Ryusuke Ono

Agents that reverse UV-induced immune suppression and photocarcinogenesis affect DNA repair

UV exposure induces skin cancer, in part, by inducing immune suppression. Repairing DNA damage, neutralizing the activity of cis-urocanic acid, and reversing oxidative stress abrogate UV-induced immune suppression and skin cancer induction, suggesting that DNA, UCA, and lipid photo-oxidation serve as UV photoreceptors. What is not clear is whether signaling through each of these different photoreceptors activates independent pathways to induce biological effects or whether there is a common checkpoint where these pathways converge. Here, we show that agents known to reverse photocarcinogenesis and photoimmune suppression, such as platelet-activating factor (PAF) and serotonin (5-HT) receptor antagonists, regulate DNA repair. Pyrimidine dimer repair was accelerated in UV-irradiated mice injected with PAF and 5-HT receptor antagonists. Nucleotide excision repair (NER), as measured by unscheduled DNA synthesis, was accelerated by PAF and 5-HT receptor antagonists. Injecting PAF and 5-HT receptor antagonists into UV-irradiated Xeroderma pigmentosum complementation group A-deficient mice, which lack the enzymes responsible for NER, did not accelerate photoproduct repair. Similarly, UV-induced formation of 8-oxo-deoxyguanosine was reduced by PAF and 5-HT receptor antagonists. We conclude that PAF and 5-HT receptor antagonists accelerate DNA repair caused by UV radiation, which prevents immune suppression and interferes with photocarcinogenesis.

Increased Expression of Versican in the Inflammatory Response to UVB- and Reactive Oxygen Species-Induced Skin Tumorigenesis

Excessive exposure to UV radiation is a major risk factor for developing skin cancer. UV-induced reactive oxygen species (ROS) cause accumulation of DNA damage products such as 8-oxoguanine (8-oxoG) in the skin. We have previously shown that mice lacking the repair enzyme 8-oxoguanine glycosylase (Ogg1 knockout mice) are highly susceptible to skin cancer after long-term UVB exposure. To investigate the genes involved, we performed gene profiling of Ogg1 knockout mouse skin after UVB exposure. Among the up-regulated genes in UVB-treated Ogg1 knockout mice, inflammatory response pathway-related genes were most affected. The Vcan gene, which encodes the large extracellular matrix proteoglycan versican, was continuously up-regulated in UVB-treated Ogg1 knockout mice, suggesting that versican is a mediator of skin cancer development. We examined the expression pattern of versican in skin tumors from wild-type mice and UVB-treated Ogg1 knockout mice, and also analyzed 157 sun-related human skin tumors. Versican was strongly expressed in malignant skin tumors in both mice and humans, and especially in Ogg1 knockout mice. Additionally, infiltrating neutrophils strongly colocalized with versican in UVB-treated Ogg1 knockout mouse skin. These data demonstrate that inflammatory responses, particularly neutrophil infiltration and versican up-regulation, are closely involved in UVB/ROS-induced skin tumorigenesis.

Inhibitory effects of dietary Spirulina platensis on UVB-induced skin inflammatory responses and carcinogenesis.

Reactive oxygen species produced in response to UVR are important in skin tumor development. We have previously reported that deficiency of the Ogg1 gene, encoding the repair enzyme for 8-oxo-7,8-dihydroguanine (8-oxoG), increases skin tumor incidence in mice upon repetitive UVB exposure and modulation of UVB-induced inflammatory response. Spirulina platensis is used as a human food supplement because it contains abundant nutritional and antioxidant components. Therefore, we investigated the inhibitory effects of S. platensis on UVB-induced skin tumor development in Ogg1 knockout-(KO) mice and the wild-type (WT) counterpart. Dietary S. platensis suppressed tumor induction and development in both genotypes compared with our previous data without S. platensis. Induction of erythema and ear swelling, one of the hallmarks of UVB-induced inflammatory responses, was suppressed in the skin of Ogg1-KO mice and albino hairless mice fed with dietary S. platensis. Compared with untreated mice, S. platensis-administered mice showed significantly reduced 8-oxoG formation in the skin after UVB exposure. Moreover, we found that S. platensis effectively downregulated the signal proteins p38 mitogen-activated protein kinase, stress-activated protein kinase/c-Jun N-terminal kinase, and extracellular signal-regulated kinase after UVB exposure especially in Ogg1-KO mice. Our results suggest that S. platensis exerts antitumor effects against UVB irradiation in the skin through its anti-inflammatory and antioxidant effects.

Suppressive effect of recombinant human thioredoxin on ultraviolet light-induced inflammation and apoptosis in murine skin.

Thioredoxin (TRX) is a small ubiquitous protein, which regulates cellular redox status and scavenges reactive oxygen species. The present study was conducted to investigate the effect of TRX on ultraviolet (UV)‐B‐mediated inflammatory and apoptotic responses. Ear swelling after UV‐B irradiation was significantly reduced in TRX‐transgenic mice compared to wild‐type mice. Administration i.p. of recombinant human TRX also reduced acute skin inflammatory reaction, such as skin erythema and swelling. Histologically, numbers of inflammatory cells including neutrophils and lymphocytes were significantly reduced and the average size of the caliber of blood vessels were also reduced in recombinant human TRX‐injected mice. The number of apoptotic keratinocytes, in terms of sunburn cells, activated‐caspase‐3‐positive cells and terminal deoxynucleotidyl transferase dUTP nick end labeling‐positive cells were all significantly reduced in recombinant human TRX‐injected mice. Immunohistochemical intensity of 8‐hydroxy‐2′‐deoxyguanosine was strikingly reduced in recombinant human TRX‐injected mouse. Western blotting showed that administration of recombinant human TRX attenuated duration of phosphorylation of p38 mitogen‐activated protein kinases and intensity of phosphorylation of c‐Jun N‐terminal kinase in the early phase, which play important roles in inflammatory and apoptotic signaling. Collectively, these findings indicated that recombinant human TRX attenuated inflammatory and apoptotic responses caused by UV‐B. Possible mechanisms for this might be via redox regulation of stress signaling and reduction of reactive oxygen species.

Skin tumours induced by narrowband UVB have higher frequency of p53 mutations than tumours induced by broadband UVB independent of Ogg1 genotype

Different wavelengths of ultraviolet (UV) light have different promoting effects on skin carcinogenesis. Narrowband UVB (NB-UVB) has a single-peak wavelength of 311 nm and is widely used for treating skin diseases. Our previous work showed that, in comparison with conventional broadband UVB (BB-UVB), long-term exposure to NB-UVB induces higher frequency of skin cancer in mice, and it suggested that this is mediated through the formation of cyclobutane pyrimidine dimers (CPDs). To explore whether the frequency of p53 mutations in skin tumours correlates with CPD-induced mutations, we compared the frequency and types of p53 mutations between NB-UVB-induced and BB-UVB-induced malignant skin tumours produced in wild-type and Ogg1 knockout mice, which are deficient in repair of oxidative 8-oxoguanine (8-oxoG), a DNA damage mediated by reactive oxygen species (ROS). The frequency of p53 mutation was significantly higher in NB-UVB-induced than in BB-UVB-induced tumours in both wild-type and Ogg1 knockout mice. Most of the p53 mutations found were G:C → A:T transitions at dipyrimidine sites in both the NB-UVB- and BB-UVB-exposed groups. However, G:C → T:A mutations caused by 8-oxoG did not increase in Ogg1 knockout mice exposed to either NB-UVB or BB-UVB. Our results strongly suggest that NB-UVB induces highly malignant tumours caused by p53 dipyrimidine mutations through the formation of CPDs.

Hydrochlorothiazide Enhances UVA-Induced DNA Damage

The UVA is currently thought to be carcinogenic because, similar to UVB, it induces the formation of cyclobutane pyrimidine dimers (CPDs). Various drugs have been reported to cause photosensitive drug eruptions as an adverse effect. Although the precise mechanism of photosensitive drug eruption remains to be elucidated, it is generally accepted that free radicals and other reactive molecules generated via UV‐irradiated drugs play important roles in the pathogenesis of photosensitive drug eruptions. The waveband of concern for photo‐reactive drugs is UVA‐visible light, but some extend into the UVB region. We tested whether photosensitive drugs could enhance CPD formation after UVA exposure by using isolated DNA in the presence of several reported photosensitive drugs using high‐performance liquid chromatography. We found that the diuretic agent hydrochlorothiazide (HCT) significantly enhanced the production of TT dimers over a wide range of UVA. Furthermore, we investigated whether UVA plus HCT could enhance CPD production in xeroderma pigmentosum model mice defective in nucleotide excision repair. Immunofluorescence studies showed that CPD formation in the skin significantly increased after 365 nm narrow‐band UVA irradiation in the presence of HCT, compared with that in wild‐type mice. HCT could be used with caution because of its enhancement of UVA‐induced DNA damage.

Differences in Clinical Phenotype among Patients with XP Complementation Group D: 3D Structure and ATP-Docking of XPD In Silico

TO THE EDITOR Xeroderma pigmentosum (XP) is an autosomal recessive hereditary disease that is classified into seven genetic complementation groups, A through G, of nucleotide excision repair (NER)deficient types and one NER-proficient variant type (DiGiovanna and Kraemer, 2012). Patients with XP complementation group D (XPD) display photosensitivity, proneness to skin cancer and neurological symptoms, but the severity varies. In XPD from Western countries, the single amino-acid change R683W is found in 73% of the patients and most of them suffer from neurological symptoms (Takayama et al., 1995; Kobayashi et al., 1997; Taylor et al., 1997; Viprakasit et al., 2001; Kobayashi et al., 2002; Boyle et al., 2008; Emmert et al., 2009; Ueda et al., 2009). In contrast, Japanese patients display only skin manifestations without neurological symptoms (Kobayashi et al., 1997; Taylor et al., 1997; Kobayashi et al., 2002). XPD protein is an ATP-dependent 50-30 DNA helicase, which exerts unwinding of a damaged DNA strand to facilitate repair of the DNA. Although the human XPD protein remains refractory to crystallization, the crystal structure of XPD proteins from the archea has been solved (Fan et al., 2008; Liu et al., 2008). These structures revealed that XPD functions as a helicase with two helicase motifs separated by an ATP-binding cleft and two additional domains, a Fe–S cluster and an Arch domain. In this study, we present six cases of Japanese XPD without neurological abnormalities in four families. All patients showed severe photosensitivity since birth, and their skin in the sunexposed areas was hyperpigmented and covered with numerous pigmented maculae with color variations from light brown to dark brown. Four patients developed skin cancer, but none of them show neurological signs. XPD1KO and XPD4KO were assessed by a neurologist and an otorhinologist. Their clinical characteristics have been summarized in Table 1. To determine the ability to repair UV-induced DNA damage, a colonyformation assay after UV irradiation and UV-induced unscheduled DNA synthesis (UDS) was assessed (Materials and Methods are described in Supplementary data). The dose range giving 37% cell survival and UV-induced UDS in patients’ cells was much lower compared with that in the healthy subjects but was higher than that in XP-A cells (Table 1). Genetic complementation tests were carried out by means of a host-cell reactivation assay. Luciferase activity was increased specifically when XPD cDNA was cotransfected into each patient’s cells. Therefore, we concluded that all cases belonged to XPD. Direct sequence analysis was performed on each exon of the genomic DNA of Accepted article preview online 13 January 2014; published online 13 February 2014 Abbreviations: NER, nucleotide excision repair; UDS, unscheduled DNA synthesis; XP, xeroderma pigmentosum; XPD, xeroderma pigmentosum complementation group D E Nakano et al. Genotype–Phenotype and Protein Structure of XPD

Thioredoxin ameliorates cutaneous inflammation by regulating the epithelial production and release of pro-inflammatory cytokines.

Human thioredoxin-1 (TRX) is a 12-kDa protein with redox-active dithiol in the active site -Cys-Gly-Pro-Cys-. It has been demonstrated that systemic administration and transgenic overexpression of TRX ameliorate inflammation in various animal models, but its anti-inflammatory mechanism is not well characterized. We investigated the anti-inflammatory effects of topically applied recombinant human TRX (rhTRX) in a murine irritant contact dermatitis (ICD) induced by croton oil. Topically applied rhTRX was distributed only in the skin tissues under both non-inflammatory and inflammatory conditions, and significantly suppressed the inflammatory response by inhibiting the production of cytokines and chemokines, such as TNF-α, Il-1β, IL-6, CXCL-1, and MCP-1. In an in vitro study, rhTRX also significantly inhibited the formation of cytokines and chemokines produced by keratinocytes after exposure to croton oil and phorbol 12-myristate 13-acetate. These results indicate that TRX prevents skin inflammation via the inhibition of local formation of inflammatory cytokines and chemokines. As a promising new approach, local application of TRX may be useful for the treatment of various skin and mucosal inflammatory disorders.

Co-regulation of Cxcl1 and versican in the inflammatory response to UVB induced reactive oxygen species in skin photo-tumorigenesis

8-Oxo-7,8-dihydroguanine (8-oxoGua), oxidatively damaged DNA base modification which can be induced by ultraviolet radiation (UV), is partly responsible for the development of skin cancers in humans and in other animals [1,2]. We have previously demonstrated that Ogg1 knockout mice exhibit an impaired ability to excise 8-oxoGua from DNA in epidermal cells after ultraviolet B (UVB) exposure and that Ogg1 knockout mice reveal significantly higher skin tumor formation and faster tumor induction after long-

Peristomal skin ulcer with intestinal metaplasia.

tations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern. Proc Natl Acad Sci USA 2007; 104:13450–4. 9 Bourdeaut F, Herault A, Gentien D et al. Mosaicism for oncogenic G12D KRAS mutation associated with epidermal nevus, polycystic kidneys and rhabdomyosarcoma. J Med Genet 2010; 47:859–62. 10 Hafner C, Toll A, Real FX. HRAS mutation mosaicism causing urothelial cancer and epidermal nevus. N Engl J Med 2011; 365:1940–2.