Chikako Nishigori

Role of Reactive Oxygen Species in Skin Carcinogenesis

Reactive oxygen species (ROS) are associated not only with initiation, but also with promotion and progression in the multistage carcinogenesis model. In the present review, we will focus on the involvement of ROS in skin carcinogenesis, especially that induced by ultraviolet (UV) radiation. UV-specific DNA damage has been well studied thus far. However, recent reports have revealed the previously unknown participation of oxidative stress in UV-induced skin carcinogenesis. Indeed, in addition to transition-type mutations at dipyrimidine sites, G:C to T:A transversions, which may be induced by the presence of 8-oxoguanine during DNA replication, are frequently observed in the ras oncogene and p53 tumor suppressor gene in human skin cancers of sun-exposed areas and in UV-induced mouse skin cancers. Recent studies have shown that not only UV-B, but also UV-A is involved in UV-induced carcinogenesis. A wide variety of biological phenomena other than direct influence by UV, such as inflammatory and immunological responses and oxidative modifications of DNA and proteins, appear to play roles in UV-induced skin carcinogenesis. Furthermore, it has become clear that genetic diseases such as xeroderma pigmentosum show deficient repair of oxidatively modified DNA lesions. The involvement of ROS in skin carcinogeneisis caused by arsenic and chemical carcinogens will also be discussed.

Interleukin-23 and Interleukin-27 Exert Quite Different Antitumor and Vaccine Effects on Poorly Immunogenic Melanoma

Recent studies revealed that two novel interleukin (IL)-12-related cytokines, IL-23 and IL-27, have potent antitumor activities. However, the antitumor effects were mainly evaluated in relatively highly immunogenic tumors and have not been fully evaluated against nonimmunogenic or poorly immunogenic tumors. In this study, we investigated the antitumor efficacies of IL-23 and IL-27 on poorly immunogenic B16F10 melanoma and found that the antitumor responses mediated by IL-23 and IL-27 were clearly different. In syngeneic mice, mouse single-chain (sc) IL-23-transfected B16F10 (B16/IL-23) tumors exhibited almost the same growth curve as B16F10 parental tumor about until day 20 after tumor injection and then showed growth inhibition or even regression. In contrast, scIL-27-transfected B16F10 (B16/IL-27) tumors exhibited significant retardation of tumor growth from the early stage. In vivo depletion assay revealed that the antitumor effect of B16/IL-23 was mainly mediated by CD8+ T cells and IFN-gamma whereas that of B16/IL-27 mainly involved natural killer cells and was independent of IFN-gamma. We also found that antitumor effects of B16/IL-23 and B16/IL-27 were synergistically enhanced by treatment with IL-18 and IL-12, respectively. Furthermore, B16/IL-23-vaccinated mice developed protective immunity against parental B16F10 tumors but B16/IL-27-vaccinated mice did not. When combined with prior in vivo depletion of CD25+ T cells, 80% of B16/IL-23-vaccinated mice completely rejected subsequent tumor challenge. Finally, we showed that the systemic administration of neither IL-23 nor IL-27 induced such intense toxicity as IL-12. Our data support that IL-23 and IL-27 might play a role in future cytokine-based immunotherapy against poorly immunogenic tumors.

8-Oxoguanine Formation Induced by Chronic UVB Exposure Makes Ogg1 Knockout Mice Susceptible to Skin Carcinogenesis

8-Oxoguanine is one of the oxidative DNA damages that can result in stable mutations. The Ogg1 gene encodes the repair enzyme 8-oxoguanine-DNA glycosylase, which removes the oxidized base from DNA. In this study, we investigated the role of 8-oxoguanine in skin carcinogenesis induced by UVB irradiation using Ogg1 knockout mice (C57Bl/6J background). We examined the effect of UVB irradiation on the formation of 8-oxoguanine in epidermal cells using immunostaining and found that the level of 8-oxoguanine in Ogg1 knockout mice 24 hours after UVB irradiation remained high compared with that in wild-type and heterozygous mice. To verify the effect of chronic UVB irradiation on 8-oxoguanine formations in epidermal cells, we irradiated wild-type, heterozygous, and Ogg1 knockout mice with UVB at a dose of 2.5 kJ/m2 thrice a week for 40 weeks. We found that the mean number of tumors in Ogg1 knockout mice was 3.71, which was significantly more than in wild-type and heterozygous mice, being 1.71 and 2.28, respectively. The rate of developing malignant tumors in Ogg1 knockout mice was also significantly higher (88.5%; squamous cell carcinomas, 73.1%; sarcomas, 15.4%) than in wild-type mice (50.0%; squamous cell carcinomas, 41.7%; sarcomas, 8.3%). Moreover, the age of onset of developing skin tumors in Ogg1 knockout mice was earlier than in the other types of mice. These results clearly indicate that oxidative DNA damage induced by sunlight plays an important role in the development of skin cancers.

Docetaxel: a therapeutic option in the treatment of cutaneous angiosarcoma: report of 9 patients.

Effective treatment options are limited for patients with cutaneous angiosarcoma (AS). Docetaxel, a member of the taxane family of drugs, reportedly has been effective in the treatment of lung, head and neck, and breast cancers. Another taxane drug, paclitaxel, reportedly had unique activity in the treatment of AS of the scalp and neck and acquired immunodeficiency syndrome‐related Kaposi sarcoma. Therefore, the authors hypothesized that docetaxel may be of value in the treatment of cutaneous AS that is resistant to conventional therapy. However, there were only 3 case reports of the successful treatment of AS in elderly patients using docetaxel in combination with surgery and radiotherapy.

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.

In vivo elimination of CD25+ regulatory T cells leads to tumor rejection of B16F10 melanoma, when combined with interleukin‐12 gene transfer

Abstract:  CD4+CD25+ T cells are an important population that plays a crucial role in the maintenance of peripheral self‐tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti‐CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25+ regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti‐CD25 mAb administration alone but tumor rejection resulted in all mice when the administration was combined with IL‐12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8+ T cells cancelled the tumor rejection. NK‐cell depletion allowed IL‐12‐transfected B16F10 melanoma (B16/IL‐12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL‐12 than that in mice without administration of anti‐CD25 mAb. On the other hand, CD8+ T‐cell depletion did not affect the tumor growth in the early phase but allowed B16/IL‐12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti‐CD25 mAb. In a previous study, we showed that the elimination of CD4+ T cells enhanced the antitumor effect of B16/IL‐12 and induced vitiligo‐like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo‐like coat color in mice showing tumor rejection caused by CD25+ T‐cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25+ T cells did not induce vitiligo‐like coat color changes, though that of CD4+ T cells induced the change in 60% of mice. Furthermore, we confirmed that elimination of CD25+ T cells did not affect the T‐helper (Th) 1/Th2 cytokine profile, while that of CD4+T cells abrogated the Th2 cytokines (IL‐4 and IL‐10) and resulted in a Th1‐dominant cytokine profile in the tumor‐draining lymph nodes (TDLNs) of B16/IL‐12‐bearing mice. These results indicate that in vivo depletion of CD25+ regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL‐12 gene transfer and that the enhancement of the antitumor effect by CD25+ T‐cell depletion is mediated through CD8+ T cells and may differ from the enhancing mechanism caused by CD4+ T‐cell depletion.

Prostaglandin E2 is required for ultraviolet B-induced skin inflammation via EP2 and EP4 receptors

Keratinocytes are the major target of sunlight, and they produce prostaglandin (PG) E2 upon ultraviolet (UV) exposure. Although indomethacin, one of cyclooxygenase inhibitors, is known to suppress UV-induced acute skin inflammation, it remains uncertain whether endogenous PGE2 is responsible for UV-induced skin inflammation, and which subtype of PGE2 receptors mediates this process. UV-induced skin inflammation was investigated by using genetically and pharmacologically PGE2 receptor-deficient mice. We applied UV-induced skin inflammation model to genetical and pharmacological PGE2 receptor-deficient mice. We exposed UVB on these mice at 5 kJ/m2, and examined the ear swelling and the histological findings. We also measured the blood flow using a laser doppler device to assess the intensity of UVB-induced inflammatory change. The UV-induced ear swelling at 48 h after exposure was significantly reduced in EP2−/−, EP4−/− or wild-type mice treated with the EP4 antagonist compared to control mice. Consistently, inflammatory cell infiltration into the local skin, and local blood flow after UV exposure were significantly reduced by EP2 or EP4 signaling blockade. These data suggest that PGE2-EP2/EP4 signaling is mandatory in UV-induced acute skin inflammation, presumably by enhancing blood flow in the microenvironment.

Role and Regulation of STAT3 Phosphorylation at Ser727 in Melanocytes and Melanoma Cells

The transcription factor signal transducer and activator of transcription 3 (STAT3) has two important phosphorylation sites, Tyr705 and Ser727, for its activation. Ser727 phosphorylation has been considered to be a secondary event after Tyr705 phosphorylation. In this study, the role and regulation of Ser727 phosphorylation in STAT3 in melanocytic cells were examined. STAT3 was phosphorylated on Ser727 in the absence of Tyr705 phosphorylation in melanocytes. 12-O-tetradecanoylphorbol-13-acetate-induced increase in cell survival activity and nuclear translocation of STAT3 was associated with Ser727 phosphorylation. Ser727 was constitutively phosphorylated in all melanoma cell lines examined irrespective of Tyr705 phosphorylation. The possible involvement of Ser727 phosphorylation in STAT3 in cell survival activity and nuclear translocation of STAT3 in melanocytes was demonstrated also in melanoma cells. The constitutive Ser727 phosphorylation in melanoma cells was partially mediated by the B-Raf-MEK-ERK1/2 pathway. Immunohistochemical studies on specimens of primary lesions of acral lentiginous melanoma revealed that Ser727 phosphorylation precedes Tyr705 phosphorylation in the early stages of melanoma progression. Our results indicate that Ser727 phosphorylation on STAT3 is not necessarily a secondary event after Tyr705 phosphorylation and suggest that it has a role in the regulation of cell survival activity and nuclear translocation of STAT3 in melanocytic cells.

Molecular Clocks in Mouse Skin

Clock genes in the skin exhibit day-night changes in expression; however, whether these changes are brought by external light or intrinsic mechanisms is unclear. In this study, we demonstrated that expression of the clock and clock-controlled genes showed robust rhythms in mouse skin under constant dark conditions, whereas these rhythms were completely lost in Cry1/Cry2 knockout mice lacking a molecular clock. At the cellular level, the main oscillatory protein in the mammalian molecular clock, PER2, was expressed in the nuclei of keratinocytes in the epidermis and hair follicles, with expression peaking at CT16 (subjective dusk), 4-8 hours after expression of its mRNA. These expression patterns in the skin stopped after the ablation of the central clock in the suprachiasmatic nucleus (SCN), which was not recovered even in animals housed in 12 hour-light/12 hour-dark conditions. These findings demonstrate that the intrinsic oscillating molecular clock exists in the epidermis, and that signaling from the SCN is essential for the maintenance of the epidermal clock, and cannot be compensated by external light.

Src Homology 2 Domain-Containing Protein Tyrosine Phosphatase Substrate 1 Regulates the Migration of Langerhans Cells from the Epidermis to Draining Lymph Nodes

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein family in which the extracellular region interacts with its ligand, CD47. Recent studies have demonstrated that SHPS-1 plays an important role in cell migration and cell adhesion. We demonstrate in this study, using immunohistochemical and flow cytometric analyses, that murine Langerhans cells (LCs) express SHPS-1. Treatment of mice ears with 2,4-dinitro-1-fluorobenzene significantly reduced the number of epidermal LCs, and that reduction could be reversed by pretreatment with mAb to SHPS-1 or the CD47-Fc fusion protein. Treatment with the SHPS-1 mAb in vivo reduced the number of FITC-bearing cells in the lesional lymph nodes after the application of FITC to the skin. The SHPS-1 mAb inhibited the in vivo TNF-α-induced migration of LCs. The emigration of dendritic cells expressing I-Ab+ from skin explants to the medium was also reduced by the SHPS-1 mAb. We further demonstrate that the chemotaxis of a murine dendritic cell line, XS52, by macrophage inflammatory protein-3β was significantly inhibited by treatment with the SHPS-1 mAb or CD47-Fc recombinant protein. Finally, we show that migration of LCs was attenuated in mutant mice that lack the intracellular domain of SHPS-1. These observations show that the ligation of SHPS-1 with the SHPS-1 mAb or with CD47-Fc abrogates the migration of LCs in vivo and in vitro, which suggests that the SHPS-1-CD47 interaction may negatively regulate LC migration.