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23rd Annual Meeting of the Japanese Society for Pigment Cell Research (JSPCR-2010) November 27, 28 Tokyo, Japan

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CONFERENCE ORGANIZER Hidemi Nakagawa (The Jikei University, Japan)

MEETING SECRETARIAT Masaaki Kawase (The Jikei University, Japan)

ORGANIZING INSTITUTIONS Department of Dermatology, The Jikei University School of Medicine, Tokyo, Japan

Invited Speakers

Special lecture

Induction mechanisms of aberrant DNA methylation

Toshikazu Ushijima Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan

Aberrant DNA methylation is causally involved in cancers, and possibly in other acquired disorders, such as neurodegenerative, mental, immune, and metabolic disorders. Chronic inflammation is one of the few known inducers of it, but what genes are methylated and how are still unknown. We showed that specific genes are methylated by specific inducers, such as Helicobacterpylori infection (gastric mucosae) and smoking (esophageal mucosae), [Nakajima, IJC, 2009; Oka, Cancer, 2009]. Genes with histone H3 lysine 27 trimethylation and without stalled RNA polymerase II were susceptible to DNA methylation induction [Takeshima, Genome Res, 2009; Takeshima, Epigenetics, 2010]. To identify genes involved in methylation induction, we analyzed temporal profiles of inflammation-related gene expression in gastric mucosae during Helicobacterpylori infection, and Cxcl2, Il1b, Nos2, and Tnf were suggested to be involved [Niwa, Cancer Res, 2010]. We compared five different inducers of inflammation in gastric mucosae (infection of three Helicobacter strains, 50% ethanol (EtOH), and saturated sodium chloride solution), and suggested that Il1b, Nos2, and Tnf might be involved in methylation induction [Hur, submitted]. Therefore, one (or combination) of Il1b, Nos2, and Tnf is likely to be involved in methylation induction by chronic inflammation. Once it is identified, it will contribute to clarification of mechanisms of methylation induction in various disorders.

Educational lecture

JSPCR preceptorship: advances in biochemistry of melanin and biology of melanocyte

EL-1

Biochemistry of mixed melanogenesis

Shosuke Ito Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan

Melanin pigments consist of dark-brown to black eumelanin (EM) and yellow to reddish-brown pheomelanin (PM). EM is photoprotective while PM is believed to be phototoxic. In this seminar, the process of mixed melanogenesis is reviewed. The common precursor L-tyrosine is oxidized by tyrosinase (Tyr) to form dopaquinone (DQ). DQ is the key intermediate that regulates mixed melanogenesis. DQ is converted to dopachrome (DC) through cyclodopa. During this process, dopa is formed from DQ as a by-product. DC is converted to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by the action of dopachrome tautomerase (DCT/Tyrp2). With low DCT activity, 5,6-dihydroxyindole (DHI) is preferentially produced. These indole intermediates are oxidized by Tyr or Tyrp1 to give rise to EM. During this process, DQ may oxidize DHI. In the presence of cysteine (cys), addition of cys to DQ gives cysteinyldopa isomers, which are then oxidized by DQ to form PM. The point of divergence between EM- and PM-genesis is determined to be a cys concentration of 0.8 μM. Based on these results, it is suggested that mixed melanogenesis proceeds through CD-genesis, PM-genesis, followed by EM-genesis. Further, ‘casing model’ is proposed, in which EM is deposited on preformed PM in melanin granules. This model is supported by measuring oxidation potential of melanosomes.

EL-2

How are the proliferation and differentiation of melanocytes regulated?

Tomohisa Hirobe Radiation Effect Mechanism Research Group, National Institute of Radiological Sciences, Chiba and Graduate School of Science, Chiba University, Chiba, Japan

The skin color and coat color are determined by melanin (eumelanin and pheomelanin). Melanin is synthesized in melanocytes and accumulated in melanosomes. Mature melanosomes are transferred to keratinocytes and the skin color and coat color are determined. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from neural crest cells. We have been studying the mechanism of regulation of proliferation and differentiation of mouse melanocytes. Regulation by the tissue environment, especially by keratinocytes is indispensable for the regulation of melanocyte proliferation and differentiation. We have suggested that endothelins, steel factor, hepatocyte growth factor, leukemia inhibitory factor, and granulocyte-macrophage colony-stimulating factor are the keratinocyte-derived factors and regulate the proliferation and differentiation of mouse epidermal melanocytes. Moreover, α-melanocyte stimulating hormone secreted from intermediate lobe of the pituitary and sex hormones secreted from sex organs are stimulators of final differentiation of mouse melanocytes. On the other hand, numerous coat color genes are involved in regulating the expression of coat color in mice. These coat color genes regulate the proliferation and differentiation of melanocytes by controlling the proliferative rate, melanosome formation and maturation, melanosome distribution, melanosome transfer, and switch from eumelanin to pheomelanin synthesis. The expression of keratinocyte-derived melanocyte mitogens and melanogens is also regulated by the genes. The regulation of gene expression, protein expression, and activity of proteins is important for melanocyte proliferation and differentiation.

EL-3

What are melanocytes distributed across the body doing? -Exploring new functions of melanocytes

Hiroaki Yamamoto1, Shigeyuki Uehara2 1Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan; 2Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, Japan.

In mammals, melanocytes contributing to the skin and hair pigmentation are classified as classical melanocytes whereas non-classical melanocytes are localized in a variety of tissues and organs such as eye, inner ear, meninges, bone, heart, etc. What are these melanocytes doing? Because different migration routes from the neural crest and homing microenvironments should impose specific gene expression profiles on each group of melanocytes, they should have a wide range of roles in a spatiotemporal fashion. For example, cochlear melanocytes in the stria vascularis is indispensable for hearing ability but, intriguingly, they do not necessarily need to produce melanin, one of the most prominent characteristics of melanin pigment cells. What is the role of melanin synthesis in melanocytes? Recently we found that melanocytes in the inner ear specifically express Gsta4, which is strongly involved in anti-stress responses (against oxidative stress) (Uehara et al., 2009). Loud noises stimulate melanization (Gratton and Wright, 1992), and induce oxidative stress (Henderson et al., 1999). We suppose that melanocytes become (have evolved) to play a variety of roles depending on their localization but still retain common signature functions in which melanin synthesis could essentially contribute.

EL-4

Molecular basis of the skin-specific albinism in medaka

Shoji Fukamachi Department of Chemical and Biological Sciences, Japan Women's University, Tokyo, Japan

The orange-red variant of medaka (Oryzias latipes) lacks melanin in the skin and has been kept in Japan for over 200 years. Mendelian inheritance of this phenotype reported in 1921 (Aida Genetics), which was only 20 years after rediscovery of Mendel's law and 10 years after Morgan's report on sex inheritance of the white eyes of Drosophila. Thus, the potential of medaka as a model organism had been demonstrated at the dawn of genetics. However, little researchers used it for their studies before this century. Positional cloning of the gene responsible for the orange-red variant (Fukamachi et al. 2001 Nat Genet) dramatically changed the situation. Experimental tools for forward/reverse genetics, including the whole-genome sequences, were rapidly established, which made medaka one of the most powerful model vertebrates.

The gene mutated in the orange-red medaka encodes a membrane-bound protein called Slc45a2. The protein might be a transporter, but little is known about its function in melanogenesis. Whereas the skin of the orange-red medaka is amelanotic, its eyes (and peritoneum) are melanized. Molecular mechanism of this was recently revealed; (i) the medaka Slc45a2 has two promoters that function in the skin or the eyes, and (ii) the orange-red mutation occurs on the cutaneous promoter (Fukamachi et al. 2008 Genetics). In this talk, I’ll also report a strain (Sukesuke) with the transparent skin that allows intact observation of internal organs, genetic pollution by artificial release of the orange-red mutants, and preliminary analysis of a ‘variegated’Slc45a2 mutant.

2009 JSPCR Incentive award lecture

AL-1

Heterogeneity of human fetal melanocytes: the unique development and differentiation of sole melanocytes

Yuji Yamaguchi Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Little is known how human melanocytes locate at their suitable positions during embryogenesis and whether the characters of melanocytes are similar or different among the various sites of body. We conducted studies using biopsies taken from different sites (scalp, back, abdomen and sole) of the 31 aborted fetuses. We proved the topographical diversity in human fetal melanocytes during embryogenesis based on the following 4 major findings; (i) location of melanocytes within the skin, (ii) maturation process of melanocytes, (iii) gene profile analyses and (iv) secretory factors from sole derived melanocytes but not from the other sites derived melanocytes. (i) Human fetal melanocytes first localize at epidermis in hair buds, as described in mice, and spread throughout epidermis not from dermis. Sole melanocytes localize at eccrine sweat gland ducts and show different expression levels of melanocyte markers. (ii) The expression of melanocyte marker, gp100, comes earlier than that of tyrosinase, DCT and MART-1 during melanocyte maturation, which is completed in scalp, back, abdomen and sole in that order. (iii) We investigated gene expression profiles among the fetal melanocytes from four different anatomical sites. The results indicate the fetal melanocytes are topographically diverse, especially between sole-derived melanocytes and the other three sites-derived melanocytes. (iv) Several factors including FGF show the higher expression levels in sole-derived melanocytes. We conclude that there exist heterogenic expression profiles of human fetal melanocytes in vivo and in vitro, suggesting that acral melanomas are genetically different from the other types of melanoma.

AL-2

Characteristic haplotype pattern of MC1R gene in the Japanese population

Tomonori Motokawa1, Hiroaki Yamamoto2, Tomomi Kato1, Takayuki Katagiri1, Koji Yokoyama1, Yoshi Kawamoto3 1POLA Chemical Industries, Yokohama, Japan; 2Nagahama Institute of Bio-Science and Technology, Nagahama, Japan; 3Primate Research Institute, Kyoto University, Inuyama, Japan

The melanocortin 1 receptor (MC1R) regulates the production of black eumelanin and red pheomelanin in melanocytes. The ratio of these two pigments primarily determines skin and hair color. The coding region of the MC1R gene is known to have numerous single nucleotide polymorphisms (SNP). In the present study, we focused on a 1.5-kb region of the MC1R gene and its promoter region. We identified eight polymorphic variants (>1%); three in the promoter region and five (four non-synonymous, and one synonymous) in the coding region of the gene. Using haplotype analysis, we found that six polymorphic haplotypes and three major types (J-1, J-2 and J-3) account for almost all (92.3%) haplotypes. Surprisingly, we found that the number of segregating sites between J-1 and J-2 or J-3 is between 4 and 6, and that there are very few intermediates among the three haplotypes. If the second- or third-most common haplotype (J-2 or J-3) had evolved from the most common haplotype (J-1), many more intermediates would be expected. This implies that Japanese MC1R haplotypes have evolved from more than one ancestral haplotype.

Lunchoen seminar 1 current treatment strategy for vitiligo

LS1-1

Review of the pathogenesis of vitiligo vulgaris and our hypothesis regarding the function of proinflammatory cytokines

Atsushi Tanemura Department of Dermatology, Integrated Medicine Graduate School of Medicine Osaka University, Osaka, Japan

Vitiligo vulgaris is an acquired de-pigmentation disorder affecting cosmetic appearance. It affects all races and occurs in 0.3–1.0% of the world population. Although pathogenic mechanisms for induction of de-pigmentation are not fully unveiled, non-segmental vilitigo has been supposed as one of autoimmune disease because of high frequency of complication with thyroid disease, that is Hashimoto's disease, type I diabetes, collagen diseases with anti-nuclear antibodies, and so on. The infiltration of cytotoxic T cells targeting melanocyte-specific antigen into vitiligo lesion has been thought to play a critical role for hypopigmentation. Spritz et al clearly showed that a notable genetic change of NALP1 gene regulating innate immune response and others were associated with vitiligo. Some reports suggested the effect of proinflammatory cytokines such as IL-1, IL-6, and TNF-alpha for inhibition of melanogenesis and melanocyte survival. Recently, Th17 cells are reported to play roles in inflammatory skin diseases such as psoriasis and atopic dermatitis and we found that Th17 cells surprisingly infiltrate in vitiligo. In this seminar, we would like to present the complexity of the pathogenesis for vitiligo and discuss the possible role of Th17 cells in induction of vitiligo with special reference to our latest experimental data. We also would like to discuss preliminary guideline for the diagnosis and management of congenital and acquired hypopigmentary diseases proposed by study group of Japanese ministry of health, labour and welfare.

LS1-2

Generalized vitiligo susceptibility genes and the treatment

Naoki Oiso Department of Dermatology, Kinki University Faculty of medicine, Osaka, Japan

Vitiligo is an acquired patchy depigmented disorder. It is classified into generalized, segmental and localized types. Generalized vitiligo is a disorder in which depigmented macules results from autoimmune loss of melanocytes. Generalized type is frequently associated with other autoimmune diseases, particularly including autoimmune thyroid disease (Hashimoto's thyroiditis and Graves’ disease), rheumatoid arthritis, adult-onset type 1 diabetes mellitus, psoriasis, pernicious anemia, systemic lupus erythematosus and Addison's disease.

Recent genomewide association studies identified multiple generalized vitiligo susceptibility genes, several ones associated with other autoimmune diseases and TYR encoding tyrosinase which may mediate target-cell specificity. The autoimmune-related genes includes major-histocompatibility-complex class I molecules, class II molecules, PTEN22, NALP1 and others.

Generalized vitiligo is treated with (i) topical steroid, vitamin D3 derivatives and immunomodulatory tacrolimus, (ii) phototherapy including psoralen plus ultraviolet A (PUVA), narrowband ultraviolet B (NB-UVB) and excimer laser, (iii) grafting techniques and (iv) cosmetic camouflage. NB-UVB therapy is as effective as PUVA therapy, and is now prevalent for the generalized vitiligo. However, NB-UVB may induce more carcinogenic skin tumors in mice than broad-band UVB. The treatment for vitiligo should be decided by the type and area of vitiligo and the age of the patient.

Lunchoen seminar 2 treatment of acquired pigmentary disorders using medical equipments

LS2-1

Treatment of acquired pigment abnormalities with Q-switched ruby laser

Yasutaka Kobayashi Department of Dermatology, The Jikei University School of Medicine, Tokyo, Japan

The treatment results of acquired pigment abnormalities using Q-switched ruby laser in our site, predominantly acquired dermal melanocytosis, will be presented. The characteristics and performances of novel Q-switched Ruby Laser, ‘The Ruby Z1’ (JMEC, Japan), will also be discussed. Additionally, the treatment experience of fractional laser resurfacing will be mentioned as a latest topic.

LS2-2

Possibility of skin disease treatment with a 308-nm excimer lamp

Toshihiro Ito Department of Dermatology, The Jikei University School of Medicine, Tokyo, Japan

Previous phototherapy centered on external or oral PUVA treatment, but, since the development of the 311-nm narrow band (NB-UVB) fluorescence tube in 1984, NB-UVB therapy has become the main treatment because irradiation can be readily performed without psoralen, while exhibiting a sufficient effect. There are various NB-UVB irradiators, such as those that irradiate the whole and a part of the body by a single irradiation. Lesions vary from small to large. When a small lesion is irradiated using an irradiator with a wide irradiation area, normal regions are also irradiated, and so the risks of pigmentation, skin disorder, and skin cancer increase. Thus, a device capable of irradiating only lesions is necessary. Excimer lamps are characterized by a simple structure and flexible shape, and the irradiated surface can be reduced. It has been reported that there was no difference in the effect between the currently available 308-nm excimer lamps and 311 nm NB-UVB. In this study, we used VTRACTM of PhotoMedex Co. Its irradiation area is small, and the area can be adjusted to the lesion size. In addition, the irradiating part is mobile, and, thus, patients do not need to change posture during treatment. The power is high, facilitating a short irradiation time. Our department uses it for various diseases, and its requirement for the treatment of vitiligo is particularly marked. Herein, we present the effects on various skin diseases, including vitiligo vulgaris, involving clinical cases.

Oral presentation

OP- 1

Quantum chemical calculations reveal heterodimerization in the cooxidation of 5, 6-dihydroxyindole and its 2-carboxy derivative

Hidekazu Okuda1, Kazumasa Wakamatsu2, Shosuke Ito2, Takayuki Sota1 1Department of Electrical Engineering and Bioscience, Waseda University,Tokyo, Japan; 2Department of Chemistry, Fujita Health University School of Health Sciences, Aichi, Japan

5,6-Dihydroxyindole (DHI) and its 2-carboxy derivative (DHICA) are precursors of eumelanin. Several homooligomers of them were isolated. However, only one heterooligomer was identified as the cooxidation of DHI and DHICA. Heteropolymerization mechanism is less understood than homopolymerization one although eumelanin is considered to be built from the heteropolymer of DHI and DHICA. We report the possible heterodimerization of DHI and DHICA based on the quantum chemical calculations.

The quantum chemical calculations have been carried out for the 4 nucleophiles and 8 electrophiles. The heterodimerization may be the electron-transfer-controlled reaction similar to polymerization of DHI, and so the electron transfer probability from a nucleophile to an electrophile is used as the reaction index. The general-purpose reactivity indices have been evaluated to find the reactive positions with high reactivity. The predicted heterodimer well agrees with the previous experimental results. The present calculations also suggest the structure of heterodimer which was not identified experimentally.

OP- 2

Elucidation of the biosynthetic pathway of a brown pigment ‘neuromelanin’ in the substantia nigra in human brain

Takaya Murase1, Shosuke Ito2, Kazumasa Wakamatsu1,2 1Fujita Health University Graduate School of Health Sciences, Aichi, Japan; 2Department of Chemistry, Fujita Health University School of Health Sciences, Aichi, Japan

Neuromelanin (NM) is a brown insoluble pigment particularly abundant in catecholaminergic neurons of the substantia nigra and locus caeruleus in brains of human and mammalian species. We have previously reported that NM is composed of a polymer derived oxidatively from DA and Cys in a ratio of 4–1. However there still remain many unknown points regarding its structure and function. In order to elucidate the structure and biosynthetic pathway of NM, we at first synthesized four standard compounds, Cys-DA, DHBT-1, ODHBT-1 and BZ-1, which are considered as intermediates in NM synthesis. We next prepared the model compounds of NM by tyrosinase oxidation at various ratio of DA and Cys under the physiological conditions (pH 7.4, 37°C). To elucidate stages of NM formation, chemical degradation method of alkaline hydrogen peroxide oxidation and reductive hydrolysis with hydroiodic acid were applied to the reaction mixtures. The time course of reaction was followed by HPLC analyses of chemical degradation products and the intermediates in NM synthesis. The results suggest that DA is oxidized to DHBT-1 via Cys-DA to give NM with gradual conversion of ODHBT-1 and BZ-1 units.

OP- 3

Membrane formation surrounding melanosomes in keratinocytes may be an important step for melanosome transfer

Hideya Ando1,2, Yoko Niki1, Masaki Yoshida1,2, Mary S. Matsui3, Daniel B. Yarosh3, Masamitsu Ichihashi1,2 1Kobe Skin Research Institute, Kobe, Japan; 2Skin Aging and Photo-aging Research Center, Doshisha University, Kyoto, Japan; 3The Estee Lauder Companies Inc., NY, USA

The mechanism of melanosome transfer has not been fully clarified. Substantial melanosome transfer was elicited in a melanocyte-keratinocyte co-culture system in which cells are separated by a microporous membrane filter through which only melanocyte dendrites can penetrate. Electron microscopic observations under these conditions revealed that melanosomes incorporated in keratinocytes were packed in clusters enclosed by a double membrane. When melanocytes were cultured on the filter with no recipient keratinocytes, pigment globules containing multiple melanosomes were observed on the opposite side of the filter. When those globules were collected and incubated with keratinocytes, the incorporated melanosomes in keratinocytes were enclosed by a double membrane in a manner similar to the co-culture system. In contrast, when individual melanosomes, not in globules, were isolated from melanocytes and then added to keratinocytes, they were phagocytosed, but were enclosed by a single membrane in a manner distinct from the co-culture system. These results suggest that the mechanism operating melanosome transfer in the co-culture system is different from methods typically used in vitro, in which isolated melanosomes or beads are used. This may indicate that the formation of an enclosing membrane is an important step for melanosome transfer.

OP-4

Differentially expressed genes reveal the novel mechanism of melanogenesis regulation in hirsein-treated B16 melanoma cells

Myra O. Villareal1, Junkyu Han1,2, Kenjiro Ikuta3, Hiroko Isoda1,2 1Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2Alliance for Research on North Africa (ARENA), University of Tsukuba, Tsukuba, Ibaraki, Japan; 3Yokohama Corporate Research Laboratories, Mitsubishi Rayon Co., Ltd, Yokohama, Kanagawa, Japan

Hirsein compounds can inhibit melanogenesis in B16 murine melanoma cells by inhibiting the expression of the microphthalmia associated transcription factor gene (Mitf) and its regulated genes, Tyr, Trp1 and Dct causing a decrease in the melanin content of the B16 murine melanoma cells. However, the overall molecular mechanism by which melanogenesiswas inhibited is unclear. To elucidate the mechanism involved in the observed anti-melanogenesis effects, DNA microarray was performed. DNA chips of 528 spots, loaded with 272 genes prepared by Mitsubishi Rayon, Ltd. (Genopal), were used to determine the gene expressions of genes for melanogenesis, membrane-bound receptors, tyrosine kinase regulation, melanosome transport, and other cell signal regulation–related genes (includes the housekeeping and the negative control genes). Results showed a down-regulation of melanogenesis-related genes and melanosome transport genes. The two highly down-regulated genes were Sorbs3 and Mc1r and these genes most likely, caused Mitf gene inhibition. The highly upregulated genes are those that are involved in the MAPK and cell signal transduction. We report for the first time that the novel hirsein compounds can inhibit melanogenesis by down-regulating the expression of genes thatregulate Mitf gene expression.

OP-5

Hypomelanization in endothelin receptor B2 mutated birds

Toyoko Akiyama1, Ai Sinomiya1, Keiji Kinoshita2, Makoto Mizutani2, Takao Namikawa2, Shin’ichi Ito3, Yoichi Matsuda2 1Department of Biology, Keio University, Yokohama, Japan; 2Bioagric. Sci., Avian Biosci. Res. Center, Nagoya University, Nagoya, Japan; 3Department of Agric. Sci., Appl. Biol. Sci., Gifu University, Gifu, Japan

A signal transduction system of Endothelins and their receptors is known to be one of the strong factors to induce pigmentation in vertebrates. A panda quail strain (s/s) with a white plumage mutation had been reported the responsible gene as EDNRB2. In this study, we report chicken lines with EDNRB2 mutation and compare the pigmentation between these mutated birds and their wild types. In a chicken line, Minohiki-white covered all over with white plumage, the mutation site was determined as EDNRB2 by linkage mapping analysis of 93 F2 progeny. Pigment production in integument and feather bud of these mutated quail and chicken during development was strongly suppressed. From the observation of organ culture and during development, our results clearly demonstrate that EDNRB2 is an indispensable factor in melanocyte development in birds. In compared with the EDNRB mutated mouse (piebald spotting; s/s), a main feature of these EDNRB2 mutated quail and chickens is only white plumage. Since birds have EDNRB2 in addition to –B, these facts suggest that these receptors in birds may share the main responsibility between pigmentation and other roles.

OP-6

Glucosamine attenuates endothelin-1+stem cell factor-stimulated pigmentation in human epidermal equivalents by specifically abolishing the CREB protein levels within melanocytes

Yuki Wakabayashi1, Hiroaki Nakajima1, Katsunori Fukasawa1, Kazumasa Wakamatsu2, Genji Imokawa1 1School of Bioscience and Biotechnology, Tokyo University of Technology, Tokyo, Japan; 2School of Health Sciences, Fujita Health University, Toyoake, Japan

We have found the inhibitory effects of a glycosylation inhibitor glucosamine (Glc) on the stimulation of pigmentation in a human epidermal equivalent and analyzed its biological mechanism. Epidermal equivalents were cultured in DMEM medium supplemented with endothelin-1 (ET-1) (10 nM) and stem cell factor (SCF) (5 nM) and were treated with or without Glc (1 mg/ml) for 14 days. The addition of Glc elicited a marked depigmenting effect on the stimulation of pigmentation after 14 days of culture, which was accompanied by significantly decreased eumelanin content. Real-time RT-PCR and western blotting revealed that increased gene and protein expression of MITF, tyrosinase and Pmel17 were significantly down-regulated at days 7–10, which suggested an impairment in intracellular signaling upstream of their gene expression. Western blotting of intracellular signaling intermediates revealed that in Glc-treated human melanocytes in culture, there is a marked deficiency in the ET-1 + SCF-stimulated phosphorylation level of CREB due to its decreased protein levels but not of ERK and MITF compared with the non-treated control. These findings indicate that Glc attenuates the ET-1 + SCF-stimulated pigmentation by down-regulating the levels of CREB activation within melanocytes.

OP-7

In vitro analysis of the interaction between pigment cells in surface patterning of zebrafish

Hiroaki Yamanaka, Yuji Amihama, Shigeru Kondo Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan

The striped pigmentation of zebrafish is constituted with two types of pigment cells, arranged in a pattern. The mechanism by which the pigment cells are arranged so beautifully is unknown. When the pigment cells were ablated by laser to erase the pattern, immediate regeneration of pigment cells formed a transient pattern which gradually transformed to the original pattern. This result suggested that the surface pattern does not depend on another pre-existing pattern such as morphogen gradient, but have the ability to form pattern autonomously even in the later stage. In vivo observation of the regeneration process suggested that the recovery of the pattern occurred by the interaction among the pigment cells that mutually controls the localization and survival. However how they interact is still unknown. So, we made primary culture of pigment cells to examine the interaction in vitro. We harvested pigment cell from fins and cultured them in plastic dish. In the primary culture, pigment cells showed dynamic cell movements. When a xanthophore (yellow pigment cell) came close to a melanophore (black pigment cell), xanthophore extended the filopodia and touched the melanophore. And the melanophore suddenly ran away from the xanthophore. So we could see the chasing between the two pigment cells for a while.

We assumed that this direct interactions and chasing movements have a pivotal role in the arrangement of pigment cells in zebrafish.

OP-8

A novel mutation of ruby-eye 2 (ru2d/Hpsd) elicits reduced melanocyte differentiation and dilute coat color in mice, and L-tyrosine restores its reduced differentiative activity

Tomohisa Hirobe1,2, Chihiro Yoshihara3, Sakae Takeuchi3, Hiyoyuki Abe4, Kazumasa Wakamatsu5, Shosuke Ito5, Yoko Kawa6, Yoshinao Soma6, Masako Mizoguchi6 1Radiation Effect Mechanism Research Group, National Institute of Radiological Sciences, Chiba, Japan; 2Graduate School of Science, Chiba University, Chiba, Japan; 3Graduate School of Science and Technology, Okayama University, Okayama, Japan; 4Graduate School of Science and Technology, Yamagata University, Yonezawa, Japan; 5Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Japan; 6Department of Dermatology, St. Marianna University School of Medicine, Kawasaki, Japan

In our laboratory, spontaneous autosomal recessive ruby-eye-like hypopigmentation mutant was obtained in C57BL/10JHir black mice in 2006. RT-PCR analysis revealed that this new mutant possessed deletion mutation of one base of the ru2 gene. To know the mechanism of the action of this gene (ru2d/Hps5d) on pigmentation, the proliferation and differentiation of cultured ru2d melanoblasts and melanocytes were compared to black. The proliferation of ru2d melanoblasts and melanocytes did not differ from that of black. However, the differentiation of ru2d melanocytes was greatly inhibited. Tyrosinase activity, TRP1 and TRP2 expression, eumelanin content, and stage IV melanosomes were greatly decreased. Indeed, eumelanin content in ru2d hair was about 1/3 that of black. Excess L-tyrosine (Tyr) added to the culture medium restored the reduced melanocyte differentiation. L-Tyr also increased TYR activity, TRP1 and TRP2 expression, eumelanin content, and stage IV melanosomes. These results suggest thatthe ru2d gene inhibits the differentiation of melanocytes and hair pigmentation, and its action is related to the transport or utilization of L-Tyr.

OP-9

Lipocalin-type prostaglandin D synthase as a regulator of the retinoic acid signaling in melanocytes

Kazuhisa Takeda, Na-Ho Takahashi, Miki Yoshizawa, Shigeki Shibahara Tohoku University School of Medicine, Sendai, Japan

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the formation of prostaglandin D2 (PGD2) and also functions as a transporter for lipophilic ligands, including all-trans retinoic acid (RA). Here we show that human epidermal melanocytes produce and secrete L-PGDS and PGD2 in culture medium, whereas L-PGDS is not expressed in human melanoma cell lines, HMV-II, SK-MEL-28, 624 mel, and G361. Treatment with RA (1 or 10 μM) for 4 days decreased the proliferation of melanocytes (30% decrease), but not melanoma cells. We therefore isolated L-PGDS-expressing cell lines from 624 mel cells. Treatment with RA decreased the proliferation of L-PGDS-expressing cells by 20%, but not mock-transfected cell lines lacking L-PGDS expression. RA induced expression of a cyclin-dependent kinase inhibitor p21Cip1 in L-PGDS-expressing cells, but not mock-transfected cells. Moreover, RA increased the transient expression of a reporter gene carrying the RA-responsive elements in L-PGDS-expressing cell lines (at least five-fold activation), compared to the two-fold activation in mock-transfected cell lines. These results suggest that L-PGDS may increase the sensitivity to RA. In conclusion, L-PGDS may fine-tune the RA signaling in melanocytes.

OP-10

Involvement of Mitf in the retinal pigment epithelium development

Daisuke Nishihara1,2, Akiha Kawasaki-Nishihara1,2, Nagaharu Tsukiji3, Ichiro Yajima4, Kazuhisa Takeda5, Shigeki Shibahara5, Harukazu Nakamura1, Hiroaki Yamamoto2 1Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Japan; 2Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Japan; 3Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Japan; 4Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Japan; 5Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Japan

The retinal pigment epithelium (RPE), one of the components of the vertebrate eye, consists of a monolayer of melanin-pigment cells. Although RPE is known to be indispensable for adult visual function, little is known about the molecular mechanisms underlying the RPE development.

Mitf, a basic/helix-loop-helix/leucine zipper motif transcription factor, is known to be essential for the normal development of melanin-pigment cells in homeothermal animals. Mitf mutant mice show malformations in RPE development. Although these phenotypes indicate necessity of normal Mitf function in the RPE development, it is not easy to understand in detail how Mitf functions in the RPE development in vivo only by analyzing Mitf mutant mice. To elucidate such unknown functions, we conducted gain or loss-of-function experiments in chick embryo by electroporation.

OP-11

Protective effect of Kit signaling for melanocyte stem cells against irradiation induced genotoxic stress

Hitomi Aoki, Takahiro Kunisada Department of tissue and organ development, Gifu University graduate school of medicine, Gifu, Japan

Stem cells are maintained in a specific microenvironment known as niche. It was reported that stem cells of the melanocyte lineage were found in the lower permanent portion of mouse hair follicles called bulge area, throughout the hair cycle. The Kit receptor is expressed on a variety of stem cells including melanocytes stem cells (MSC) and also known to behave a growth and differentiation factor in melanocyte lineage cells. Recently, it is reported that irreparable DNA damage caused by ionizing radiation, abrogates renewal of MSC in mice and the DNA-damage response triggers MSC differentiation into mature melanocytes in the niche, rather than inducing their apoptosis or senescence (Inomata et al., 2010). In this study, we investigate the effect of Kit signaling for MSC located in niche under the irreparable DNA damage induced by ionizing radiation by using various Kit mutants and hk14-SCF mice exogenously express Kitl in the skin. We observed that Kit mutants were more sensitive for the irradiation induced genotoxic stress than wild type littermates. Conversely, hk14-SCF mice were less sensitive or resistant for the irradiation. We previously proposed the existence of two major melanocyte populations: KIT-dependent cutaneous melanocytes in the epidermis and ET3 and/or HGF-dependent non-cutaneous and dermal melanocytes (Aoki et al. 2009). While both transgenic mouse strains were resistant to the irradiation, hk14-SCF mice were more significant resistance than hk14-ET3 mice, indicating that Kit signaling has a clear protection effect to the irradiation induced genotoxic stress for MSC.

OP-12

Specific differentiation of human sole melanocytes during embryogenesis

Motoki Nakamura1, Yuji Yamaguchi1, Hiroshi Kato1, Mayumi Sugiura2, Akimichi Morita1 1Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 2Department of Obstetrics and Gynaecology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

Melanocytes are known to originate from neural crest and to migrate from dorsal neural tube to ventral site during embryogenesis. However, little is known about the mechanisms by which human melanocytes locate at their suitable positions during embryogenesis. We took biopsies from 4 different sites (scalp, back, abdomen and right sole) of the aborted fetuses under the university approval. The following results were obtained in vivo using frozen sections. Gp100 expression comes earlier than tyrosinase, DCT and MART-1. Fetal melanocytes in hair follicles are supplied not from dermis but via epidermis. In palmoplantar areas, melanocytes originally localize around eccrine sweat gland ducts. In vitro using cultured fetal melanocytes from four different sites, those cells were not stained positively for HMB-45, αPEP-7h, αPEP-8h and Melan-A, although those were stained positively in vivo. Only nestin was positive for the cultured melanocytes. However, when co-cultured with normal human adult keratinocytes and fibroblasts, those cells were stained positively for HMB-45. We further investigated gene profile differences among the fetal melanocytes from the 4 different anatomical sites. The results indicate the fetal melanocytes were topographically diverse, especially between sole-derived melanocytes and the other three sites-derived melanocytes. We conclude that cultured fetal melanocytes had the immature phenotype although microarray results indicate the heterogeneity of melanocytes derived from the different sites of body and that cultured fetal melanocytes gain the expression levels of gp100 when co-cultured with keratinocytes and fibroblasts.

OP-13

Biological and biochemical activities of low-molecular-weight polyphenol (Oligonol) in melanocytes

Kazuya Hagiwara, Masae Ohkura, Tokimasa Hida, Akihiro Yoneta, Kenji Yanagisawa, Toshiharu Yamashita Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan

Oligonol is a lychee-derived low-molecular-weight polyphenol, which reportedly can be absorbed into the body more efficiently than conventional polyphenols. Anti-oxidant and anti-apoptotic activities of Oligonol have been reported in various cell systems: however, its biological and biochemical activities in melanocytes have not yet been elucidated. Here, we present the effect of Oligonol on ROS production and melanogenesis in human epidermal melanocytes, pigmented MM418 and non-pigmented SK-mel-24 melanoma cells. The amount of ROS production after antimycin-A treatment and Oligonol's effect on melanogenesis were analyzed by cellular fluorescence using DCF reagent and dopa reaction, respectively. As a result, Oligonol suppresses antimycin-A mediated ROS production in melanocytes and melanoma cells as efficiently as arbutin. Dopa activity was reduced when human melanocytes were cultured in the presence of arbutin or Oligonol. These results suggest that Oligonol suppresses ROS-mediated cytotoxicity and tyrosinase activity in melanocytic cells, and that topical application of Oligonol might prevent aging-associated skin changes including senile lentigo.

OP-14

Establishment of a novel mouse model for de novo melanoma

Mayuko Y. Kumasaka, Ichiro Yajima, Machiko Iida, Masashi Kato Department of Biomedical Sciences, Unit of the Environmental Health Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi, Japan

Human carcinoma is categorized to two types. One is multi-step carcinoma that arises from a pre-existing benign lesion, and the other is de novo carcinoma that arises without a pre-existing lesion. Human melanoma develops from pre-existing benign lesions (multi-step melanomagenesis) and in the absence of benign lesions (de novo melanomagenesis). Since most melanoma develops de novo and the patients have shorter survivals and a higher percentage of metastasis, the development of effective therapies has been an important theme. However molecular mechanisms of de novo melanomagenesis are still largely unknown.

RET-transgenic mice (RET-mice) spontaneously develop systemic skin melanosis, benign melanocytic tumors and melanoma stepwise. In this study, we newly established RET-mice with heterozygously deleted Ednrb[Ednrb (+/­);RET-mice] and analyzed their melanocytic tumors. Surprisingly, more than 80% of primary tumors were malignant in Ednrb (+/­);RET-mice. Further analysis showed Ednrb (+/­);RET-mice have a shorter life span and a higher lung metastasis ratio than RET-mice after tumor development suggesting a possibility that the melanoma developed in Ednrb (+/­);RET-mice is de novo melanoma.

OP-15

c-RET-associated signaling and melanomagenesis in oncogenic RET-carrying transgenic mice and human cell lines

Ichiro Yajima1, Yuichiro Ohshima1,2, Kozue Takeda1, Machiko Iida1, Mayuko Kumasaka1, Yoshinari Matsumoto2, Masashi Kato1 1Department of Biomedical Sciences, Units of Environmental Health Sciences, College of Life and Health Sciences, Chubu University, Japan; 2Department of Dermatology, Aichi Medical University School of Medicine, Aichi, Japan

The incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer. Since melanoma is the most serious skin cancer, malignant melanoma is a threat for human life. We previously reported that constitutively activated RFP-RET-carrying transgenic mice (RET-mice) spontaneously develop malignant melanoma. In this study, we found that endogenous expression levels of c-Ret and c-Ret-associated genes in malignant melanomas from RET-transgenic mice were significantly upregulated compared with those in benign melanocytic tumors suggesting that endogenous c-Ret/Gdnf are involved in murine melanomagenesis in RET-mice. We then observed that expression levels of some c-RET-associated genes in human malignant melanoma cell lines were higher than those in primary cultured normal human epithelial melanocytes (NHEM). Our results suggest that c-RET-associated siganlng is associated with the pathogenesis of malignant melanoma.

OP-16

TPA inhibits melanoma growth by inactivation of STAT3 through PKC-activated tyrosine phosphatase(s)

Naoko Sumita1, Masahiro Oka1, Masanobu Sakaguch1, Tetsushi Iwasaki2, Toshinori Bito1, Ken-ichi Sato3, Yasuo Fukami2, Chikako Nishigori1 1Division of Dermatology, Department of Internal Related, Kobe University Graduate School of Medicine, Kobe, Japan; 2Research Center for Environmental Genomics, Organization of Advanced Science and Technology, Kobe University, Kobe, Japan; 3Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kyoto, Japan

The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by siRNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCα, PKCδ, and PKCε. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Since TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).

OP-17

Effect of Riluzole, Caffeine, and Forscolin on development and growth of melanoma in metabotropic glutamate receptor 1 transgenic mouse

Yoko Funasaka1, Abdel-Daim Mohamed2, Seiji Kawana1, Chikako Nishigori2 1Department of Dermatology, Nippon Medical School, Tokyo, Japan; 2Division of Dermatology, Kobe University Graduate School of Medicine, Kobe, Japan

We have previously shown that ectopic expression of metabotropic glutamate receptor subtype 1 (mGluR1) in melanocytes is essential for both development and in vivo growth of melanoma in transgenic mice. Riluzole is a glutamate release inhibitor and is approved by FDA for the treatment of amyotropic lateral sclerosis. Caffeine is a non-specific cyclic AMP-phosphodiesterase inhibitor and is known to show anticancer activity in photocarcinogenesis and B16 melanoma cells. Forscolin is an activator of adenyl cyclase and prevent photocarcinogenesis by enhancing eu-melanin synthesis. We examined the effect of these chemicals on melanoma formation and growth using mGluR1 transgenic mice. Riluzole inhibited development and growth of melanoma, however Caffeine and Forscolin enhanced them. As cAMP is known to phosphorylate ERK1/2 in mGluR1 signaling, we examined pERK by immunohistochemistory. Caffeiine and Forscolin enhanced pERK expression, however, the enhancement of pERK by Forscolin was inhibited by MEK inhibitor, U0126 with the inhibition of melanoma development. These results indicate that Riluzole inhibits development and growth of melanoma in mGluR1 expressing melanoma, however, Caffeine and Forscolin enhance them via activation of ERK1/2 signaling.

OP-18

Polyclonality of BRAF mutations in primary melanoma and the selection of mutant alleles during progression

Jingrong Lin1, Yasufumi Goto2, Hiroshi Murata2, Toshiaki Saida2, Minoru Takata3 1Department of Dermatology, The 1st Affiliated Hospital of Dalian Medical University, Dalian, China; Department of Dermatology, Shinshu University School of Medicine, Matsumoto, Japan; 3Department of Dermatology, Okayama University Graduate School of Medical, Dentistry and Pharmaceutical Sciences, Okayama, Japan

Background: Oncogenic BRAF mutation had been considered to be a founder even in the formation of melanocytic tumors; however, we recently argued against this notion by showing marked polyclonality of BRAF mutations in acquired melanocytic nevi (Lin et al., J Nat Cancer Inst, 2009; 101:1423-7). Here, we tested whether similar heterogeneity of BRAF mutations exists in primary melanomas.

Methods: We isolated and sequenced single melanoma cells from five primary melanoma tissues using antibodies against human high molecular weight-melanoma associated antigen. We also examined 10 primary melanomas by the sensitive Mutector assay detecting the BRAFV600E mutation, as well as cloning and sequencing of separated alleles. Furthermore, we estimated the frequency of BRAF mutant alleles in paired samples of primary tumor and recurrence or metastasis in three patients.

Results: Single-cell mutation analyses revealed that four of five primary melanomas contained both BRAF-wild-type and BRAF-mutant tumor cells. Tumor heterogeneity in terms of BRAF mutations was also shown in eight of 10 primary melanomas. Selection of BRAF mutant alleles during progression was demonstrated in all three patients.

Conclusion: The results underscore the importance of BRAFV600E-selective drugs in the treatment of melanoma and of genotyping metastatic tumors to identify patients who are likely to respond.

OP-19

Lack of inverse correlation between IGFBP7 expression and BRAFV600E in malignant melanoma in Japanese population

Koji Chiyomaru, Tohru Nagano, Yoko Funasaka, Chikako Nishigori Division of Dermatology, Department of Internal Related, Kobe University Graduate School of Medicine, Kobe, Japan

Recent report shows that oncogenic BRAF could induce senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Whether there is the expression of IGFBP7 or not, the report explained the difference between malignant melanomas and pigmented nevi (both have high frequency of BRAFV600E), i.e. lack of IGFBP7 expression is closely related to melanomagenesis in BRAFV600E melanoma. Whereas more recently, it was reported that IGFBP7 is not required for BRAF-induced melanocyte senescence. Currently, the relation between BRAF and IGFBP7 remains a matter of debate. In our study, we try to elucidate the relation between BRAFV600E and IGFBP7 expression in both malignant melanomas and pigmented nevi in Japanese population.

Twenty-one cases of melanomas and 27 cases of nevi were investigated. Using a microdisection method, we extracted the DNAs from the paraffin embedded samples and detected BRAFV600E mutation by direct sequence analysis. Immunohistochemical analysis also performed to stain IGFBP7 protein.

The number of BRAFV600E melanoma with IGFBP7 expression (19.0%) is more than that without IGFBP7 (9.5%). And the number of BRAFV600E pigmented nevus with IGFBP7 expression (23.1%) is more than that without IGFBP7 (3.8%).

Our results indicate that lack of IGFBP7 expression is not involved in BRAFV600E melanomagenesis.

OP-20

Analysis of serum 5-S-cysteinyldopa level in the patients with malignant melanoma

Yoshimasa Nobeyama, Hidemi Nakagawa The Jikei University School of Medicine, Minato-ku, Tokyo, Japan

The level of serum 5-S-cysteinyldopa is in widespread use as a tumor progression marker in malignant melanoma. Now we analyzed the association between serum 5-S-cysteinyldopa level and clinical course in the 73 patients with malignant melanoma referred to the Jikei University School of Medicine from 2001 to 2010.

OP-21

Noble melanoma discrimination index derived from hyperspectral data

Haruka Okutani1, Atsushi Nakamura1, Takashi Nagaoka1, Yoshio Kiyohara2, Takayuki Sota1 1Waseda University, Tokyo, Japan; 2Shizuoka Cancer Center, Shizuoka, Japan;

ABCD-rule of dermoscopy has increased the diagnostic accuracy of differentiating melanoma from other pigmented skin lesions. However, some training is required in using it efficiently and the diagnosis based on morphology is to a large extent subjective as with any clinical diagnosis. Morphology comes from pigmentation of skin, i.e. pigment molecular species and their concentration. Optical spectra reflected from skin carry information on them. An essential feature of melanoma is variegation in morphology of the lesion, which is equivalent to spatial variegation in spectrum over the lesion. Hyperspectral imaging technique enables one to measure hyperspectral data (HSD) within a short period, which consist of both spatial and spectral information. In this paper, we propose a single melanoma discrimination index derived from HSD using entropy concept and demonstrate its usefulness. Correspondence between histopathological examination and value of index will be also discussed.

OP-22

Efficacy of dendritic cell vaccination therapy for advanced melanoma

Akihiro Yoneta, Takahiro Nishizaka, Toshiharu Yamashita Department of Dermatology, Sapporo Medical University School of Medicine, Sapporo, Japan

The establishment of an effective therapy against advanced melanoma remains a major endeavor. Although various kinds of chemotherapies and immunotherapies have been tried, to date limited efficacy has been observed. Herein, we report a case of advanced melanoma successfully treated by peptide-pulsed dendritic cell vaccination therapy. A 67-year-old woman with a lentiginous melanoma on her right sole visited our clinic in May 2005. The tumor was resected and a sentinel lymph node biopsy was performed (tumor thickness: 12 mm, pT4bN2aM0, Stage IIIb). After adjuvant therapy with DTIC/vincristine/nimustine hydrochloride/interferon-β was administered three times, the tumor metastasized to the right external iliac lymph nodes, and then to the lung and liver in June 2006. A combination of IFN-β and dendritic cell vaccination therapy using gp100, MAGE-1 and NY-Eso-1 peptides was initiated in June 2006. The spots on the lung and liver gradually decreased in size and finally disappeared. Levels of expression of MHC class I and gp100 of the metastatic tumor cells were higher than those of the primary ones. After treatment, depigmented spots appeared on the patient's face and extremities. These results suggest that expressions of both MHC class I molecules and melanoma-associated antigen on the metastatic tumor cells determine the efficacy of the immunotherapy against metastatic melanoma.

OP-23

Gene delivery to melanoma cells by combination of ultrasound and phospholipid microbubbles

Reiko Naito1,2, Loreto B. Feril Jr2, Yoshimi Harada2, Katsuro Tachibana2, Juichiro Nakayama1 1Department of Dermatology, Fukuoka University School of Medicine, Fukuoka, Japan; 2Department of Anatomy, Fukuoka University School of Medicine, Fukuoka, Japan

Objective: Gene therapy has a potential in the treatment of melanoma and gene transfection by ultrasound is one method. However, the efficiency is still very low. Microbubbles improve transfection efficiency of ultrasound-mediated gene delivery (sonotransfection). We have created microbubbles made from phospholipid and perfluoropropane gas and investigated the possible use of this microbubbles for sonotransfection.

Methods: Microbubbles were prepared from phospholipid suspension and perfluoropropane gas. The melanoma cells (C32 cell line) were seeded. Phospholipid microbubbles and plasmid DNA (pEGFP-N1) were added to the medium of C32 cells before sonication under various conditions. To determine the transfection rates, microphotographs were taken and cells expressing GFP after sonication were counted and divided by the total number of cells visualized. Cell viabilities were also analyzed by MTS assay.

Results: C32 cells sonicated with pEGFP and phospholipids microbubbles showed GFP expression by fluorescent microscopy. The optimal sonication condition for sonotransfection was at oscillation frequency of 1.011 MHz, intensity (ISATA) of 0.11 W/cm2, burst frequency of 0.5 Hz with 25% duty factor and sonication for 20 s. Cell viability under this optimal condition was 50% or higher.

Conclusion: Combination of ultrasound and phospholipids microbubbles could be used for sonotransfection to melanoma cells.

OP-24

Coat color and Mc1r variation in Izu Island populations of the large Japanese wood mouse (Apodemus speciosus)

Morihiko Tomozawa, Hirotake Ono Department of Biology, Keio University, Tokyo, Japan

Characterizing coat color variation and its causative genetic variation in wild populations will be useful to understand not only the molecular mechanism but also the association between local adaptation and genetic variability. We assessed coat color variation of the large Japanese wood mouse (Apodemus speciosus) on Izu Islands and a phylogenetically related population on Sado Island. The mice on one of the Izu islands (Miyake Island) showed significantly darker coat color than Sado population. The difference of coat color associated with changes in lightness of the pigments in light colored region in a hair and changes in proportion of the light colored region to an entire hair. Since the Mc1r and Agouti genes are the strong candidates of this phenotypic change, the nucleotide sequences of these genes were compared between the Miyake population and a neighbor island population with different coat color. We found a mutation C293T in the Mc1r coding region in Miyake population, which changes an amino acid from threonine to lysine, while no diagnostic mutation was found in the Agouti. The C293T mutation was not found from the neighbor population. In addition, phylogeographic analyses suggested a close relationship between the populations. The Mc1r C293T mutation and/or mutations linked to the C293T may be associated with the dark coat color in Miyake Island.

OP-25

Association of the P gene polymorphism with skin pigmentation phenotypes in Japanese women

Yuko Abe1, Hozumi Yutaka1, Tamiya Gen2, Tamio Suzuki1 1Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan; 2Advanced Molecular Epidemiology Research Institute, Yamagata University School of Medicine, Yamagata, Japan

Oculocutaneous albinism (OCA) is a group of autosomal recessive hypopigmentary disorders of the skin, hair and eyes. Mutations in four genes have been reported in different subtypes of human OCA. OCA2 is caused by mutations in the P gene, which is the most common type of albinism and accounts for approximately 50% of OCA worldwide. On the other hand, in the Japanese OCA patients, type 2 patients are <10%. As we previously reported, the p.A481T is a relatively common SNP of the P gene, not only in patients with OCA2, but also in normally pigmented Japanese (allele frequency (q) = 0.12) (Suzuki et al. 2003). Furthermore, the transfection experiment using p null mouse melanocytes with p.A481T human P cDNA revealed a decrease in functional melanogenesis activity to approximately 70% of the level of the normal allele, indicating that this SNP should cause OCA as a pathologic allele (Sviderskaya et al, 1997). However, there have been few statistical analyses on the relationship between amount of melanin in human skin and the SNP. Then, we investigated how the SNP, p.A481T, would influence the melanogenesis in the human skins among 432 Japanese women.

The skin color was measured using a portable spectrophotometer (CM-2600d; Konica Minolta Sensing Inc., Osaka, Japan) with an analysis program (CM-SA, Konica Minolta Sensing Inc.). As a new unit containing the spectrometer and the program has been developed for measurement of melanin content in Japanese skin. Our study showed that the mean melanin index of heterozygotes of the Thr481 allele significantly decreased from that of homozygotes for wild type (Ala481).

In conclusion, it is suggested that the p.A481T may be one of significant SNPs on P gene for skin pigmentation phenotypes in Japanese women.

OP-26

Mutation analyses of three patients with dyschromatosis symmetrica hereditaria

Masahiro Hayashi1, Ichidai Murata1, Yutaka Hozumi1, Yoshihiko Mitsuhashi2, Yasuyuki Fujita3, Tamio Suzuki1 1Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan; 2Department of Dermatology, Tokyo Medical University, Tokyo, Japan; 3Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Dyschromatosis symmetrica hereditaria (DSH) shows an autosomal dominant pattern of inheritance with high penetrance, which is characterized by hyper- and hypopigmented macules on the face and dorsal aspects of the extremities. We described that the responsible gene for DSH isadenosine deaminase acting on RNA1 gene (ADAR1) in 2003. More than 90 mutations have been reported from East Asian countries to date. These data indicate that ADAR1 is responsible for DSH not only in Japanese but also in other ethnic groups. Here we present three Japanese cases who have been clinically diagnosed as DSH and unexpected results of ADAR1 mutation.

Case 1 and 2 are 5 and 10 year-old unrelated boy, respectively. They demonstrated typical rash of DSH on his dorsal aspect of hand and cheek since they were about 2 years old. SSCP and direct sequence analyses of ADAR1 revealed a novel missense mutation c.T2878A (p.Y960N) in case 1 and a recurrent missense mutation c.C2746T (p.R916W) in case 2. Case 3 is a 9 year-old boy. He also showed hyper- and hypopigmented macules on the dorsal aspects of extremities and cheek since several years ago. However, we could find any pathological mutation neither in SSCP nor sequencing of all exons and flanking regions of ADAR1. We also examined ADAR1 mRNA expression level in peripheral blood cells from the patient and his family using real-time quantitative RT-PCR method. Interestingly, amounts of the expression in this family were similar levels as healthy control, indicating that the promoter region of ADAR1 may not be affected. These results suggest that alternative genes might be implicated for the development of DSH.

OP-27

Five novel ADAR1 mutations in patients with dyschromatosis symmetrica hereditaria and a known mutation in a patient developing her skin manifestation after viral encephalitis

Michihiro Kono1, Taisuke Kondo1, Tamio Suzuki2, Mari Kaneda3, Sakuhei Fujiwara4, Akihiko Shibaki5, Amarillis Sanchez-Valle6, Hirotaka Akita7, Joseph Lam8, Yasushi Tomita1 1Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan; 3Department of Dermatology, Osaka University Graduate School of Medicine, Suita, Japan; 4Department of Dermatology, Faculty of Medicine, Oita University, Yufu, Japan; 5Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan; 6Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA; 7Department of Dermatology, Fujita Health University School of Medicine, Toyoake, Japan; 8Department of Pediatrics and Department of Dermatology, University of British Columbia, Vancouver, BC, Canada

Dyschromatosis symmetrica hereditaria (DSH) is a pigmentary genodermatosis of autosomal dominant inheritance. It is characterized by hyperpigmented and hypopigmented macules on the backs of the hands and feet. We had clarified that the disease is caused by a mutation of the RNA-editing enzyme of adenosine deaminase acting on the RNA 1 (ADAR1) gene in 2003. The pathomechanism has not been clarified yet. We investigated the ADAR1 mutations causing DSH in three familial and three sporadic cases with DSH. And we clarified that five novel pathological mutations such as p.K1167fsX1178, p.R534X, p.N398fsX401, p.M1034I and p.D1147fsX1152, and a known mutation of p.C893X of a patient developing her skin manifestation after viral infection. The patient with p.C893X in the gene is 4 year old Hispanic female and was healthy until 11 month of age at that time she had seizure after having viral exanthema for a few days and stopped sitting up. She was diagnosed as post viral encephalitis. After 1 month, at 12 months of the age, she started developing hypopigmented macules on the extremities. We cannot account that the mutation directly causes his developing delay, because our previously-reported Japanese patient with p.C893X was healthy. We have shown that DSH may be caused by the defect of 150 kDa ADAR1 that is induced by interferon which is easily increased with any virus infection. Therefore it may be very suggestive observation to suspect the pathomechanism on DSH.

OP-28

Analysis of MC1R gene in patients of numerous freckles on sun-exposed skin

Natsumi Hama, Hiroshi Fujiwara, Masaaki Ito Department of Dermatology, Niigata University School of Medicine, Niigata, Japan

Human MC1R gene, which encodes melanocortin 1 receptor, a.k.a. melanocyte-stimulating hormone receptor, is a single exon gene, and of 954 bp of its CDS. MC1R is a highly polymorphic gene, and is associated with increased risk of developing skin cancers, such as malignant melanoma, basal cell carcinoma; Most of the polymorphisms are well documented in Caucasians, however, the reports were scant in Japanese patients. We sequenced entire coding region of MC1R gene in 16 Japanese patients who developed numerous freckles on sun-exposed area. Among them all the patients revealed R163Q polymorphism, 12 patients with 1583 A to A/G, which is a silent polymorphism, one with V92M, and the other one with V92M, L136V and I155T; L136V was an unknown mutation. The biopsies of pigmented lesions disclosed a hyperkeratotic epidermis and basal pigmentation; none suggested malignant change. Some of our patients also possessed malignant melanoma or non-melanoma skin cancer. Although the familial tendency of skin cancer was not clear in our series, the MC1R polymorphism can be related to the development of skin tumors in Japanese.

OP-29

New approach to repigmentation of vitiligo: treatment with CUSA, seed-grafting and narrowband UVB therapy

Katsuhiko Tsukamoto, Takamitsu Matsuzawa, Miyuki Matsuzawa, Asuka Komatsu, Atsushi Osada Department of Dermatology, Yamanashi Prefectural Central Hospital, Yamanashi, Japan

Introduction: Vitiligo vulgaris is a common disease throughout the world. The most frequent treatment is PUVA, narrowband UVB, and topical steroids. But against stable refractory vitiligo, various other surgical techniques have been developed. To overcome the deficiencies of current surgical treatment, we have discovered a new method using ultrasonic abrasion, seed-grafting and narrowband UVB therapy.

Methods: We use the CUSA Excel or the Sonopet UST-2000. A local or systemic anesthetic is administered, and then, the operator presses the tip of the handpiece onto the epidermis of the vitiligo lesions and moves it around in brush like motions. A very thin piece of epidermal graft skin is taken from a normally pigmented donor site. The graft skin is then minced into fragments <1–2 mm2. These minced skin pieces are placed onto the epidermal-abraded vitiligo lesions and are covered with wound dressing. The wound dressing is removed 5–7 days after grafting. Narrowband UVB treatment is started 1 month after grafting.

Results: Two patients with stable segmental vitiligo were treated using this method. Each operation was very successful, and excellent repigmentation was observed at all grafting sites without leaving a scar.

Conclusion: Ultrasonic abrasion easily and safely removes only the epidermis, even on spotty lesions or intricate regions. Epidermal seed-grafting can cover more area than sheet-grafting, and subsequent narrowband UVB treatment can enlarge the area of pigmentation with coalescence of adjacent grafts. Our results show that this new method is an easy, safe, and very effective treatment for stable vitiligo.

OP-30

The evaluation of recent therapy of vitiligo vulgaris

Takakazu Shibata Shibata Clinic of Dermatology, Osaka, Japan

I examine about 7000 patients of vitiligo vulgaris every year in my clinic. This time I’ll present diagnoses, classifications, results of blood examinations,methods and effects of therapyies for 1000 patients of vitiligo vulgaris for about 6 years.

I also present methods, risk and effects of narrow-band UVB (DERMARAY-400), excimer lamp (VTRAC) therapies. Fuethermore I analyze the effect of various ointments, vitamin D3, tretinoin tocoferil, tacrolimus, prostaglandin and ViTiX.

I propose the 4 conditions of better repigmentation of vitiligo vulugaris after therapies. Face,child,early phase and smaller lesions (FACES) are the 4 key-words.

OP-31

Serum 5-S-cysteinyldopa levels in psoriasis and vitiligo patients undergoing narrowband ultraviolet B phototherapy

Kanako Kikuchi1, Kazumasa Wakamatsu2, Yayoi Tada1, Shosuke Ito2 1Department of Dermatology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; Department of Chemistry, Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan

Narrowband UV-B (NBUVB), a new light source, is effective for treating generalized psoriasis and vitiligo without the use of psoralens. First, we measured the time course of changes in serum levels of the melanin-related metabolite 5-S-cysteinyldopa (5-S-CD) in psoriasis patients undergoing NBUVB phototherapy. Eleven Japanese patients with generalized psoriasis vulgaris received NBUVB treatment five times per week, with an initial dose of 0.1 J/cm2. The dose was increased by 10% to 20% per treatment for more than 20 treatments. Serum samples were taken before, and 3, 7, 10, 14, 28 days after the phototherapy. After 4 weeks of NBUVB treatment, nine of 11 patients were in remission. Two patients were dropped from the investigation before day 28 because of other complications. The Mean level of 5-S-CD in serum was significantly elevated on days 7, 10 14, and 28 compared with that before the phototherapy. The serum 5-S-CD level peaked on day 10. We also treated vitiligo outpatients with NBUVB twice a week. We measured serum 5-S-CD of vitiligo patients undergoing NBUVB phototherapy. Serum 5-S-CD level of vitiligo patients was significantly lower than that of normal controls or psoriasis patients. Also serum 5-S-CD level of psoriasis patients was significantly higher than that of normal controls. Frequent sun-exposure of psoriasis patients as home-remedy might explain this phenomenon.

OP-32

A new therapy for vitiligo by Q-switch alexandrite laser irradiation

Mayuko Araki, Takafumi Etou Tokyo Teishin Hospital, Tokyo, Japan

We report a 80-year-old woman who suffered from enlarging vitiligo on face. She had been treated with topical steroid and vitamin D analogues for twenty years, but they had not cause color. However Narrowband UVB therapy is presently one of the most effective therapies for vitiligo, it may not be helpful in repigmenting white patches, particularly if they are applied in the late stages of the disease.The main goal of treating vitiligo is to improve appearance, so we decided to treat hyperpigmented remaining area among vitiligo with Q-switced alexandrite laser, in order to improve the color contrast between vitligo and normal skin. After therapy of four times laser irradiation, the color contrast was completely improved, and also her Quality of Life was dramatically improved.

Poster presentation

PP-1

Skin conditions of patients of pollen-induced allergic diseases such as Kawasaki disease etc., Parkinson disease patients and hearing-impaired persons who are supposed to be weak in melanin (a biodefense substance) synthesis & metabolism systems (MSMS), and melanocyte activity – increased activity of search for useful materials to activate MSMS and utilization of the fruits of the exploration–

Akira Awaya Dermatology & Epidemiology Research Institute (DERI), Yokohama, Japan

Background: Since 2002, the author et al. have obtained the melanocyte immunological findings that ‘resistance or sensitivity to allergic diseases (below, Allg) correlates with more or less numbers of nevi.’ i.e., ‘nevi (forming activity) are phenotypic characters (predisposition) of resistance to Allg such as Kawasaki disease, allergic rhinitis, etc which are pollen-induced diseases (PID)’, and gotten the epidemiological observations in each patient association etc. that ‘individuals of Parkinson's disease (PD) and deafness whose skin conditions of head and neck are soft, and who hardly exhibit nevi and are inactive at nevi-forming systems, are a large majority of these patients’, and reported at each conference etc.(M & I, 47 (1), 101-, 2003) by proposing that these diseases are syndromes of weakness of MSMS based on common genetic background.

Experiments & Discussion: The author examined the response to UV of synthesized pyrimidine compounds, eiwanei·A1A·MS-818, etc. at the beginning by using HaCaTcells and B16 melanoma cells. Although A1A etc. had been shown to be weak at the other use, we found newly their neurotrophic activity, and further elucidated their promoting effects on restoration & regeneration, and wound healing in neural injuries, muscle injuries, cerebral ischemic injuries, bone fractures, organ injuries, etc. by animal tests cooperatively done with many Drs, and then he gave A1A etc. a position of a new category of medicine by defining them as ‘growth factor potentiators’. For the purpose of acquisition of resistance to Allg, delaying the onsets and development of PD and deafness, the author has been recommending practicing the daily way to keep fit such as ‘to activate MSMS’, and ‘being exposed to sunlight, skin rubbing, swimming and taking a sand bath’. It is essential for us to conduct intensive validation, such as cohort long-run follow-up epidemiological studies of protective benefit of UV exposure (irradiation) against Allg in general from childhood to adults, and back-up animal studies, etc. The search for whitening ingredients originated from huge amounts of herbal materials is the main target in cosmetic companies, but at the same time substances promoting MSMS have been found and so, the application of these components through skin tests in efficacy is greatly expected as preventive agents for Allg etc. which the author has been intending them.