by increasing glutathione availability but was independent of the transcription repressor of XPC. Adding XPC or glutathione restored the DNA repair capacity in Nrf1-inhibited cells. Finally, we demonstrate that Nrf1 levels are significantly reduced by UVB radiation in mouse skin and are lower in human skin tumors than in normal skin. These results indicate a novel role of Nrf1 in UVB-induced DNA damage repair and suggest Nrf1 as a tumor suppressor in the skin.

Abstract 1599: Grape seed proanthocyanidins inhibit UV-induced tolerogenic T-cell response through DNA repair-dependent functional activation of Langerhans cells

Abstract It is well documented that ultraviolet (UV) radiation-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPD) diminishes the antigen presenting capacity of epidermal Langerhans cells (LCs) and this leads to the suppression of immune system. Previously, we have shown that dietary grape seed proanthocyanidins (GSPs) inhibit UVB-induced immunosuppression in mice via activation of CD8+ T cells. However, it is not understood whether (1) GSPs target LCs while exerting its immunomodulatory effects on T cells in response to the application of cutaneous contact sensitizer, 2,4-dinitrofluorobenzene (DNFB) onto the UV-exposed skin, and whether (2) the ability of GSPs to activate dendritic cells/LCs correlates with their DNA repair properties. To understand this mechanism, we first determined DNA repair ability of GSPs in UV-exposed dendritic cells for which CD11c+ cells were prepared from bone marrow of xeroderma pigmentosum complementation group A-knockout (XPA-KO) mice which are deficient in DNA repair, and their wild-type (WT) counterparts. CD11c+ cells were exposed to UVB radiation with or without pretreatment with GSPs. Treatment of GSPs resulted in significant reduction in the number of CPD-positive cells in CD11c+ cells from WT mice, but this DNA repair effect of GSPs was not seen in the CD11c+ cells from XPA-KO mice. We then analyzed effects of dietary GSPs (0.5%, w/w) on secretion of cytokines by CD11c+ cells that were isolated from lymph nodes and spleens of mice (WT and XPA-KO) that were exposed to UVB [150 mJ/cm2 (WT), 20 mJ/cm2 (XPA-KO)]. Our results showed that in comparison to CD11c+ cells isolated from WT control mice (GSP-untreated), CD11c+ cells isolated from mice that received GSPs in diet secreted a significantly higher level of Th1 cytokines (IL-12, IFNα), while the levels of Th2 cytokines (IL-4, IL-10) were significantly lower, but this effect of GSPs was not observed in CD11c+ cells isolated from XPA-KO mice. Finally, we used an adoptive transfer approach, wherein draining lymph nodes were harvested from mice that were exposed to UVB with and without GSPs supplementation in diet and sensitized by DNFB onto the UVB-exposed skin. CD11c+ cells were positively selected and transferred into naïve mice that were subsequently challenged by application of DNFB on the ear skin. Naïve recipients that received CD11c+ cells from WT, GSPs-treated, UVB-irradiated donors exhibited full contact hypersensitivity (CHS) response, whereas no significant CHS was observed in XPA-KO mice. These data suggest that dietary GSPs inhibit UVB-induced immunosuppression in mice by improving the ability of LCs to mount an immune response via activation of T cells in a manner which correlates with its capacity to enhance repair of UVB-induced DNA damage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1599. doi:1538-7445.AM2012-1599

PTEN Positively Regulates UVB-Induced DNA Damage Repair

Nonmelanoma skin cancer is the most common cancer in the United States, where DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared with skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCCs, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin through its ability to positively regulate XPC-dependent GG-NER following DNA damage.

Proanthocyanidins Inhibit Photocarcinogenesis through Enhancement of DNA Repair and Xeroderma Pigmentosum Group A–Dependent Mechanism

Dietary grape seed proanthocyanidins (GSP) inhibit photocarcinogenesis in mice; however, the molecular mechanisms underlying this effect have not been fully elucidated. As ultraviolet B (UVB)-induced DNA damage in the form of cyclobutane pyrimidine dimers (CPDs) has been implicated in skin cancer risk, we studied whether dietary GSPs enhance repair of UVB-induced DNA damage and, if so, what is the potential mechanism? Supplementation of GSPs (0.5%, w/w) with AIN76A control diet significantly reduced the levels of CPD(+) cells in UVB-exposed mouse skin; however, GSPs did not significantly reduce UVB-induced CPD(+) cells in the skin of interleukin-12p40 (IL-12) knockout (KO) mice, suggesting that IL-12 is required for the repair of CPDs by GSPs. Using IL-12 KO mice and their wild-type counterparts and standard photocarcinogenesis protocol, we found that supplementation of control diet with GSPs (0.5%, w/w) significantly reduced UVB-induced skin tumor development in wild-type mice, which was associated with the elevated mRNA levels of nucleotide excision repair genes, such as XPA, XPC, DDB2, and RPA1; however, this effect of GSPs was less pronounced in IL-12 KO mice. Cytostaining analysis revealed that GSPs repaired UV-induced CPD(+) cells in xeroderma pigmentosum complementation group A (XPA)-proficient fibroblasts from a healthy individual but did not repair in XPA-deficient fibroblasts from XPA patients. Furthermore, GSPs enhance nuclear translocation of XPA and enhanced its interactions with other DNA repair protein ERCC1. Together, our findings reveal that prevention of photocarcinogenesis by GSPs is mediated through enhanced DNA repair in epidermal cells by IL-12- and XPA-dependent mechanisms.

Hypoxia-inducible factor-1α regulates the expression of nucleotide excision repair proteins in keratinocytes

The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1α (HIF-1α) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1α increased XPC mRNA expression due to competition between HIF-1α and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1α protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1α also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1α is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1α downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1α in the repair of UVB-induced DNA damage.

RETRACTED: IL-12 Deficiency Exacerbates Inflammatory Responses in UV-Irradiated Skin and Skin Tumors

IL-12 deficiency has been shown to promote photocarcinogenesis in mice. As UVB-induced inflammation is an important tumor-promoting event in the development of skin tumors, we determined the effects of IL-12-deficiency on UVB-induced inflammatory responses in mice. For this purpose, IL-12-knockout (IL-12 KO) and their wild-type counterparts were subjected to a photocarcinogenesis protocol; skin and tumor samples were collected at the termination of the experiment, and analyzed for biomarkers of inflammation and their mediators. We found that the levels of infiltrating leukocytes, myeloperoxidase, proliferating cell-nuclear antigen (PCNA), COX-2, PGE2, and the proinflammatory cytokines IL-1beta, TNF-alpha, and IL-6 were higher in the UVB-exposed skin of IL-12 KO than in that of wild-type mice. In a short-term experiment, pretreatment of IL-12 KO mice with rIL-12 (50 ng per mouse) before each exposure to UVB increased the repair rate of UVB-induced cyclobutane pyrimidine dimers, while inhibiting UVB-induced increases in myeloperoxidase, COX-2, PGE2, PCNA, TNF-alpha, and IL-1beta in the skin as compared with non-rIL-12-treated IL-12 KO mice. Similarly, tumors of IL-12 KO mice expressed higher levels of inflammatory responses than those of wild-type mice. Together, our data suggest that IL-12 KO mice are more susceptible to both UVB-induced inflammation and photocarcinogenesis because of the deficiency in the repair of UVB-induced DNA damage.

Xenoestrogens modulate genotoxic (UVB)-induced cellular responses in estrogen receptors positive human breast cancer cells

Human populations and wildlife are exposed to mixtures of both anthropogenic and natural chemicals. Some of these compounds are known to interact principally with the endocrine function, whereas others act mainly on genomic DNA. Given this evidence, we wanted to address the question of whether concomitant exposure of such chemicals was able to interact at the cellular level. We have previously shown that 17β-Estradiol (E 2) modulates the DNA repair capacity of cells. In this work, we wanted to examine if other xenoestrogens (i.e. industrial compounds, pesticides or pharmaceuticals) were able to interact with the UVB-induced cellular response as E 2 does. Here, we show that xenoestrogens modulate the capacity of cells to repair their DNA damage according to the type of compounds. For example, the oral contraceptive 17α-Ethinylestradiol down-regulated the repair of UVB-induced DNA damage whereas the UV filter Eusolex 6007 up-regulated this pathway. The notion that xenoestrogens could interact with a genotoxic stress is reinforced by the modulation of the estrogens-dependent luciferase reporter gene expression when cells are UVB-irradiated. Finally, these observations suggested the potential role of xenoestrogens in carcinogenesis by their capacity to modulate cells responses to genotoxic stress.

Mechanisms and preclinical efficacy of silibinin in preventing skin cancer

Eukaryotic cellular machineries including the genome face continuous challenge from environmental deleterious agents, as well as from the by products of their own metabolism. Our skin is the most important barrier. It protects us from xenobiotic and genotoxic agents including ultraviolet (UV) solar radiation and potential carcinogens, which are notorious for causing skin cancer. There is a rise in non-melanoma skin cancer (NMSC), which is diagnosed in more than a million people every year in the United States alone, and is also prevalent in the other Western countries. In addition to sunscreens, chemoprevention of skin cancer by natural non-toxic compounds is suggested as an effective strategy to prevent the incidence of skin cancer. Our extensive animal studies on silibinin, a non-toxic bioactive component in milk thistle, suggest that it has a strong potential to prevent skin cancer incidence, promotion and progression in response to chemical carcinogens and tumour promoters as well as UV radiation. Our data suggest that silibinin has multiple targets in the cell, and can be protective against the harmful effects of cytotoxic agents such as reactive oxygen species and inflammation. Further, silibinin modulates mitogenic and survival signalling, p53, Cip1/p21 and other cell cycle regulatory molecules to prevent UVB-induced skin carcinogenesis. Our ongoing studies also suggest the positive effect of silibinin on the repair of UVB-induced DNA damage in mouse skin. Overall, the protective efficacy of silibinin against skin cancer is supported by sound mechanistic rationale in animal and cell culture studies, and suggests its potential use for humans.

The expression of p16INK4a, the product of a tumor suppressor gene for melanoma, is upregulated in human melanocytes by UVB irradiation

Background: The genetic locus CDKN2A has been linked to familial melanoma, and mutations or deletions in its coding sequence are seen in some cases of sporadic and familial melanomas. The protein encoded by CDKN2A, p16INK4a, functions as a negative regulator of cell cycle progression and as a tumor suppressor, but the regulatory mechanisms involved in controlling its expression remain poorly defined. Objective: This study tested the hypothesis that UVB irradiation, which transiently inhibits the growth of human melanocytes, is one of the regulators of p16INK4a expression. Methods: Cultured human melanocytes were irradiated with UVB over a sublethal dosage range, and p16INK4a protein and mRNA levels were quantified at varying times thereafter by quantitative immunostaining and by Western and Northern blotting. Results: Levels of p16INK4a protein in melanocytes increased significantly after sublethal UVB irradiation as compared with nonirradiated cells. Northern analysis indicated that p16INK4a messenger RNA coordinately increased in a dose-dependent manner more than 2-fold in irradiated cells at the tested doses. Conclusion: UVB irradiation transcriptionally activates the expression of p16INK4a in cultured human melanocytes. Therefore the growth arrest that occurs with irradiation of melanocytes could be mediated, in part, by upregulation of p16INK4a. This transient arrest may allow repair of UVB-induced DNA damage before cell division. Conversely, hereditary or acquired defects in CDK4A that give rise to functional insufficiency of p16INK4a could permit the premature propagation of melanocytes harboring potentially carcinogenic DNA damage. (J Am Acad Dermatol 2000;42:741-5.)

Repair of Ultraviolet B and Singlet Oxygen-Induced DNA Damage in Xeroderma Pigmentosum Cells

Ultraviolet B (UVB) (290-320 nm) is capable of damaging the DNA molecule directly by generating predominantly pyrimidine dimers. UVA (320-400 nm) does not alter the DNA molecule directly. However, when it is absorbed by cellular photosensitizers, it can damage the DNA molecule indirectly, e.g., by mediation of singlet oxygen, generating predominantly 8-hydroxyguanine. These indirect effects have been implicated in the mutagenic, genotoxic, and carcinogenic effects of UVA. To study the processing of directly and indirectly UV-induced DNA damage in intact, DNA-repair-proficient and -deficient human cells, we used the replicating plasmid pRSVcat, either irradiated with up to 10 kJ/m2 UVB or treated with the photosensitizer methylene blue plus visible light (which generates singlet oxygen). These treated plasmids were introduced into lymphoblast lines from normal donors or from patients with xeroderma pigmentosum (XP) complementation groups A, C, D, E, and variant. DNA repair was assessed by measuring activity of reactivated chloramphenicol-acetyl-transferase enzyme, encoded by the plasmid's cat gene, in cell extracts after 3 d. As expected, the repair of UVB-induced DNA damage was reduced in all XP cell lines, and the degree varied with the complementation group. XP-A, -D, -E, and -variant cells were normally efficient in the repair of singlet oxygen-induced DNA damage. Only three of four XP-C cell lines showed a markedly reduced repair of these lesions. This indicates differential DNA-repair pathways for directly and indirectly UV-induced DNA damage in human cells and suggests that both may be affected in XP-C.

Processing of directly and indirectly ultraviolet-induced DNA damage in human cells.

Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in the tumorigenesis of UV-induced skin cancer. Different wavelengths of UV radiation cause different kinds of DNA damage and mutations. UVB (290-320 nm) generates pyrimidine dimers by direct excitation of the DNA molecule. UVA (320-400 nm) can damage the DNA only indirectly through a photosensitized reaction. This indirect action is mediated mainly by singlet oxygen, which generates purine base modifications, and has been implicated in the carcinogenic effects of UVA. In order to study the processing of directly and indirectly UV-induced DNA damage in human cells, we first treated the replicating plasmid pRSVcat with up to 10 kJ/m2 UVB or with the photosensitizer methylene blue plus visible light (which generates singlet oxygen) in vitro. Then, the damaged plasmid was transfected into normal or repair deficient xeroderma pigmentosum complementation group A (XP-A) cells. DNA repair was assessed by measuring activity of reactivated chloramphenicol acetyltransferase (CAT) enzyme, encoded by the plasmid's cat gene, in cell extracts after 3 days. While XP-A cells exhibited a significantly reduced repair of UVB-induced DNA damage, they showed a normal repair of singlet oxygen-induced DNA damage. This indicates a differential DNA repair pathway for directly and indirectly UV-induced DNA damage in human cells. Irradiation of the plasmid with UVA alone did not result in a genotoxic effect. Only in conjunction with a cell extract, which provides all candidate cellular photosensitizers, did we find a reduced CAT activity after transfection. This indicates that the genotoxicity of UVA is mediated by a cellular photosensitizer.