Article first published online: 1 SEP 2006
Pigment Cell Research
Volume 19, Issue 5, pages 490–548, October 2006
How to Cite
(2006), Abstracts. Pigment Cell Research, 19: 490–548. doi: 10.1111/j.1600-0749.2006.00339_2.x
- Issue published online: 1 SEP 2006
- Article first published online: 1 SEP 2006
IS01 - DERIVATION OF MELANOCYTE PRECURSORS FROM EMBRYONIC STEM CELLS AND THEIR DIFFERENTIATION IN VIVO
Tsutomu Motohashi, Hitomi Aoki, Kairi Chiba and Takahiro Kunisada.
Department of Tissue and Organ Development, Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Yanagido, Gifu 501-1194, Japan
Generation of specific cell types from mouse embryonic stem (ES) cells usually reflects their normal development in vivo. We have recently shown that melanocytes, which are the exclusive descendant of neural crest cells, were differentiated from mouse ES cell by the culture system using stromal cells. Melanocyte precursors could be identified as c-Kit and/or Sox10 positive cells prospectively by flow cytometry. In this culture condition, some of the purified cells showed potentials to differentiate into neurons and glial cells, indicating that these cell populations comprise multipotential neural crest-like stage. When transplanted in vivo, ES cell derived precursors differentiated into mature melanocyte in uveal part of the adult eyes. These results suggest that the present melanocyte precursors derived from ES cells were the outcome of normal developmental pathway and likely to be replaceable with normal melanocytes in vivo.
IS02 - A SENSITIZED MOUSE MUTAGENESIS SCREEN FOR NOVEL LOCI REGULATING MAMMALIAN NEURAL CREST DEVELOPMENT
Dawn Watkins-Chow, Debra Silver, Stacie K. Loftus, Ivana Matera, Denise Larson, Kristina Buac, Cecelia Rivas, Eugene Elliot and William J. Pavan.
Genetic Disease Research Branch, National Human Genome Research Institute, National Institutes of Health, Betheda, MD, USA
A mouse mutagenesis program is in progress to screen for mutations disrupting mammalian neural crest cell development and to develop an archived resource of mutagenized mice. The focused phenotype screen has been designed to detect mutations specifically affecting melanocyte and peripheral nervous system development. Progeny of N-ethyl-N-nitrosourea (ENU) treated mice are being bred to Sox10LacZ/+ mice (Britsch et al.,) carrying a disruption in a transcription factor important for neural crest cell development. These mice manifest sub clinical neural crest defects due to haploinsufficiency for SOX10. The sensitized screen uncovers mutations that act synergistically with Sox10LacZ/+ resulting in clinically visible phenotypes such as white coat color spotting. Additionally, third generation embryos are being generated in a backcross to screen for embryonic phenotypes that alter expression of the LacZ reporter gene in Sox10 expressing cells. Our three-generation breeding strategy utilizes two different mouse strains, BALB/cJ and C57BL/6J, to facilitate mapping of both dominant and recessive phenotypes and allows for subsequent recovery of lethal phenotypes. To date, we have identified two heritable phenotypes from the dominant screen of 100 pedigrees and five heritable mutations from a recessive embryonic screen of 60 pedigrees. The phenotypes observed in the embryonic screen include ectopic expression of Sox10-LacZ, abnormal glial cell patterning and migration, and loss of Sox10-LacZ expression in subsets of peripheral nervous system cell lineages. None of the loci localize to genes for the major mouse spotting mutants, demonstrating the feasibility of this approach for identifying novel loci regulating neural crest development.
IS03 - ROLES OF TFIID SUBUNITS TAF4 AND TBP IN MELANOCYTES IN VIVO
Gabrielle Mengus1, Dominique Kobi1, Anas Fadloun1, Lionel Larue2 and Irwin Davidson1.
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, 67404 Illkirch, France;
2Developmental Genetics of Melanocytes, UMR CNRS 146, Institut Curie, 91405 Orsay France
General transcription factor TFIID comprises the TATA-binding protein TBP and 14 TBP-associated factors TAFs. We have previously shown that the TAF4 subunit plays an important role in retinoid and TGF-βsignalling in fibroblasts and EGF signalling in basal keratinocytes of the mouse epidermis where it has tumour suppressor activity. Given its important role in controlling cell proliferation in these different tissues we have addressed TAF4 function in the melanocyte lineage where it has been inactivated the using the Tyr-Cre transgenic line. Taf4mel-/- mice show diminished pigmentation associated with a white belly phenotype suggesting that TAF4 is required for proper proliferation and/or migration of melanocytes. We have also established melanocyte cultures from taf4lox/- and taf4mel-/- mice with the aim of comparing their proliferation in vitro and identifying affected target genes. Inactivation of TBP, thought to be an essential gene, leads to a white coat phenotype indicating a complete ablation of neural crest derived melanocytes.
IS04 - SHADING IN THE FUNCTION OF ANAPLASTIC LYMPHOMA KINASE IN PIGMENT CELL DEVELOPMENT
Xueyan Yang, Jeanette Müller, Susana Lopes, Thomas J. Carney and Robert N. Kelsh.
Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
Pigment cells are important derivatives of the neural crest. Melanocytes are ubiquitous throughout the vertebrates, but the neural crest of fish, but also amphibians and reptiles, generates other pigment cell-types, including shiny iridophores. What factors underlie the choice between melanocyte and other pigment cell fates? Some factors driving melanocyte development have been identified, but the requirements for other pigment cell-types are less clear. Anaplastic lymphoma kinase (ALK) is a proto-oncogenic receptor tyrosine kinase underlying some human lymphomas, but for which the endogenous function is unknown in mammals. The zebrafish shady gene encodes an alk orthologue. Shady mutants lack iridophores, but we see no defects in any other neural crest derivative. Shady/alk expression in early embryos is ubiquitous, but by 24 h post fertilisation is restricted to some neural crest cells. Subsequently, alk expression demarcates iridoblasts and differentiating iridophores. In shady mutants, shady/alk expression in the neural crest is dramatically reduced. Iridophore development is rescued in shady mutants expressing a constitutively active ALK protein, which also drives overproduction of iridophores or their precursors. This, together with analysis of the iridophore phenotype of colourless/sox10mutants, may suggest roles for shady/alk function in iridophore specification and proliferation.
IS05 - THE ROLE OF MITF DURING EYE AND NEURAL CREST DEVELOPMENT
Kapil Bharti, Alessandro Nodari, Stefano Bertuzzi and Heinz Arnheiter.
Mammalian Development Section, NINDS, NIH, USA
Although neural crest-derived melanoblasts and neuroepithelium-derived retinal pigment epithelium (RPE) cells both depend on the transcription factor MITF for differentiation, they differ fundamentally in the response to a variety of MITF mutations with respect to cell survival and proliferation: melanoblasts usually stop proliferating and die, and RPE cells usually hyperproliferate and survive. It is conceivable that this difference is due to cell type-specific expression of distinct MITF isoforms. In fact, MITF is expressed from at least nine different promoters, six of which give rise to RNA isoforms that encode MITF proteins with different aminotermini. Here, we determined by quantitative RT-PCR assays that of the nine RNA isoforms, five are not or only barely detectable in the developing eye, three are detectable at similar levels in the developing RPE and retina, one is found predominantly in the RPE and one predominantly in melanocytes. We then generated mice in which specific isoforms were eliminated by targeted mutagenesis. These knock-out mice are currently being assessed for direct effects on eye and melanocyte development and for interactions with MITF regulatory genes such as Chx10,Vax, and Pax. These studies will give us insights into the regulation of individual MITF isoforms during eye and neural crest development and into their relative importance in cell lineage determination.
IS06 - BAC TRANSGENE RESCUE EXPERIMENTS AND POST-TRANSLATIONAL MODIFICATIONS OF THE MITF TRANSCRIPTION FACTOR
Bryndis K. Gisladottir1, Norene O’Sullivan2, Jon H. Hallsson1, Christian Praetorius1, Neal Copeland2, Nancy Jenkins2 and Eirikur Steingrimsson1.
1Department of Biochemistry and Molecular Biology, University of Iceland, Reykjavík, Iceland; 2Mouse Cancer Genetics Program, National Cancer Institute, Frederick, MD; USA
The Mitf transcription factor is a key regulator of melanocyte development and has been shown to affect differentiation, survival and proliferation. The activity of Mitf has been shown to be regulated by various different post-translational modifications including SUMOylation, ubiquitination, phosphorylation and acetylation. For example, activation of the tyrosine kinase receptor Kit leads to the MAPK-mediated phosphorylation of Mitf at Ser73 and Ser409 resulting in effects on transcription activation and stability of the protein. Furthermore, SUMOylation of Mitf affects transcription activation potential of promoters containing multiple Mitf binding sites. However, at present, the in vivo roles of these modifications are unknown. We are using BAC transgene rescue experiments to investigate the roles of all these post-translational modifications in vivo. We have used BAC recombineering to modify the post-translational modification sites in a BAC clone and generated transgenic mice on an Mitf mutant background; the BAC clone contains the entire Mitf gene, except exon 1A, and is able to rescue the phenotype of the Mitfmi-vga9 loss-of-function mutation. The phenotypes of mice carrying mutant BACs will be reported. These mice will reveal novel insights into the role of post-translational modifications in transcription factor function.
IS07 - REGULATION OF HUMAN SKIN PIGMENTATION AND RESPONSES TO UV
Yoshinori Miyamura1, Rainer Wolber2, Kazumasa Wakamatsu3, Shosuke Ito3, Christoph Smuda2, Jan Batzer2 and VincentJ. Hearing1.
1Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD, USA;
2Department of Skin Research, Beiersdorf AG, Hamburg, Germany;
3Department of Chemistry, Fujita Health University, Toyoake, Japan
We previously reported that a single one MED dose of UVA/UVB stimulates visible pigmentation of the skin but without significantly increasing the expression of melanogenic proteins or melanin content within 1 week (FASEB J 2003; 17:1177). The visible tanning of the skin resulted from a redistribution of existing melanin from lower layers of the epidermis towards the upper layers (JID 2005; 124:1326). In this study, we chronically irradiated human skin with UV-A/ UV-B, UV-A or UV-B for 2 weeks and then assessed visible pigmentation, melanin content, absorption characteristics of the skin and expression of melanogenic markers. The results demonstrate that even after chronic UV irradiation, the amounts of melanin (assessed by chemical analysis and by Fontana Masson staining) are increased only by 50% or less despite the dramatic increase of visible skin pigmentation. Similarly, the density of melanocytes in the skin, as well as the expression of melanocyte-specific markers (e.g. Mitf, Tyr and Pmel17) was increased. These results emphasize the stable nature of melanocyte density and differentiation in the skin and the important role of pigment distribution for response to UV irradiation.
IS08 - FUNCTIONAL STATUS AND TRAFFICKING PATTERNS OF THE RED HAIR COLOUR-ASSOCIATED MELANOCORTIN 1 RECEPTOR VARIANTS
José Carlos García-Borrón.
Dep. de Bioquimica y Biología Molecular B e Inmunología. Facultad de Medicina, University of Murcia, Spain
The α-melanocyte stimulating hormone receptor (MC1R) is a Gs protein coupled receptor serving key roles in melanocytes. MC1R signalling increases tyrosinase activity, promotes eumelanin synthesis, and participates in the UV tanning response. Several MC1R alleles (the RHCs) are associated with red hair, fair skin, poor tanning and increased melanoma risk. We have compared the function of the frequent RHC alleles R151C, R160W and D294H. R151C and R160W are expressed less efficiently than wild type (WT) on the plasma membrane due to intracellular retention in the endoplasmic reticulum and the Golgi apparatus. Several other second intracellular loop mutants are similarly retained, thus identifying this domain as a key determinant for correct processing. Conversely, membrane expression of D294H is higher than WT. Receptor internalization following agonist binding is similar for WT, R151C and R160W, but strongly impaired for D294H, which may account for its cell surface accumulation. Although less efficient than WT in triggering cAMP synthesis, R151C and R160W display high residual activity and show reduced desensitization kinetics. Conversely D294H is basically inactive. The rank of affinity for NDP-MSH is R151C ≅ R160W > D294H. Thus, D294H function is more severely impaired than either R151C or R160W. This shows that the phenotypic effects of RHC alleles do not correlate strictly with their degree of functional loss. Moreover, they are paradoxical in that the form with the highest degree of functional impairment is efficiently processed through the secretory pathway, whereas other forms with relatively minor functional losses are recognized as aberrant.
IS09 - STRUCTURAL MARKERS FOR PHEOMELANIN ANALYSIS: ADVANCES AND PERSPECTIVES.
Alessandra Napolitano1, Lucia Panzella1,2, Paola Manini1, Giuseppe Monfrecola2 and Marco d’Ischia1.
1Dept Org. Chem. Biochemistry;
2Dept. of Systematic Pathol., Univ. Naples, Federico II, Naples, Italy
The peculiar traits of red haired individuals, i.e. the UV susceptibility and proneness to skin cancer, have greatly contributed to the recent shift of interest from eumelanin to pheomelanin pigmentation. The relationship of loss-of-function variants of the MC1R gene with red hair colour (RHC) phenotype has been documented and the photosensitizing properties of the pigments in a variety of models have been assessed. There are, however, a number of open questions that need to be addressed. These include: the relationship of RHC phenotype with the actual nature of the pigment; the way in which pheomelanin properties might be related to the structural characteristics; whether differences in levels or even in the structure of pheomelanin pigments might account for the apparent discrepancies observed in the sun sensitivity of low phototypes RHC individuals; and finally whether is pheomelanin or its biogenetic pathway the actual determinant of the observed response. Settling of these and other issues depends on, and awaits for, a better knowledge of the origin, structure and chemistry of pheomelanins. Because of the notorious difficulties in the direct investigation of pheomelanins, analysis of specific products arising by chemical degradation of tissues remains the most rewarding approach. We previously showed that alkaline hydrogen peroxide degradation of pheomelanic tissues and synthetic pigments leads to 1,3-thiazole-2, 4,5-tricarboxylic acid (TTCA) and 6-(2-amino-2-carboxyethyl)-2-carboxy-4-hydroxybenzothiazole (BTCA) which provided the basis of an analytical procedure for identification and quantitation of true pheomelanic pigments. We have now improved and simplified the analytical procedure and have succeeded in identifying new degradation products of pheomelanin of potential use as markers. The structure of these products and their origin in synthetic and natural pheomelanins was investigated, and the potential of these markers for routine prediction of high-risk individuals and the relationship with MC1R variants alleles is being evaluated.
IS10 - MOUSE MODELS TO UNDERSTAND THE ROLE OF TELOMERES AND TELOMERASE IN CANCER AND AGING
María A. Blasco.
Telomeres and Telomerase Group, Molecular Oncology Program, Spanish National Cancer Centre (CNIO), Madrid, Spain
Telomeres protect the chromosome ends from unscheduled DNA repair and degradation. Telomeres are heterochromatic domains composed of repetitive DNA (TTAGGG repeats) bound to an array of specialized proteins. The length of telomere repeats and the integrity of telomere-binding proteins are both important for telomere protection. In addition, we have recently shown that telomere length is regulated by a number of epigenetic modifications, thus pointing to a higher-order control of telomere function. A key process in organ homeostasis is the mobilization of stem cells out of their niches. Defects in organ homeostasis are present both in cancer and in aging-related diseases. Here we will discuss that telomere length and the catalytic component of telomerase, Tert, are critical determinants for the mobilization of epidermal stem cells. On one hand, we will show that telomere shortening in the absence of telomerase negatively impacts on the mobilization of epidermal stem cells. On the other hand, Tert over-expression in the absence of changes in telomere length significantly increases the mobilization of epidermal stem cells, thus providing a mechanism by which Tert may promote tumorigenesis independently of telomere length. Finally, we will describe the generation and characterization of mice with constitutive expression of the telomere-binding protein TRF2 in the skin. TRF2 mice show a remarkable phenotype in the skin consisting of hyper-pigmentation, hair loss, dry skin, as well as increased skin tumors, all of which are reminiscent of the skin abnormalities characteristic of Xeroderma pigmentosum (XP) syndrome. We propose that the XP-like skin phenotypes described here for TRF2 mice are the result of a combination of defective DNA repair together with short telomeres, thus pinpointing to the roles of TRF2 in the context of the organism. In addition, this new mouse model demonstrates the impact of altered TRF2 expression both on cancer and aging.
IS11 - ARF, P53 AND THE CONTROL OF CELLULAR SENESCENCE IN MOUSE MELANOCYTES.
Elena V. Sviderskaya1, Takeshi Ichikawa2, Linan Ha2, Glenn Merlino2 and Dorothy C. Bennett1.
1Centre for Molecular and Metabolic Signalling, Division of Basic Medical Sciences, St George's, University of London, London, UK;
2Laboratory of Cell Regulation and Carcinogenesis, NCI, Bethesda, MD, USA
The Ink4a-Arf tumour suppressor locus encodes two unrelated cell growth inhibitors, p16Ink4a and Arf, activators respectively of Rb and p53. Human germline defects in the INK4A-ARF locus are associated with familial melanoma. We have already shown that the Ink4a-Arf locus controls cell senescence and differentiation in mouse and human melanocytes (Sviderskaya et al, J Natl Cancer Inst, 2002 and 2003). We have also examined the effect of p16Ink4a deletion, with retention of Arf, on mouse melanocyte senescence (Sviderskaya et al, Pigment Cell Res, 2004). Now we continue our studies on the relative roles of specific members of the p16/Rb and Arf/p53 pathways in the control of melanocyte senescence. We have derived melanocyte lines from mice nullizygous for Arf, with retention of p16, and also nullizygous for the p53 gene. We showed previously that Ink4a-Arf-null mouse melanocytes did not senesce, while wild-type melanocytes senesced within a month of culture (Sviderskaya et al., J Natl Cancer Inst, 2002). Interestingly, Arf-null melanocytes grow significantly faster then Ink4a-Arf-null melanocytes and readily become immortal. p53-null melanocytes, however, initially grow slowly compared to Arf-null and Ink4a-Arf-null melanocytes, although later in culture their growth becomes comparable to that of Arf-null melanocytes. Thus the activation of Arf is not entirely p53-dependent. Relations to the p16/Rb pathway will be discussed.
This work was supported by the Welcome Trust.
IS12 - FUNCTION OF THE ZINC-FINGER TRANSCRIPTION FACTOR SNAI2 IN HEALTH AND DISEASES
Carolina Vicente-Dueñas, Camino Bermejo-Rodríguez, María Pérez-Caro and Isidro Sánchez-García.
Laboratorio 13, Instituto de Biología Molecular y Celular del Cáncer, CSIC/ Universidad de Salamanca, Campus Unamuno s/n, 37007-Salamanca, Spain.
The elucidation of the molecular mechanisms that underlie disease development remains a tremendous challenge for basic science, but also represents an essential step in the development of new and more potent drugs, in particular with the emergence of disease-specific, targeted therapies. The origin of disease within a particular tissue is often impossible to determine, due to the advanced stages of many diseases when patients enter the clinic. Our knowledge about the etiology of disease is therefore derived from animal models that recapitulate human disease as accurate as possible. This article highlights the recent advances in the function of Snai2, a member of the Snail family of zinc-finger transcription factors, and discusses its possible role in disease development. Snai2 has been implicated in diseases of melanocyte development and cancer in humans. In this regard, recent data suggest many malignancies arise from a rare population of cells that exclusively maintain the ability to self-renew and sustain the tumor (i.e., ‘cancer stem cells’). Snai2 controls key aspects of stem cell function in mice and humans, suggesting that similar mechanisms control normal development and cancer stem cell properties. These insights are expected to contribute significantly to the genetics of disease and its impact on both therapy and new methods for assessing treatment efficacy.
IS13 - SIGNALLING AND TRANSCRIPTION NETWORKS REGULATING MELANOCYTE AND MELANOMA PROLIFERATION AND INVASIVENESS
Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK
The signalling pathways operating during melanocyte development are highly related to those that are deregulated in melanoma and most likely reflect the fact that while melanoblasts proliferate and migrate, melanoma cells proliferate and metastasise. We have been focused for the past few years on trying to understand the melanocyte-specific molecular mechanisms that control proliferation. Our work has revealed that a key role is played by the Microphthalmia-associated transcription factor, Mitf that acts in both a pro- and anti-proliferative fashion depending on the level of its activity. More recently we have been able to demonstrate that Mitf, through its ability to regulate the actin cytoskeleton also controls the invasive properties of melanoma cells as well as melanosome transport. The results suggest a dynamic epigenetic mechanism for controlling melanoma metastasis and suggest that melanomas will contain a subset of cells with properties reminiscent of melanocyte stem cells that are quiescent and highly invasive. At the heart of understanding how Mitf controls melanocyte and melanoma behaviour is the question of what we mean by Mitf ‘activity’ and how that activity is regulated. Our recent results on the regulation of Mitf and its downstream targets will be presented.
IS14 - ROLE OF MITF IN DIFFERENTIATION AND SURVIVAL OF MELANOCYTES AND MELANOMAS
Corine Bertolotto, Laurent Beuret, Roser Busca, Christine Chiaverini, Christophe Denoyelle, Enrica Flori, Lionel Larribère and Robert Ballotti.
Microphthalmia Associated Transcription Factor (MITF) is the master gene of melanocyte differentiation. MITF stimulates the transcription of the genes coding for the melanogenic enzymes, and thus controls the melanin pigment production by the melanocytes. Noteworthy, MITF is also involved in survival of melanocytes, since MITF loss of function leads in human and in mouse to severe pigmentation disorder due to the absence of mature melanocytes in the skin. Recently, survival and cell cycle regulation function has been ascribed to MITF through its ability to regulate the transcription of gene involved in apoptosis and cell growth such as (i) BCL2 that encode an anti-apoptotic protein involved in the control of mitochondrial apoptosis pathway and (ii) CDK2, p16 and p 21 that control cell cycle. Recently, we have undertaken an exhaustive study of the genes regulated by MITF in melanocytes and melanoma cells. MITF appears to plays a pivotal role in melanocytes differentiation program by controlling, in addition to melanin synthesis, melanosome genesis and movements as well melanocyte dendricity. Further, MITF control the expression several other genes that might be involved in the development of melanoma. Among these genes, HIF1A play a key role in angiogenesis and in cell survival. These observations make of MITF a potential target of new anti-melanoma therapy.
IS15 - BRAF SIGNALLING IN CANCER: BIOLOGY AND THERAPY
The Institute of Cancer Research, Cancer Research UK Centre for Cell and Molecular Biology, 237 Fulham Road, London, UK
The protein kinase BRAF is a component of a signalling cascade that regulates cell fate decisions in response to extracellular signals. In melanocytes this protein regulates proliferation in response to mitogenic growth factors. However, BRAF is also mutated in 70% of melanomas and in these cells it stimulates proliferation and survival. The most common mutation in BRAF in melanoma is a glutamic acid for valine substitution at position 600 (V600E); this mutation accounts for over 90% of all mutations observed in melanoma. V600EBRAF is 500 fold activated, it stimulates constitutive signalling and is required for melanoma maintenance and progression. Thus BRAF is an important therapeutic target. BRAF signalling is targeted indirectly by agents such as the HSP90 poison 17AAG and agents that inhibit the BRAF effector protein MEK. It is also the direct target in several drug discovery programmes. A key target of BRAF in melanoma is the transcription factor MITF, which is down-regulated by BRAF. MITF produces an anti-proliferative signal when expressed at high levels and its down-regulation by BRAF is essential for proliferation. Other targets of BRAF include the cell cycle inhibitory protein p16INK4A. We are currently attempting to understand BRAF biology in melanoma and to use this information to develop novel therapeutic strategies with which to treat this disease.
IS16 - DIMERIZATION AND INTRACELLULAR TRANSPORT OF KIT-LIGAND
Frédérique Paulhe1, Monique Wehrle-Haller1, Beat A. Imhof2 and Bernhard Wehrle-Haller1.
1Department of Cellular Physiology and Metabolism, Centre Médical Universitaire, University of Geneva, Switzerland;
2Department of Pathology and Immunology, Centre Médical Universitaire, University of Geneva, Switzerland
Kit-ligand (Kitl) also known as Stem Cell factor or mast cell growth factor binds to the receptor tyrosine kinase c-kit expressed on melanocytes. Kitl is required for migration, survival and proliferation of embryonic and adult melanocytes. The receptor-binding domain of Kitl is a non-covalently bound dimer expressed as a transmembrane precursor in basal keratinocytes of the skin. Kitl carries essential basolateral and ER-export determinant in its cytoplasmic domain, but it is not known when dimerization occurs. In order to analyze the mechanisms leading to Kitl dimerization we employed a fluorescence complementation assay co-expressing Kitl proteins fused to either the N-terminal or C-terminal half of yellow fluorescent protein. Using this system we analyzed the role for the extracellular, transmembrane and cytoplasmic domain for dimerization and transport of Kitl. We determined (i) that the extracellular domain of Kitl is not required for dimerization (ii) that dimerization of Kitl was dependent on the transmembrane domain of Kitl and (iii) that Kitl heterodimers expressing only one functional ER-export motif were retained in the ER. This suggests that the transmembrane domain-induced dimerization of Kitl occurs in the ER and is required for the efficient cell surface transport of Kitl dimers.
IS17 - CELL BIOLOGY OF HUMAN DISORDERS OF LYSOSOME-RELATED ORGANELLES
MarjanHuizing1, Amanda Helip-Wooley1, Wendy Westbroek1, Heidi Dorward1, Richard Hess1, Patrice Held1, Raymond Boissy2 and William Gahl1.
1NHGRI, NIH, Bethesda, MD;
2Department Dermatology, University of Cincinnati, OH, USA
Altered biogenesis of lysosome-related organelles (LROs) such as melanosomes, platelet delta and alpha granules, lamellar bodies, and lytic granules has been implicated in human pathologies. We investigated patients with rare disorders of LRO biogenesis, including Hermansky-Pudlak (HPS), Griscelli, and Chediak-Higashi (CHS) syndromes. These patients generally presented with hypopigmentation, prolonged bleeding times, and occasional other symptoms such as immunodeficiency, granulomatous colitis or pulmonary fibrosis. Apart from comprehensive clinical and genetic characterization of each patient, we collected patients’ cells for cell biological studies. These studies provided valuable insights into the complex mechanisms of LRO biogenesis, which will be reviewed in this presentation. For example, immunocytochemistry demonstrated altered localization of the melanogenic markers tyrosinase, TYRP-1, and Pmel17 in affected HPS and CHS melanocytes. Ultrastructural studies revealed that HPS-3 and HPS-5 melanocytes contain an abundance of small 50-nm tyrosinase-positive vesicles. HPS patients’ platelets revealed that only delta granules are affected, and that the numbers and shapes of alpha granules (also considered to be an LRO) are normal in this disorder. The extent of larger and aberrantly distributed melanosomes and lysosomes in CHS cells appeared to be LYST-mutation dependent. We recorded other findings from live cell imaging, 2-hybrid interactions, FRET, immunoEM, transfections, and immunoprecipitation studies which all contributed to further understanding the intriguing mechanisms of LRO biogenesis. Our growing group of patients with unclassified defects in LROs should provide additional opportunities to search for novel genes and concepts of LRO biogenesis.
IS18 - THE ROLE OF RAB GTPASES IN THE REGULATION OF MELANOSOME BIOGENESIS AND MOTILITY IN MELANOCYTES AND RETINAL PIGMENT EPITHELIAL CELLS
Miguel C. Seabra.
Molecular and Cellular Medicine, Division of Biomedical Sciences, Imperial College London, UK
Rab GTPases are critical regulators of membrane identity, organelle motility and trafficking pathways in cells. We have been studying the role of Rabs in melanosome biology in both skin melanocytes and the retinal pigment epithelium (RPE), with a focus on Rab38 and Rab27. A mutation in the small GTPase Rab38 gives rise to the coat colour phenotype ‘chocolate’ (cht), implicating Rab38 in the regulation of melanogenesis. We report that cht mutation, Rab38G19V is inactive, and that pigmentation in cht melanocytes results from functional compensation by the closely related Rab32. In cht cells treated with Rab32-specific siRNA a dramatic loss of pigmentation is observed. In these cells, we observed a block in transport of melanogenic enzymes, in particular tyrosinase, from the Trans-Golgi to melanosomes resulting in their retention in the Trans-Golgi region. This suggests that Rab38 and Rab32 regulate an early and critical step in the trafficking of melanogenic enzymes. Rab27a operates at a later step to connect melanosomes with the peripheral actin cytoskeleton in preparation for melanosome transfer. In skin melanocytes, this is performed by recruiting Melanophilin and Myosin Va to mature melanosomes. In RPE, Rab27a is proposed to recruit a related set of effectors, namely Myrip and Myosin VIIa. Our results using Myrip-specific siRNA in cultured primary RPE confirmed that the complex is indeed functional. Altogether, our studies suggest a critical role of Rab38/32 in melanosome biogenesis and Rab27 in melanosome motility of skin melanocytes and RPE cells.
IS19 - DEFINITION AND ASSESSMENT OF VITILIGO: A PROGRESS REPORT OF THE VITILIGO EUROPEAN TASK FORCE
AlainTaïeb, on behalf of the VETF: A. Alomar (Barcelona), D. C Bennett (London), M. Böhm (Münster), Y. Gauthier and A. Taïeb (Bordeaux), D. Gawkrodger (Sheffield), G. Leone, M. Pelliciotta and M. Picardo (Rome), S. Moretti (Florence), J. M. Naeyaert, K. Ongenae and N. van Geel (Ghent), M. J. Olsson (Uppsala), G. Orecchia (Pavia), T. Passeron and J. P. Ortonne (Nice), K. Schallreuter (Bradford), J. P. Wietze van der Veen and W. Westerhof (Amsterdam)
Dept of Dermatology, CHU de Bordeaux and Mauro Picardo, San Gallicano Dermatological Institute, Roma
This communication summarizes the work done by the Vitiligo European Task Force (VETF) to assess vitiligo and treatment outcomes using a system, which combines analysis of extent, stage of disease (staging), and disease progression (spreading). A workshop with patients allowed this system to be validated, after it had been tested at several European institutions and discussed at three previous workshops. This system can be easily handled in clinical practice. However, variations between scorer profiles indicate a need for training to decrease inter-observer variability. Further steps are envisaged, namely (1) build a global index including staging and spreading for the initial assessment of vitiligo patients, usable as a guidance for therapeutic indications and prognosis, which could be interpreted as an equivalent of the TNM system for cancer; (2) implement large-scale tests necessary for clinical trials (to check reproducibility and sensitivity); (3) studies of automated devices to assess extent more precisely; (4) set up a teaching tool for scoring vitiligo, which could be posted on web sites such as the ESPCR or EADV sites, and (5) set up an international conference on classifying, staging and scoring vitiligo, through the IFPCS Special Interest Group on Vitiligo.
IS20 - NEW TRENDS IN VITILIGO RESEARCH
San Gallicano Dermatological Institute, Rome, Italy
The multifactorial pathogenesis of vitiligo needs a broad and multidisciplinary approach for in vivo and in vitro study. Beside from the practical aspects of the pathogenesis study, the opinion has changed during the last few years. The emerging knowledge on cellular and molecular processes leading to the autoimmune diseases, focuses on the possible immune-mediated killing of the melanocytes in white vitiligo lesions: the occurrence of antibodies anti-melanocytic antigens and melanocyte-specific T clones was further proved by phage library and HLA/epitope tetramers methods. However, it appears more and more relevant that primitive melanocyte damage could occur. The understanding of the molecular pathways involved in cell survival and melanogenesis suggests the direct deregulation of c-Kit-MC1R/p38-ERK-CREB/MITF axis. A mouse model for MITF-dependent vitiligo has also been provided. Clinical, biochemical, cellular and molecular evidence indicate the occurrence of generalized non-melanocyte-specific alteration. An increase in the release of toxic by-products can be associated with biopterin and catecholamine metabolism, melanin synthesis, or energy production. The toxic species are able to inhibit the crucial c-Kit/kinases/MITF pathway, providing a possible link between the two events. More exhaustive knowledge of the cellular impairment is provided by the description of a initial lipidic defective arrangement in the membrane bilayer leading to exposure of new antigen, altered receptor expression and signal transduction, and impaired mitochondrial energy production. The current genetic, cellular, molecular and functional expertise represent an adequate background for a new approach, based on the metabolomic method.
IS21 - MELANIN AND THE REGULATION OF RETINAL DEVELOPMENT
Glen Jeffery, Marc Tibber and Alan Whitmore.
Institute of Ophthalmology, University College London.
The albino visual system is abnormal. There are profound deficits in specific cellular populations, abnormal pathways between the eyes and the brain and central retinal region fail to develop fully. During development many cell are retained in the cell cycle longer than they should be and divide abnormally. Further, there are abnormal patterns of cellular communication between the retinal pigment epithelium (RPE) and the neural retina. We have demonstrated that dopa which is present in the synthetic pathway of melanin in the RPE appears to play a key role in regulating critical aspects of cell division and cell cycle exit during development. Its addition during development corrects many of the abnormal facets seen in developing albino retinae.
IS22 - RESCUING ALBINO VISUAL DEFICIENCIES IN THE ABSENCE OF MELANIN
Alfonso Lavado1, Glen Jeffery2, Victoria Tovar1, Marta Cantero1, Juan Jose Lazcano1, Pedro de la Villa3 and Lluís Montoliu1.
1Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologia (CNB-CSIC), 28049 Madrid, Spain;
2University College London, Institute of Ophthalmology, Bath Street, London EC1V 9EL UK;
3Department of Physiology, University of Alcala, 28871 Madrid, Spain
Albino mammals have profound retinal abnormalities, including photoreceptor deficits and misrouted hemispheric pathways into the brain, demonstrating that melanin or its precursors are required for normal retinal development. Tyrosinase, the primary enzyme in melanin synthesis commonly mutated in albinism, oxidises L-Tyrosine to L-Dopaquinone using L-3, 4-dihydroxyphenylalanine (L-DOPA) as an intermediate product. L-DOPA is known to signal cell cycle exit during retinal development and plays an important role in the regulation of retinal development. We have mimicked L-DOPA production by ectopically expressing tyrosine hydroxylase in mouse albino retinal pigment epithelium cells. Tyrosine hydroxylase can only oxidise L-Tyrosine to L-DOPA without further progression towards melanin. The resulting transgenic animals remain phenotypically albino, but their visual abnormalities are corrected, with normal photoreceptor numbers and hemispheric pathways and improved visual function, assessed by an increase of spatial acuity. Our results demonstrate definitively that only early melanin precursors, L-DOPA or its metabolic derivatives, are vital in the appropriate development of mammalian retinae. They further highlight the value of substituting independent but biochemically related enzymes to overcome developmental abnormalities (Lavado et al. J. Neurochem. 2006, 96(4): 1201–11).
IS23 - ALBINISM AS A DEFECT OF MELANOSOME NUMBER AND MATURATION AND NOT OF MELANIN PRODUCTION: A LESSON FROM THE OA1 GENE
Francesca Giordano1, Francesco Vetrini1, Roberta Tammaro1, Katia Cortese2, Enrico M. Surace1, Andrea Ballabio1, Carlo Tacchetti2 and Valeria Marigo1,3.
1Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy;
2MicroSCoBiO Research Center and IFOM Center of Cell Oncology and Ultrastructure, Department of Experimental Medicine, University of Genoa, Genoa, Italy;
3Department of Biomedical Sciences, University of Modena and Reggio Emilia, Modena, Italy
Ocular Albinism type 1 is an X-linked form of albinism isolated to the eye. The disease causes a severe visual handicap in affected males, manifesting foveal hypoplasia, horizontal and rotatory nystagmus, strabismus, photophobia and lack of stereoscopic vision. Contrary to other forms of albinism, melanin is synthesized but melanosome biogenesis is impaired. The disease is caused by mutations in the OA1 gene encoding a protein localized in the melanosomal membrane and acting as a G-protein coupled receptor however the ligand has not been identified yet. We demonstrated that the tissue specific expression of OA1 in pigment cells lies within 617 bp upstream to the transcription start site and is regulated by MITF.
Our studies on the murine model of this disease defined ocular albinism type 1 as a defect in melanosome organellogenesis and not in melanin production. Furthermore, we unravelled that OA1 functions at two different steps of melanosome maturation controlling first the number of organelles and later their size. Finally, we recently developed a new approach to revert the mutant phenotype in pigment cells from patients bearing a splicing mutation in the OA1 gene. We successfully applied the technology of exon-skipping morpholino oligos to recover protein expression in patient mutant cells. Our data open new applications of morpholino oligos as a potential therapeutic tool for retinal pigment epithelium diseases.
IS24 - MODEL OF EARLY DEVELOPMENT OF MALIGNANT MELANOMA
Institute of Pathology, Molecular Pathology, University of Regensburg, Germany
Crosstalk between melanocytes and keratinocytes is important in human epidermis. It is known that normal melanocytic phenotype and control of proliferation of melanocytes is strictly regulated by keratinocytes via E-cadherin. Malignant transformation of melanocytes frequently coincides with loss of E-cadherin expression and the upregulation of N-cadherin. This leads to the loss of regulatory dominance by keratinocytes. Further, melanoma cells can now get into contact with fibroblasts via N-cadherin and induce e.g. MMP expression.
Previously, we could show that loss of E-cadherin expression by upregulation of the transcriptional repressor snail in melanoma cells leads to a consistently NFkappaB expression and after transiently transfection of full length E-cadherin the NFkappaB activity goes down. Furthermore, transiently transfected cytoplasmatic β-catenin into the melanoma cells leads to upregulated NFkappaB activity. Additionally, experiments show that melanoma cells down regulate endogenous N-cadherin expression after transiently transfection of full length E-cadherin. We further analyzed the effect of E-cadherin derivatives on endogeneous N-cadherin expression and revealed that the cytoplasmic domain of E-cadherin leads to down regulation of N-cadherin after stably transfection of a soluble and membrane-anchored cytoplasmatic domain into the melanoma cell line Mel Im. In summary, we conclude that loss of expression of E-cadherin induces NFkappaB activity which could lead to inhibition of apoptosis and induced proliferation of melanoma cells. Additionally, it leads to an up regulation of endogenous N-cadherin in melanoma cells and this plays an important role in regulating contacts of melanoma cells to their environment and therefore, tumorigenesis of malignant melanoma.
IS25 - USING A MOUSE MODEL TO FIND NEW RELEVANT MOLECULES INVOLVED IN CUTANEOUS MALIGNANT MELANOMA
Medical Oncology Research Program, Vall d’Hebron Research Institute, Vall d’Hebron Hospital Barcelona 08035, Spain
Even though melanoma is the less frequent skin cancer, it is very likely to metastasize and lead to fatal consequences. During the pass 30 years its incidence has increased significantly and that it has a very poor response to the currently available therapies. To date different mouse models has been used to describe cutaneous melanoma, however, most of these tumors do not have the epidermal component that characterize the conventional human melanomas. Moreover, a number of these models used chemical carcinogens (TPA, DMBA) to induce tumor's development. Ultra violet (UV) radiation has been epidemiologically related to the melanoma acquisition. The UV induced melanoma tumors raised in the HGF transgenic melanoma mouse model resemble human cutaneous melanoma with respect etiology histopathology and molecular pathogenesis. A number of different studies have demonstrated the importance of receptor tyrosine kinases (RTK) in human melanoma including c-MET (HGF receptor). In addition to this, several identified genetics alterations such as activating mutations in BRaf and NRas, CCND1, CDK4, MITF gene amplifications and CDKN2A (p16Ink4a, p19ARF) and PTEN deletions, should play a role in melanoma development and progression. We also know from mouse models that Ras pathway activation is very important in melanoma development. However, the analysis of all the data suggest that the contribution of the HGF signaling in this mouse melanoma model can not be explained just with the Ras pathway activation. The specific contributions made by the UV irradiation and the HGF signaling in these melanoma model that make this animal model so interesting, are unknown. Using a proteomic and a genetic approach we’ll try to find novel relevant molecules involved in HGF mouse model melanomagenesis.
IS26 - GENETICS OF MELANOMA AND NEVI PREDISPOSITION
Servei de Bioquímica i Genètica Molecular Hospital Clínic de Barcelona, Spain
Malignant melanoma (MM) is a multifactorial and polygenic disease. The main risk factors are number of nevi, familial predisposition and skin phototype related to ultraviolet radiation exposition. Three high susceptibility genes have been implicated in MM: CDKN2A/p14ARF and CDK4. CDKN2A mutations are detected in 20–60% of MM families, although this prevalence varies in different geographic regions (i.e. 20% in Australia and 57% in Europe), while CDK4 mutations are present in 1% of families. We have studied the implication of these genes in melanoma susceptibility in Spain and detected mutations in nearly 20% of families with 2 or more cases of melanoma, in 16.3% of multiple primary (MPM) patients, but also in 2.3% of sporadic cases. Spanish families with two or three melanoma cases have the same risk of being mutation carriers, but the mutation rate increases with four or more cases, MPM patients or presence of pancreatic cancer. Polymorphisms in melanocortin receptor (MC1R) gene (specially those responsible of red hair) have been consistently related to melanoma susceptibility. Polymorphisms in CDKN2A (mainly A148T) could also modulate MM predisposition. Although presence of nevi is one of the main risk factors for MM, no gene has been associated with nevus predisposition. The presence of dysplastic nevi in mutation positive families is similar between carriers and non-carriers. Nevertheless, 9 p21, where CDKN2A is located, has been associated with nevi susceptibility. Our group has described a candidate gene in this region (c9orf14) and has described polymorphisms in this gene related to an increased risk of developing nevi.
IS27 - MELANOMA CLINICS, GENETIC SUSCEPTIBILITY AND ENVIRONMENTAL FACTORS
Melanoma Unit, Dermatology Department, Hospital Clínic, IDIBAPS, Barcelona, Spain
Malignant melanoma is a multifactorial disease in which the interaction of genetic susceptibility and environmental factors are crucial in the development. There are well-defined sub-types of melanoma epidemiologically, clinically and pathologically but recently this concept gains significance since the genetic pathways seem to be different in the different sub-types with different genetic susceptibility background and environmental influence. One type of melanoma is directly related with intermittent sun exposure, frequently developed in trunk or extremities, with a superficial spreading histological subtype. This type of melanoma is developed in individuals with high susceptibility to develop melanoma in relationship with an increased susceptibility to develop multiple nevi induced by sun exposure in childhood. In this subtype of melanoma patients, an increased proliferation of melanocytes is present and BRAF/NRAS mutations frequently detected. The second type of melanoma is associated with very high chronic sun exposure, histologically being Lentigo Maligna and Lentigo Maligna Melanoma. This type of melanoma is independent of the presence of nevi and hardly ever shows mutations in BRAF/NRAS, while activation of Cyclin D1 is quite common. Interestingly, this kind of melanoma seems to have a better prognosis. Finally, another group of melanomas exist, developed with independence of sun exposure, including acral and mucosal melanoma. Better knowledge of these kinds of melanoma and genetic pathways implicated in their development may have an important impact in future strategies of prevention, early diagnosis and specific therapy.
IS28 - CURRENT EXPERIMENTAL THERAPIES AND FUTURE DIRECTIONS IN METASTATIC MELANOMA
LOCE, Bordet Institute, Free University of Brussels, Belgium
Despite intensive research efforts at least within the past 15 years, the prognosis of advanced melanoma remains rather poor with about 30% 5 year survival for patients with limited regional lymph node involvement. The barrier of 20% responses to any therapy has not been crossed yet. The large number of clinical trials over the last couple of years, still going or planned, also reflect the need for innovative treatment strategies. Most if not all of these trials are supported by dozens of papers with original experimental data. Thus, more than 200 trials are conducted 55% of which deal with immunotherapy. While vaccination did not yield but modest results, boosting the immune system seems to hold some promises particularly with GM-CSF, Toll-like receptor 7 agonists and specially anti-CTLA-4 mAb. Chemotherapy is in the second line with 22% of the trials mostly based on new combinations and improved drug delivery with e.g. the newly designed nanocells. 7% of the trials use mAbs mostly against integrins. Molecules directed against specific molecular targets represent 14% with about 20 aimed pathways. VEGF-R tyrosine kinase, PKC, cdk and mTOR inhibitors take the lead followed by inhibitors of proteasome, topoisomerase, histone deacetylase, HSP, mTOR, Farnesyl transferase,… Brachytherapy could also be an interesting alternative by targeting somatostatin receptors. Considering the small but real improvement that some of the experimental drugs are showing, finding the right drug combinations, dose and administration schedules seems today the key to a successful treatment.
IS29 - EPIDERMAL HOMEOSTASIS AND ULTRAVIOLET RADIATION: LEADS AND LESSONS FROM THE MELANOCORTIN SYSTEM
Markus Böhm, Agatha Kokot, Meinhard Schiller and Thomas Luger.
Dept. of Dermatology and Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, University of Münster, Münster, Germany
Epidermal cells are constantly exposed to various environmental stressors among ultraviolet (UV) light is most abundant. The biological impact of UV irradiation on epidermal cells are complex and include generation of reactive oxygen species (ROS) and DNA damage, apoptosis, and induction of proinflammatory cytokines and mediators. The melanocortin (MC) system appears to be crucially involved in maintaining epidermal homeostasis via the immunomodulatory actions of pro-opiomelanocortin (POMC)-derived peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH), and moreover, by the cytoprotective activity of the latter. Accordingly, we recently demonstrated that alpha-MSH prevents UV-B-induced apoptosis in epidermal cells and reduces DNA damage as shown by reduced amounts cyclopyrimidine dimers. In light of these biologic activities of alpha-MSH and the profound inductive effect of UV exposure on the POMC system it is tempting to speculate on the impact of ROS per se on the MC system. Surprisingly, hydrogen peroxide at doses present in human skin after UV A/B exposure led to a dramatic down regulation of several components of the POMC system in human melanocytes. Down regulation of the MC system was likewise observed in non-melanocytic cutaneous cell types after treatment with additional oxidative stressors. These data highlight an astonishingly complex regulation of the cutaneous MC system by UV light and ROS per se. Moreover, down regulation of the cellular MC system by excessive ROS may be relevant for the pathogenesis of certain cutaneous disorders including vitiligo.
IS30 - ACTIVATION OF MC1R MODIFIES THE UV RESPONSE OF HUMAN MELANOCYTES
Zalfa A. Abdel-Malek.
Department of Dermatology, University of Cincinnati, Cincinnati, Ohio, USA
The melanocortin 1 receptor (MC1R) and melanocortins play an important role in the diversity of human pigmentation and in determining the risk for melanoma. Human melanocytes (hMC) respond to a α-MSH and ACTH with increased proliferation and eumelanogenesis. Loss-of-function mutations in the MC1R are associated with red hair phenotype, which predisposes to skin cancer, including melanoma. We reported that activation of the cAMP pathway that mediates the effects of melanocortins and the activated MC1R is pivotal for UV-induced melanogenesis in hMC. Additionally, we observed that cultured hMC that express loss-of-function MC1R are more sensitive to UV-induced cytotoxicity, consequent to their reduced ability to repair DNA photoproducts and overcome oxidative stress. We reported that activation of the MC1R reduced UV-induced apoptosis and generation of reactive oxygen species, and enhanced nucleotide excision repair in normal hMC. These effects were absent in hMC expressing loss-of-function MC1R alleles, or normal hMC treated with agouti signaling protein. Microarray data comparing hMC with functional versus non-functional MC1R revealed that in the former 2676 genes, compared to only two in the latter, were altered in expression by α-MSH. In hMC with functional MC1R, α-MSH reversed the effects of UV on many genes that regulate apoptosis, cell cycle, DNA repair, oxidative stress and melanogenesis. These findings suggest that melanocortins counteract the genotoxic effects of UV, and explain the role of MC1R as a melanoma susceptibility gene.
IS31 - GENE EXPRESSION REGULATION IN RESPONSE TO UV-LIGHT STIMULATION
M. D. Galibert, S. Corre, Y. Barona and N. Mouchet.
CNRS UMR 6061 Génétique et Dévelopment, Université de Rennes 1, Groupe Régulation Transcriptionnel et Oncogénèse, IFR140 GFAS, Faculté de médecine, 2 avenue du Pr Léon Bernard, CS 34317, 35043 Rennes Cedex, France
Skin exposure to solar radiation initiates complex molecular processes. These include the protective tanning response, local inflammation, immune suppression, and DNA damage that can lead to skin carcinogenesis. The tanning response protects against UV-mediated DNA damage, with UV irradiation triggering production of melanin in specific melanocytes organelles the melanosomes, which are then transferred to the neighboring keratinocytes. Using tissue culture models, we have investigated the molecular mechanisms involved in pigmentation process, allowing the identification of the stress-responsive p38 kinase and the p38-activated USF-1 transcription factor as key regulators of the tanning response (Galibert et al., EMBO J. 2001; Corre et al., JBC. 2004). Gene expression regulation of upstream components of the pigmentation process, POMC and MC1R, as well as genes involved in pigment manufacturer, Tyrosinase, TYRP-1, Dct, have been shown to be dependant on the p38-activated USF-1 transcription factor and the presence of critical cis-DNA binding elements within their promoter genes. To examine UV-induced gene expression with respect to cell organization and cooperation, we investigated the in vivoUV response, using irradiated skin biopsies and irradiated cultured skin explants (Corre et al., J. Invest. Dermatol. 2006). Taken together, gene expression analysis, using in vitro and in vivodata, lead to get further insight into the UV-epidermal biology.
IS32 - THE COMPLEXITY OF HUMAN ALBINISM
Richard A. King1,3, C. Gail Summers2 and William S. Oetting1,3.
Departments of 1Medicine;
3Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota, USA
Melanin synthesis is associated with macroscopic visible variation and microscopic retinal and optic nerve development. Melanin in the skin, hair and irises can vary dramatically among and between individuals showing that normal development of these tissues tolerates large differences in melanin content. This contrasts with the visual system where melanin in the developing retina and optic system is critical to normal development. Gene mutations that reduce melanin formation in melanocytes produce a clinical spectrum of cutaneous and ocular hypopigmentation, and the term albinism defines the genetic syndrome of hypopigmentation associated with incomplete (hypoplasia) or absent (aplasia) development of the foveal region of the retina, resulting in reduced acuity, and with altered optic nerve connections between the retina and the brain, resulting in reduced stereovision and strabismus. The types of human albinism are best separated by the gene involved, including genes expressed in melanocytes [tyrosinase (TYR), P protein (P), tyrosinase related protein 1 (TYRP1), membrane associated transported protein (MATP), ocular albinism 1 (OA1)], and genes expressed in melanocytes and other tissues [Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome (CHS)]. The phenotypic variation of each type of albinism is broad, and depends on: (1) the mutation and the residual function of the gene product; (2) the effect of modifying genes on amount and type of melanin synthesized; and (3) environmental factors. The majority of human albinism genes have probably been identified. The major challenge will be to understand how melanin or a component of the melanin pathway drives normal development of the retina and optic nerves.
IS33 - OCULOCUTANEOUS ALBINISM: ITS GENOTYPE AND PHENOTYPE CORRELATION
Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya, Japan
Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders caused by mutations of melanogenic genes such as TYR for OCA1, P for OCA2, TYRP1 for OCA3 and SLC45A2 (MATP/AIM-1) for OCA4. OCA also develops as a manifestation of Hermansky-Pudluk Syndrome (HPS) 1–8 by mutations of HPS1, ADTB3A, HPS3-6, DTNBP1, and HPS8, respectively, Chediak-Higashi Synd. by mutation of CHS1, and Griscelli Synd. 1–3 by mutations of MY5A, RAB27, and MLPH, respectively. We have been analyzing the genes of OCA patients since we first reported a pathological mutation of the tyrosinase gene of a patient with OCA in 1989. According to the results of the sequence or single -strand conformation polymorphism analyses, we classified 105 Japanese OCA patients into 37 (35%), OCA1; 10 (10%), OCA2; 0 (0%), OCA3; 28 (27%), OCA4; 11 (10%), HPS1; 0 (0%), HPS4 and 18 (18%), undetected. It is remarkable that OCA4 is the second major type, and that there are as many HPS1 patients as OCA2 ones in Japan. Individuals with OCA1a, the tyrosinase-negative type, are born with a complete absence of pigment in the hair, eyes and skin. In other types, the skin phenotypes are less severe than in those with OCA1a, and display no clinical finding characteristic for their classification except for traumatic purpura, which is often recognized in patients with HPS1.
IS34 - THE OCULAR ALBINISM TYPE 1 (OA1) PROTEIN IS A RESIDENT LYSOSOMAL/MELANOSOMAL G PROTEIN-COUPLED RECEPTOR
M. V. Schiaffino, R. Piccirillo, G. Innamorati, P. Bagnato and I. Palmisano.
Scientific Institute San Raffalele, Via Olgettina 58, 20132 Milan, Italy
Ocular albinism type 1 is an X-linked recessive disorder characterized by severe reduction of visual acuity, nystagmus, strabismus, photophobia, retinal hypopigmentation, iris translucency, foveal hypoplasia, optic misrouting and the presence of macromelanosomes in the skin and eyes, suggesting a defect in melanosome biogenesis. The gene responsible for this disorder encodes a pigment cell-specific membrane glycoprotein, named OA1, displaying structural and functional features of G protein-coupled receptors (GPCRs). In fact, in addition to possess seven transmembrane domains and share sequence similarities with GPCRs, OA1 exhibits two fundamental properties of this family of receptors, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins. However, in contrast to canonical GPCRs, OA1 is not found at the cell surface, but is exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Furthermore, OA1 contains at least two independent sorting signals, i.e. an unconventional dileucine-based motif and a novel cytosolic motif, which are responsible for its efficient intracellular retention and delivery to lysosomes and melanosomes, further supporting the notion that OA1 is a resident lysosomal/melanosomal GPCR. These unique characteristics suggest that OA1 functions as the ‘sensor’ for a yet unidentified intra-melanosomal ligand and regulates melanosome biogenesis by triggering a signal transduction cascade on the cytoplasmic side of organelle membrane.
IS35 - NEW INSIGHTS IN ‘IN VIVO’ IMAGING TECHNIQUES FOR THE EVALUATION OF MELANOMA AND BENIGN MELANOCYTIC LESIONS
J. Malvehy, S. Segura, C. Carrera, I. Kolm and S. Puig.
Melanoma Unit, Dermatology Dept. Hospital clinic, IDIBAPS Barcelona, Spain
The preoperative evaluation of skin tumors by new imaging techniques has been introduced in recent time. Dermoscopy and sonography are imaging techniques used for the study of skin tumors that allow a better recognition of architecture and render a new semiology of major interest in the study of melanocytic lesions. In addition new techniques such as in vivo confocal reflectance microscopy, a new tool that makes feasible the observation in vivo of the skin at ‘quasi histopathological’ resolution, opens a new era in the study of melanocytic tumors. The combination of different imaging techniques is complementary to the static image given by conventional histopathology. Correlation of the findings obtained by different imaging techniques and histopathology is needed to understand their significance. Moreover the concept of the in vivo mapping of the tumors to guide the selection of specific areas for molecular analysis will be relevant in the field of melanocytic lesions.
IS36 - IMAGE AND MELANOMA
Université de la Méditerranée et Service de Dermatologie, Hôpital Ste Marguerite, Marseille, France
IS37 - ANOMALIES OF THE P53 PATHWAY IN MELANOCYTES AND MELANOMA
Dorothy C Bennett1, D. Alastair MacKenzie Ross1, Martin G Cook2 and Elena V Sviderskaya1.
1Division of Basic Medical Sciences, St George's, University of London, UK;
2Department of Histopathology, Royal Surrey County Hospital, Guildford, UK
The tumour suppressor p53 is mutated in over 50% of human cancers, but in only around 10% of melanomas, although p53 protein is often expressed. This suggests something unusual about the p53 pathway in melanocytes or melanoma - why does p53 not suppress melanoma growth? We are investigating whether there is a connection with cell senescence, the growth arrest seen in normal cells consequent on extended proliferation. Senescence can be mediated by either the p16-RB pathway or the telomere-p53 pathway, where chromosomal telomeres become short and activate DNA-damage signalling through ATM. ATM phosphorylates CHK2 and CHK2 phosphorylates p53. P53 is activated, normally leading to growth arrest through p21/CDKN1A. We and others have shown that cell senescence occurs in benign melanocytic naevi, involving the p16 but not p53 pathway, as in cultured human melanocytes. Mouse melanocytes with germline p53 deletion initially undergo senescence (see talk by E.V.S.), further evidence that p53 has reduced importance in melanocytes. We have assessed markers of the ATM-p53 pathway in melanoma progression, by immunostaining of benign and malignant pigmented lesions, compared to other senescence markers. Unexpectedly, this pathway appears particularly active in advanced primary (VGP) melanomas; yet p21 is not detected and the cells proliferate. Activation of p21 thus appears deficient.
IS38 - TOPICAL KHELLIN PHOTOTHERAPY: 20 YEARS OF EXPERIENCE
Hospital Sant Pau, Autonomous University Barcelona, Barcelona, Spain
The capacity to induce repigmentation by Khellin , a flurochrome similar to psoralens but without phototoxic action was described by Honigsmann and Ortel 20 years ago. The treatment with topical Khellin formulation, plus controlled sun exposure in moderate cases of vitiligo is a very useful treatment in sunny countries such as Spain or other Mediterranean countries. We will present the experience after several years using this technique.
IS39 - THE INK4/ARF LOCUS IN SENESCENCE, CANCER AND AGING
Spanish National Cancer Center (CNIO), Madrid, Spain
The INK4/ARF locus encodes three tumour suppressors, namely, p15INK4b, ARF, and p16INK4a, and is among the most frequently inactivated loci in human cancer. Our current understanding of the function of this locus indicates that it is a sensor of oncogenic signals, which, upon activation, triggers either senescence or apoptosis. Oncogene-Induced Senescence (OIS) is considered a relevant cellular response against cancer. We have identified new molecular markers for OIS using DNA microarrays and we have tested them in murine pre-malignant and malignant neoplasias driven by an endogenous K-ras oncogenic allele (1). We have found that pre-malignant lesions (adenomas) in lung and pancreas are positive for senescence markers, including p15INK4b, ARF, and p16INK4a, whereas malignant lesions (carcinomas) are negative. We conclude that senescent cells are a defining feature of pre-malignant tumors with diagnostic and/or prognostic potential (2). We have recently identified a Regulatory Domain (RD) in the INK4/ARF locus that is able to control the expression of the entire locus (3). This element is co-opted by the replication machinery playing a dual role in transcription and in the initiation of replication. Interestingly, RD has the capacity to control the status of the chromatin of the INK4/ARF locus. In particular, high levels of Cdc6 result in recruitment of histone deacetylases, heterochromatinization, and silencing of the locus. In support of this model, human lung carcinomas with high levels of Cdc6 are associated with low levels of p16INK4a. Finally, we have generated mouse strains with increased gene dosage of the INK4/ARF locus. These mice are cancer resistant and age normally (4). Surprisingly, mice with combined increased gene dosage for INK4/ARF and p53 show delayed aging. This delayed aging cannot be explained by their reduced incidence of cancer, indicating that these tumor suppressors have an intrinsic anti-aging activity. I will discuss the potential role of the ARF/p53 pathway in providing anti-oxidant defenses.
1. Collado et al. Nature, 436, 642 (2005).
2. Collado and Serrano. Nat. Rev. Cancer, 6, 472–476 (2006).
3. Gonzalez et al. Nature, 440, 702–706 (2006).
4. Matheu et al. Genes Dev., 18, 2736–2746 (2004).
SHORT ORAL PRESENTATION
OP01 - INDUCIBLE TRANSGENIC EXPRESSION OF EDN3 PARTIALLY RESCUES THE PIGMENTATION PHENOTYPES OF EDN3ls, KITw-v, AND Ay MUTANT MICE
Roman J. Garcia1, Avner Ittah1, Sheyla Mirabal1, Jessica Figueroa1, Lidice Lopez1, Adam B. Glick2 and Lidia Kos1.
1Department of Biological Sciences, Florida International University, Miami, FL 33199, USA;
2The Department of Veterinary Science, Pennsylvania State University, University Park, PA 16802, USA
Neural crest cells (NCC) are a transient population of cells situated over the dorsal neural tube. Some NCC undertake a dorsolateral migratory route between the epidermis and the dermamyotome and eventually differentiate into the melanocytes found in the skin. Endothelin 3 (Edn3)encodes for a ligand important to developing NCC and is allelic to the spontaneous mouse mutation occurring at the lethal spotting (ls) locus. Edn3ls/ls mutants give rise to a phenotype consisting of hypopigmentation and aganglionic megacolon. In this study we show that when Edn3 is driven by a keratin promoter and ectopically placed proximal to migrating melanoblasts and differentiated melanocytes in the skin, adult mice manifest pigmented skin harboring a population of dermal melanocytes. Using a tetracycline inducible system we show that the postnatal expression of Edn3 is required for the maintenance of dermal melanocytes, and that the early expression of transgenic Edn3 is important to the onset of the hyperpigmented phenotype. Transgenic embryos showed increased numbers of dermal and epidermal melanoblasts when compared to controls as well as ectopic cells that remain at the dorsal aspect of the neural tube. Crosses into Edn3ls/ls mutants demonstrate that the ectopic surface ectoderm expression of Edn3 is not able to fully compensate for the endogenous mesenchymal expression pattern. Crosses into tyrosine kinase receptor KitWv mutants indicate that Edn3 can partially compensate for Kit’s role in early development. Crosses into Ay mutant mice considerably darkened their yellow coat color suggesting that endothelin signaling may also play a role in pigment switching.
OP02 - RALP, A NOVEL MEMBER OF THE SHC FAMILY, IS INVOLVED IN CELL MIGRATION AND DIFFERENTIATION
Ernesta Fagiani1, Giuseppina Giardina1, Allegra Pianaroli1, Lucilla Luzi1, Antonio Simeone2 and Luisa Lanfrancone1.
1European Institute of Oncology, Department of Experimental Oncology, Milano, Italy;
2CEINGE, Napoli, Italy
RaLP is a recently isolated member of the Shc family of adaptor proteins characterized by a modular organization (CH2, PTB, CH1 and SH2 domains). Its expression is confined to cells of melanocytic origin, in particular to melanomas. Analysis of RaLP expression during melanoma progression revealed that RaLP is expressed in vertically growing and metastatic melanomas, but not in nevi and radially growing melanomas, suggesting a role of RaLP in the invasive and migratory phenotype of this tumor. Down-regulation of RaLP expression in melanoma by RNA interference inhibits cell migration and induces anoikis. Biochemically, RaLP is a physiological substrate of the IGF-1 and EGF receptor tyrosine kinases in melanoma. Analysis of RaLP expression by in situ hybridization during mouse embryonic development revealed a specific positivity of the territories that are site of neurogenesis. In vitro experiments showed high levels of RaLP protein product in neural stem cells and migrating melanoblasts, which decrease in the differentiated melanocytes. Preliminary data suggest the RaLP is expressed in stem cells of different embryological origin. To dissect the role of RaLP in self-renewal and differentiation, we have used the embryonic stem (ES) cells induced to differentiate in vitro in several lineages. Results will be discussed.
OP03 - NOTCH/RBP-J SIGNALING CONTROLS MELANOCYTE LINEAGE DEVELOPMENT
Geneviève Aubin-Houzelstein1, Florence Bernex1, Johanna Djian1, Véronique Delmas2, Ichiro Yajima2, and Jean-Jacques Panthier1,3.
1Cellular and Molecular Genetics UMR 955 INRA-ENVA, Ecole Nationale Vétérinaire d’Alfort, 7 avenue du Général-de-Gaulle, 94704 Maisons-Alfort cedex, France;
2Developmental Genetics of Melanocytes, UMR146 CNRS-Institut Curie, Centre Universitaire, Orsay, France;
3Mouse functional Genetics Unit, URA CNRS 2578, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France
Notch signaling is an evolutionarily conserved mechanism that regulates numerous cell fate decisions. Upon ligand binding, the intracellular domain of Notch receptor is translocated to the nucleus where it interacts with the recombination signal binding protein-J (RBP-J) within a multiproteic complex, which activates various target genes. We first thought that Notch signaling may be important for mouse melanocytes when we found that overexpression of Strawberry notch (mSno) gene in either patchwork (pwk) mutant mice or in transgenic mice led to lack of differentiated melanocytes in the hair matrix of newborn1,2,3. We examined the effect of inhibition and activation of Notch signaling in melanocytes using a melanocyte-specific gene targeting approach. Inhibition of the Notch pathway had several effects. In the embryo, it resulted in a reduced number of melanoblasts migrating through the epidermis. Postnatally, it induced precocious melanocyte differentiation in the bulge region and ectopic pigmentation in the hair follicle. It further led to progressive loss of differentiated melanocytes within the hair matrix and to premature hair whitening. Activation of Notch led to altered expansion of melanoblasts in the embryonic skin, variable body spotting in newborn. However activation of Notch signaling did not lead to accelerated hair greying. Thus, Notch signaling is involved in the expansion of migrating melanoblasts in the embryo and in the maintenance of melanocyte stem cells in the adult hair follicle. These data are consistent with the contention that Notch maintains the progenitor state and inhibits differentiation.
1. Aubin-Houzelstein G, Bernex F, Elbaz C, Panthier JJ (1998). Survival of patchwork melanoblasts is dependent upon their number in the hair follicle at the end of embryogenesis. Dev Biol. 198(2): 266–76.
2. Aubin-Houzelstein G, Panthier JJ (1999). The patchwork mouse phenotype: implication for melanocyte replacement in the hair follicle. Pigment Cell Res. 1999 Jun;12(3):181–6.
3. Aubin-Houzelstein, G et al. (unpublished data).
OP04 - FUNCTIONAL AND STRUCTURAL DISSECTION OF DNA REGULATORY ELEMENTS FOUND WITHIN THE LOCUS CONTROL REGION OF THE MOUSE TYROSINASE GENE
Angel García-Diaz1, Lucia Regales1, Hector Rincón-Arano2, Martín Escamilla-del Arenal2, Félix Recillas-Targa2 and Lluis Montoliu1.
1Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología (CNB-CSIC), 28049 Madrid (Spain);
2Instituto de Fisiología Celular, Departamento de Genética Molecular, Universidad Nacional Autónoma de México (UNAM), Apartado Postal 70-242, México D.F. 04510, México
Previous studies have identified a boundary element at the Locus Control Region (LCR) of the mouse tyrosinase gene in an open chromatin genomic region surrounded by highly methylated repetitive sequences (LINE elements). These approaches showed an insulator element located within the LCR (Giraldo et al. Nucleic Acids Res. 2003, 31(21): 6290–6305). Insulators or boundaries have been functionally defined by at least one of the following two properties. First, they have a barrier activity, protecting a gene against chromosomal position effects, such as the spreading of chromatin condensation and heterochromatinization into expression domains. Second, insulators can also have an enhancer blocker activity, interfering with the trans-activation of a given promoter by a distal enhancer when placed between these two regulatory elements. We have carried out different type of assays in order to describe and analyse the properties of this boundary element of the mouse tyrosinase locus, focusing into possible nuclear/transcription factors associated with it and the corresponding chromatin environment, using the following methods: electrophoretic mobility shift assay, chromatin immunoprecipitation, enhancer-blocker assay, DNAse I Hypersensitivity analysis, position effect essays in cells and chromatin immunoshifts. A summary of results from all these analyses will be presented and discussed in terms of the mouse tyrosinase chromatin domain organization and regulation.
OP05 - MOLECULAR AND GENETIC ANALYSIS OF MITFA FUNCTION IN ZEBRAFISH MELANOPHORE DEVELOPMENT
James A. Lister and AnhThu Nguyen.
Department of Human Genetics, Virginia Commonwealth University, Richmond, Virginia, USA
The mitfa gene encodes a zebrafish ortholog of the microphthalmia-associated transcription factor (Mitf), which, like its counterparts in other species, is absolutely required for development of neural crest melanocytes. To better understand the function of this gene, we are characterizing several hypomorphic alleles of nacre/mitfa. We have identified mutations in three such alleles, which together with two previously characterized alleles form a series with wild type > z25 > w35 > fh53 > b692 = w2 (null). The z25 and fh53 alleles are single base pair changes producing amino acid substitutions in the first helix of the helix-loop-helix dimerization domain and basic DNA-binding domain, respectively, while the w35 allele is a mutation in a splice donor site that reduces the level of correctly spliced transcripts. For each allele we are examining the number of differentiated melanophores and the number of dct-positive melanoblasts present during the first several days of development. These studies will provide insight to the kinetics of the melanophore progenitor population and the role of mitfa in the regulation of differentiation. In a second approach, we are constructing and analyzing chimeras between Mitf proteins, which can rescue melanophore development in mitfa null mutants when expressed under the mitfa promoter, and the Tfe3a protein, which cannot. Our results suggest that important determinants for this specificity lie in the amino termini of these proteins.
This work was supported by American Cancer Society grant IRG-99-225-04.
OP06 - CONTROL OF GENE EXPRESSION IN MELANOCYTES AND RPE BY CONSERVED DISTAL REGULATORY ELEMENTS
Fabien Murisier, Sabrina Guichard, and Friedrich Beermann.
ISREC (Swiss Institute for Experimental Cancer Research), Epalinges, Switzerland
Pigment cells of mammals originate from two different lineages: melanocytes arise from the neural crest, whereas cells of the retinal-pigmented epithelium (RPE) originate from the optic cup of the developing forebrain. Previous studies have suggested that pigmentation genes are differentially regulated in melanocytes and RPE. In transgenic mice, the promoters of the tyrosinase and Tyrp1 genes were shown to drive detectable lacZ reporter gene expression only to the RPE, even though these genes are also expressed in melanocytes. This indicates that some of the regulatory elements required for gene expression in melanocytes and RPE are located outside of the promoter such as in introns or in more distal regions. We have now used comparative sequence analysis and transgenic experiments to study the involvement of distal regulatory elements in the control of tyrosinase and Tyrp1 gene expression. Comparison of orthologous sequences from multiple species identified several stretches of evolutionarily conserved non-coding sequences (CNS) that might represent putative regulatory elements. Functional analysis of these CNS in transgenic mice and cell culture experiments revealed that some of them behave as melanocyte or RPE-specific enhancers and are required for expression in these cell types. The identification and characterization of these tyrosinase and Tyrp1 distal regulatory elements supports the idea that separate regulatory sequences mediate differential gene expression in melanocytes and RPE and might furthermore contribute to our understanding of the evolution of pigment cells.
OP07 - USE OF HUMAN MELANOCYTIC CELLS OF DEFINED SLC45A2/MATP AND SLC24A5/NCKX5 GENOTYPE IN THE ANALYSIS OF MELANOGENESIS
A. L. Cook1, T. G. Bladen1, W. Chen1, D. J. Smit1, J. H. Leonard2, J. L. Stow1 and R. A. Sturm1.
1Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
2Queensland Institute of Medical Research, Brisbane, Australia
Polymorphism within the MATP protein-coding region of the SLC45A2 gene has been associated with the degree of pigmentation in human skin. The 374Leu allele is considered to be the ancestral form of the protein, and is highly associated with darker skin, hair and eye colour compared to the 374Phe variant. Similarly, population specific polymorphism within the coding region of the zebrafish golden gene homologue SLC24A5/NCKX5 at amino acid position 111 has been reported, with the 111Ala and 111Thr alleles being associated with darker and lighter skin pigmentation respectively. We have established a bank of primary human melanocytic cells from neonatal foreskin and have characterised the MATP and NCKX5 alleles at these and other SNPs potentially impacting human pigmentation. We are currently examining these cell strains for patterns in melanin content, tyrosinase activity and expression of genes involved in melanogenesis. Several human melanoma cell lines have also been genotyped for these changes and are being used in over-expression and siRNA experiments to gain insight into MATP and NCKX5 function. We are now in a position to study the contribution of several human pigmentation gene variants to the process of melanogenesis at the molecular level and ultimately these studies will reveal mechanisms for regulating cutaneous pigmentation.
OP08 - MELANOGENESIS AND OXIDATIVE STRESS ARE BOTH ELEVATED IN PATIENTS UNDERGOING HEMODIALYSIS
Shosuke Ito1, Kazutaka Murakami2, Kazumasa Wakamatsu1, Yukiko Nakanishi1, Hiroki Takahashi2 and Satoshi Sugiyama2.
1Department of Chemistry, Fujita Health University School of Health Sciences, Aichi, Japan;
2Department of Internal Medicine, Fujita Health University School of Medicine, Aichi, Japan
Diffuse hyperpigmentation occurs in a majority of patients with chronic renal failure (CRF) undergoing hemodialysis (HD). Oxidative stress is also suggested to be one of the most important complications during long-term HD therapy. We have examined serum levels of 5-S-cysteinyldopa (5SCD), pheomelanin, and oxidative stress markers in CRF patients during HD treatment. Protein-bound (PB) forms of DOPA and 5SCD are produced by hydroxyl radical attach on tyrosine residues. HI reductive hydrolysis of pheomelanin and 3-nitrotyrosine residues in proteins produces 4-amino-3-hydroxyphenylalanine (4-AHP) and 3-amino-4-hydroxyphenylalanine (3-AHP), respectively. Free 5SCD levels in sera of HD patients (n = 16) and healthy controls (n = 16) were 37.9 and 4.05 nmol/L, respectively. Levels of 4-AHP and 3-AHP in patients were 217 and 1379 nmol/L while those in controls were 85.3 and 241 nmol/L. Levels of PB-DOPA and PB-5SCD in patients were 789 and 45.7 nmol/L while those in controls were 506 and 23.1 nmol/L. Thus, levels of 5SCD, pheomelanin, and oxidative stress markers were found to be significantly elevated in HD patients compared with controls. These results suggest that pheomelanin accumulates in the skin of HD patients due to the high serum levels of free 5SCD.
OP09 - MELANIN SYNTHESIS ININ VITRO MELANOCYTES ASSOCIATED WITH CATALASE ACTIVITY
Vittoria Maresca1, Enrica Flori1, Stefania Briganti1, Arianna Mastrofrancesco1, Alessandra Crisi2, Anna Maria Mileo3, Paola Grammatico2, Marco Giorgio Paggi3 and Mauro Picardo1.
1San Gallicano Dermatological Institute, Rome, Italy;
2Medical Genetics, Experimental Medicine and Pathology Dept, University La Sapienza, Rome, Italy;
3Laboratory ‘C’, Department for the Development of Therapeutic Programs, Regina Elena Cancer Institute, Rome, Italy
We previously demonstrated, in in vitro and ex vivo models, a correlation between cutaneous phototype and catalase activity. Considering the main role of this enzyme in neutralizing hydrogen peroxide in melanocytes, we investigated on primary cultures of human melanocytes, the possible correlation between melanogenic and catalase activity. Total melanin concentration was directly correlated with the original degree of pigmentation, and the proportional contribution of pheomelanin became progressively negligible, as the degree of pigmentation increased. mRNA, protein levels and activity of catalase resulted directly associated with total melanin. Moreover when this enzyme was inhibited, an increase in reactive oxygen species was observed, directly correlated to the original degree of pigmentation. These results demonstrate that melanin synthesis is strictly associated with catalase expression and activity. Thus, darkly pigmented melanocytes possess two protective strategies, acting synergistically to counteract the deleterious effects of ultraviolet radiation. These are represented by the filter exerted by melanins, as well as catalase activity. By contrast, lightly pigmented melanocytes, possessing lower levels of melanogenic and catalase activity, are therefore more susceptible to accumulate damages, after ultraviolet exposition.
OP10 - FUNCTIONAL CHARACTERIZATION OF THE GREY COAT COLOUR GENE IN HORSES
Anna Golovko1, Gerli Pielberg1, Elisabeth Sundström2 and Leif Andersson1,2.
1Inst. of Medical Biochemistry and Microbiology, Uppsala University, Sweden;
2Dept. of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Sweden
Greying with age is a dominant coat colour mutation found in several horse breeds. Grey horses are born coloured but with age the hair becomes progressively white. The skin colour is usually retained. This phenotype is strongly associated with a high incidence of melanoma and vitiligo. Grey horse melanomas are heavily pigmented; usually show slow growth and histological characteristics reminiscent of human pigment synthesizing melanomas. By using Identical-By-Descent mapping we have identified a region harbouring the Grey gene and mutation. The region contains five known genes with no established role in pigmentation or melanoma development. No coding mutations have been identified; however a specific haplotype showing a complete association with the Grey phenotype was detected. We initiated several experiments to evaluate possible roles of these genes in pigmentation and melanoma development. We found that all the genes are expressed in melanoma and skin. We are currently evaluating the role of the genes in cell proliferation by overexpression and RNAi experiments. In situ hybridizations and immunohistochemistry are used to investigate the expression pattern of the genes and their products. Construction of a knock-out and knock-in mice and yeast 2-hybrid studies will gain further knowledge on the function of the Grey gene and the phenotype.
OP11 - DOSE-DEPENDENT EFFECT OF NOTCH1 AND NOTCH2 RECEPTORS ON MELANOCYTE-MEDIATED HAIR PIGMENTATION
Karine Schouwey1, Lionel Larue2, Véronique Delmas2, Ulla Zimber-Strobl3, Lothar Strobl3, Freddy Radtke1 and Friedrich Beermann1.
1ISREC (Swiss Institute for Experimental Cancer Research), Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland;
2Developmental Genetics of Melanocytes, UMR 146 CNRS-Institut Curie, Centre Universitaire, F-91405 Orsay, France;
3Institute of Clinical Molecular Biology and Tumor Genetics, National Research Center for Environment and Health, GSF, D-81377 Munich, Germany
The Notch signaling pathway is a conserved cell-interaction mechanism which is involved in many biological processes as cell fate decisions or stem cell maintenance. Notch signaling via Hes1 has been recently demonstrated to play a critical role in hair pigmentation. In this study we dissected the Notch signaling pathway using Notch1 and Notch2 conditional knockout mice. Targeted deletion of these receptors in the melanocyte lineage using Tyr::Cre mice led to a gradual, dose and receptor type-dependent coat color dilution. As in Tyr::CreRBP-Jκflox/flox mice, hair graying was mediated by a complete elimination of Dct-expressing cells in mutant hair follicles. Moreover, this phenotye was proportional to the number of floxed Notch alleles, with the most pronounced effect seen in Tyr::CreNotch1flox/floxNotch2flox/flox mice. Deletion of Notch1 and Notch2 in melanocytes did not affect the number of Dct-expressing melanoblasts at embryonic stages, but melanocytes located within the hair matrix disappeared during the first regeneration of hair follicle. In addition, non-follicular melanocytes and pigmentation in the dermis (ear) and in the eye (choroid) were not or only slightly affected by removal of Notch1 and Notch2. We suggest that both Notch1 and Notch2 receptors are independently involved in maintenance of melanoblasts and melanocyte stem cells, and thus mainly required for proper hair pigmentation.
OP12 - POSITIONAL CLONING OF GENES UNDERLYING AUTOIMMUNE VITILIGO IN A UNIQUE CHICKEN MODEL
AnnaStina Sahlqvist, Olov Ekwall, Olle Kämpe and Susanne Kerje.
Department of Medical Sciences, Uppsala University, University Hospital, Uppsala, Sweden
Vitiligo is a human autoimmune disorder characterized by depigmentation of the skin due to a loss of epidermal melanocytes. The Smyth line (SL) chicken represents an animal model for vitiligo in which 70–90% express a post-hatch autoimmune destruction of melanocytes leading to feather depigmentation at 6–14 weeks of age. An interesting aspect of this model is that the incidence of vitiligo is dramatically increased (from ∼15% to ∼85%) after immunization with a Herpes virus vaccine. It is generally believed that a virus infection may trigger autoimmune disorders in humans. In parallel to vitiligo in humans other autoimmune disorders are over represented in SL chickens, which tend to develop alopecia, autoimmune thyroiditis and blindness. The genetic background of the vitiligo in SL chickens is still unknown. Initially, we have crossed the vitiligo susceptible SL chicken line with the resistant Brown line and intercrossed the F1 generation to generate a total of 800 F2 birds. All F2 chickens will be carefully phenotyped and a genome scan will be performed using 384 informative single nucleotide polymorphisms (SNPs). The phenotype and genotype data will be combined in the statistical analysis for identification of genomic regions harbouring genes underlying autoimmune vitiligo.
OP13 - THE MOLECULAR MECHANISM OF MC1R ASSOCIATION WITH SKIN CANCER RISK PHENOTYPES
K. A. Beaumont1, R. A. Newton1, D. J. Smit1, J. L. Stow1, J. H. Leonard2 and R. A. Sturm1.
1IMB, The University of Queensland;
2QRI Research Unit, QIMR, Brisbane, QLD, Australia
We have quantified the contribution of individual MC1R alleles to pigmentary phenotypes using allelic modelling. Four common (D84E, R151C, R160W and D294H) and two rare (R142H and I155T) alleles were designated as R as they were strongly associated with red hair, fair skin and skin cancer risk (RHC phenotype). Three alleles (V60L, V92M and R163Q) designated as r had lower penetrance. Using immunofluorescence and ligand binding studies, we have found that melanocytic cells exogenously or endogenously expressing MC1R show strong surface localisation of the wildtype, R142H and D294H receptors but markedly reduced cell surface expression of the other R variants. Conversely r alleles were expressed with normal or intermediate cell surface receptor levels. Functional cAMP assays in transfected melanoma and HEK cells revealed that all R variants had reduced functional responses with the I155T and the D294H variants showing the most severe loss of function. The V60L and R163Q variants had intermediate signalling ability, while the V92M variant showed no impairment. These functional results correlate with the genetic association of these MC1R variants with RHC and indicate that impaired G-protein coupling together with altered receptor localisation are contributing factors. Preliminary experiments also indicate that a dominant negative effect on wildtype MC1R may underlie the MC1R variant heterozygote effect on skin colour, freckling and melanoma risk.
OP14 - MITF AND METASTATIC POTENTIAL IN MELANOMA.
Keith Hoek, Natalie Schlegel, Ossia Eichhoff, and Reinhard Dummer.
Department of Dermatology, University Hospital of Zürich, Switzerland
Until recently, the progression of melanoma from in situ to metastatic stages was thought to be directly linked to the fixed metastatic potential of the progenitor cell. We present data, which suggests that metastatic potential is not a fixed characteristic, but rather is controlled by variations in signalling which underlie the changing expression of metastasis genes. We have recently completed a gene expression taxonomy for melanoma cultures and this has indicated that transcriptional differences between melanomas underlie differences in metastatic potential. Part of this study used resistance to TGFβ-mediated inhibition of proliferation as a marker for high metastatic potential. We have sought to identify the factors, which lie at the heart of resistance or susceptibility to the growth inhibitory effects of TGFβ. We are investigating the role of Micropthalmia-associated transcription factor (Mitf), which was highlighted by our DNA microarray investigations. Our results show that TGFβ-susceptible melanomas (low metastatic potential) express Mitf and subsequent siRNA knockdown of Mitf in these melanomas partially rescue them from TGFβ-mediated inhibition of proliferation. We also show immunohistochemical evidence, which indicates that in vivo low metastatic melanomas can transform to become high metastatic variants and vice versa. These studies suggest a new model for melanoma biology, where stage progression is separated from metastatic potential, which may serve to explain why melanomas continue to escape targeted therapies.
OP15 - HSP70 PROTECTS AGAINST UV-B INDUCED APOPTOSIS BY PREVENTING RELEASE OF CATHEPSINS AND CYTOCHROME C IN HUMAN MELANOCYTES.
CeciliaBivik1, Inger Rosdahl1 and Karin Öllinger2.
1Department of Biomedicine and Surgery, Division of Dermatology;
2Department of Neuroscience and Locomotion, Division of Experimental Pathology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden
Stress-induced heat-shock protein 70 (Hsp70) protects human melanocytes against UV-B induced apoptosis. After exposing cells to heat and UV-B irradiation the Hsp70 level increased. The apoptosis protective effect of Hsp70 was eliminated by Hsp70 siRNA transfection. UV-B induced apoptosis was accompanied by lysosomal and mitochondrial membrane permeabilization, detected as release of cathepsin D and cytochrome c, respectively, which were prevented by heat pre-treatment. In purified fractions of mitochondria and lysosomes, recombinant Hsp70 attached to both lysosomal and mitochondrial membranes. Moreover, in apoptotic cells Bax was translocated from a diffuse cytosolic location into punctate mitochondrial-like structures, which was inhibited by Hsp70 induction. These findings show Hsp70 to rescue melanocytes from UV-B induced apoptosis by preventing release of cathepsin D from lysosomes, Bax translocation and cytochrome c release from mitochondria.
OP16 - THE EFFECTS OF GLYCOSYLATION OVER THE TRAFFICKING AND TRANSFER OF PMEL17/GP100 REVEALED BY A NEW ANTIBODY, αPEP25H.
JulioC. Valencia, Francois Rouzaud, Kevin G. Chen, Gertrude E. Costin, Hiroshi Yamaguchi, Lisa M. Miller Jenkins, Ettore Appella and Vincent J. Hearing.
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
Pmel17 is a melanocyte/melanoma-specific protein that is essential for the maturation of melanosomes to form fibrillar and pigmented organelles. Among melanosome-specific markers, Pmel17 is the only one reported as a secretory protein. Pmel17 contains a mixture of complex, hybrid and mannose-rich carbohydrate N-glycan chains that distinguish its secreted and cytoplasmic forms. Recently, we reported that a partially glycosylated immature form of Pmel17 (iPmel17) is sorted via the plasma membrane (PM) in a manner distinct from mature Pmel17 (mPmel17) which is sorted directly to melanosomes (J.Cell Sci. 2006; 119:1080). To understand the mechanisms underlying the processing and sorting of Pmel17, we generated a highly specific antibody (termed αPEP25h) against the core region of Pmel17. We used αPEP25h to demonstrate that iPmel17 differs from mPmel17 due to the presence of more mannose-rich complex\ hybrid N-glycans and fewer O-glycan chains, both modified with sialic acid. αPEP25h stained specifically the cytoplasm of epidermal melanocytes and melanoma cells. Interestingly, it also exhibited a fine granular pattern in the extracellular spaces between keratinocytes located in the near basal and middle epidermal layers of human skin samples, a pattern that may result from the detection of Pmel17 inside exosomes or intralumenal vesicles near the PM as confirmed by immuno-electron microscopy. Thus, these results raise the possibility that posttranslational modifications may define new functions, such as cellular communication, for Pmel17 in addition to its well-known structural role in melanosomes.
OP17 - KERATINOCYTE GROWTH FACTOR DIFFERENTLY REGULATES THE PHAGOCYTIC PROCESS IN LIGHT AND DARK SKIN DERIVED KERATINOCYTES
G. Bolasco1,2, N. Aspite1, G. Lucania2, L. V. Lotti2, G. Cardinali1, M. R. Torrisi1,2,3 and M. Picardo1.
1Istituto Dermatologico San Gallicano, IRCCS, Rome;
2Dipartimento di Medicina Sperimentale e Patologia;
3Azienda Ospedaliera Sant’Andrea, Università di Roma ‘La Sapienza’, Rome, Italy
Melanin transfer from melanocytes to keratinocytes is necessary to protect skin from UV damage. This process is upregulated by UV itself and modulated by autocrine and paracrine factors, such as the keratinocyte growth factor (KGF/FGF7). We have recently demonstrated that KGF promotes melanosome transfer through stimulation of the phagocytic process. Moreover, we have suggested a direct role of KGF receptor in regulating the uptake and the intracellular traffic of the phagosomes (Cardinali et al. 2005). To search for possible differences in the phagocytic behaviour of keratinocytes from different skin colour, we compared the effect of KGF on the uptake kinetics and distribution pattern of latex beads in human primary keratinocytes (NHKs) from light and dark skin. Using a 3D image reconstruction carried out by fluorescence microscopy, we observed differences in the distribution pattern of the fluorescent latex particles in light and dark NHKs. To verify that the mechanism of beads internalization occurred via actin-dependent phagocytosis, we performed treatments with cytochalasin D, demonstrating that actin-depolymerization was effective in inhibiting particles uptake. In addition, confocal microscopy using fluorescent latex beads and FITC-dextran, a marker of phagosomal compartments, showed co localization of the two signals after internalization. Phagocytosis of the beads promoted by KGF treatment appeared more significant in light than in dark skin derived keratinocytes. These findings were lastly supported by time-lapse videomicroscopy and ultrastructural analysis, both revealing that beads within light and dark skin derived keratinocytes are differently distributed.
OP18 - TRANSFER OF MELANOSOMES FROM XENOPUS LAEVIS MELANOPHORES TO FIBROBLASTS
Daniel Hedberg, Sara Aspengren and Margareta Wallin.
Zoophysiology, Department of Zoology, Göteborg University, Sweden
In order to investigate an exocytosis/endocytisis model for transfer of melanosomes a cell culture method using melanophores and fibroblasts from the African clawed frog, Xenopus laevis, was established. This system was then used for characterization and quantification of transfer using fluorescent dyes, immunocytochemistry, Western blot and electron microscopy. The results where also compared to observations, by light and electron microscopy, of melanosomes primarily in the dermis of Xenopus laevis skin. In vitro transfer of pigment could be observed both in the close proximity of pigment cells but also without apparent cell-cell interaction. Specific groups of fibroblasts appeared to have an increased capability to absorb pigment. The export could be increased by adding α-MSH. The transferred pigment also co localized with fluorescent dyes marking melanophore membranes and cytoplasm. Transferred pigment was stored either in individual vesicles or in groups of vesicles surrounded by a larger membrane. Extracellular melanosomes with outer membranes could be found in the dermis of adult Xenopus laevis. These melanosomes where often close to or appeared to be taken up by extremely flat cells or fibroblast like cells in the dermis. Our results indicate that melanophores are transferred in an exocytosis/endocytosis like fashion but with a retained outer membrane during the process.
OP19 - 308 NM EXCIMER LASER IN THE REPIGMENTATION OF VITILIGO
Agustín Alomar, Lucía Pimentel and Adriana Ribé.
Hospital Sant Pau, Autonomous University of Barcelona, Barcelona, Spain
Excimer Laser (308 nm) has been accepted as a useful tool for the treatment of moderate vitiligo, with quite good responses. Nevertheless the mechanism of action it is not clearly understood. We have conducted an inmunohistochemical study, biopsing perifollicular areas before and after treatment. The post treatment biopsy was done at the early signs of repigmentation to try to define the modulation of the response with 308 nm irradiation. We were able to observe early refill of new melanocytes, Melan-A 81.2% increase expression in the basal layer, and a rise in C-Kit expression in 87.5% and Bcl 2 in 68.7% of the biopsies.
OP20 - PAR-2 EXPRESSION IN VITILIGO AND HALO NEVUS.
Silvia Moretti1, Antonella Simoni2, Paolo Fabbri1, Samantha Berti1 and Daniela Massi2.
1Department of Dermatological Sciences;
and 2Department of Human Pathology and Oncology, University of Florence, Florence, Italy
Protease-activated receptor (PAR)-2 is a member of a novel G-protein-coupled receptor superfamily, which is activated by the proteolytic cleavage of amino terminal domain. In the epidermis PAR-2 is expressed by \keratinocytes, being activated by trypsin-like enzymes such as mast-cell tryptase. PAR-2 seems to affect pigmentation via modulation of melanosome uptake in vivo, and appears involved in epidermal pigmentation. Since vitiligo and halo nevus are skin disorders characterized by disappearance of pigmented melanocytes, we evaluated PAR-2 expression in these conditions. Sixteen skin specimens from vitiligo patients with active disease and six halo nevi were examined by means of immunohistochemistry. PAR-2 protein was expressed on the epidermis (basal and suprabasal layers) of both vitiligo lesional and perilesional skin, with a trend toward a weaker staining in lesional skin, whereas normal control epidermis was more intensely stained. Halo nevi exhibited a strong reaction on nevus cells, especially in junctional nests, while overlying epidermis showed a weak staining. Common nevi revealed a good reactivity on both nevus cells and overlying epidermis. These data confirm that PAR-2 expression in the skin is involved in the pigmentation process and that its down-regulation may be associated with pigment loss in vitiligo and halo nevus.
OP21 - VITILIGO-INFILTRATING T CELLS KILL AUTOLOGOUS MELANOCYTES WITHIN THE SKIN TISSUE
Jasper G. van den Boorn1, Debby Konijnenberg1, Wanda Douwenga1, Trees A. M. Dellemijn2, Jan D. Bos1, J. P.Wietze van der Veen1, Cornelis J. Melief 3, Florry A. Vyth-Dreese2 and Rosalie M. Luiten1.
1Netherlands Institute for Pigment Disorders and dept of Dermatology, University of Amsterdam;
2The Netherlands Cancer Institute, Amsterdam;
3Leiden University Medical Center, The Netherlands
The autoimmune hypothesis for the pathogenesis of vitiligo vulgaris is based on the presence of autoimmune responses against melanocyte self-antigens that cause disappearance of melanocytes from the skin. To provide evidence for this hypothesis, we have previously shown that melanocyte antigen-specific CD8+ T cell infiltrate in vitiligo perilesional skin of vitiligo patients. Using skin explants we have developed one of the first ex vivo models of human autoimmune disease, in which we can dissect the precise cellular events causing melanocyte destruction. We have previously shown in this system that HLA-A2/tyrosinase-specific T cells can kill melanocytes in the non-lesional skin of vitiligo patients, whereas control HLA-A2/influenza virus-specific CTL infiltrated the skin explants, but did not induce apoptosis in melanocytes. We are now investigating the functional activity of the autologous T cells found in the perilesional vitiligo skin. Perilesional vitiligo T cells infiltrated the autologous non-lesional skin explants and caused apoptosis of epidermal cells, including melanocytes. Skin explant assays with sorted CD8+ en CD8- perilesional T cells showed that the cytotoxicity was mainly mediated by the CD8+ T cells. The skin incubated with perilesional T cells showed tissue destruction at the basal epidermal layer, leaving cavities at the site of the melanocytes. This effect was also seen in explant assays with HLA-A2/gp100-specific T cells, indicating that high doses of melanocyte-specific T cells can mediate this type of epidermal destruction. Further research in this model will provide direct evidence of T cell-mediated depigmentation in vitiligo vulgaris.
OP22 - ANALYSIS OF THE AUDITORY FUNCTION IN AN ANIMAL MODEL OF OCULOCUTANEOUS ALBINISM TYPE I
Silvia Murillo1, Marta Cantero2, Juan Jose Lazcano2, Isabel Varela-Nieto1 and Lluis Montoliu2.
1Instituto de Investigaciones Biomedicas ’Alberto Sols’, CSIC-UAM, 28029 Madrid, Spain;
2Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologia (CNB-CSIC), 28049 Madrid, Spain
Hearing impairment is often present in hypopigmented congenital diseases, such as albinism. Different types of albinism have also been related to auditory defects although its precise relevance and pathophysiological mechanism have not been yet established. There have been some reports describing auditory defects, for example, abnormal decussation of auditory pathways in albino animals, presumably correlated with its lack of pigmentation. Inside the cochlea, at the stria vascularis, there are neural crest-derived melanocytes. Cochlear hypopigmentation might cause these auditory alterations. In albino animals, these inner ear melanocytes are also non-pigmented. Here, we have used a genetically well-defined animal model of oculocutaneous albinism type I (OCA I) to specifically address the potential role of tyrosinase in ear development and hearing function, as it has been established in the retina, for the normal development of the visual system. Animal analysed are transgenic mice expressing a functional genomic-type tyrosinase construct that are proven to be phenotypically and functionally undistinguishable from pigmented mice, as compared with albino counterparts. In this case, albino and pigmented mice only differ in the presence of the tyrosinase transgene. Both pigmented and albino animals have been analysed by testing the auditory brainstem response (ABR), at different ages. Observed differences will be presented and discussed.
OP23 - HUMAN OCA2 LOCALIZES TO MATURE MELANOSOMES AND REQUIRES N-TERMINAL DILEUCINE MOTIFS FOR ITS TRAFFICKING
A. Sitaram1, A. C Theos1, D. C Harper1, D. Altimare2, M. V. Schiaffino2, and M. S. Marks1.1Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA;
2DIBIT, Scientific Institute San Raffaele, Milan, Italy
The mouse P gene and its human homolog OCA2 play an important but still undefined role in the biogenesis of melanosomes and in melanocyte pigmentation. Defects in these genes affect melanosome morphology and the maturation of tyrosinase. The site of OCA2 function is under debate. Our evidence suggests that OCA2 functions primarily at the mature melanosome. We show here that human OCA2 is N-glycosylated. Metabolic pulse/chase assays coupled with endoglycosidase H digestion of human OCA2 transiently expressed in HeLa cells or endogenously expressed in MNT-1 melanoma cells show that OCA2 rapidly acquires terminal glycosylation, indicating efficient export from the ER to at least the medial Golgi. Immunofluorescence data indicate that human OCA2 localizes to the lysosome or the mature melanosome when expressed in HeLa cells or mouse melanocytes, respectively. This localization depends upon dileucine-based sorting sequences in the cytoplasmic N-terminal region of the protein. Mutation of these sequences results in a redistribution of the protein to the cell surface. These results indicate a requirement for the dileucine motifs for melanosomal trafficking of OCA2, as has been noted for other melanosomal proteins, and suggest that OCA2 functions predominantly, if not exclusively, from mature melanosomes.
OP24 - TEN NOVEL MUTATIONS OF THE ADAR1 GENE IN PATIENTS WITH DYSCHROMATOSIS SYMMETRICA HEREDITARIA
Yasushi Tomita, Noriyuki Suzuki, Tamio Suzuki, Katsuhiko Inagaki, Shiro Ito and Michihiro Kono.
Department of Dermatology, Nagoya University Graduate School of Medicine
Dyschromatosis symmetrica hereditaria (DSH), an autosomal dominant inherited disease, characterized by hyperpigmented and hypopigmented macules on the face and dorsal aspects of the extremities that appear in infancy or early childhood. We for the first time clarified that a heterozygous mutation of the adenosine deaminase acting on RNA 1 (ADAR1) gene causes DSH in 2003, and so far 42 pathological mutations in the ADAR1 gene have been reported. ADAR1 protein catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates, which results in the creation of alternative splicing sites or alternations of the codon and thus leads to functional changes in the protein. Human ADAR1 is known to present two forms; an interferon-inducible full-length 150-kDa protein and a constitutively expressed 110-kDa protein. The synthesis of ADAR1-p150 and -p110 start from the initiation codon located in Exon1A and Exon2, respectively. The molecular pathogenesis of DSH has not been clarified yet. In this study, we report 10 novel mutations responsible for DSH: p.Q102fsX123, p.T369fsX374, p.S664fsX677, p.R892L, p.I913R, p.R916Q, p.P990fsX1016, p.C1081S, p.C1169F, and p.K1187X. Furthermore, we speculate that the full-length 150-kDa protein, one of the two forms of ADAR1, is possibly involved in DSH, because a novel p.102fs mutation and previously-reported p.216fs can probably produce the 110-kDa protein but not the full-length 150-kDa protein.
OP25 - DNA METHYLATION, GENE EXPRESSION AND GROWTH REGULATION IN MELANOMA CELLS
Daniela Kovacs1,Elaine Cheng2, Mario Sznol3, Stephan Ariyan4, Harriet Kluger3, Yuval Kluger5, Sherman Weissman6, Mauro Picardo1 and Ruth Halaban2.
1San Gallicano Dermatological Institute, Rome, Italy;
2Department of Dermatology;
3Comprehensive Cancer Center Section of Medical Oncology;
5Cell Biology, New York University School of Medicine, NewYork, NY, USA;
6Genetics, Yale University School of Medicine, New Haven, CT, USA
Epigenetic alterations in chromatin play an important role in malignant progression by affecting the expression of critical genes such as those regulating differentiation, growth and survival of tumor cells. For example, aberrant DNA methylation of cytosine residues in CpG rich islands of promoter regions, commonly observed in tumors including melanoma, is known to suppress gene expression. The reversibility of epigenetic changes is the rationale for novel cancer therapies involving epigenetic modifiers, such as the demethylating agent azacitidine and its analogue 5-aza-2’-deoxycytidine (5-Aza-CdR) that are currently approved in clinical trials. We therefore assessed the growth inhibitory effect of 5-Aza-CdR and its effect on gene expression employing whole genome microarrays. Most melanoma cells displayed high sensitivity to the drug that was associated with changes in the expression of common genes, some belonging to the apoptotic/growth arrest, immune responses and metabolic pathways. We assessed changes in the CpG methylation pattern at the promoter regions of selected genes and confirmed that changes in gene expression was associated with the epigenetic effect of the drug. Furthermore, we also identified novel genes silenced by DNA methylation in melanoma cells compared to normal melanocytes. Our analysis may reveal novel candidate tumor suppressor genes that can be re-activated by 5-Aza-CdR. In addition, the methylation status of these genes can be used as markers for the efficacy of demethylating agents in the treatment of melanoma.
OP26 - β-CATENIN SUPPRESSES P16INK4A EXPRESSION TO INDUCE IMMORTALISATION OF MELANOCYTES AND IT COOPERATES WITH N-RAS IN MELANOMA DEVELOPMENT
Véronique Delmas1, Friedrich Beermann2, Silvia Martinozzi1, Suzanne Carreira3, Julien Ackermann2, Mayuko Kumasaka1, Laurence Denat1, Jane Goodall3, Nese Demirkan1, Colin R. Goding3 and Lionel Larue1.
1Institut Curie, Orsay Cedex, France;
2ISREC, Epalinges, Switzerland;
3Marie Curie Research Institute, Oxted, UK
As activated RAS and RAF promote senescence in primary cells, senescence bypass is a critical event in cancer progression and occurs through inactivation of the Rb pathway, frequently by mutation or silencing of the p16INK4a gene. Malignant melanoma, a highly metastatic and increasingly common cancer, represents an important model for understanding senescence bypass in transformation, as nevi bearing activating NRAS or BRAF mutations comprise a mass of senescent cells. Loss of expression or mutation of the p16INK4alocus is one of the key events during melanoma progression, yet the mechanism underlying transcriptional silencing of p16INK4a in melanoma is unknown. Many melanomas and other cancers exhibit constitutive activation and nuclear accumulation of β-catenin, a critical component of the Wnt signalling pathway. Here we show that synergy between the Wnt and MAP kinase pathways in melanocytes leads to melanomagenesis in vivo. Activated β-catenin silences the p16Ink4a promoter, leading to highly efficient immortalization of primary skin melanocytes, and consequently bypasses the requirement for p16INK4a loss in melanoma predisposition. The results provide a direct molecular link between β-catenin and the melanoma susceptibility gene p16Ink4a, and reveal a novel pathway for inactivation of p16Ink4a expression and senescence bypass relevant to a wide range of cancers.
OP27 - THERAPEUTIC EFFICACY OF ANTIGEN-SPECIFIC VACCINATION AND TOLL-LIKE STIMULATION AGAINST ESTABLISHED TRANSPLANTED AND AUTOCHTHONOUS MELANOMA IN MICE
Damià Tormo, Aleix Ferrer, Pilar Bosch, Evelyn Gaffal, Etiena Basner-Tschakarjan and Thomas Tüting.
Laboratory of Experimental Dermatology, University of Bonn, Germany
Antigen-specific immunotherapeutic approaches for melanoma have been primarily investigated using the transplantable B16 melanoma cell line in mice. To more closely mimic the clinical situation in patients with melanoma, we use genetically modified mice overexpressing hepatocyte growth factor (HGF) and carrying an oncogenic mutation in the cyclin dependent kinase 4 (CDK4R24C). HGF x CDK4R24C mice rapidly develop multiple invasive melanomas in the skin following neonatal carcinogen treatment, which spontaneously metastasize to lymph nodes and lung. We used a vaccine strategy consisting of recombinant adenovirus encoding human tyrosinase-related protein 2 (Ad-hTRP2) and application of CpG DNA and synthetic dsRNA against engrafted B16 melanoma or primary autochthonous melanomas in the skin and in the lung of HGF x CDK4R24C mice. Both Ad-hTRP2 vaccination and peritumoral injections of TLR ligands were required for rejection of established B16 melanoma in the skin. Vaccination with Ad-TRP2 followed by injections of TLR ligands resulted in delayed growth of autochthonous primary melanomas in the skin and a reduction in the number of spontaneous lung metastases but did not induce tumor regression. Carcinogen-treated HGF x CDK4R24C mice bearing multiple autochthonous melanomas did not reject transplanted B16 melanoma despite treatment with Ad-hTRP2 and TLR ligands. These resultssuggest the development of a profound tumor immune tolerance in mice carrying primary autochthonous melanomas.
OP28 - VITILIGO DEVELOPMENT IN METASTATIC MELANOMA PATIENTS FOLLOWING VACCINATION WITH AUTOLOGOUS GM-CSF-TRANSDUCED TUMOR CELLS
RosalieM. Luiten1, Esther W.M. Kueter2, Wolter Mooi4, Maarten P.W. Gallee4, Elaine M. Rankin3, Winald R. Gerritsen3, Shirley M. Clift8, Willem J. Nooijen5, Pauline Weder2, Willeke F. van de Kasteele2, Johan Sein2, Paul C. M. van den Berk2, Omgo E. Nieweg6, Anton M. Berns7, Hergen Spits2 and Gijsbert C. de Gast3.
1Netherlands Institute for Pigment Disorders and Dept. of Dermatology AMC, University of Amsterdam, The Netherland;
Departments of 2Immunology;
and 7Molecular Genetics of the Netherlands Cancer Institute, Amsterdam, The Netherlands;
8Cell Genesys, Foster City, California, USA
To determine the feasibility, toxicity and immunological effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients. Sixty-four patients were randomized to receive three vaccinations of high dose or low dose tumor cells at three weeks intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but due to progressive disease the well-tolerated vaccination was completed in only 28 patients. We analyzed the priming of T cells against melanoma antigens, MART-1, tyrosinase, gp100, MAGE-A1 and MAGE-A3, using HLA/peptide tetramers and in functional assays. The high dose vaccination induced the infiltration of T cells into the tumor tissue. Three of 14 patients receiving the high dose vaccine showed an increase in MART-1 or gp100-specific T cells in the peripheral blood during vaccination. Six patients experienced disease free survival for more than 5 years, and two of these patients developed vitiligo at multiple sites after vaccination. MART-1 and gp100-specific T cells were found infiltrating in vitiligo skin. Upon vaccination, the T cells acquired an effector phenotype and produced IFNγ upon specific antigenic stimulation. We conclude that vaccination with GM-CSF-transduced autologous tumor cells has limited toxicity and can enhance T cell activation against melanocyte differentiation antigens, which can lead to vitiligo. Whether the induction of autoimmune vitiligo may prolong disease free survival of metastatic melanoma patients that are surgically rendered, as having no evidence of disease prior to vaccination is worthy of further investigation.
OP29 - INCORPORATION TO MELANOMA CELLS AND INDUCTION OF CELL DEATH BY MAGNETITE-CONJUGATED N-PROPIONYL CYSTEAMINYLPHENOL (NPRCAP/M) NANOPARTICLES FOR MELANOMA CTI THERAPY
Makito Sato1, Toshiharu Yamashita1, Masae Ohkura1, Akiko Sakemoto1, Tomoaki Takada1, Hidenobu Matsusaka1, Ichiro Ono1, Yasuaki Tamura2, Noriyuki Sato2, Akira Ito3, Hiroyuki Honda4, Kazumasa Wakamatsu5, Shosuke Ito5 and Kowichi Jimbow1.
Departments of 1Dermatology;
and 21st Pathology, Sapporo Medical University;
3Department of Chemical Engineering, Kyusyu University;
4Department of Biotechnology, Nagoya University;
5Department of Chemistry, Fujita Health University, Japan
Cysteaminylphenol is efficiently incorporated into melanoma cells and reacts with tyrosinase, resulting in melanoma-cell death. In order to establish melanogenesis-targeted chemo-thermo-immuno therapy (CTI) through conjugating NPrCAP with magnetite (M) nanoparticles, we studied i) by what method NPrCAP is most appropriately conjugated with M in order to be efficiently incorporated into melanoma cells, ii) how NPrCAP/M is selectively incorporated into melanoma cells, and iii) by what manner NPrCAP/M with exposure to AMF (alternating magnetic field) can disintegrate melanoma cells. The in vitro uptake study showed the direct conjugation of NPrCAP with M (NPrCAP/M) to be much better than that with neutral or cationic magneto-liposomes (ML or CML). Electron microscopic examination showed that NPrCAP/M nanoparticles were selectively incorporated into melanoma cells, often showing large conglomerations whereas they were aggregated often non-specifically around the non-melanoma cell surface. Flow cytometric and agarose gel electrophoresis using p63/53O chimera cDNA indicated that the cell death of NPrCAP/M with AMF is mediated not by apoptosis, but necrosis, the most efficient immune reaction manner for CTI.
OP30 - GENE EXPRESSION PROFILING OF HUMAN MELANOCYTES FROM DIFFERENT SKIN PIGMENTATION AFTER REPEATED UV-A+B IRRADIATION
Alonso S1, Smith-Zubiaga I2, Smit N3, Boyano MD4, J, Díaz-Pérez JL5, García I1, Izagirre N1 and de la Rúa C1.
1Dept. Genetics, Physical Anthopology and Animal Physiology. Faculty of Science and Technology. University of the Basque Country. 48940 Leioa, Bizkaia, Spain;
2Dept. Zoology and Animal Cell Biology. Faculty of Science and Technology. University of the Basque Country. 48940 Leioa, Bizkaia, Spain;
3Dept. Clinical Chemistry. Leiden University Medical Center Albinusdreef 2 2333 AZ Leiden, the Netherlands;
4Department of Cell Biology and Histology.Faculty of Medicine and Dentistry. University of the Basque Country. 48940 Leioa, Bizkaia, Spain;
5Dermatology Service. Cruces Hospital. 48903 Barakaldo, Bizkaia, Spain
We have investigated the gene expression profile of seven cultured human melanocytes from donors of light (n = 4) and dark (n = 3) pigmentation, and compared them to the expression profiles of the same cells after repeated irradiation with daily doses of approximately 50 mJ/cm2 of UV-A and UV-B (once a day for consecutive 6 days). mRNA was hybridized to Affymetrix U133Av2 chips. After normalization (using smoothing curves), we used SAM software for pattern discovery by means of principal components. Functional profiles were obtained using ONTO-EXPRESS software. While melanoytes from lightly pigmented individuals were visually less pigmented than those melanocytes from more darkly pigmented donors, unexpectedly, the major difference in gene expression among within the data is not associated to differences in the donor´s pigmentation or the irradiation status. Instead the major variance in overall expression is associated to the different behaviour of the cells regarding proliferation and differentiation. Less differentiated and more proliferating cells show an increased expression of the major pigmentation genes (TYR, TYRP1, DCT, MC1R, OCA2, AIM-1, EDNRB3, PKC-b, HPS, GPR143, AP3D1, RAB27, KIT, MITF, MLANA, MYO5Aa and MYO10 among others) (at a FDR 5%). This expression is also associated to increased expression of DNA repair genes like BRCA1, MDM2 or ATM. Although this association might suggest a role for pigmentation in photo protection against skin cancer, however, a greater expression level of pigmentation genes was not associated to greater pigmentation within the cell. We speculate that senescence or the accumulation of melanin within the melanocyte might also act as inhibitors of the expression of pigmentation genes.
OP31 - CONTRIBUTION OF MELANOCORTIN 1 RECEPTOR MEDIATED PIGMENTARY AND NON-PIGMENTARY MECHANISMS IN IN VIVO SKINRESPONSES TO ULTRAVIOLET RADIATION
S. Robinson, S Dixon, P. S. Friedmann and E. Healy.
Dermatopharmacology, University of Southampton, UK
An association between melanocortin 1 receptor (MC1R) variants and skin cancer is well established. MC1R variants may predispose to skin cancer through their effects on pigmentation (via phaeomelanin mediated phototoxicity or reduced photoprotection). Alternatively, based on case control and in vitro studies, MC1R variants may increase skin cancer risk through non-pigmentary mechanisms. We have generated a MC1R transgenic Mc1r null hairless pigmented and albino mouse line to define the relative contribution of MC1R mediated pigmentary and non-pigmentary effects in vivo in skin responses to ultraviolet radiation (UVR) / skin cancer susceptibility. Following a single exposure to UVR, the number of epidermal nuclei positive for cyclobutane pyrimidine dimers (CPDs) in UV-naïve skin was similar in the MC1R+Mc1r-/-pigmented, Mc1r-/-pigmented, MC1R+Mc1r-/-albino and Mc1r-/-albino groups. No difference in numbers of CPD positive epidermal nuclei after UVR was observed in UV-acclimatised skin between these four groups. By contrast, fewer p53 epidermal clones were noted in the MC1R+Mc1r-/-albino mice than in the Mc1r-/-albino animals following repetitive UVR exposure (P < 0.05), suggesting the MC1R mediated non-pigmentary mechanisms protect against chronic repetitive UVR. Fewer epidermal p53 clones were also seen in the Mc1r-/-pigmented than in the Mc1r-/-albino mice (P < 0.05), consistent with protection by a ‘phaeomelanin phenotype’ in vivo. Furthermore, the MC1R+Mc1r-/-pigmented animals contained lower numbers of p53 clones than the Mc1r-/-pigmented and MC1R+Mc1r-/-albino groups (P < 0.05). The results demonstrate that MC1R protects skin against UVR through a combination of pigmentary and non-pigmentary effects.
OP32 - UV-A-INDUCED APOPTOSIS IN NORMAL HUMAN MELANOCYTES IS ASSOCIATED WITH DOWN-REGULATION OF β-CATENIN AD WITH REDUCED TRANSACTIVATION POTENTIAL OF THE TCF/LEF-β-CATENIN COMPLEX
Barbara Bellei1, Veronica Albanesi1, Lionel Larue 3, Vincent J Hearing2 and Mauro Picardo1.
1Istituto Dermatologico S. Gallicano, Rome, Italy;
2Laboratory of Cell Biology, NCI, Bethesda, MD 20892, USA;
3Developmental Genetics of Melanocytes, CNRS-Institut Curie, Orsay, Cedex, France
Human skin is exposed daily to UV irradiation leading to cutaneous aging and skin cancer. To protect the skin from accumulating damage or mutated cells, human skin cells have developed a highly regulated mechanism of eliminating damaged cells through apoptosis which is normally initiated within 6–9 h after UV irradiation Preliminary experiments showed that UV-A-induced apoptosis (16 J/cm2) is associated with the down-regulation of β-catenin in normal human melanocytes (NHM) and that caspase-3 is the major protease involved in this process. Interestingly, down-regulation of Cyclin D1, a Wnt target gene, is only partially blocked by caspases inhibitors, suggesting that following UV-A irradiation, β-catenin is regulated not only at the protein level but also by inhibiting Tcf/Lef-dependent transcription. We analyzed Cyclin D1 mRNA by RT-PCR and the activity of Tcf-Lef/β-catenin complex on the TOP-FLASH reporter plasmid and confirmed that following UV-A irradiation β-catenin-mediated transcription is down regulated. Starting 1 h after irradiation, the inhibitor of β-catenin and T-cell factor (ICAT) co-immunoprecipitated with β-catenin suggesting that multiple mechanisms regulate β-catenin activity. Altogether, these results suggest that Wnt signalling has a crucial role in the regulation of stress-induced mechanism activated by UV-A in NHM.
OP33 - DLQI SCORE IN VITILIGO AND ITS IMPACT ON THE TREATMENT OUTCOME
D. Parsad and A. J. Kanwar.
Department of Dermatology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.
Background: Vitiligo has a major impact on patient's quality of life. A number of studies have demonstrated the impact of vitiligo on quality of life, however there is no study to reflect its impact on the treatment outcome in vitiligo.
The purpose of this study was to assess the nature and extent of the social and psychological difficulties associated with vitiligo and its impact on treatment by using dermatology life quality index (DLQI).
Patients and Methods: One hundred and fifty patients with vitiligo vulgaris involving more than 10% body surface area were enrolled for this study. They were given Dermatology life quality index (DLQI) questionnaire to fill. These patients were treated as per protocol of our pigmentary clinic. Two parameters; repigmentation and arrest of progression of disease activity were considered to evaluate the response of treatment. The patients were grouped into two groups depending upon response to treatment, group A showing successful response and group B showing treatment failure.
Scores on the DLQI ranged from 2 to 21 (mean 10.67, SD = 4.56). At the end of treatment period, 141 patients could be evaluated. 91 patients were considered to be showing successful treatment outcome and remaining 50 as failure. Mean DLQI was 7.06 of patients with successful treatment outcome (Group A) whereas it was 13.12 for patients of patients with treatment failure (Group B) (P < 0.0001).
Conclusion: Our study clearly demonstrated that patients with high DLQI scores responded less favorably to a given therapeutic modality. These results suggest that psychological approaches may be particularly helpful in this respect.
OP34 - THE VITILIGO MELANOCYTORRHAGIC HYPOTHESIS TESTED ON PIGMENTED RECONSTRUCTED EPIDERMIS STRESSED WITH VARIOUS (IMMUNE-RELATED, PRO-OXIDATIVE AND NEUROGENIC) AGENTS
M. Cario-André, C. Pain, D. Parsad, Y. Gauthier and A. Taieb.
Department of Dermatology, Bordeaux University Hospitals; INSERM E217; University V Segalen, Bordeaux, France
Background: Common generalized vitiligo is an acquired depigmenting disorder characterized by a chronic and progressive loss of melanocytes from the epidermis and follicular reservoir. We have previously proposed a new theory integrating autoimmune, neural and impaired redox status theories associated with mechanical trauma, which supports the chronic detachment and transepidermal loss of melanocytes. In this study, we tested catecholamines, hydrogen peroxide and sera from non-segmental vitiligo (NSV) patients on pigmented reconstructed epidermis. Our reasoning was that in this model, in which epidermis is weakly attached to a dead deepidermised dermis, friction is not necessary to induce detachment.
Methods: Cells were obtained from patients with NSV and normal controls (plastic surgery specimen). Vitiligo was classified according to extent, stage, and spread. Stressors were chosen according to the major current theories of NSV, i.e. decomplemented sera from patients to investigate a possible immune-related loss of melanocytes, H2O2 (from 0.0001% to 0.1%) as a natural potent oxidant, and catecholamines (0.5 mg/ml dopamine or 0.02, 0.1 or 0.2 mg/ml adrenalin or 0.2 or 0.4 mg/ml noradrenalin) as potential noxious effectors of peripheral nerves. Epidermis was reconstructed with various combinations of melanocytes and keratinocytes from normal skin (n = 20) and non lesional NSV skin (n = 15). Experiments were analyzed 3, 6, 16, 24, 48 or 72 h after stress.
Results: Under unstressed conditions, the number of melanocytes located in the basal layer was significantly lower in reconstructs made with melanocytes from non lesional NSV skin and normal keratinocytes as compared to control made with the same normal keratinocytes and autologous melanocytes (P = 0.012). In contrast with catecholamines and H202, some sera induced melanocyte detachment unrelated to the extent/spread of NSV in donors (in heterologous conditions).
Conclusion: We did not succeed to obtain a reproducible transepidermal loss of melanocytes in vitro with the stressors investigated. However, our results suggest that the adhesion defect of NSV melanocytes is mostly intrinsic.
OP35 - MEMBRANE-ASSOCIATED IMPAIRMENT IN MELANOCYTE: A NEW POINT OF VIEW FOR THE MELANOCYTE DEGENERATION IN VITILIGO
Maria Lucia Dell’Anna, Monica Ottaviani, Andrea Paro Vidolin, Carlo Cannistraci, Giovanni Leone and Mauro Picardo.
San Gallicano Dermatological Institute Rome Italy
Epidermal melanocytes from vitiligo subjects are reported to be highly susceptible to physical and chemical oxidative stress. Previously, we proposed that the mitochondrial impairment can be the biochemical marker of the oxidative stress and the basis for the disappearance of melanocytes. The aim of the present study was to better characterize the site of the possible mitochondrial defect and the degree of susceptibility to peroxidative stimuli in primary epidermal melanocytes of five vitiligo patients and healthy subjects. We evaluated the pattern of the total membrane fatty acids (GC-MS method), the transmebrane distribution of the cardiolipin (FACS analysis of the fluorescence intensity of NAO), and the degree of membrane lipoperoxidation (FACS and confocal analysis of the fluorescence pattern of C11-BODIPY581/591). Moreover, we evaluated the modulatory activity of two different pro-oxidant stimuli (7–30 mJ/cm2 UV-B and 50–500 μM tBOOH) on the intracellular pathway associated with Ca++ fluxes. We observed a different transmembrane cardiolipin distribution and lipid peroxidation in vitiligo melanocytes. The exposure to the peroxidative stimuli gave rise to a pronounced alteration of these parameters and affected the intracellular Ca++ fluxes. In conclusion, we described a possible intracellular pathway, involving the structure and the function of the lipid envelope and the kinetic of calcium, leading to the increased susceptibility to mild peroxidative stimuli.
OP36 - ASSOCIATION OF GLUTATHIONE S-TRANSFERASE GENE POLYMORPHISMS (GSTM1 AND GSTT1) OF VITILIGO IN KOREAN POPULATION
Mu-Hyoung Lee1, Yoon Kyung Uhm2, Seo Hyun Yoon2, Ik Joon Kang1, Joo-Ho Chung2 and Sung-Vin Yim2
1Department of Dermatology;
2Kohwang Medical Research Institute, Department of Pharmacology, College of Medicine, Kyunghee University, Seoul 130-702, Korea
Vitiligo is an acquired pigmentary disorder of the skin involving melanocyte dysfunction. It has been reported that melanocyte impairment could be related to increased oxidative stress. The glutathione S-transferases (GSTs) are group of enzymes that are important in protection against oxidative stress. To address the possible association between oxidative stress and vitiligo susceptibility, GSTM1 and GSTT1 (homozygous deletion vs. non-deleted) polymorphisms between vitiligo patients (n = 310) and healthy controls (n = 549) were analyzed and compared. We observed a significant association in null alleles of the GSTM1 (P < 0.001, OR = 2.048, 95% CI = 1.529–2.743!). GSTM1 null type was also statistically different between vitiligo subtypes (Focal P < 0.001, OR = 2.224, 95% CI = 1.499–3.298; Generalized P = 0.001, OR = 1.974, 95% CI = 1.342–2.904) and controls. However, no significant association in GSTT1 (P = 0.869, OR = 1.024, 95% CI = 0.775–1.353) was observed with vitiligo. In combined analysis of GSTM1 and GSTT1, both null type and GSTM1/GSTT1 (±) group showed significant differences between controls and vitiligo patients! . These results suggest that GSTM1 null type might be associated with vitiligo susceptibility in Korean population.
OP37 - IMPROVED TECHNOLOGIES FOR TRANSLATING CULTURE OF HUMAN MELANOCYTES TO THE CLINIC
Paula Eves1, Alison Beck1, Alex Shard1, Nial Bullett2, David Haddow2 and Sheila MacNeil1.
1Department of Engineering Materials, University of Sheffield, Sheffield, S3 7HQ, UK;
2CellTran Limited, The Innovation Centre, Sheffield, S1 4DP, UK
Obtaining pigmentary function in autologous skin grafts is a current challenge in the healthcare industry as is developing reliable robust grafting strategies for patients with vitiligo. In this paper we present ongoing work aimed at developing a simple methodology for delivering cultured cells to the patient that is clinically effective, low risk for the patient but also user friendly for the surgeon. So far, we have demonstrated that chemically defined substrates can be used to culture both melanocytes and keratinocytes in medium (Greens) currently used in the clinic (which contains fetal calf serum sourced from New Zealand), but also in a serum free alternative - M2. We have also achieved successful transfer of melanocyte: keratinocyte co-cultures from flexible plasma polymerized silicone carriers onto an in vitro human wound bed model. However, in order to progress this work into a clinical environment we need to establish culture protocols within a GMP clean room and develop a cell transport media based on agar/hyaluronic acid for the transport of cells over a wide geographical area. In conclusion we have developed a pre-clinical methodology for the delivery of a co-culture of autologous melanocytes and keratinocytes using a flexible and user-friendly chemically defined carrier surface. We now hope to translate this work from the laboratory into the clinic.
OP38 - 11.000 SESSIONS WITH THE 308 NM EXCIMER LASER IN VITILIGO PATIENTS. AN EVIDENCE BASED, RETROSPECTIVE STUDY
A. Overbeck1 and I. C. le Poole2
1Lumiderm, Madrid Spain;
2Dept of Pathology/ Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL, USA
Background: Together with other concepts of phototherapy and in combination with chemotherapy the excimer laser plays an increasing role in repigmenting Vitiligo
Aim: To show retrospectively the limits and possibilities of the laser, proposing guidelines for its successful use and outline situations where the laser doesn't fulfill the expectation.
Results: Based on the results of over 11.000 sessions performed by the author with 330+ patients during 2.5 years it seems evident that by respecting a rigid selection and limitations known from other methods of phototherapy and combining the laser with chemotherapy it is possible to reach a high success-rate of pigmentation. Nevertheless the laser is not a miracle and should not be misused to create false hopes in patients. Also it seems clearly possible to correlate therapeutical success with conditions of the disease and thus predict a possible success in advance.
OP39 - FUNCTIONAL REGULATORY T-CELLS ARE PRESENT IN PERIPHERAL BLOOD BUT ABSENT FROM SKIN IN VITILIGO PATIENTS
IC le Poole1, C. Hernandez2 and A Overbeck3
1Dept of Pathology/ Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL,USA;
2Dept of Dermatology, University of Illinois at Chicago. 3Lumiderm, Madrid, Spain
Aim: To define the role of regulatory T cells in persistent autoimmune reactivity to melanocytes by immunohistochemistry, fluorescence activated cell sorting and proliferation assays involving vitiligo skin and blood.
Background: Autoreactive CD8+ T cells responsive to melanocyte specific antigens are abundant in perilesional skin from progressive vitiligo patients. Thus it appears that an immune response to self-antigens is not properly kept in check in patients with active disease. In skin from control donors, regulatory T cells (Treg) infiltrate the skin to maintain a healthy balance between immune reactivity and tolerance. Others have recently shown that the abundance of Treg in melanoma tumors can interfere with an effective immune response to the tumor. Thus we hypothesized that depigmentation of vitiligo skin will progress in the absence of Treg activity
Results: We can show, that Tregs are virtually absent from vitiligo skin. By contrast, the number of circulating Tregs is elevated in vitiligo patients compared to controls. Circulating vitiligo Tregs are equally effective in suppressing autologous Th proliferation compared to control Treg. The absence of Treg in vitiligo skin cannot be explained by lack of CLA expression by Treg, or lack of CCL1 expression in vitiligo skin. A potential defect in CCR8 expression among circulating vitiligo Tregs responding to CCL1 remains to be further explored.
OP40 - LONG-TERM RESULTS OF MINIGRAFT TRANSPLANTATION IN PATIENTS WITH VITILIGO VULGARIS AND SEGMENTALIS
A. Fongers, L. Nieuweboer-Krobotova, A. Wolkerstorfer and J. P. W. van der Veen
Netherlands Institute for Pigment Disorders, Amsterdam, the Netherlands
In this cohort study, with a mean period of follow-up of 4.8 years, the outcome of repigmentation after minigrafting in 67 vulgaris lesions and nine segmental lesions was evaluated. The degree of repigmentation was measured with the use of a digital image analysis system. In the group of vulgaris lesions, excellent; good; fair and poor repigmentation was found in 26.9%; 23.9%; 22.4% and 26.9%, respectively. In the group of segmental vitiligo, 78% of the lesions showed excellent repigmentation. We found a relationship between disease activity assessed by Koebner phenomenon of the donor site and the outcome of repigmentation. In the vulgaris group, lesions on the joints showed the highest amount of repigmentation, with a mean of 92.5%, in contrast to the inner side of the wrists, which showed a repigmentation of 30.3%. In conclusion, minigrafting provides long-lasting good to excellent results in half of the patients with vitiligo vulgaris and in 89% of the patients with segmental vitiligo. Results are better in patients with stable disease, without Koebner phenomenon at the time of minigrafting.
OP41 - PROPOSAL FOR A NEW CLASSIFICATION OF SEGMENTAL VITILIGO OF THE FACE
Y. Gauthier and A. Taïeb.
Dept of Dermatology, CHU de Bordeaux, France
Segmental vitiligo (SV) is usually localized to one dermatome, and is characterized by a relatively stable course and low prevalence of associated autoimmunity. The pathogenesis of SV remains unclear. We evaluated 130 SV patients with facial involvement. In 45 cases, SV was distributed to a trigeminal dermatome, ophthalmic (V1) in 20 cases, maxillary(V2) in 20 cases, and mandibular (V3) in 5 cases. In 85 patients SV did not follow exactly dermatomes and was overlapping one, two or three dermatomes. According to distribution, we thus propose a new classification of facial SV according to five topographic patterns:
Type 1 - Corresponding to V1, ophthalmic branch
Type 2 - V2, maxillary branch
Type 3 - V3, mandibular branch
Type 4 - mixed distribution either V1+V2 or V2+V3 or V1+V2+V3
Type 5 - Cervicofacial.
Several observations have led to the hypothesis that the primary disturbance of SV lies in the function of sympathetic nerves of the affected areas, e.g. degeneration changes in terminal cutaneous nerves, increase in cutaneous blood flow, increase cutaneous ( and ( adrenoreceptors responses, increased sweating. In some cases SV has developed in areas supplied by nerves damaged in an injury, suggesting that a ‘sympathetic repercussion’ may result in the onset of leucoderma. Our own observations suggest that irritation or chronic compression of the sympathetic nerve endings of one or several sensitive roots can trigger inappropriate reflexes, which result in depigmentation, of true dermatomal distribution in some cases, but also in the majority of cases crossing two or more dermatomes. The possibility of another predisposing local factor such as a somatic mosaic gene susceptibility to develop vitiligo would explain the limited, segmental distribution of most cases of SV.
OP42 - FACTORS AFFECTING AUDITORY DISTURBANCES IN VITILIGO
A. J. Kanwar and D. Parsad.
Department of Dermatology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
Background: Audiological abnormality in vitiligo patients is a controversial subject. Studies conducted on this subject have shown variable results. Melanocytes in inner ear seem to play role in hearing. Purpose of this study was to determine occurrence of audiological abnormalities in vitiligo patients in Indian population and to find out its relation to various disease parameters.
Patients and Methods: One hundred patients of vitiligo and fifty controls were included in this study. Pure tone thresholds were determined at frequencies between 250 and 12 000 Hz.
Results: Twenty-two patients (22%) patients of vitiligo had sensorineural hypoacusis and among controls only four (8%) had sensorineural hypoacusis. The difference was statistically significant. Majority of patients had audiological abnormalities at higher frequencies. Audiological abnormalities in vitiligo patients were significantly higher in older age group, with longer duration of illness, more extensive disease and patients who had leukotrichia.
Conclusion; Vitiligo seems to be an effective factor in hearing loss The mechanism for this condition might be the absence of the protective function of melanin containing cells in the inner ear
PP001 - ENDOTHELIN RECEPTOR B2 MUTATED JAPANESE QUAIL WITH PANDA PLUMAGE COLOR SHOWS HYPOPIGMENTATION DURING DEVELOPMENT
T. Akiyama1, M. Miwa2, M. Inoue-Murayama2, M. Mizutani3, Y. Kayashima1 and S. Ito2.
1Dept. of Biology, Keio University, Yokohama, Japan;
2Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan;
3Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
Autosomal recessive allele (s) in the Japanese quail (Coturnix japonica) controls white with spots of wild type (+) plumage (Panda) and the mutation site in the responsible gene was identified at the 332His on the 6th transmembrane region of the endothelin receptor B2 (EDNRB2) (Miwa et al, 2005). The association of EDNRB2 with plumage color in Aves was firstly reported. In this study, we analyzed pigmentation during developmental process and effects of the mutated EDNRB2. These s/s quail developed normally without any malformation and delay but hypopigmentation from embryogenesis. In 5–8 day-embryos, two lines of pigmented feather buds were appeared in the dorsal central region parallel to craniocaudal axis in +/+ but very faintly pigmented in s/s and remained white color in other trunk region. Organ cultures of these skin pieces from embryos, a very low number of melanocytes migrated out from s/s even pigmented area and disappeared after migration but many melanoblasts were found in both cases with immunofluorescence. The s/s quail with wild-type feather in dorsal region (wild pattern) implies the existence of other regulation systems preceded than ETs-EDNRB2 systems for pigmentation.
PP002 - WNT3A REGULATES THE DEVELOPMENT OF MELANOCYTE PRECURSORS IN MURINE NEURAL CREST CELLS AND NEURAL CREST-DERIVED CELL LINES
Chang Chung-Hsing1,3, Shie Jia-Heng1,3 and Tsai Rong-Kung2,3.
1Department of Dermatology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan;
2Department of Ophthalmology, Buddhist Tzu Chi General Hospital, Hualien, Taiwan;
3Graduate Institute of Medicine, Tzu Chi University, Hualien, Taiwan
Melanocytes originate from neural crest. Wnt 3a and the Frizzled 3 receptor are expressed in the dorsal neural tube and ectoderm. We investigated the expression of Frizzed 3 on melanocyte lineage and the effect of Wnt3a on melanocyte development by murine embryo sections, neural crest explant cultures and three neural crest-derived cell lines NCCmelb4M5 (GFAP+, Kit-, TRP-1+, tyrosinase-, DOPA-, without melanosomes), NCCmelb4 (Kit+, TRP-1+, tyrosinase-, DOPA-, with only stage I melanosomes) and NCCmelan5 (Kit+, TRP-1+, tyrosinase+, DOPA+, with stage II and III melanosomes). We identified TRP-1+ melanocyte precursors express the Frizzled 3 receptor in embryo sections, NCC explants and three melanocyte precursor cell lines. Wnt3a increases the TRP-1+ cell population, and also increases the DOPA+ cell number in NCC explants. Total dendrite number and dendrite length of melanocytes are significantly increased by Wnt3a treatment in NCC explants. Wnt3a stimulates the proliferation of three cell lines by a dose dependent manner. Wnt3a stimulates the tyrosinase activity and melanin formation in NCCmelan5 cells, but not in NCCmelb4M5 or NCCmelb4 cells. Cells of three cell lines express MITF with the fluorescence intensity of NCCmelb4M5 < NCCmelb4 < NCCmelan5. Wnt3a differentially regulates the mRNA of MITF in cells with various developmental stages.
PP003 - CHARACTERIZATION OF THE MELANOCYTE LINEAGE IN PATCHWORK HAIR FOLLICLES
J. Djian-Zaouche1, G. Aubin-Houzelstein1, F. Bernex1, Da Silva N.1, P. Salaün1 and J-J. Panthier1,2.
1Cellular and Molecular Genetics UMR 955 INRA-ENVA, Ecole Nationale Vétérinaire d’Alfort, 7 avenue du Général-de-Gaulle, 94704 Maisons-Alfort cedex, France;
2Mouse functional Genetics Unit, URA CNRS2578, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France
Mice homozygous for the patchwork (pwk) recessive mutation are salt and pepper: their coat contains a mixture of unpigmented and fully pigmented hairs, but no partially pigmented hairs (1). The number and spatial pattern of melanocytes and their precursors in pigmented hair follicles of pwk/pwk and wild-type mice do not differ. By contrast, there are no mature melanocytes in the matrix of pwk/pwk unpigmented hair follicles. Several mechanisms could account for the absence of mature melanocytes in the unpigmented hair follicles: (1) complete absence of the stem cells, (2) impaired differentiation of stem cells into transit amplifying cells, (3) inhibition of the proliferation and/or migration of the transient amplifying population, or (4) death of melanoblasts within the proliferative compartment. To address this issue, we use immunohistological methods with antibodies specific for the melanocyte lineage subpopulations (TRP-2, TRP-1 and MITF) and transgenic mice expressing reporter genes in the melanocyte lineage. In unpigmented hair follicles, stem cells and transit amplifying cells are present in the outer root sheath in the bulge region and below. However, they never reach the bulb region. Which process (survival, proliferation and/or migration) is deficient in the transit amplifying population is currently under investigation.
1. Aubin-Houzelstein G, Bernex F, Elbaz C, Panthier JJ (1998). Survival of patchwork melanoblasts is dependent upon their number in the hair follicle at the end of embryogenesis. Dev Biol. 198(2):266–76.
PP004 - THE ROLE OF C-MYC IN MELANOCYTE DEVELOPMENT
Irina Pshenichnaya1,Véronique Delmas2, Lionel Larue2, Andreas Trumpp1 and Friedrich Beermann1.
1Swiss Institute for Experimental Cancer Research, CH-1066 Epalinges, Switzerland;
2UMR 146 CNRS, Institut Curie, Orsay, France
c-Myc is over expressed in a variety of primary and metastatic cutaneous melanomas, which is correlated to a poor prognosis for this type of cancer. It is known that c-Myc is one of the key factors, which control cell differentiation in various tissues. Thus, c-Myc expression is increased when cells are triggered from the stem cell compartment to the transit amplifying pool, followed by decreased expression in differentiated cells. It might thus be proposed that tumors with c-Myc over expression are highly proliferative and undifferentiated. In this study, we wanted to address the role of c-Myc in melanocyte development and the maintenance of the melanocytes in the postnatal period, as well as the impact of c-Myc on melanomagenesis. To address these issues a conditional knockout allele of c-Myc was used to specifically deplete c-Myc in melanocytes. Mice which are heterozygous mutant for a melanocyte-specific deletion of c-Myc (c-Mycflox/+, Tyr::Cre) did not show any phenotype whereas complete c-Myc removal in melanocytes leads to a hair greying phenotype. The greying phenotype can be observed directly after birth, when pigmentation starts to be appear. We are currently analyzing the phenotype of these mice in detail to determine whether c-Myc depletion will affect the melanocyte progenitors in the bulge region and whether c-Myc deficient melanocytes are still present. Nevertheless, we can already conclude that c-Myc is an important factor for melanocytes/melanoblasts.
PP005 - FOXD3 REPRESSES MELANOGENESIS BY REPRESSING EXPRESSION OF MITF
Aaron J. Thomas and Carol A. Erickson.
University of California, Davis, Davis, California, USA
In the avian trunk, neural crest cells migrate in two temporal waves separated by about 24 h, with cells of the first wave differentiating into neurons and glia and cells of the second wave differentiating into melanocytes. Many neural crest cells are fate-specified at the time they delaminate from the neural tube. Two transcription factors, Mitf and FoxD3 play important roles in the lineage switch from neural and glial precursors to melanoblasts. Mitf is known to be required for melanoblast specification in many model systems. We determined the Mitf expression pattern in chick embryos by immunohistochemistry and found that Mitf is present in presumptive melanocyte precursors in the migration staging area. FoxD3 is expressed in the early migrating neural and glial precursor cells, but not in the later migrating melanocyte precursors. Miss expression of FoxD3 represses melanogenesis and loss of FoxD3 causes premature melanoblast specification. Misexpression of FoxD3 in B16 melanoma cells represses expression of endogenous Mitf and represses expression of luciferase driven by the chick Mitf promoter. FoxD3 was shown to interact with the Mitf promoter by EMSA. Thus, in early migrating neural crest cells, FoxD3 represses melanogenesis by repressing expression of Mitf, and thus permits neural and glial specification. Later, when FoxD3 is downregulated, melanoblast specification is permitted.
PP006 - PIGMENTATION, MIGRATION AND MORPHOLOGY; A CHEMICAL GENETIC APPROACH TO DISSECTING MELANOCYTE DEVELOPMENT IN XENOPUS
Matthew. L. Tomlinson1, Robert. A. Field2 and Grant. N. Wheeler1.
1School of Biological Sciences, University of East Anglia, Norwich, UK;
2School of Chemistry and Pharmacy, University of East Anglia, Norwich, UK
With the mounting quantity of genomic information becoming available, there is a growing desire to understand how genes function at a protein level; an increasingly important tool for doing this is chemical genetics. Which uses small molecules to intervene with and modulate biological activity. We have used the long established developmental model organism Xenopus laevis, previously shown to be highly amenable to this approach (Tomlinson, 2005),to perform a forward chemical genetic screen on developing embryos in a high throughput fashion. Results from this screen show highly specific development effects, illustrating the value of using diverse compound libraries to intervene in biological pathways. The screen has been mainly focused on looking at developmental effects concerning melanocyte migration, morphology and pigment production. Melanocytes in the developing Xenopus embryo originate from a pluripotent neural crest cell population on the dorsal side of the neural tube; the cells then take ventral-medial and lateral migratory pathways. Our results include sets of compounds that cause a loss of dendricity, inhibition of pigment production. An inhibition of melanocyte migration, firstly from the dorsal side of the neural tube itself (normally giving rise to both dorsal and lateral pigment stripes) and secondly from one half of the somite area (typically giving rise to the dorsal stripe). These results demonstrate the value of using chemical genetics in a systematic fashion to dissect biological processes in-vivo. The results also indicate the potential for the discovery of novel biological information, molecular tool sets and drug leads, the use of this approach could bring.
Tomlinson, M., Field, R. A., Wheeler, G. N. (2005). Xenopus as a model organism in developmental chemical genetic screens. Molecular BioSystems 1, 223–228.
PP007 - PHENOTYPIC INSTABILITY AT THE PATCHWORK LOCUS: AN EPIGENETIC EFFECT?
Geneviève Aubin-Houzelstein1, Pierrick Salaün1, Laurent Guillaud1, and Jean-Jacques Panthier1,2.
1Cellular and Molecular Genetics UMR 955 INRA-ENVA, Ecole Nationale Vétérinaire d’Alfort, 7 avenue du Général-de-Gaulle, 94704 Maisons-Alfort cedex, France;
2Mouse functional Genetics Unit, CNRS URA2578, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France
PRM/Alf mice are homozygous for patchwork (pwk), a mutation conferring a salt-and-pepper coat color phenotype. In their coat, black hairs are intermingled with white hairs, with no gray hairs (1). pwk is also characterized by its phenotypic instability. Indeed, two thirds of pwk/pwk mice carry areas of wild-type pigmentation on their coat, referred to as reversion spots. No reversion event can be transmitted via the germline. Micro arrays experiments showed a number of genes from the pwk region that were differentially expressed in the skin of pwk/pwk fetuses compared to controls. Among them, Mbd3, encoding Methyl Binding Protein 3, was overexpressed in pwk/pwk skin. MBD3 is part of a CpG-methylated DNA binding complex and is involved in chromatin remodeling. Moreover, we performed fluorescent in situ hybridations on PRM/Alf pwk/pwk and C57BL/6N embryonic fibroblasts with three adjacent probes from the patchwork region. We found that chromatin seemed to be more condensed in the pwk region of PRM/Alf fibroblasts compared to C57BL/6N controls. We hypothesized that pwk DNA methylation status may be involved in patchwork coat color phenotype and reversion rate. To test this hypothesis, dams were given a diet supplemented with methyl precursors, that was shown to increase CpG methylation at the Avy locus (2). We have produced 90 and 107 PRM/Alf mice fed with a methyl precursors supplemented diet and a control diet respectively. The number and size of reversion spots in both populations are under analysis.
1. Aubin-Houzelstein G, Bernex F, Elbaz C, Panthier JJ (1998). Survival of patchwork melanoblasts is dependent upon their number in the hair follicle at the end of embryogenesis. Dev Biol. 198(2):266–76.
2. Waterland RA, Jirtle RL (2003). Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol. 23(15): 5293–300.
PP008 - SNPS DISCOVERY IN COAT COLOUR GENES FOR GOATS BREEDS TRACEABILITY: A COMPARATIVE APPROACH
Letizia Nicoloso, Elisabetta Milanesi, Francesca Fornarelli and Paola Crepaldi.
Istituto di Zootecnia Generale, Facoltà di Agraria, Università degli Studi di Milano, Italy
Coat colour genes are useful candidates for breed's traceability of farm animals. In order to identify SNPs in genes involved in pigmentation of goat, we carried out in silico studies in human, mouse and cattle. We analysed 40 genes and we found: i) in mouse (Mouse Genomics Informatics) 88 SNPs, 29 synonymous and 59 non synonymous; ii) in humans (Children Hospital Informatics) 279 SNPs, 121 synonymous and 158 non synonymous of which 78 validated; iii) in cattle (NCBI and IBISS) 147 putative SNPs, 62 synonymous and 75 non synonymous of which 10 validated. In these species, in order to identify the more interesting coding regions, we collected information on pigmentation traits or on pathologies associated to different mutation. In goat, molecular information is available only on seven genes and no SNPs are reported, moreover mutation associated to different coat colour are limited. We obtained PCR products for 60 exons in 36 genes. The PCR products were sequenced and checked for omology with the target sequences. Sequences on eight animals from four breeds (Alpine, Saanen, Blonde of Adamello and Orobica) characterised by different coat colour phenotypes have been compared, revealing 25 SNPs (11 synonymous, 14 non synonymous) in 21 genes. The usefulness of comparative approach is discussed.
PP009 - A WHOLE GENOME SNP ASSOCIATION STUDY IDENTIFIES NON-SYNONYMOUS SNPS IN SLC24A5, MATP AND TYR ASSOCIATED WITH NATURAL VARIATION IN SOUTH ASIAN SKIN COLOUR
Tony Dadd1, Amelia Fereday1, Carl Jarman1, Wendy Filsell1, Rebecca Ginger1, Renee Stokowski2, David Hinds2, Krishna Pant2, David R. Cox2, Frans van-der-Ouderaa1 and Martin R. Green1.
1Unilever Corporate Research, Colworth Park, Sharnbrook, Bedfordshire, UK;
2Perlegen Sciences Inc., Mountain View, California, USA
We conducted a high-density genome scan to identify genes associated with natural human skin colour variation in a population of South Asian ancestry. DNA was obtained from 778 UK volunteers, chosen from the approximate 20% tails of the skin lightness distribution. Skin colour was defined using chromameter readings taken from six sites on the volunteers’ arms. To minimise the effects of admixture a genetic stratification test was carried out which resulted in 286 darker and 285 lighter samples being selected to form two DNA pools. These pools were used to estimate the relative allele frequencies of 1.5M SNPs covering the human genome at an average density of 1 SNP per 2kb. The 30K SNPs showing the greatest allele frequency difference between the pools were individually genotyped for all 778 samples. Results were replicated by individually genotyping 1.6K SNPs in 232 independent volunteers. At least three genomic regions were found to be associated with natural skin colour variation once replication, linkage disequilibrium, and admixture were taken into account. These regions centred on non-synonymous (ns) SNPs in SLC24A5 (rs1426654), MATP (rs16891982), and TYR (rs1042602). The allele frequency differences between darker and lighter volunteers for these SNPs are 38.7% (SLC24A5), 12.3% (MATP), and 11.2% (TYR). Loss of function mutations in MATP and TYR are associated with human oculocutaneous albinism and SLC24A5 was recently identified as being involved in both zebra fish and human pigmentation. Our work confirms the important contribution made by polymorphisms in these genes to natural human skin colour variation.
PP010 - INHERITANCE AND NATURAL VARIATION OF ADULT MALE ORNAMENTS OF THE GUPPY, P. RETICULATA
Christine Dreyer1, Nicolas Langlade2, Margarete Hoffmann1, Namita Tripathi1, Stephen Russell1, and Detlef Weigel1.
1Max-Planck-Institut für Entwicklungsbiologie, Abteilung Molekularbiologie, Spemannstr. 37-39/VI, D-72011 Tuebingen, Germany;
2John Innes Centre, Norwich Research Park, Colney Lane, Norwich, NR4 7UH, UK
The inheritance of the highly variable male ornaments has been studied in laboratory strains of the guppy Poecilia reticulata since the beginning of the 20th century. At least 35 genes for adult phenotypic traits have been localized to the sex chromosomes and about 50% of these are apparently closely linked to the male sex-determining region of the gonosomal part of the Y chromosome. We have crossed wild type guppies from populations from Trinidad and Venezuela that have been isolated from each other since at least 200 000 years, yet without speciation. We analyse F1 and F2 individuals of reciprocal crosses for mapping of male colour traits, using EST-linked SNP markers and microsatellites. Colour traits of adult males after puberty were documented and assigned to male or female inheritance and the complex traits were subjected to principal component analysis (PCA). In addition the variation of male ornaments in a mapping cross between a female from central Cumaná (Venezuela) and a male from the Quare river (East Trinidad), was captured in a statistical appearance model. The first 12 PCs of this genetically based model explained 69% of the variance in appearance between 60 F2 males. Most PCs contribute to several phenotypic components, which suggests that complex male ornaments arise from independently inherited traits. Furthermore we propose an X-linked allele of a trait may at instances dominant over a Y-linked allele and by this mechanism contribute to the maintenance of natural variation.
PP011 - CONTROL OF MICROPHTHALMIA TRANSCRIPTION FACTOR LOCALIZATION THROUGH REGULATED ISOFORM-SPECIFIC NUCLEAR SHUTTLING
Magdalena Dziembowska, Natacha Cigna, Oceane Anezo, Fabrice P. Cordelieres and Simon Saule.
Institut Curie, CNRS UMR 146, Bâtiment 110, Centre Universitaire Orsay, 91405 France
Mitf encodes a basic helix-loop-helix transcription factor essential for the differentiation of the retinal-pigmented epithelium (RPE) and of neural crest-derived melanocytes. Mitf produces several isoforms that differ at their N-terminus. The A-Mitf protein bears the largest N-terminus and is found in both the nucleus and cytoplasm. M-Mitf, which contains only 18 specific aminoacids is predominantly nuclear. Here we show that export of A-Mitf from the nucleus is exportin (CRM-1) dependent since it can be blocked by leptomycin B. We have identified the leucine-rich nuclear export sequence (NES) downstream from the leucine zipper domain. Despite containing the NES, the M-Mitf isoform is entirely nuclear. Clearly, a specific N-terminus is required to facilitate the A-Mitf nucleo-cytoplasmic shuttling. Using FRAP methodology we measured the import and exort rates of these molecules. In addition, we show that glycogen synthase kinase 3βGSK3β phosphorylates Ser298 (Ser399 in A-Mitf) adjacent to NES. Phamacological inhibition of GSK3β resulted in A-Mitf, remaining in the nucleus, suggesting that this kinase contribute to the nucleo-cytoplasmic distribution of the Mitf products.
PP012 - MELANOMA RISK IN THE SPANISH POPULATION: SEQUENCING THE MELANOCORTIN-1 RECEPTOR (MC1R) GENE
L. PFernandez1, J. M. Lopez2, R. L. Milne2, J. Bravo3, J. A. Avilés4, M. I. Longo4, J. Benítez1,2, P. Lázaro4 and G. Ribas1.
1Human Genetics Group, Human Cancer Genetics Program;
2CeGen, Human Cancer Genetics Program;
3Signal Transduction Group, Structural Biology and Biocomputation, CNIO, Spain;
4Dept. of Dermatology, General Universitario Gregorio Marañón Hospital, Madrid, Spain
The human melanocortin-1 receptor gene (MC1R), which plays a crucial role in pigmentation, also seems important in determining melanoma risk. This case-control study included 115 consecutive melanoma patients from the Dermatology Unit of the Gregorio Marañón Hospital and 200 control subjects matched for sex and age. Phenotypic information was colleted using a standardized questionnaire. Sequence analysis of the entire coding region of MC1R was performed. Because several subjects had two or more variants in the MC1R gene we cloned them and obtained haplotype information and also performed unphased haplotype calculations using PHASE v2.0. We identified 20 variants, all of them having been previously reported, except for two novel non-synonymous changes. Structural analysis of these latter two were performed using recently generated 3D models of MC1R (Zhang Y. et al., 2006). We observed that carrying MC1R variant alleles was associated with an increased risk of sporadic melanoma. These results support the contribution of MC1R variant alleles to genetic predisposition to sporadic cutaneous melanoma in Spain.
Zhang Y. et al., 2006 PLOS Comput Biol. 2(2):e13
PP014 - THE EUMELANIN AND PHEOMELANIN CONTENTS IN DORSAL HAIRS OF FEMALE RECESSIVE YELLOW MICE ARE GREATER THAN IN MALE
Tomohisa Hirobe1,2, Kazumasa Wakamatsu 3 and Shosuke Ito3.
1Radiation Effect Mechanism Research Group, National Institute of Radiological Sciences, Chiba 263-8555, Japan;
2Graduate School of Science and Technology, Chiba University, Chiba 263-8522, Japan;
3Department of Chemistry, Fujita Health University School of Health Sciences, Aichi 470-1192, Japan
The murine recessive yellow (Mc1re) is a loss-of-function mutation in the receptor for α-melanocyte-stimulating hormone (MSH), melanocortin receptor 1 (MC1R), and produces yellow coats by inducing pheomelanin synthesis in hair follicular melanocytes. However, it is not known whether Mc1re mutation affects pheomelanin synthesis in other skin sites. The eumelanin contents in the epidermis and dermis of newborn wild-type (Mc1r+/Mc1r+) mice (0.5, 3.5, 5.5 and 7.5 days) were much greater than those of mutant (Mc1re/Mc1re) mice, whereas the pheomelanin contents in the epidermis and dermis of mutant mice were much greater than those of wild-type mice. No sex differences in the contents of eumelanin and pheomalanin in the epidermis and dermis both in mutant and wild type mice were observed. The eumelanin contents in mutant hairs (5-week-old) was much smaller than in wild-type hairs, whereas the pheomelanin contents in mutant hairs was much greater than in wild-type hairs. However, the eumelanin and pheomelanin contents in mutant female hairs were greater than in male. These sex differences were not observed in wild-type mice. These results suggest that the Mc1re gene stimulates pheomelanin synthesis in the epidermis, dermis and hair follicles. In addition, Mc1re gene may affect eumelanin and pheomelanin contents in hairs by influencing through sex-related mechanisms.
PP015 - GENE VARIATIONS ASSOCIATED WITH HAIR AND EYE COLOURS IN HUMANS
Jonas Mengel-Jørgensen, Claus Børsting, Juan J. Sanchez, and Niels Morling.
Department of Forensic Genetics, Institute of Forensic Medicine, University of Copenhagen, Denmark
DNA analyses of human traces found at crime scenes are used to identify a person or establish/rebut a connection between a potential suspect and a crime scene. In cases without suspects, the DNA profiles from the crime scene are matched against DNA profiles from known offenders or DNA profiles from traces of unknown origin found at other crime scenes. Without a match in the database, the DNA profiles are not informative for the police before the same DNA profile turns up in a future case. For investigative purposes, it would be very useful, if the DNA profiles were supplemented with additional markers that may reveal information on physical traits. We developed a multiplex PCR and a multiplex single base extension (SBE) reaction for typing of 16 mutations in MC1R and two mutations in MATP and OCA2, respectively. Genotypes from 392 Danes were determined and compared to the hair- and eye-colours of each individual. Furthermore, the frequencies of the mutations in additional populations were determined. The DNA from individuals with two mutations in MC1R were amplified by allele specific PCR and the haplotypes determined.
PP016 - PRESENCE OF A SECOND HIT MUTATION IN MELANOCYTES OBTAINED FROM NEUROFIBROMATOSIS TYPE 1 (NF1) CAFÉ-AU-LAIT MACULES.
Sofie De Schepper1, Tom Callens2, Ophelia Maertens3, Jean-Marie Naeyaert1, Jo Lambert1 and Ludwine Messiaen2.
1Department of Dermatology, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
2Department of Genetics, University of Alabama at Birmingham , Birmingham 35294, AL, USA;
3Department of Medical Genetics, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium
Café-au-lait macules (CALM) are major cutaneous manifestations of NF1. The hypothesis that alterations in the NF1 gene play a crucial role in CALM pathogenesis, remains poorly examined. Only one study has addressed this question and no LOH was observed in the CALM melanocytes (MC) of 11 NF1 patients. We prelevated CALM skin biopsies from 13 NF1 patients with known germline mutation, from which 11 fibroblast (FB), 3 keratinocyte (KC) and 6 MC cultures were obtained. First, allelic imbalance and LOH was assessed by comparing constitutional DNA extracted from white blood cells with DNA extracted from the MC, FB and KC cultures. Genotype analysis was performed on amplified products using four intragenic microsatellite markers. No LOH was observed in any of the cell types, in agreement with previous findings. Long-range RT-PCR and direct sequencing of the entire NF1 coding region revealed a germline mutation only in the 11 FB cultures as well as in the 3 KC cultures. In contrast, in 5 MC cultures a differentsecondhit was identified: three nonsense mutations, one splice mutation and finally one loss of expression of wild type allele without identifiable mutation at cDNA level. The gDNA alteration causing the loss of expression of the wild-type allele is currently under further investigation. These data improve our understanding of the molecular mechanisms underlying the formation of CALMs and will boost further research aiming to understand the phenotypes associated with somatic mutations in melanocytes and their precursors during development and their association with segmental NF.
PP017 - EFFECT OF VAL92MET AND ARG163GLN VARIANTS OF THE MC1R GENE ON THE MELANOGENIC PHENOTYPE IN THE JAPANESE POPULATION
Tomonori Motokawa1, Tomomi Kato1, Yuki Hashimoto2 and Takayuki Katagiri1.
1Cutaneous Drug Research Laboratories, POLA Chemical Industries, Inc.,Yokohama, Japan;
2First Department of Dermatology, Toho University School of Medicine, Tokyo, Japan
Melanocortin 1 receptor (MC1R) is a highly polymorphic gene and specific polymorphic variants (e.g., Arg151Cys, Arg160Trp, and Asp294His) were common in the Caucasian individuals with skin cancer, red hair, fair skin, and pigmented lesions. These MC1R variants (denoted as RHC for red hair color) showed a remarkably reduced response to agonist in vitro. Recently, very high proportions of Val92Met and Arg163Gln were observed in the Asian population. These variants are also common in Caucasians. However, several association studies have shown that these variants had very little or no impact on phenotype when compared to RHC. This suggests that Val92Met and Arg163Gln are not important variants in Caucasians. In the Asian population, we thought that Val92Met and Arg163Gln might be notable variants because Asian people have very few RHC variants; this might blur the effect of Val92Met and Arg163Gln. In the previous study, we found that the Japanese could serve as ideal models to elucidate the phenotype effects of the variants because they showed a high proportion of Val92Met and Arg163Gln and no RHC variant. In the present study, we performed an association study in the Japanese population to elucidate the impacts of these variants on the melanogenic phenotype. We found that Val92Met allele was positively associated with freckles, severe solar lentigines, and brown hair color, whereas, Arg163Gln allele was negatively associated with freckles and severe solar lentigines. These results suggested that the Val92Met and Arg163Gln variants, which might not be significant in Caucasians, significantly affect the melanogenic phenotype in Asians.
PP018 - ANALYSIS OF REGULATION OF HUMAN TYROSINASE PROMOTER IN RETINAL PIGMENT EPITHELIAL CELLS (RPE) BY USING REVERSE TRANSFECTION METHOD
M. Reinisalo1, A. Urtti2 and P. Honkakoski1.
1Department of Pharmaceutics, University of Kuopio, Kuopio, Finland;
2Drug Discovery and Development Technology Centre, University of Helsinki, Helsinki, Finland
Purpose: Studying the function of gene regulatory elements in ocular cells can be laborious. Our purpose was to develop a simple reverse transfection method to study the human tyrosinase promoter in RPE cells.
Methods: Reverse transfection of various promoter fragments was performed as a co-transfection with MITF-A or MITF-M expression plasmids in ARPE-19 cells, bovine primary RPE cells and G-361 melanoma cells. After optimization of transfection efficiency of DNA/polyethylenimine (PEI25) complexes, the optimal ratio +5 was used thereafter for reverse transfection plates. DNA/PEI complexes were pipetted on the 48-well plates with a direct freezing and freeze-drying overnight. After freeze-drying, plates were stored at 4oC in a desiccator. On the day of transfection, cells were seeded on the plates, and reporter activities were determined 48 h later.
Results: Charge ratio +5 was used in all cell types with good efficiency. All transfections were performed within two months after preparation of the plates. Promoters containing the TDE binding element for MITF displayed highest activity in melanoma cells whereas in RPE cells the activity of all promoters was quite constant. Co-transfection with MITF-A enhanced the activity of all promoter fragments in every cell line. MITF-M showed strong activation only in ARPE-19 cells.
Conclusions: Reverse transfection was efficient transfection method for RPE and melanoma cells. Importantly, the biological activity of complexes on the plates was stable over several months. Tyrosinase promoter was differentially regulated between RPE and melanoma cells.
PP019 - AUGMENTED VENTILATORY RESPONSES TO HYPOXIA AND HYPERCAPNIA IN BLACK-EYED WHITE MITF MUTANT MICE
Shigeki Shibahara1, Kazuhisa Takeda1, Tetsuya Adachi1, Feng Han1, Satoru Yokoyama1, Wataru Hida2 and Hiroaki Yamamoto3.
1Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Japan;
2Health Administration Center, Tohoku University;
3Department of Developmental Biology and Neuroscience, Graduate School of Life Sciences, Tohoku University
Appropriate ventilatory responses to hypoxia and hypercapnia are essential for survival. Microphthalmia-associated transcription factor (Mitf) is responsible for differentiation of melanocytes. A homozygous black-eyed white Mitfmi-bw (bw) mouse is characterized by white coat color and deafness, due to the lack of skin and inner ear melanocytes. To search for the hitherto unknown role of melanocytes, we analyzed respiratory functions of bwmice. Breathing frequency, tidal volume and minute ventilation were measured in unanesthetized mice by whole body plethysmography. The bw mice showed the lowest respiratory frequency and the largest tidal volume, among the nine mouse strains examined, including A/J, AKR/N, BALB/c, C3H/He (control for bw), C57BL/6, DBA/2, NZW, SWR/J, and 129Sv. However, minute ventilation of bw mice is similar to that of control mice. Interestingly, only bw mice exhibited augmented ventilatory responses to acute hypoxia (10% O2) and hypercapnia (10% CO2). Moreover, there are no noticeable differences in the expression levels of Mitf mRNAs in the brain, heart, and lung between bw and control mice. These results suggest that melanocytes may modulate the function of the central respiratory controller.
PP020 - POSSIBLE INVOLVEMENT OF TYROSINASE DEGRADATION IN THE MECHANISM OF MELANOGENESIS INHIBITORY AGENTS DERIVED FROM BOTANICAL EXTRACTS
Hideya Ando1, Hirofumi Kondoh1, Tomoyuki Asano2, Mitsunori Kirihata2 and Masamitsu Ichihashi3.
1Bio-Research, Inc., Kobe, Japan;
2Osaka Prefecture University, Osaka, Japan;
3Sun-Clinic, Osaka, Japan
There are multiple pathways to control melanin synthesis through tyrosinase regulation, e.g. alteration of tyrosinase mRNA levels, changes of tyrosinase maturation, and competitive or non-competitive inhibition of tyrosinase activity. It was also reported that acceleration or deceleration of tyrosinase degradation is an effective way to control melanogenesis. Recently, it has been revealed that chemical chaperones that bind to enzyme can affect its stability and degradation. It is likely that some chemicals that bind to tyrosinase (chelators for copper atoms in tyrosinase active site or competitive inhibitors for tyrosinase activity) can act as chemical chaperones and accelerate tyrosinase degradation, as observed in phenylthiourea. On the other hand, many reports showed that botanical extracts includes various kinds of chelators and competitive inhibitors that bind to tyrosinase. In this study, we found that the extract of sugar cane inhibited tyrosinase activity that lead to inhibition of melanin synthesis and the possible involvement of tyrosinase degradation induced by chemical chaperones in the botanical extracts is under investigation.
PP021 - PROTECTION OF HUMAN RETINAL PIGMENT EPITHELIAL CELLS FROM UV-A IRRADIATION BY ZINC IS MELANIN DEPENDENT
Biesemeier Antje, Kokkinou Despina and Schraermeyer Ulrich.
Section of Experimental Vitreoretinal Surgery, University of Tuebingen, Schleich Str. 12/1, 72076 Tuebingen, Germany
Purpose: To investigate the antioxidant role of zinc in human amelanotic (ARPE-19) and native pigmented (HRPE) retinal pigment epithelial cells against ultraviolet A (UV-A) induced oxidative damage.
Methods: Human ARPE-19 and pigmented HRPE cells were treated with zinc (100 μM for 2 h) and subjected to oxidative stress by UV-A irradiation (20 mW/cm2). Intracellular H2O2 formation was measured by a fluorescence dye (2´,7´-dichlorofluorescein diacetate) using a fluorescence oxidation assay. Additionally, apoptosis and cell survival assays were performed.
Results: Addition of zinc increased H2O2 formation by factor 1.2 in ARPE cells compared to untreated ARPE cells. HRPE cells were significantly more protected (2–8 fold) than the amelanotic. Zinc enhanced the short and long-term resistance of HRPE cells to UV-A mediated cytotoxicity (factor 1.2).
Conclusion: Melanin pigmentation by itself is important for the protection of human RPE cells from oxidative damage. This effect is enhanced significantly in pigmented HRPE cells by zinc supplementation.
PP022 - DETERMINATION OF CA2+ BINDING PROPERTIES WITHIN SEPIA EUMELANIN
WilliamD. Bush and John D. Simon.
Department of Chemistry, Duke University, Durham, NC, USA
Isothermal titration calorimetry (ITC) was used to measure the binding properties of calcium ions to eumelanin extracted from the ink sac of Sepiaofficinalis cuttlefish. The binding constant (KB) was measured at pH = 7.1 as 3.2 × 103± 0.2 × 103 M-1. Infrared spectroscopy data are supportive of Ca2+ binding to the deprotonated carboxylic acid residues within the eumelanin pigment. This strength of binding is relatively weak compared to cellular calcium transport proteins, which does not support a role for melanin as a calcium ion scavenger but may support a role for melanin in the maintenance of intracellular calcium homeostasis.
PP023 - ANTI-AGING AND DEPIGMENTING PROPERTIES OF A MALVA SYLVESTRIS EXTRACT
Jocelyne Franchi, Mathide Bonnet-Duquenoy, Robin Kurfurst and Sylvianne Schnebert.
LVMH Recherche, Parfums et Cosmétiques, Saint Jean de Braye, France
Photoaging is characterized clinically by wrinkles, mottled pigmentation, rough skin and loss of skin tone. Previous results invite us to consider an oligosaccharide fraction rich in galacturonic acids from the flowery heads of Malva sylvestris as a potent candidate for anti-aging programs. This extract, studied in the human reconstructed epidermis model, induced a gene expression profile very similar to that observed with retinoids (cDNA macro-array system containing skin-related genes). This was confirmed by studies on senescent keratinocytes and fibroblasts showing restored proliferation capacities and synthesis. However, the effect on melanogenesis is not known. In this study, we investigated the action of Malva sylvestris extract on pigmentation using a pigmented reconstructed epidermis. The treatment of this pigmented reconstructed epidermis model shows a reduced pigmentation comparable to a reference molecule, as shown by the Fontana Masson staining. No histological alterations were noticed. We confirmed these data using the same 3D pigmented epidermis model with a topically applied whitening formula containing or not the extract. Chromametric measurement revealed a lightening effect of the tested formula, which is increased in presence of the Malva sylvestris extract. These results may prove to be an answer to the harmful effects of aging on the skin, including hyper pigmentation events.
PP024 - TYROSINASE AND PEROXIDASE IN AMPHIBIA KUPFFER CELL MELANOSOME
A. Gallone1, A. Sagliano1, V. Capozzi1, G. Perna1, D. Fiorentino1, M. Lastella1, C. Frassanito1, E. Mezzenga1 and R. Cicero2.
1Dipartimento Scienze Biomediche, Università degli Studi, via L. Pinto - 71100 Foggia, Italy;
2DIBIFIM, Università degli Studi, Piazza G. Cesare, 70124 Bari, Italy
The melanosome proteins extracted from the Kupffer Cells of R. esculenta L. possess a tyrosine-hydroxylase (TH) activity. The reaction depends on dopa as cofactor and is not affected by catalase or H2O2.The enzyme is activated by Cu++ ions. The presence of both the TH and dopaoxidase activity shows that melanin pathway is carried out by a tyrosinase, in these cells. Observations that TH reaction is not affected by addition of catalase or H2O2 show that it is catalyzed by a tyrosinase and not by the peroxidase present in melanosomes. Western blotting shows that anti-KC-tyrosinase and anti-KC-peroxidase antibodies recognize bands of different molecular weight. Extracts likely only containing peroxidase showed a protein band of 85 kDa. On the contrary, samples prevalently containing tyrosinase showed bands with apparent molecular weights in the range of 254 kDa and 172 kDa. SDS-PAGE also demonstrated the presence of bands with molecular weights corresponding to those of the tyrosinase and peroxidase in the protein patterns. Results of studies on hepatic melanin by Atomic Force Microscopy and Scanning Near-Field Optical Microscopy are reported.
PP025 - THE RATIONAL SYNTHETIC DESIGN OF MELANOGENIC INHIBITORS
JuliusL. Harp1 and Jesse J. Nicholson2.
1North Carolina A&T State University, Greensboro, NC, USA;
2Howard University, Washington, DC, USA
The primary focus of this work has involved the selective synthetic structural manipulations of a variety of Dopa and pre-Dopa mimetics acting as potential substrates and cofactors for the enzyme, tyrosinase (Tyr-ase). The structuring of the mimetics is designed to down-regulate the rate of melanin formation as well as the intermediates responsible for generating the complexed polymer, melanin. It is commonly characteristic for nonmalignant or normal melanocytes to produce biologically optimum levels of melanin, whereas increased levels of melanin production is known to be co-expressive with malignant melanoma. The structuring methods are specifically designed to maximize the interactions of the potential substrates or mimetics with Tyr-ase by maximizing the Dopa and pre-Dopa mimicking abilities of these same compounds while simultaneously inhibiting the formation of melanotic intermediates essential in the production of melanin. Preliminary success in the synthesis of a variety of Dopa mimetics commonly related to a model structure has lead to the proposition of synthetically manipulating the active units of related analogs, and homologs of Dopa in order to regulate selective phases of melanogenesis. Prevention of the amino-cyclization step or the formation of the leuko Dopa-chrome is proposed to halt the formation of melanin, as well as simplify the kinetics involved in phase I of melanogenesis. This should allow for the analysis of speculated intermediates and alternate biochemical pathways that are ordinarily difficult to detect spectroscopically. The analyses of the melanogenic process will be aided by radiolabeled substrates as well as molecular modeling calculations.
PP026 - CHEMICAL INVESTIGATION OF A KEY INITIAL STEP IN THE MELANOGENESIS PATHWAY
LanyingQ. Hatcher and John D. Simon.
Department of Chemistry, Duke University, Durham, NC, USA
The oxidation of 3,4-dihydroxyphenylalinine (L-DOPA) to its reactive quinine form is a key step in the melanogenesis pathway. Therefore, we pursued a detailed, mechanistic, study of the actual species involved in L-DOPA oxidation. The reactivity of L-DOPA was studied in the presence, and absence, of O2, and Cu2+, in water. In the absence of O2, Cu2+ was found to be unreactive towards L-DOPA over 3 days. Within the same timeframe, the reaction of L-DOPA with O2, in the absence of Cu2+, did produce a black precipitate indicating melanogenesis presumably via the quinine intermediate. The reaction of L-DOPA in the presence of both Cu2+, and O2, also produced a black pigment. These combined results indicate that Cu2+ alone, is not able to oxidize L-DOPA, but that O2 is the actual oxidant responsible for quinine formation. The same reactions were done in the presence of NaOH. Under strict anaerobic conditions, L-DOPA is stable at basic pH for over several hours. In the presence of NaOH and O2, L-DOPA rapidly oxidizes to a black, water-soluble, pigment.
PP027 - INDUCTION OF PHEOMELANIN SYNTHESIS IN CULTURED MURINE MELANOCYTES
Tokimasa Hida1,4, Kazumasa Wakamatsu2, Gregory Barsh3, Kowichi Jimbow4, Shosuke Ito2 and Dorothy Bennett1
1Division of Basic Medical Sciences, St George's, University of London, London, UK;
2Department of Chemistry, School of Health Sciences, Fujita Health University, Toyoake, Japan
3Department of Genetics, Stanford University, Stanford, USA;
4Department of Dermatology, Sapporo Medical University, Sapporo, Japan
In the hair cycle of agouti mice, follicular melanocytes switch the type of melanin synthesized from eumelanin to pheomelanin and back, resulting in black-yellow-black striped hairs. This involves agouti signal protein (ASP), which inhibits eumelanogenesis by melanocortin-1 receptor antagonism, inhibiting cAMP-dependent signalling and melanogenic protein expression. However, the mechanism that increases pheomelanin production is not understood. No conditions are yet known that induce cultured mouse melanocytes to synthesize predominantly pheomelanin. We are seeking such conditions by culturing melan-a melanocytes with various factors reported to affect pheomelanogenesis. Growth for 9 days with ASP reduced eumelanin content several fold, but also somewhat reduced pheomelanin content. However, in the presence of cysteine and mercaptoethanol as well as ASP, the pheomelanin content and the pheomelanin to eumelanin ratio were substantially increased. Surprisingly, addition of the tyrosinase inhibitor phenylthiourea could further increase pheomelanin content under some conditions, while reducing eumelanin. Light brown melan-a cell pellets were generated by combining these factors. These results suggest that i) optimal pheomelanogenesis induced by ASP requires high cysteine levels and reducing conditions, and ii) tyrosinase inhibition can also enhance pheomelanogenesis.
PP028 - POTENTIAL ROLE OF MC1R AGONIST-INDUCED UBIQUITINATION IN RECEPTOR INTERNALIZATION AND TRAFFICKING
Ana B Pérez Oliva, Berta L Sánchez-Laorden, Mª Carmen Turpín, José C García-Borrón and Celia Jiménez-Cervantes
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Murcia, Murcia, Spain
G protein-coupled receptors (GPCR) activation is followed by a series of regulated events involving second messenger synthesis, desensitization and internalization. Recent data have demonstrated the involvement of ubiquitination in many of these events, particularly internalization, trafficking and turnover. Protein ubiquitination is the result of the concerted action of three enzymes, the last one (termed E3) catalyzing the formation of an isopeptide bond between lysine side chains of the target and ubiquitin. The possibility that α-melanocyte stimulating hormone receptor (MC1R) might be regulated by ubiquitination was underscored by the finding that the Mahogunin (Mgrn1) gene encodes for an E3 ubiquitin ligase. In the mahoganoid mouse mutation, Mgrn1 loss-of-function is associated with a pigmentation phenotype. It has been postulated that by catalyzing the ubiquitination of MC1R, Mgrn1 might promote receptor down regulation and decrease signalling. We are analyzing the role of ubiquitination in the regulation of MC1R receptor, as related with the fate of the internalized receptor. In HEK 293 cells expressing MC1R, agonist binding promoted receptor internalization and decreased the half-life of the receptor protein, suggesting that the internalized receptor is targeted for degradation. This was confirmed in human melanoma cells. Receptor ubiquitination was suggested by immunoprecipitation of MC1R with anti-ubiquitin antibodies. MC1R displays four potential ubiquitination sites. Ablation of each of these targets individually did not abrogate MC1R immunoprecipitation with anti-ubiquitin antibodies, suggesting that ubiquitination may occur in more that one site or, alternatively, that MC1R might interact and co-inmunoprecipitate with an ubiquitinated partner. These possibilities are currently under study.
PP029 - RAB7 INTERACTS WITH THE MELANOSOMAL MATRIX PROTEIN GP100/PMEL17/SILV AND REGULATES ITS MATURATION IN MMAC HUMAN MELANOMA CELLS
Akinori Kawakami1, Fumio Sakane2, Shin-ichi Imai2, Hideo Kanoh2, Masae Okura1, Kuninori Hirosaki1, Toshiharu Yamashita1 and Kowichi Jimbow1.
1Department of Dermatology, Sapporo Medical University School of Medicine;
2Department of Biochemistry (Section II), Sapporo Medical University School of Medicine
Rab7 is a small GTPase that plays a crucial role in lysosomal/endosomal traffic in mammalian cells. We have previously identified Rab7 as a melanosome-associated protein involved in the intracellular transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes. In order to identify a potential partner for Rab7-binding, we performed yeast two-hybrid screening using a cDNA library of the human pigmented melanoma cell line MMAc. We found that wild-type Rab7 and its dominant-active mutant (Rab7-Q67L) bound to the carboxyl-terminal one-third of gp100/Pmel17/Silv (amino acid 422-661). Rab7-Q67L and wild-type Rab7 were co-localized in the perinuclear region with granules containing stage I-melanosomes. Next, using western blot analysis, we quantified proteolytically processed, mature gp100 in MMAc cells over expressing Rab7 mutants. Interestingly, we found that Rab7-Q67L and, to a lesser extent, wild-type Rab7 increased the amount of the mature gp100. Conversely, reduction of Rab7 expression by RNA interference caused a significant decrease of the amount of the mature gp100. These results collectively suggest that Rab7 specifically interacts with gp100, playing an important role in melanogenesis cascade through the regulation of gp100 maturation.
PP030 - COMPARATIVE ANALYSIS OF CULTURE CONDITIONS FOR HUMAN MELANOCYTES
Jin Hwa Kim1, Ki-Ho Kim2, Dong Han Ko3 and Tae Jin Yoon3.
Department of Dermatology, School of Medicine, 1Chungnam National University, Daejeon, Korea;
2Dong-A University, Busan, Korea;
3Gyeongsang National University, Jinju, Korea
Melanocytes, highly specialized cells for skin pigmentation, can be grown in defined culture media. However, the behavior of melanocytes cultured in vitro is influenced by the components of media. To determine the physiologically relevant culture condition, we performed comparative analysis using several parameters such as melanin content, tyrosinase activity and the expression level for melanocyte-specific genes. We used two culture media; commercial melanocyte growth medium (CMGM) and physiologic melanocyte growth medium (PMGM). CMGM contains fetal bovine serum (FBS), 12-o-tetradecanoylphorbol-13-acetate (TPA), hydrocortisone, heparin, bovine pituitary extract (BPE), transferrin, insulin and recombinant human fibroblast growth factor (rh-FGF). In contrast, PMGM contains rh-FGF, endothelin-1, and melanocyte stimulating hormone (α-MSH). Melanocytes cultured in CMGM showed accelerated proliferation, higher dendricity, and the increase in tyrosinase activity and melanin content. In contrast, melanocytes in PMGM showed larger cytoplasm, lower dendricity, and the decrease in tyrosinase activity and melanin content. RT-PCR and western blot analysis showed that the expression level of MITF, TRP1 was slightly higher in CMGM, while that of tyrosinase, TRP2, and GP100 was not significantly different between two culture conditions. We then determined the expression level of several malignancy markers including HLA-DR alpha, ICAM-1, MART-1 and p53. There was little difference in the expression level of HLA-DR alpha and ICAM-1 between CMGM and PMGM. However, the expression level of MART-1 and p53 was markedly increased in CMGM. These results suggest that MGM should be carefully selected for a further application, such as the cell transplantation therapy.
PP031 - REGULATION OF MELANIN SYNTHESIS IN HUMAN MELANOCYTES BY CAMP AND RESVERATROL: A ROLE FOR POST-TRANSCRIPTIONAL MECHANISMS
R. A. Newton1, D. W. Roberts1,2, J. H. Leonard2 and R. A. Sturm1.
1Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
2Queensland Institute of Medical Research, Brisbane, Australia
Microphthalmia-associated transcription factor (MITF) is implicated as a key regulator of pigmentation due to its ability to recognise E-box motifs in the promoter regions of melanogenic enzymes. Up-regulation of MITF expression has been proposed to mediate melanogenesis stimulated by cAMP, whereas down-regulation of MITF activity has been suggested to underlie the depigmentary effects of resveratrol. We have assessed the contribution of MITF to the regulation of pigmentation by treating primary human melanocyte cultures with either resveratrol or the adenylate cyclase activator forskolin, then quantifying levels of MITF mRNA and protein. The expression of the melanogenic enzymes tyrosinase, TYRP1 and DCT were also quantified at both the mRNA and protein levels and related to changes in activity of tyrosinase. Inhibition of tyrosinase activity by resveratrol was not explained by alterations in MITF but instead appeared to be due to a post-transcriptional effect that reduced the amount of mature tyrosinase protein. Elevation of intracellular cAMP concentration by forskolin increased expression of MITF but this did not result in a concomitant increase in tyrosinase mRNA indicating that elevated levels of MITF were not sufficient to promote tyrosinase transcription. The increase in mature tyrosinase protein elicited by cAMP therefore appears to be predominantly mediated by a post-transcriptional process in cultured human melanocytes.
PP032 - A NEW RAPID METHOD FOR ANALYSIS OF AHPS IN MELANIN
Dzeneta Nezirevic, Kerstin Årstrand, and Bertil Kågedal.
Department of Biomedicine and Surgery, Linköping University, Linköping, Sweden
In the present work we have simplified the treatment of hydriodic acid (HI) hydrolysate for HPLC analysis of aminohydroxyphenylalanines (AHPs) from pheomelanins (Wakamatsu 2003). We also developed a mass spectrometry friendly LC technique. An SPE method with a strong cation exchanger (SCX) was used. At acidic pH, AHP is positively charged while HI is neutral. We used these properties for binding AHPs to the sorbent until HI acid was washed out. Analytes (4-AHP and 3-AHP) were eluted by acetonitrile:0.2M NH4Ac (50:50), an eluent suitable for further HPLC analysis, into tubes containing 1 % of glacial HAc. HILIC (hydrophilic interaction liquid chromatography) is a technique suitable for separation of polar and hydrophilic compounds. Typical eluents for HILIC consist of acetonitrile in water or a volatile buffer. We used these advantages and developed a new HPLC method for analysis of the AHPs. We used the ZIC®-HILIC column with the stationary phase consisting of a covalently bonded, permanently zwitter-ionic functional group of sulfabetaine type. The analytes were eluted with a mobile phase consisting of acetonitrile:0.1M acetate buffer pH 4.5: (85:15). Ion-pairing reagents were completely avoided. The compounds were identified with ED at +0.4 V. The method gave good linearity and precision. Recovery of AHPs added to hydrolysate samples was satisfactory.
Conclusion: Our simplified sample preparation method and a new HPLC method are suitable for use in AHP analysis.
PP033 - PHOTOCHEMICAL PROPERTIES OF CHLOROFORM-SOLUBLE FRACTION OF AGED HUMAN RPE MELANOSOMES
A. Pawlak1, G. Szewczyk1, A. Zadlo1, M. Zareba2, J. M. Burke2, M. B. Rozanowska3, M. E. Boulton3 and T. Sarna1.
1Dept. of Biophysics, Jagiellonian University, Krakow, Poland;
2Dept. of Ophthalmology, The Eye Institute, Medical College of Wisconsin, Milwaukee, USA;
3School of Optometry and Vision Science, Cardiff University, Cardiff, UK
Human RPE melanosomes exhibit age-dependent aerobic photo reactivity. To date, the molecular nature of the chromophores responsible for the observed photoreactivity remains unknown. The main goal of our study was to compare photochemical properties of the chloroform-soluble fraction of melanosomes (CSM) and RPE lipofuscin. RPE melanosomes and lipofuscin granules from different age-group donors and from bovine eyes were purified by ultracentrifugation in a discontinuous sucrose gradient. Photo reactivity of human and bovine CSM was analyzed in selected organic solvent and in liposomes by time-resolved singlet oxygen phosphorescence at 1270 nm, laser flash photolysis and ESR oximetry and ESR spin-trapping. When irradiated with blue light human CSM generated singlet oxygen with quantum yield: 0.08–0.14 dependent on oxygen concentration and age of donors. The data suggest that the increased photochemical reactivity of aged RPE melanosomes is due to an accumulation of chloroform-soluble photosensitizing compounds. It appears that photogeneration of singlet oxygen by CMS involves short-lived triplet excited state of an unknown photosensitizer perhaps similar to that observed for human lipofuscin.
PP034 - PULSE RADIOLYSIS DETECTION AND COMPUTATIONAL ANALYSIS OF QUINONES FROM 5,6-DIHYDROXYINDOLE DIMERS: IMPLICATION FOR EUMELANIN BUILDS UP
Alessandro Pezzella1, Lucia Panzella1, Alessandra Napolitano1, Orlando Crescenzi2, Vincenzo Barone2, Marco d’Ischia1 and Edward J. Land3.
1Dept Org. Chem. Biochem.;
2Dept of Chem. Univ. Naples Federico II, Naples, Italy; 3Dept of Chem.Phys, Keele Univ., Keele, England, UK
Eumelanin structure and build up is still a challenge. Two models have been proposed to account for the unique properties of eumelanin: one put forward by Simon (Clancy and Simon, 2001) envisages a planar oligomer of 5,6-dihydroxyindole (DHI) /5,6-dihydroxyindole-2-carboxylic acid (DHICA) units which assembles through π-stacking and side-on interactions, while the other (Cheng et al, 1994) depicts five to seven monomers of 5,6-indolequinone arranged in a plane, forming 3-4 layers stacked 3.4 Å apart. To probe either of these hypotheses, we underwent a systematic characterisation of oligomers of DHI and DHICA and their oxidation chemistry. Here we report the use of combined pulse radiolysis and quantum mechanical calculations to inquire into the nature of the species formed by oxidation of three dimers of DHI, namely 5,6,5’,6’-tetrahydroxy-2,2’-biindolyl, 5,6,5’,6’-tetrahydroxy-2,4’-biindolyl and 5,6,5’,6’-tetrahydroxy-2,7’-biindolyl. Pulse radiolytic oxidation of 2,4’- and 2,7’-biindolyls led to semiquinones absorbing around 450 nm, which decayed to give the corresponding quinones (500–550 nm). 2,2’-Biindolyl, on the other hand, furnished a semiquinone (λmax 480 nm) which disproportionated to give a relatively stable quinone (λmax 580 nm). A quantum mechanical investigation of o-quinone, quinone imine and quinomethane structures of the dimers suggested that in the oxidised forms they exist mainly as 2-substituted extended quinomethane tautomers stabilised by intramolecular hydrogen bonds, reaching a planar conformation which is not favoured for the reduced forms. Overall, these results indicate that oxidation of oligomers to quinonoid species is critical to achieve planar assemblies which can interact by π-stacking.
PP035 - ULTRASTRUCTURAL FINDINGS IN PROGRESSIVE MACULAR HYPOMELANOSIS INDICATE DECREASED MELANOSME PRODUCTION BY MELANOCYTES
G. N. Relyveld MD1,4, K. P. Ding emans PhD2, H. E. Menke MD, PhD3, J. D. Bos MD, PhD 4 and W. Westerhof MD, PhD1.
1Netherlands Institute for Pigment Disorders, Academic Medical Center, University of Amsterdam, the Netherlands;
2Academical Medical Center, Department of Pathology, University of Amsterdam, the Netherlands;
3Sint Franciscus Gasthuis, Department of Dermatology, Rotterdam, the Netherlands;
4Academical Medical Center, Department of Dermatology, University of Amsterdam, the Netherlands
Background: The pathogenesis of progressive macular hypomelanosis (PMH) is unknown. Recently, however, Westerhof et al. (1) hypothesized that Propionibacterium acnes produces a depigmenting factor that interferes with melanogenesis in the skin, resulting in hypopigmented spots. The purpose of the study was to gain an insight into the pathogenesis of PMH.
Materials and methods: We took two biopsies of two mm diameter each from normal and lesional skin in eight PMH patients. Using electron microscopy, we compared melanization of melanosomes, melanosome transfer and amount of epidermal melanin in normal and lesional skin.
Results: Patients had skin type III to VI. Compared to non-lesional skin, we observed a decrease of epidermal melanin and less melanized melanosomes in the lesional skin of all patients. Furthermore, in lesional skin of patients with skin type V and VI (brown to black skinned people) we observed a change in melanosome size, from single transferred stage IV melanosomes (as seen in skin type V and VI) to aggregated stage I, II and III transferred melanosomes (as seen in skin type I to IV).
Conclusion: Hypopigmentation in PMH seems to be the result of an altered melanogenesis based on a change in structure and distribution of melanosomes and a transfer of less melanized melanosomes and aggregated melanosomes instead of single melanosomes from melanocytes to keratinocytes in lesional skin of skin type V and VI. This results in a decreased epidermal melanin. Further investigations are needed to determine if Propionibacterium acnes play a role in this alteration of melanogenesis.
PP036 - ANTIOXIDANT PROPERTIES OF PHOTOBLEACHED RPE MELANOSOMES
Andrzej Zadlo1, Grzegorz Szewczyk1, Mariusz Zareba1, Tadeusz Sarna1, Michele Henry2 and Janice Burke2.
1Department of Biophysics, Jagiellonian University, Krakow, Poland;
2Department of Ophthalmology, Medical College of Wisconsin, Milwaukee, WI, USA
Melanin in the retinal pigment epithelium (RPE) is believed to act as an antioxidant and photo protective agent. We postulate that human RPE melanosomes undergo in situphoto oxidation that may compromise their biological functions. To test our hypothesis, purified melanosomes, isolated from cow and porcine RPE cells, were subjected to aerobic photolysis using intense visible and near UV light. Progress of melanosome photobleaching was monitored by UV-VIS absorption and electron spin resonance (ESR) spectroscopies. Antioxidant capacity of untreated and bleached melanosomes, determined by the melanosome ability to inhibit peroxidation of lipids, induced by iron/ascorbate was analyzed by ESR-oximetry, ESR-spin trapping and iodometric assay of lipid hydroperoxides. We found that illuminated melanosomes have lower melanin content and reduced ability to bind iron ions. We also found that control, untreated melanosomes show a concentration-dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolyzed melanosomes lose their antioxidant efficiency and even became prooxidant. Compared to untreated melanosomes, photobleached melanosomes exhibited increased photogeneration of superoxide anion. Our study suggests that visible light irradiation is responsible for some changes of human RPE melanosomes with ageing. It further suggests that the normal antioxidant properties of human RPE melanin become compromised with aging.
PP037 - INFLUENCE OF ZINC DEFICIENCY ON PIGMENTATION
Schraermeyer Ulrich and Kokkinou Despina.
Section of Experimental Vitreoretinal Surgery, University of Tuebingen, Schleich Str. 12/1, 72076 Tuebingen, Germany
Introduction: Age-related changes in age-related macular degeneration (AMD), the leading cause of blindness in the western countries among the elderly Caucasian, are associated with degeneration of the retinal pigment epithelium (RPE), accumulation of amorphous material in Bruch's membrane, called drusen, and accumulation of lipofuscin and melano-lipofuscin granules. Drusen, lipofuscin and macular changes are less prevalent in humans with black fundus pigmentation (black Africans) than in white Caucasians suggesting a possible correlation with melanin pigment. Zinc is known to bind to melanin in pigmented tissues and to enhance antioxidant capacity by function as a cofactor or gene expression factor of important enzymes in the eye. It is the most abundant trace element in the eye and plays a key role in the metabolism of the retina. In this study, we investigated the effect of zinc deficiency in pigmented (Long Evans, L.E.) and non-pigmented (Wistar) rats.
Materials-Methods: Animals fed with normal nutrition (control, five L.E. rats and five Wistar rats) and animals kept on a zinc-free diet (six L.E. rats and seven Wistar rats, diet no: C1040, Altromin, Lage, Germany) for 6 months were compared. The concentration of zinc was measured in the choroid/RPE complex of all animals by inductively coupled plasma mass spectrometry (ICP-MS) assay after the 6 months. Electroretinography, light, fluorescence and electron microscopy were performed. Additionally, immunohistochemistry was performed in sections of the choroid of the L.E rats where some heavily pigmented cells were observed.
Results: The concentration of zinc in control and zinc deficiency animals showed no significant difference measured by ICP-MS. The direct comparison of the mean value of the b-wave by electroretinography in the zinc-deficiency groups resulted in a statistically significant difference with a greater impairment in the pigmented rats. The results indicated a greater dysfunction of the rods and cones of the L.E. compared to the Wistar rats. The number of photoreceptor nuclei significantly decreased in the zinc deficiency animals (n = 9, P < 0.001). The number of lipofuscin granules was significantly increased only in the L.E. deficiency rats (n = 6, P = 0.001). Additionally, the fluorescence intensity of these granules was also increased (P < 0.05) quantified by fluorescence microscopy. Interestingly, the number of the lipofuscin granules in the control Wistar was increased compared to the control L.E. rats (P < 0.05). Immunohistochemistry proved that the atypical heavily pigmented cells observed in the choroid tissue of the zinc deficiency Long Evans rats were macrophages and their number was significantly higher than in the choroid of the control L.E (n = 6, P = 0.001). Electron microscopy showed giant melanosomes and lyposomes containing homogenous material and melanosomes in the large round cells of the choroid of the zinc deficiency L.E. rats.
Conclusions: Melanin is a great antioxidant for the eye tissues as shown by the comparison of the control pigmented and non-pigmented animals. However, its strong association with zinc seems to involve also the antioxidant function of melanin in zinc deficiency conditions. As elderly people are prone to zinc deficiency, we assume that an enlargement of fundus pigmentation by gene-therapy and zinc substitution can be considered as a possible therapeutical approach in patients suffering from AMD.
PP038 - THE SURFACE OXIDATION POTENTIAL OF MELANOSOMES MEASURED BY FREE ELECTRON LASER PHOTOELECTRON EMISSION MICROSCOPY
John D. Simon.
Duke University, Durham, NC, 27708, USA
A technique for measuring the photo ionization spectrum and the photoelectron emission threshold of melanosomes will be presented. The relationship between the photo ionization threshold and the electrochemical potential referenced to the normal hydrogen electrode (NHE) is used to quantify the surface oxidation potential of the melanosome. Human red and black melanosomes reveal different oxidation potentials; the red pigment is more reductive, consistent with recent observations comparing eumelanin and pheomelanin reactivity. The effect of iron chelation on the surface oxidation potential of sepia melanosomes is examined. The surface oxidation potential is insensitive to bound Fe (III) up to saturation, suggesting that the metal is bound to the interior of the granule. This latter result will be discussed in relation to the age-dependent accumulation of iron by melanosomes in both the eye and brain and the fact that the oxidation potential of human melanosomes from the retinal pigment epithelium show changes with age.
PP039 - FURTHER INSIGHTS ON THE ACTIVE SITE OF TYROSINASES. A BACTERIAL TYROSINASE WITH HIGH TYROSINE HYDROXYLASE/DOPA OXIDASE RATIO
Francisco Solano1 , Diana Hernández-Romero2 and Antonio Sanchez-Amat2.
1Dept of Biochemistry & Molecular Biology B;
and 2Dept of Genetics & Microbiology, Univ. Murcia, Spain
The availability of a number of bacterial genomes revealed several genes that putatively code for polyphenol oxidases (PPOs) with two sequence profiles, tyrosinase-like and laccase-like. Ralstonia solanacearum is a soil-borne pathogenic bacterium that withers a wide range of plants. We detected the expression of two PPO genes (accession numbers NP_518458 and NP_519622) with high similarity to tyrosinases, both containing the six conserved histidines required to bind the pair of type-3 copper ions. Generation of null mutants in those genes and protein purification allowed us to correlate each gene with the enzymatic activity of the protein they encode. In contrast with all tyrosinases so far described, the enzyme NP_518458 shows higher TH than DO activity. On the other hand, protein NP_519622 is an enzyme with a clear preference to oxidize o-diphenols and only residual TH activity, behaving as a catechol oxidase. These catalytic characteristics of preferred TH or DO are discussed in relation to: i) the putative presence of a seventh histidine which interacts with the carboxyl group on the substrate and controls the preference for carboxylated and decarboxylated substrates; ii) the size of the residue isosteric with the aromatic F261 reported in model sweet potato catechol oxidase which acts as a gate to control accessibility to CuA at the active site.
PP040 - REJECTION OF SECONDLY INOCULATED MELANOMA AND PROLONGATION OF LIFE SPAN OF MELANOMA-BEARING MICE BY MELANOGENESIS-TARGETED CHEMO-THERMO-IMMUNO (CTI) THERAPY USING NPRCAP-MAGNETITE NANO-PARTICLES
Tomoaki Takada1, Toshiharu Yamashita1, Makito Sato1, Akiko Sakemoto1, Hidenobu Matsusaka1, Ichiro Ono1, Yasuaki Tamura2, Noriyuki Sato2, Akira Ito3, Hiroyuki Honda4, Kazumasa Wakamatsu5, Shosuke Ito5 and Kowichi Jimbow1.
Departments of 1Dermatology;
and 2Pathology, Sapporo Medical University;
3Faculty of Engineering, Kyushu University;
4Department of Biotechnology, Nagoya University;
5Department of Chemistry, Fujita Health University, Japan
N-propionyl cysteaminylphenol (NPrCAP) is a good substrate for tyrosinase and selectively incorporated into melanoma tissues, and inhibits the in vivo and in vitro melanoma. In this study NPrCAP was conjugated with magnetite (NPrCAP/M) and its in vivoanti-melanoma effects were evaluated. We found that i) daily administration of NPrCAP/M for three times without AMF caused a statistically significant inhibition of growth of melanoma cells (B16F1) grown s.c. in C57/BL mice, and ii) the repeated exposures to AMF produced complete abolishment of melanoma. Importantly when mice with melanoma inoculation on the flank were treated initially by either NPrCAP/M with or without AMF and then received second inoculation of melanoma cells at the opposite site of flank, they showed a significant growth inhibition or almost complete rejection of secondly inoculated, melanoma cells, revealing histologically the total necrosis and a marked prolongation of life span, 100% survival more than 78 days by Kaplan Meier. Thus our NPrCAP/M with AMF established a novel, melanogenesis-targeted DDS and CTI therapy for malignant melanoma.
PP041 - THE SPECIFIC P38 MITOGEN-ACTIVATED PROTEIN KINASE INHIBITOR SB203580 STIMULATES MELANOGENESIS IN HUMAN MELANOMA CELLS
M. CTurpín, C. Jiménez-Cervantes and J. C. García-Borrón.
Dep. de Bioquímica y Biología Molecular B e Inmunología. Facultad de Medicina, University of Murcia. Spain
Many extracellular stimuli are converted into specific cellular responses by mitogen-activated protein kinases (MAPKs). The p38 MAPKs are activated by most environmental stresses and by certain hormonal stimuli. In mouse melanoma cells, it has been shown that p38 is activated by ultraviolet (UV) radiation and α-melanocyte stimulating hormone (αMSH), suggesting that the kinase may play key roles in the tanning response and in the melanogenic action of melanocortins. We are analyzing the role of p38 in the regulation of basal melanogenesis in human melanoma cells. Basal p38 phosphorylation was detected in a panel of 10 human melanoma cell lines. The level of phospho-p38 (p-p38) did not correlate with tyrosinase activity. In HBL human melanoma cells, p38 phosphorylation was stimulated by αMSH. Treatment of HBL cells with the specific p38 inhibitor SB203580 caused a concentration and time-dependent activation of tyrosinase and increased their pigmentation. Activation of tyrosinase was weaker in B16 mouse melanoma cells. In HBL cells, tyrosinase activation was due to increased protein and mRNA levels, as detected by Western blot and real-time PCR. Since the αMSH receptor (MC1R) contains a potential p38 phosphorylation target we analyzed MC1R signalling in cells treated with SB203580. The number of binding sites, receptor internalization and hormone-induced cAMP accumulation were not affected by SB203580. These data suggest that in the absence of melanogenic stimuli such as UV radiation or αMSH, p38 may inhibit basal melanogenesis by decreasing tyrosinase levels, and that this effect is not dependent on the functional status of MC1R.
PP042 - STRUCTURAL ANALYSIS OF SEPIA MELANIN EXTRACTION PRODUCTS BY LIQUID CHROMATOGRAPHY- TANDEM MASS SPECTROMETRY
WeslynWard and John Simon.
Duke University, Durham, NC 27708, USA
The insolubility of the melanin has challenged scientists in its structural determination for nearly a century.The structural features causing the insolubility of melanin are not entirely understood. In this study, the partially soluble moiety of the sepia eumelanin is analyzed using liquid chromatography and tandem mass spectrometry. Studies of these portions provide insights on attachment schematics of the larger biopolymer. In this work, eumelanin was suspended in nano-pure water and shaken for varying amounts of time. An Agilent 1100 HPLC-ion trap and Applied Biosystems QSTAR mass spectrometers were used to gain structural information and exact mass measurements. Parent ions of the extraction products were fragmented to MS3 and compared using positive and negative mode electrospray ionization. Probable structures for dimers and other higher-order units have been proposed. These extraction products of sepia melanin observed using LC/MS/MS provide insight towards determining the structural connectivity of the biopolymer. The array of different products observed indicate that there may not be one single pathway, as the Raper-Mason scheme suggests.
PP043 - FUNCTIONAL CHARACTERISATION OF THE MEMBRANE ASSOCIATED TRANSPORT PROTEIN MATP - LOCALISATION AND ACTIVITY
Steve Wilson, Rebecca Ginger, Shubana Kazi, Richard Ogborne and Martin R. Green.
Unilever Corporate Research, Colworth Park, Sharnbrook, Bedford, UK
The Membrane associated transport protein (MATP) has been identified as key to vertebrate melanogenesis, and nonsense gene mutations are responsible for human oculocutaneous albinism type IV. Population studies have also identified single nucleotide polymorphisms within the MATP gene which link it to natural variation in human hair, skin and eye colour. We have analysed the allelic frequencies of a number of these SNPs in 516 darker and 507 lighter volunteers (as defined by chromameter L* values) of Southern Asian ancestry (India, Bangladesh, Sri Lanka and Pakistan). There was strong evidence to suggest that natural skin colour variation in Southern Asians is associated with a single SNP (pF374L). The frequency of the alternate allele (pF374) was 16% in lights and 4% in darks. This indicates that although there is a strong association between this allele and lighter skin, it is present in only a small proportion of the study population. Despite its obvious importance to the processes of melanosomal maturation and melanin biosynthesis in human skin colour variation, the specific function of MATP has remained elusive (although protein sequence homology analyses have identified a pronounced similarity to plant sucrose symporters). We have raised polyclonal antibodies to MATP and generated mammalian over-expression constructs in order to attempt clarification of some of these unknowns. To date, we have shown MATP to be localised, as expected, in an intracellular compartment within cultured melanocytes. Our initial findings using these aforementioned tools will be discussed.
PP044 - BINDING CHARACTERISTICS OF MITF (MICROPHTHALMIA-ASSOCIATED TRANSCRIPTION FACTOR) WITH PROMOTER DNA OF PIGMENTARY GENES
Yang Sang-Hee1, Kim Dong-Gun1, Shin Jung-Hyun2 and Kim Eun-Ki1.
1Dept. Biological Eng. Inha University, Inchon, Korea;
2Dept. Dermatology, Inha Univ. Hospital
A protein chip containing Mitf (Microphthalmia-associated transcription factor) was developed and its potential as a screening tool for Mitf inhibitor was investigated. Mitf is a key regulatory transcriptional regulator of pigment related genes such as tyrosinase and MC1R. Binding of Mitf on its DNA binding domain, E-box of the corresponding promoter, initiates the production of melanin, a key-determining factor of skin color. Mitf was produced as a fusion protein of maltose binding protein (MBP) and was immobilized on a dextran-coated glass plate. Binding of cyt-3 labelled 8-mer E-box DNA was monitored by fluorescent. Kinetics of DNA binding with MITF showed Langumuir isotherm and its kinetic constants were determined. Binding intensity of Mitf on various DNA sequences was also investigated. Tyrosinase promoter showed more strong binding than MC1R promoter sequence. DNA sequences adjacent to E-box showed affected the binding intensity. Changes of the sequence in the E-Box (CANNTG) decreased the binding intensity more than other sequences.
PP045 - OPTICAL PROPERTIES OF HUMAN SKIN MELANIN IN VIVO
G. Zonios1, D. Galaris2, I. Bassukas3, A. Batistatou4 and A. Dimou1.
1Dept. of Materials Science and Engineering, University of Ioannina, Greece;
2Dept. of Biological Chemistry, School of Medicine, University of Ioannina, Greece;
3Dept. of Skin and Venereal Diseases, School of Medicine, University of Ioannina, Greece;
4Dept. of Pathology, School of Medicine, University of Ioannina, Greece
Melanin is perhaps the most characteristic chromophore of human skin but information about its structure and properties, in vivo, is limited. In this study, we have employed diffuse reflectance spectroscopy for the non-invasive study of human skin melaninin vivo. Skin types studied include normal type III human skin, vitiligo, melanocytic nevi, nevus spilus, dysplastic nevi, and melanoma. For each of these categories, melanin concentration was assessed, as well as the detailed spectroscopic characteristics of melanin. In particular, we found that the absorption spectrum of melanin as a function of wavelength is described very well by a single exponential function. Results also indicate that the slope of the absorption spectrum varies among the skin types studied, which may indicate differences in the structure and properties of melanin in various skin types and skin pathological conditions such as malignant melanoma. Apart from contributing to the elucidation of the detailed structure of melanin, which remains to a large, extend a mystery, these results are very promising for the development of non-invasive techniques for the early detection and characterization of skin diseases such as melanoma.
PP046 - LOSS OF MOUSE TYROSINASE ACTIVITY BY A MUTATION DESTABILIZING CUB
Thorsten Schweikardt1, Francisco Solano2, Elmar Jaenicke1, José Carlos García-Borrón2 and Heinz Decker1.
1Institute of Molecular Biophysics, University of Mainz, Germany;
2Departamento de Bioquímica, Biología Molecular (B) e Inmunología, Universidad de Murcia, Spain
Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments, which are responsible for colouring hair, skin and eyes. Mammalian tyrosinases are glycoproteins whose enzymatic activity is dependent on the presence of two copper ions at the active site. Mutation of tyrosinases can cause a decrease in melanin concentration resulting in albinism, but often the effects are not understood on a molecular level. Here we show that a double mutation of two active site residues, M374G and S375G, results in a complete loss of enzymatic activity of mouse tyrosinase. Homology modelling of mouse tyrosinase based on the crystal structures of the first recently published tyrosinase provides a structural basis to understand this observation. The residues M374 and H367, one of the six histidines coordinating the two copper atoms at the active site, are in van-der-Waals-contact, with the side chain of M374 restraining the imidazole ring of H367. The double substitution S375G and M374G leaves substantial empty space due to the absence of any side chain. This cavity leads to a local perturbation of the protein matrix. Thus, the side chain of H367 is not locked in the required orientation and may be not able to reliably bind CuB, resulting in the loss of enzyme activity. This double mutation also results in incomplete glycosylation and aberrant processing of the protein. As the genes encoding human and mouse tyrosinase have 85% overall sequence identity and 100% identity at the active site, the observed loss of activity gives clues towards understanding the structural basis of certain forms of albinism in humans as well.
PP047 - ENZYMATIC ANALYSIS OF REDUCED LEVELS OF GLUTATHIONE CONTRIBUTING TO OXIDATIVE STRESS IN FOWL VITILIGO: FURTHER STUDIES
J. E Ycaza and R. RBowers.
Dept. of Biological Sciences, California State University, Los Angeles, USA, 90032
Currently two mutant species of roosters, Barred Plymouth Rock (BPR) and White Leghorn (WL), are being used as animal models for vitiligo with the wild type rooster, Jungle Fowl (JF), serving as a control. Previous research indicates that vitiligo in our animal models is caused by low levels of SOD and glutathione causing oxidative stress. Recently our laboratory has characterized the SOD gene and found that it has a structural defect in the BPR and a regulatory defect in the WL. Glutathione (GSH) is important in the feather as a scavenger of reactive oxygen species (ROS), which are produced during melanogenesis. Therefore, roosters with a diminished antioxidant defense will have pigment cells creating ROS that result in their own death due to oxidative stress. Total GSH in both BPR and WL feathers showed approximately a 33% drop in the concentration of GSH as compared to the concentration found in JF feathers. The purpose of this study was to ascertain why GSH levels were low in the feather of the two mutant roosters. Three different enzymes, glutathione reductase (GR), glutathione synthetase (GS), and γ-glutamylcysteine synthetase (γ-GCS) involved with either the production or restoration of GSH were studied. Assays of enzyme activity of GR indicated that there was no statistical difference in enzyme activities between any of the three genotypes of roosters. Assays of enzyme activity of γGCS showed no statistical difference between the activities of JF and BPR but demonstrated a statistical increase in the activity in WL that was at least double that of the control JF. The hypothesis for this apparent anomaly is addressed; however, the activity of this enzyme is not responsible for the reduction of the total GSH in the mutant strains. Assays of enzyme activity of GS showed a statistically significant decrease in the two mutant roosters as compared to the control rooster. It appears that a reduction of total activity of GS results in reduced levels of glutathione in the mutant roosters. Whether this activity is low due to a structural defect of the enzyme or to a regulatory defect, causing a diminished quantity of the total enzyme present, will have to be determined.
PP048 - PIGMENTED AND ALBINO RATS SHOW DIFFERENT RESPONSE TO LIGHT AS STUDIED BY ELECTRORETINOGRAPHY AND VISUAL EVOKED POTENTIALS
Peter Heiduschka and Ulrich Schraermeyer.
University Eye Hospital, Experimental Vitreoretinal Surgery, Schleichstr. 12/1, D-72076 Tübingen, Germany
The goal of this study was to check if there are differences in visual function that depend on the status of pigmentation. For this purpose, electroretinography to detect retinal function and measurement of visual evoked potentials to detect function of the optic nerve and the visual cortex were utilised. As a model, we chose albino Wistar rats and pigmented Long-Evans rats. The animals were born in our facilities and grown up under identical light conditions avoiding bright light. Visual electrophysiology was performed in aged-matched animals. The amplitudes of both scotopic and photopic b-waves were found to be increased markedly in Long-Evans rats compared to Wistar rats. Amplitudes of oscillatory potentials were also increased clearly. Finally, photopic 30 Hz Flicker amplitudes also were larger in pigmented than in albino rats. Differences in amplitudes were also found in visual evoked potentials. Both photopic and scotopic visual evoked potentials had larger amplitudes. The origin of the amplitude increase in pigmented animals requires further investigation. As the electro retinographic a-waves were less enhanced than the corresponding b-waves, it could be speculated that post-receptoral processing is favoured in pigmented animals. This study demonstrates clearly that pigmentation is more important for normal vision than generally believed.
PP049 - DIABETES TYPE 2 IN ZDF RATS CAUSES DAMAGE OF MELANOCYTES-LIKE INTERMEDIATE CELLS OF THE STRIA VASCULARIS OF THE INNER EAR
Angela-M Meyer zum Gottesberge1, Thomas Massing1, Anja Lange1, Silvia Palma2, Paola Boldrini2 and Stefan Schäfer3.
1Department of Othorhinolaryngology, University of Duesseldorf, Germany;
2Department of Othorhinolaryngology and Electron Microscopy, University of Ferrara, Italy;
3Cardiovascular Research, AventisPharma, Frankfurt, Germany
Epidemiological data indicate an excess incidence of sensorineural hearing loss in patients with diabetes mellitus. However, the pathophysiological background of this diabetic otopathy remains unclear. We investigated hearing function (ABRs), inner ear morphology (electron microscopy) and functional stage of the stria vascularis (immunohistochemistry of ion pumps and channels characteristic for cells present) in the Zucker diabetic fatty (ZDF) rat, a type 2 diabetic animal model. At age 42 weeks, the homozygous animals were characterized by overt diabetes mellitus (HbA1c > 10%) and massive glucosuria and proteinuria indicating severe diabetic nephropathy. The hearing threshold was increased in the diabetic animals to 45.0 ± 2.1 dB, compared to 34.7 ± 1.0 in their heterozygous, non-diabetic littermates (n = 9). The stria vascularis of the cochlea is involved in active(energy consuming) ion transport necessary to maintain the endolymph composition and endocochlear potential essential for acoustic signal transduction. In the adult stria the marginalcells form extensive interdigitations with the basal and intermediate cells. In diabetic rats histopathological and electron microscopic analysis revealed specific damage to the melanocytes-like intermediate cells of the stria vascularis. Melanogenesis occurred in cells with less pathology, however, a majority of the intermediate cells lost their dendricity and severe degeneration was observed. In summary, we found that type 2 diabetes mellitus is associated with functional and morphological alterations of the inner ear, which occurs in parallel to typical end organ damage. In contrast to the microvascular injury, which occurs in nephropathy, diabetic otopathy is characterized by a specific damage to the melanocytes-like intermediate cells of the stria vascularis.
PP050 - THE MELIM SWINE AS A SUITABLE MODEL TO ANALYZE MOLECULAR EVENTS INVOLVED IN REGRESSION OF CUTANEOUS MELANOMA
F. Rambow1, O. Malek2, C. Geffrotin1, J-J. Leplat1, S. Bouet1, V. Horak2,G. Frelat1 and S. Vincent-Naulleau1.
1Laboratory of Radiobiology and Genome Study, CEA-INRA, Jouy-en-Josas Cedex, France;
2Institute of Animal Physiology and Genetics, Libechov, Czech Republic
Spontaneous regression is a well-defined phenomenon commonly observed in human melanoma in their partial histopathological form but sporadically in their complete expression. In the MeLiM (Melanoblastoma-bearing Libechov Minipig) model, swine exhibit cutaneous melanomas which appear around birth. These lesions are clinically and histologically comparable to their human counterparts. Nevertheless, in swine most of the highly invasive melanoma undergo complete spontaneous regression. Induction of regression is expressed by clinical features including changes in color, drying aspect and decrease in size; histologically we observed infiltration of histocyte-like cells, fibrosis and lymphocytic infiltrate later. So, this model offers the opportunity to study the molecular events, which conduct from an invasive tumor to a regressive one. We used Suppression Subtractive Hybridization (SSH) to isolate differentially expressed transcripts between regressive and growing tumor tissue and cell extracts from the two tumor states. About 1000 cDNA clones were sequenced for each SSH library. After identification by homologous search, the genes were classified by Gene Ontology means. Furthermore, we created gene networks using Ingenuity Pathways Analysis 3.1 to discover target genes involved in melanoma regression. The validation of differential gene expression was conducted by quantitative RT-PCR.
PP051 - ESTABLISHMENT AND CHARACTERIZATION OF MELANOMA CELL LINES DERIVED FROM GREY HORSES
Monika Seltenhammer1, MonikaVetterlein2, Jedrzej Kosiuk2, Josef Neumüller2, Margit Pavelka2, Otto Majdic3, Peter Steinberger3, Petra Kohl3, Udo Losert4, Eva Maria Kokoschka5, Michael Micksche6 and Gert Niebauer7.
1Dept. of Clinical Surgery and Ophthalmology, University of Veterinary Medicine, Vienna, Austria;
2Dept. of Cell Biology and Ultrastructure Research;
3Institute of Immunology;
4Core Unit for Biomedical Research;
5Dept. of Dermatology;
6Cancer Research Institute, all Medical University, Vienna, Austria;
7Ecole Nationale Véterinaire de Nantes, France
We have established and characterized two permanently growing in vitro cell lines derived from equine tumour specimens of a Lipizzan (Ho-Mel L1) and an Arabian breed (Ho-Mel A1), respectively. Of 10 specimens cultured two cell lines grew permanently. The doubling time was 24 h in Ho-Mel A1 and 50 h in Ho-Mel L1. Melanin synthesis depended on tyrosine concentrations in the growth media. Cytogenetic analysis revealed for Ho-Mel A1 various sets of chromosomes, and for Ho-Mel L1 a hyperdiploid set. Both cell lines reacted with antibodies against HMB45, S100, MITF, MelanA, Tyrosinase, and MCSP. Furthermore, we analyzed the transcription of specific melanoma associated genes by RT-PCR. Mitf, c-Kit, Ap-2, SILV, TYRP1, ASIP and MC1R were expressed in both cell lines. Finally, we investigated the expression of various leucocyte differentiation antigens by means of FACS analysis. Interestingly, high levels of B7H3 expression were determined in both cell lines.
PP052 - SYK IS EPIGENETICALLY INACTIVATED AND EXHIBITS TUMOR-SUPPRESSIVE ACTIVITY IN HUMAN MELANOMA CELLS
OlivierBailet1, Cédric Gaggioli1, Patricia Abbe1, Guillaume Robert1, Robert Ballotti1, Marcel Deckert2 and Sophie Tartare-Deckert1.
1Inserm, U597, Nice, France;
2Inserm, U576, Nice, France
The protein tyrosine kinase Syk plays critical roles in signaling through immunoreceptors and integrins, and is also a putative tumor suppressor in breast cancer. To investigate the role of Syk in melanoma, we examined its expression pattern and mechanisms of regulation during tumor development. Western blot analysis and quantitative real-time PCR assay were used to assess changes in protein and mRNA expression. We show that Syk is commonly expressed in normal epidermal melanocytes but absent or nearly undetectable in most human primary and metastatic melanoma cells tested. Using methylation-specific PCR, we demonstrated that transcriptional silencing of Syk in melanoma cells was associated with aberrant 5’ CpG island methylation of the Syk promoter, and that treatment with the demethylating agent, 5-Aza-2’-deoxycytidine, restored Syk expression in Syk-negative melanoma cells. Interestingly, even in the presence of oncogenic BRAF, reintroduction of wild type Syk kinase abrogates clonogenic growth properties of melanoma cells. Surprisingly, a kinase-deficient mutant also reduced colony formation efficiency indicating that this effect might be independent of enzymatic activity of the kinase. Suppression of tumor growth by Syk appeared to be the result of altered cell-cycle control and is associated with activation of the JNK signaling pathway. We propose that Syk is a potential tumor suppressor in human melanoma, which is silenced by means of a DNA methylation-dependent mechanism. Our results provide new insights into Syk regulation and function in cancer and indicate that loss of Syk may contribute to melanoma tumorigenesis.
PP053 - IGF-1 MEDIATES PROTECTION AGAINST TRAIL-INDUCED APOPTOSIS IN MELANOMA CELLS THROUGH A PI3K/AKT/FOXO3A-DEPENDENT PATHWAY
C. Hilmi1, M. Deckert2, L. Larribère1, K. Bille1, R. Ballotti1 and C. Bertolotto1.
1INSERM U597, 28 avenue de Valombrose, 06107 Nice Cedex 2 France;
2INSERM U576, Hôpital de l'Archet, 06202 Nice Cedex 03 France
In the skin, IGF-1, which is produced by fibroblasts of the tumor stroma, is one of the most critical factors for the growth, migration and survival of melanoma cells. IGF-1 mediates its signal through binding to its IGF1-R, which over expression has been shown to be tightly associated with melanoma progression and metastasis. However, the mechanism by which IGF-1/IGF1-R cascade exerts its effects in melanoma cells is not fully elucidated. We show that IGF-1, through activation of the PI3K/AKT pathway, protects A375 melanoma cells from TRAIL-induced apoptosis. Interestingly, we find that IGF-1 leads to the phosphorylation and nuclear exclusion of the forkhead transcription factor, FoxO3a, while the PI3K pharmacological inhibitor, LY294002, prevents IGF-1 anti-apoptotic effects and nuclear exclusion of FoxO3a and sensitizes melanoma cells to death stimuli. At the nucleus, FoxO3a controls the transcription of genes involved in the cell cycle arrest and apoptosis. Interestingly, transduction of melanoma cells with an active form of FoxO3a that is constitutively localized in the nucleus, promotes death of melanoma cells. A gene candidate approach, using real-time quantitative PCR has been undertaken to identify genes regulated by FoxO3a in melanoma cells.
PP054 - IN MELANOMA RAS MUTATIONS ARE ACCOMPANIED BY SWITCHING SIGNALLING FROM BRAF TO CRAF AND DISRUPTED CAMP SIGNALLING
Nicolas Dumaz1,2, Armand Bensussan1 and Richard Marais2.
1INSERM U659, Faculté de Médecine de Créteil, 8 rue du Général Sarrail, 94010 Créteil, France;
2The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK
Melanocytes require the RAS/RAF/MEK/ERK and the cAMP signalling pathways to maintain the fine balance between proliferation and differentiation. We have investigated how cross-talk between these pathways affects melanoma progression. We show that cAMP suppresses CRAF activity in melanocytes and that this is essential to suppress the oncogenic potential of CRAF in these cells. As a consequence, BRAF alone is responsible for signalling to MEK. However, when RAS is mutated in melanoma, the cells switch their signalling from BRAF to CRAF. This switch is accompanied by dysregulated cAMP signalling, a step that is necessary to allow CRAF to signal to MEK. Thus, a fundamental switch in RAF isoform usage occurs when RAS is mutated in melanoma and this occurs in the context of disrupted cAMP signalling. These data have important implications for the development of therapeutic strategies to treat this life threatening disease.
PP055 - TNFα BLOCKS APOPTOSIS IN MELANOMA CELLS WHEN BRAF SIGNALLING IS INHIBITED
VanessaC. Gray-Schopfer1, Maria Karasarides2, Robert Hayward1 and Richard Marais1.
1The Institute of Cancer Research, Signal Transduction Team, Cancer Research UK Centre of Cell and Molecular Biology, 237 Fulham Road, London SW3 6JB, UK;
2Current address: Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street Worcester, MA 01605, USA
The protein kinase BRAF, a component of the RAS/RAF/MEK/ERK signalling pathway, regulates cell fate in response to extracellular signals. Activating mutations in BRAF occur in approximately 70% of human melanomas. The active proteins stimulate constitutive pathway signalling, proliferation and survival. Thus, inhibition of BRAF signalling in melanoma cells causes cell cycle arrest and induces cell death through apoptosis, validating BRAF as an important therapeutic target. Here, we show that the apoptosis induced by inhibition of BRAF signalling in melanoma cells can be prevented if the cells are treated with TNFα. This allows the cells to recover from the inhibition of BRAF signalling and re-enter the cell cycle. This effect is specific to TNFα and is not observed when the cells are treated with other cytokines. The underlying mechanism is mediated by the transcription factor NFκB. These findings suggest that drugs that target the BRAF/MEK pathway could be combined with agents that target TNFα and/or NFκB signalling to provide exciting new therapeutic opportunities for the treatment of melanoma.
PP056 - MOLECULAR AND FUNCTIONAL ANALYSIS OF MC1R RECEPTOR IN MALIGNANT MELANOMA
P. Zanna1, G. Guida2, F. Susca2, C. Danisi2, V. Lattanzi3, I. Maida2, G. A. Vena3, R. Filotico3, Maria del Carmen Turpin1, C. Jimenez-Cervantes1, J. C. Garcia-Borron1 and R. Cicero2.
1Department. of Biochemistry and Molecular Biology, University of Murcia, Murcia, Spain;
2Dip. di Biochimica Medica, Biologia Medica e Fisica Medica;
3Clinica Dermatologica II Facoltà di Medicina e Chirurgia Università degli Studi di Bari, Bari - Italy
We obtained two malignant melanoma (MM) cell lines from South Italy Caucasian patients, hmel 1and hmel 9. These cultures showed histological and morphological differences. Their cariotype present genetics abnormalities, among which we found common features in both cell lines such as an isochromosome 6p and a polysomy of the chromosome 7, present only in polyploid cells. We screened these cell lines for MC1R genotype that resulted to be wildtype. We demonstrated an increase in MC1R transcripts, higher in hmel 1 than hmel 9. Functional analysis of MC1R in terms of cAMP production in response to MSH treatment revealed that hmel 1 cells are able to couple the signal at low levels in response to MSH treatment with a maximum after 15 min., differently to hmel 9 that are unable to couple the signal. This situation is not uncommon, as we have detected failure to respond to MSH in two other cell lines homozygous for wildtype MC1R, in a survey of 10 human melanoma cell lines. Tyrosinase activity was detectable in the hmel 9 cell line and absent in hmel 1, indicating loss of correlation between the functional status of MC1R and basal tyrosinase activity. These data show that the cAMP pathway may be impaired by still uncharacterised mechanisms in a sizeable fraction of human melanoma cells expressing wildtype MC1R. Correlation between histological and cytogenetic studies, MC1R genotype, its expression levels and activity in primary cell lines may help to understand how the melanocyte transformation occurs.
PP057 - HISTONE DEACETYLASE INHIBITOR, FK228 TRIGGERS MITF LEADING TO G1 ARREST/APOPTOSIS VIA DOWN- AND UP-REGULATION OF CDK2 AND P21 IN HUMAN MALIGNANT MELANOMA CELLS
GenjiImokawa1,2, Ken Futaki1, Yusuke Furukawa3, Mamitaro Ohtsuki4 and Hidemi Nakagawa1.
1Department of Dermatology, Jikei Medical University, Tokyo, Japan;
2Skin Science Research Institute, Tokyo, Japan;
3Division of Stem Cell Regulation, Jichi Medical University, Tochigi, Japan;
4Department of Dermatology, Jichi Medical University, Tochigi, Japan
Histone deacylase inhibitors (HDACi) could act as a novel anti-cancer agent for malignant melanoma because of their action mechanisms distinct from conventional anti-cancer agents. Here, we examined signaling mechanism(s) involved in HDACi FK228-induced G1 arrest/apoptosis in association with newly discovered cell regulatory roles of MITF in 5 human malignant melanoma cell lines. An addition of FK228 at concentrations of 2–10 nM elicited G1 arrest/apoptosis in all cell lines tested at 24–48 post incubation. The gene and protein expressions of MITF were markedly down-regulated by FK228, which was followed by a significant suppression of Cdk2 or cdc2, with both protein levels being well correlated with each other (r = 0.745 or 0.79), while MITF level well correlated with Cdk2 (r = 0.81) but not with cdc2 (r = 0.05) or Cdk4 (r = 0.15) in non-treated cells. In contrast, CDK2 inhibitor protein p21 not constitutively expressed in non-treated cells was markedly up-regulated at 12–24 h post-incubation with reversed correlation to MITF (r = –0.6), while Bcl2 expression remained almost unaffected. These findings suggest that FK228 exerts its anti-melanoma effects via down-regulation of MITF which leads to the suppression of CDK2 and the concomitant up-regulation of p21 without any effect on Bcl2 expression.
PP058 - HYPERGRAVITY DIFFERENTIALLY MODULATES THE EXPRESSION OF MULTIDRUG RESISTANCE PROTEINS IN HUMAN MELANOCYTIC CELLS
KrassimiraIvanova1, Britta Lambers1, Christiane Stieber1, Ingrid Block1, Pranab K. Das2 and Rupert Gerzer1.
1Institute of Aerospace Medicine, DLR, Cologne, Germany
2Department of Dermatology, AMC, University of Amsterdam, The Netherlands
Gravity alteration (micro- and hypergravity) is known to influence cell functions. In previous studies we have shown that hypergravity may differentially modulate cGMP efflux in normal human melanocytes (NHMs) and melanoma cells (MCs) in the presence of phosphodiesterase inhibitors and/or induction of cGMP synthesis by nitric oxide. In this study we have analyzed the expression of the multidrug resistance proteins 4/5 (MRP4/5) as highly selective cGMP exporters at basal (1 g) conditions and during long-term exposure of human melanocytes to centrifugal acceleration (up to 5 g for 24 h). For 5-g experiments, the mRNA levels for MRP5 were increased in NHMs and nonmetastatic MCs in comparison to 1-g controls that correlate with hypergravity-induced increase in cGMP efflux in these cells. Metastatic MCs, in contrary, were insensitive to the hypergravity effects. We further found no differences between 1-g and 5-g treated cells in the expression of mRNAs for MRP4 and other ABC-transporters like ABC-1, MRP1, MRP2, and BCRP. The present results suggest that environment created by centrifugal acceleration represents a new factor for regulating cGMP levels in human melanocytes that involve MRP5. Future studies on this aspect in microgravity may be important for residents on the International Space Station and astronauts involved in long space flights.
PP059 - A NEW SPLICE-VARIANT OF β-ARRESTIN 2 IS INVOLVED IN AGONIST-INDUCED MC1R ENDOCYTOSIS
Berta López Sánchez-Laorden, José C García-Borrón and CeliaJiménez-Cervantes.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Murcia. Murcia, Spain
The level of G protein coupled receptors (GPCRs) at the plasma membrane depends on a balance between export through the secretory pathway and constitutive or ligand induced internalization. Receptor endocytosis is intimately linked to desensitization. Current models involve rapid phosphorylation of occupied receptors by a G protein coupled receptor kinase resulting in binding of arrestin, which uncouples the receptor from G proteins and initiates endocytosis. Two ubiquitous non visual arrestins have been described (β-arrestins 1 and 2), with several splice variants. The melanocortin 1 receptor (MC1R) expressed in melanocytes and melanoma cells belongs to the GPCR superfamily. We investigated the mechanisms of MC1R internalization. We found that melanoma cells express β-arrestin 1 and a new β-arrestin 2 splice variant (variant 3) showing the presence in the coding sequence part of the intron 13–14, that is translated in frame. This yields a 12 aminoacid insertion (APTPTPPLPVPP) absent in β-arrestin 2. When cotransfected with MC1R in HEK 293 cells, this new variant was recruited to the plasma membrane even in the absence of agonist. Cotransfection of MC1R with β-arrestin 2 (variant 3), but not with β-arrestin 1, increased homologous desensitization and caused a dramatic increase in agonist-induced sequestration which was partially blocked by a dominant negative form. Confocal laser scanning microscopy demonstrated a stable association of MC1R and β-arrestin 2 (variant 3) in endocytic vesicles. Therefore, human melanoma cells express a new splice variant of β-arrestin 2, with high affinity for MC1R that potently and specifically promotes receptor desensitization and internalization.
PP060 - THE EXPRESSIONS OF MELANOSOME ASSOCIATED RABS ARE UPREGULATED IN MELANOCYTES BUT DOWNREGULATED IN NEVUS CELLS BY MSH
TsuneyoshiKamo, Yoko Funasaka and Chikako Nishigori.
Division of Dermatology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
Melanosomes are transported from the perinucleus to the peripheral region of melanocytes, eventually being transferred to adjacent keratinocytes. Rab proteins are monomeric GTPases of the Ras superfamily. In the active state, Rabs recruit a diverse group of proteins termed effector proteins to the cytoplasmic leaflet of the membrane, and recruitment of effector proteins might enable Rabs to control the main steps in vesicular transport. We have previously reported that Rab3A and Rab8A are associated with melanosomes in mouse B16 melanoma cells. Recently another secretion related Rab protein, Rab27A has been shown to bind with melanophilin/Slac-2a which associates with myosin Va. We examined the effect of MSH on Rab expression as well as configuration using cultured normal human melanocytes and nevus cells, the latter are believed to have a tendency to retain melanin. MSH upregulated the expression of Rab3A, Rab8A, and Rab27A in melanocytes with the dendrite elongation and enhanced tyrosinase expression. In contrast to melanocytes, nevus cells were down-regulated in their expression of Rabs and tyrosinase. The determination of the melanocortin-1 receptor (MC-1R) sequence showed no difference between these cell types. These results suggest that GTPase molecules involved in melanosome transport are coordinately regulated with melanin synthesis by MSH stimulation, however these are downregulated in nevus cells which are poor in the ability of melanosome transport, and these differences were not due to the variance of MC1-R gene itself, but rather the variance of signal transduction in the downstream of MSH receptor.
PP061 - DOWN-REGULATION OF MELANIN SYNTHESIS BY MB302 AND ITS MECHANISM
Jin-HeeKim1, Seung-Hwa Baek1, Eun-Sook Yoo2 and Choong-Hwan Lee1.
1Natural Medicines Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea;
2Department of Pharmacology, College of Medicine, Cheju National University, Cheju, Korea
Down-regulation of melanin synthesis is required for the recovery of pigmentary disorders and it is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, suppress melanin synthesis. MB302((R,E)-3-(3,4-dihydroxyphenyl)-1-methoxy-1-oxopropan-2-yl 3-(3,4-dihydroxyphenyl)acrylate) was synthesized and examined for melanin synthesis inhibition activity and found that MB302 down-regulated melanin synthesis effectively. In vitro experiments using treatment of Melan-a cells with MB302 (1.6–13.8 μM) for 4 days significantly reduced melanin levels in a dose-dependent manner. Many skin-whitening agents inhibit tyrosinase directly. Thus, to investigate the direct effects of MB302 on tyrosinase, we measured the tyrosinase activities of mushroom tyrosinase in a cell-free system. MB302 was found to have no direct inhibitory effect on mushroom tyrosinase activity. Furthermore, we found that MB302 decreased the protein expression of microphthalmia-associated transcription factor (MITF), which is required for tyrosinase expression. To identify the signaling pathway of MB302, we studied the ability of MB302 to influence extracellular signal-regulated protein kinase (ERK) and Akt/protein kinase B (PKB) activation. Akt/PKB activation was induced by MB302 in Melan-a cells. Thus, it seems that sustained Akt/PKB activation may lead to the suppression of cell growth and melanogenesis.
PP062 - CENTAURIN β4, A REGULATOR OF CELL INVASION AND METASTASIS, HAS NOVEL ISOFORMS IN HUMAN MELANOMA CELL LINES
R. K. Martin, A.J. Thody and T. R. Jackson.
Dermatological Sciences, University of Newcastle-upon-Tyne, UK
Centaurins are ADP Ribosylation Factor (Arf) GTPase Activating Proteins (GAPs) that regulate cyclic activation of Arfs, important for actin cytoskeletal responses. Centaurin β4 has been implicated in cell invasion and metastasis; disruption of the interaction of centaurin β4 with cortactin, reduces invasion and metastasis (1). The protein centaurin β4/ASAP1/DDEF1/PAG3/AMAP1 is produced by the DDEF1 gene locus (2) which is amplified and upregulated in various cancers including uveal melanoma (3). We have identified several isoforms of centaurin β4 in four human melanoma cell lines. Western blotting using two different antisera revealed multiple bands in all cell lysates. To determine whether they arise by alternate splicing we performed PCR across the C’ known to show alternative splicing in mouse. Two splice forms present in all four melanoma lines were orthologous to those in mouse, three further variants were found in two lines. These novel human splice forms variously lack proline rich regions or an SH3 domain implicated in interactions with cortactin and other focal adhesion proteins such as Crk-L, paxillin, FAK, POB1, Src and also with Pyk2 and CIN85. These splice forms could therefore act in several different ways to regulate adhesion and movement. We propose that centaurin β4 and its novel splice forms play pivotal roles in melanoma progression.
1. Hashimoto, S., et al., Targeting AMAP1 and cortactin binding bearing an atypical src homology 3/proline interface for prevention of breast cancer invasion and metastasis. Proc Natl Acad Sci U S A, 2006. 103(18): p. 7036–41.
2. Martin, R.K. and T.R. Jackson, Centaurin beta4 in cancer. Biochem Soc Trans, 2005. 33(Pt 6): p. 1282–4.
3. Ehlers, J.P., et al., DDEF1 is located in an amplified region of chromosome 8q and is overexpressed in uveal melanoma. Clin Cancer Res, 2005. 11(10): p. 3609–13.
PP063 - STABLE OVEREXPRESSION OF SMAD7 IN HUMAN MELANOMA CELLS INHIBITS BONE METASTASIS IN VIVO
Delphine Javelaud1, Khalid S. Mohammad2, Christopher R. McKenna2, Maria Niewolna2, Jocelyne André1, Véronique Delmas3, Lionel Larue3, Theresa A. Guise2 and AlainMauviel1.
1INSERM U697, Hopital Saint-Louis, Paris, 75, France;
2University of Virginia, Charlottesville, VA, USA;
3UMR146 CNRS, Orsay, France
Melanoma has a propensity to metastasize to bone, where it is exposed to high concentrations of TGF-β. Because TGF-β promotes bone metastases from other solid tumors such as breast cancer, we tested the role of TGF-β in melanoma metastases to bone. 1205Lu melanoma cells which stably overexpress Smad7 were studied in an experimental model of bone metastasis whereby tumor cells are inoculated into the left cardiac ventricle of nude mice. All mice (n = 6) bearing parental 1205Lu cells and 4/6 mice bearing mock-transfected 1205Lu cells developed osteolytic bone metastases 5–10 weeks post tumor inoculation, as well as metastases to adrenal glands and kidney. Only 1/14 mice bearing Smad7-transfected 1205Lu cells (2 clones) had osteolytic bone lesions on radiographs 10 weeks post tumor inoculation and only 5/14 were sacrificied after 15 weeks. The mice bearing these 1205Lu-Smad7 tumors had therefore significantly longer survival compared to parental and mock-tranfected 1205Lu mice. To determine if the reduced bone metastases observed in mice bearing 1205Lu-Smad7 clones was due to reduced expression of TGF-β target genes known to enhance metastases to bone from breast cancer cells, we analyzed gene expression of osteolytic factors, PTHrP and IL-11, the chemotactic factor, CXCR4, as well as CTGF and osteopontin, in Lu1205. Quantitative RT-PCR analysis indicated that PTHrP, IL-11, CXCR4, and osteopontin mRNA steady-state levels were robustly increased in response to TGF-β, and that Smad7, and the TβRI small molecule inhibitor, SB431542, prevented such induction. These data suggest that TGF-β promotes osteolytic bone metastases due to melanoma by stimulating the expression of prometastatic factors via the Smad pathway. Blockade of TGF-β signaling may be effective treatment for melanoma metastatis to bone.
PP064 - CELL AGGREGATION AS A POSSIBLE ESCAPE MECHANISM FROM RGD PEPTIDE-INDUCED APOPTOSIS IN MELANOMA CELLS
MorandiniRenato1, Süli-Vargha Helga2, Szabo Rita2 and Ghanem Ghanem1.
1LOCE, Bordet Institute, Free University of Brussels, Belgium;
2Eotvos University, Budapest, Hungary
Peptides containing RGD motif are ligands for Integrin receptors contributing to cell detachment from ECM components and subsequent induction of apoptosis. In melanoma cells however, this process does not end-up with significant cell death. We exposed melanoma and other cells to 10 different cyclic RGD peptides at various times and concentrations. This treatment is known to cause cell detachment and subsequent anoïkis. Using video time-lapse, we observed the formation of growing cell aggregates while single cells that were unable to rapidly join the latter, died. One logical explanation would be that cells in aggregates were able to reform their ECM and thus escape the death signal. With two immunofluorescent antibodies against fibronectin, we registered increasing signal along with the formation of the aggregates. Flowcytometry with melanoma and endothelial cells showed a correlation between fibronectin signal and the size of the aggregates and was confirmed by western blot. In cell adhesion assay on fibronectin, a strong correlation between inhibition of cell adhesion and growth rate and size of the aggregates was observed. In conclusion, RGD peptides caused cells to detach and form aggregates to survive by reforming their ECM and receiving survival signals from αv-integrins/fibronectin binding, thus escaping apoptosis. Other ECM components within the aggregates are currently under investigation.
PP065 - FUNCTIONAL STUDIES OF MC1R IN HUMAN MELANOCYTE - KERATINOCYTE COCULTURES
D. W. Roberts1,2, R A Newton1, J. H Leonard2 and R A Sturm1.
1Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
2Queensland Institute of Medical Research, Brisbane, Australia
Individuals who possess allelic variants that lead to reduced functional activity of the melanocortin-1 receptor (MC1R), present a phenotype of red hair, pale complexion and an increased sensitivity to ultraviolet radiation, each of which are strong risk factors associated with skin cancer development. We have established a serum free melanocyte - keratinocyte coculture system to examine the behaviour and functional abilities of melanocytes possessing such reduced MC1R activity and make comparison to wildtype strains. Using this model, melanocytes of similar MC1R genotype displayed differences in morphological appearance when cocultured with keratinocytes under various calcium conditions. MC1R activation of the main enzymes involved in the melanogenic pathway of melanocytes was investigated at the RNA and protein levels following stimulation with the ligand NDP-MSH, and we have also documented melanocyte morphology changes upon ligand stimulation. Direct evaluation of MC1R expression in skin cells and a range of tumor lines were undertaken in monoculture utilising transfected HEK positive and parental HEK negative controls. This has revealed that while MC1R mRNA is detectable in a variety of cell types using RT-PCR methods, the levels are up to 100 fold higher in melanocytes, with protein levels and functional stimulation of MC1R being restricted to melanocytic cells only.
PP066 - ROLE OF T-BOX FACTORS DURING MELANOMA PROGRESSION
MercedesRodriguez and Colin R. Goding.
Signaling and Development Laboratory, Marie Curie Research Institute, The Chart, Oxted, Surrey, UK
One of the great challenges in cancer research is to understand the molecular mechanisms underlying the progression from a localized tumour to metastases. Malignant melanoma (the most lethal skin cancer that arises from melanocyte) represents a good model to study these events since all stages of the disease can be diagnosed and phenotypically characterized. In the skin melanocytes are tightly controlled by the surrounding keratinocytes and a disruption of this homeostatic balance can lead to a change in the expression of cell-cell adhesion and cell-cell communication molecules. In particular, the loss of the epithelial cell-cell adhesion molecule E-cadherin appears to be one of the critical steps in the escape of malignant melanocytes from keratinocyte mediated growth control, when the tumour cells became independent from the primary focus and acquire an invasive phenotype.Traditionally, the T-box protein family has been described as key developmental transcription factors, playing a crucial role in cell cycle regulation and maintenance of cell identity. Moreover, recent studies have shown that T-box factors are misregulated in cancer, for example, the Tbx2 gene is frequently mutated in ovarian carcinomas and amplified in pancreatic tumours, as well as in breast cancer. In particular, Tbx2 and its highly related factor Tbx3 are over-expressed in human melanomas and play an important role maintaining cell proliferation by senescence suppression. Here we identified a novel role for T-box factors during melanoma progression, providing in vitro and in vivo evidence which suggest that Tbx3 and Tbx2 repress the E-cadherin promoter in malignant melanoma. The results support a model in which Tbx3 and Tbx2 play a role in the later stages of melanoma progression, during the transition from an in situ superficial tumour (radial growth phase) to a potential invasive tumour (vertical growth phase), contributing to the homeostatic disequilibrium needed for the tumuorigenic cell to become independent from the surrounding keratinocytes. Furthermore, the finding that either Tbx3 or Tbx2 can regulate E-cadherin raises the interesting possibility of a new role for these T-box factors during development and the progression of other malignancies.
PP067 - UVA/B IRRADIATION CAUSES TRANSLOCATION OF BCL-2 FAMILY PROTEINS IN BOTH APOPTOTIC AND SURVIVING MELANOCYTES
PetraLarsson1, Cecilia Bivik1, Katarina Kågedal2, Inger Rosdahl1 and Karin Öllinger2.
1Divisions of Dermatology;
2Experimental Pathology, Faculty of Health Sciences, Linköping University, S-581 85 Linköping, Sweden
We demonstrate UVA/B to induce apoptosis in human melanocytes through the mitochondrial pathway, displaying cytochrome c release, caspase-3 activation and fragmentation of nuclei. The outcome of a death signal depends on the balance between positive and negative apoptotic regulators, such as members of the Bcl-2 protein family. Apoptotic melanocytes, containing fragmented nucleus, show translocation of the pro-apoptotic proteins Bax and Bid from the cytosol to punctate mitochondrial-like structures. Bcl-2, generally thought to be attached only to membranes, was in melanocytes localized in the cytosol as well. In the fraction of surviving melanocytes, i.e. cells with morphologically unchanged nucleus, the anti-apoptotic proteins Bcl-2 and Bcl-XL were translocated to mitochondria following UVA/B. Altogether, our results emphasize translocation of Bcl-2 family proteins to have central regulatory functions of UV-induced apoptosis in melanocytes.
PP068 - CYTOKINE PRODUCTION IN MELANOCYTES
NicoSmit1, Fred Romijn1, Margo van Schie1, Marja Kersbergen1, Lester Davids2, Stan Pavel1 and Hans van Pelt1.
1Department of Clinical Chemistry, Leiden University Medical Centre, The Netherlands; 2Department of Human Biology, University of Cape Town Medical, South Africa
Melanocytes are dendritic cells strategically located in the skin on the basal layer. Next to melanin production these cells produce various cytokines, they are capable of phagocytosis and could function as potentially immunocompetent cells (Smit N, 1993; Le Poole IC, 1993). ACTH and MSH influence melanocytes via the MC1receptor and have been described to exert a variety of immunomodulating activities (Luger TA, 1999). We have investigated cytokine expression and production in cultured melanocytes. Micro arrays were used to study gene expression and a multi cytokine screening system was used to measure cytokine secretion in culture supernatants in the presence and absence of TPA or ACTH. Expression data in melanocytes revealed relatively high levels of IFN-g, Il-13, Il-6 and TNF-a. Using the cytokine protein array, the highest values were found for Il-8, VEGF and monocyte chemotactic protein-1. IL-6 production was higher in the culture supernatants of melanocytes treated with ACTH. Increased Il-6 secretion was confirmed by ELISA in 5 out of 6 different cultures treated with ACTH and for all 8 cultures treated with TPA. Signalling via Il-6 and other cytokines may be influenced by ACTH and could have, considerable consequences for melanocyte function (Swope VB, 1985; Kamaraju, AK 2002).
PP069 - ANTI-PROLIFERATIVE EFFECTS OF PPARγ AGONISTS ON MELANOMA CELLS ARE MEDIATED IN PART BY MODULATION OF β-CATENIN FUNCTION
AaronGSmith, Darren J Smit, Richard A Sturm and George EO Muscat.
Institute for Molecular Bioscience, University of Queensland, St Lucia Queensland, Australia 4072
Peroxisome Proliferator-activated Receptor-gamma (PPARγ) is a member of the nuclear hormone receptor superfamily of ligand activated transcriptional regulators. PPARγ is known to be a key regulator of adipose differentiation and function and to regulate the expression of a range of genes controlling lipid and carbohydrate homeostasis in adipose and non-adipose tissues. Commensurate with this function it has been shown that the thiazolidinedione (TZD) class of insulin sensitising drugs are high affinity ligands for PPARγ. Furthermore, accumulating evidence suggests that TZD and non-TZD PPARγ activators may prove to be useful anticancer agents exhibiting anti-proliferative and/or pro-apoptotic affects in a range of cancer cell types including melanoma, however the mechanisms underlying this effect remain unclear. Studies conducted in our laboratory using TZD compounds rosiglitazone and troglitazone and novel non-TZD compounds support previous observations of the anti-tumorigenic activity of these agents on melanoma cell lines. Furthermore siRNA mediated ablation of PPARγ in the MM96L melanoma cell line attenuates the anti-proliferative effect of all TZD and non-TZD agents suggesting this effect is mediated via activation of PPARγ. Studies aimed at identifying the molecular targets underlying the anti-proliferative effects of PPARγ in melanoma cells have revealed changes in expression of a number of cell cycle and melanocytic regulatory genes. Moreover, the expression and function of β-catenin was modulated by PPARγ activation in human melanoma cell lines. These results suggest modulation of β-catenin function may be one way by which PPARγ activators exert anti-proliferative effects in melanoma cells.
PP070 - DIACYLGLYCEROL KINASE ALPHA SUPPRESSES TNF-ALPHA-INDUCED APOPTOSIS OF HUMAN MELANOMA CELLS THROUGH ACTIVATION OF NF-KAPPA B
KenjiYanagisawa1, Kowichi Jimbow1, Hideo Kanoh2 and Fumio Sakane2.
1Department of Dermatology, Sapporo Medical University, Sapporo, Japan;
2Department of Biochemistry, Sapporo Medical University, Sapporo, Japan
Diacylglycerol kinase (DGK) is thought to regulate many cellular processes through modulating the balance between two signaling lipids, diacylglycerol and phosphatidic acid. We found that DGK alpha (type I isozyme) was expressed in several human melanoma cell lines but not in noncancerous melanocytes. To study melanoma-specific functions of DGK alpha, this isozyme was down-regulated or, conversely, overexpressed in AKI human melanoma cells. The overexpression of wild-type (WT) DGK alpha considerably suppressed tumor necrosis factor (TNF)-alpha-induced apoptosis of AKI cells (TUNEL-positive cell: 6.7%), compared with the vector control (20.3%). However, a kinase-dead (KD) mutant failed to affect the extent of the apoptosis (18.2%). Moreover, siRNA-mediated knockdown of DGK alpha markedly enhanced the apoptosis (10.2%) compared with the control RNA (4.0%). These results indicate that DGK alpha is uniquely involved in the suppression of AKI cell apoptosis induced by TNF-alpha. The overexpression of DGK alpha-WT further enhanced the TNF-alpha-stimulated transcription activity of an anti-apoptotic factor, NF-kappa B (∼2-fold increase), whereas DGK alpha-KD failed to affect the activity. Conversely, DGK alpha-knockdown considerably inhibited the TNF-alpha-dependent NF-kappa B activity (∼30% inhibition). These results collectively indicate that DGK alpha negatively regulates TNF-alpha-induced apoptosis through NF-kappa B activation.
PP071 - APURINIC/APYRIMIDINIC ENDONUCLEASE-1/REDOX FACTOR-1 (APE/REF-1), A POTENTIAL THERAPEUTIC TARGET FOR HUMAN MELANOMA
SunYang1,2, K. Irani3, S.E. Heffron4, Frances Jurnak1,4.and Frank L. Meyskens1,2,5
1Chao Family Comprehensive Cancer Center;
2Department of Medicine;
3Cardiovascular Institute, University of Pittsburgh Medical Center, Pittsburgh PA;
4Department of Physiology and Biophysics, UC Irvine School of Medicine, Irvine, CA, USA
5Department of Biological Chemistry, UCI, School of Medicine, Orange, CA;
APE/Ref-1 is a multifunctional protein involved in DNA base excision repair and redox regulation of many transcription factors. In different melanoma cell lines we found significantly elevated Ref-1 expression levels compared to normal melanocytes. Similar increases, detected by immunohistofluorescence, were also evident in nevi and malignant melanoma biopsies, which were predominantly localized in the nucleus. Using recombinant adenovirus Adref-1, encoding Ref-1, we transiently overexpressed APE/Ref-1 in human melanocytes, which protected these cells from UV-induced apoptosis and foci formation in culture. Ref-1 overexpression also protected melanoma cells from cisplatin or H2O2-induced apoptosis, while increased apoptosis was observed with Ref-1 antisense. EMSA evaluation demonstrated that both AP-1 and NF-κB DNA-binding activities were significantly diminished or shifted with addition of APE/Ref-1 antibody to deplete Ref-1 from the binding complexes. Furthermore, we built two specific Ref-1 deficient constructs, including nuclear translocation sequences deleted-Ref-1 (ΔNLS-Ref-1) and redox deficient Ref-1 (C65/93A-Ref-1), to study its effects on cell proliferation. Real-time PCR results showed that melanoma inhibitory activity (MIA) expression levels were significantly reduced by stably overexpresing ΔNLS-Ref-1 and C65/93A-Ref-1 in human melanoma cells, indicating the importance of Ref-1 on melanoma transformation and progression.
PP072 - MECHANISMS OF DIMERIZATION OF THE HUMAN MELANOCORTIN 1 RECEPTOR
P. Zanna, B. Sanchez-Laorden, A. B. Perez-Oliva, Jimenez-Cervantes C and Garcia-Borron JC.
Dep. de Bioquimica y Biología Molecular B e Inmunología. Facultad de Medicina, University of Murcia. Spain
The melanocortin 1 receptor (MC1R) mediates the responses of melanocytes to alpha-MSH. MC1R is a G protein coupled receptor (GPCR) activating the cAMP pathway. Many GPCRs function as dimers through coiled-coil interactions of C terminal extensions, hydrophobic contacts between helices, disulfide bonds or domain swapping. MC1R dimerizes in living cells and heterodimerization of wild type with mutant units modifies its intracellular traffic and functional properties. We studied the mechanisms of MC1R dimerization through C-terminal deletions. Ablations of the cytosolic extension and the entire seventh transmembrane fragment did not dissociate MC1R dimers, excluding coiled-coil interactions. Dimers resistance to SDS argued against hydrophobic contact dimerization mechanisms, but sensitivity to mercaptoethanol suggested the contribution of disulfide bonds. Mutagenesis of Cys residues to Ala suggested a model with four intermolecular disulfide bonds involving Cys35, Cys267, Cys273 and Cys275. Disruption of a single disulfide bond completely inhibited receptor function and impaired plasma membrane expression, but did not abolish dimerization. Dissociation of MC1R dimers in non-reducing SDS-PAGE required mutation of all four Cys residues. Co-immunoprecipitation studies suggested a contribution of non-covalent domain swap interactions to dimerization. We propose a model for MC1R dimerization involving non-covalent domain swap-type interactions and covalent cross-linking of monomers by four disulfide bonds.
PP073 - INHIBITION OF SERCA2 DISRUPTS MELANOSOME TRANSFER TO HUMAN KERATINOCYTES - INSIGHT INTO THE PATHOMECHANISM OF GUTTATE LEUCODERMA IN DARIER’S DISEASE
Boon-KeeGoh1, KarolienVanDen Bossche2, Veerle Van Marck3, Marc Bracke3, Jo Lambert2, Cheng-Leng Kee1, S P W Kumarasinghe1 and Jean Marie Naeyaert2.
1National Skin Centre, Singapore;
2Department of Dermatology, Ghent University Hospital, Ghent, Belgium;
3Department of Experimental Oncology, Ghent University Hospital, Ghent, Belgium
Introduction: Guttate leucoderma is an unusual feature seen predominantly in dark-skinned individuals with Darier's disease. Although the pathomechanism of Darier's disease has been characterized and is related to the disturbance of intracellular calcium homeostasis due to ATP2A2 mutations, that of guttate leucoderma remains an enigma. We have previously postulated that aberrant intracellular calcium fluxes, due to defective sarcoendoplasmic reticulum calcium ATPase isoform 2 (SERCA2) pump, may cause proteolysis or conformational change of E-cadherins and disrupt melanocyte adhesion and melanosome transfer to keratinocytes, resulting in leucoderma.
Methods: Electron microscopic examination of leucodermic lesions and non-lesional skin from Asian patients with Darier's disease was compared. Keratinocyte-melanocyte co-aggregation assay was performed in the presence of SERCA2 inhibitor, thapsigargin. Epidermal skin equivalents were constructed with and without the presence of thapsigargin to evaluate pigment transfer.
Results: Ultrastructural examination showed that in leucodermic lesions, melanocyte dendrites were filled with melanosomes, yet there was a lack of transfer to surrounding acantholytic keratinocytes. In the presence of thapsigargin, there was a significant loss of co-aggregation between melanocytes and keratinocytes, and a significant reduction of pigment transfer was demonstrated in the epidermal reconstruct models.
Conclusion: Loss of melanosome transfer, mediated by dysfunctional SERCA2, is involved in the pathogenesis of guttate leucoderma in Darier's disease.
PP074 - TYROSINASE BIOSYNTHESIS AND TRAFFICKING IN ADULT HUMAN RETINAL PIGMENT EPITHELIAL CELLS
SylvieJulien1, Norbert Kociok2, Florian Kreppel3, Veronica Brito3, Swaantje Peters4, Sigrid Henke-Fahle4, Petra Blitgen-Heinecke1, Despina Kokkinou1, Karl-Ulrich Bartz-Schmidt4, Stefan Kochanek3and Ulrich Schraermeyer1.
1Section for Experimental Vitreoretinal Surgery, University Eye Hospital Tubingen, Germany;
2Netzhautlabor, Cell Center Cologne, Josef-Stelzmann-Str.50, 50931 Cologne, Germany;
3Division of gene therapy, University of Ulm, Helmholtzstr. 8/1, 89081 Ulm, Germany;
4University Eye Hospital Dept. I, Schleichstr. 12, 72076 Tubingen, Germany
Tyrosinase (EC 188.8.131.52) is the key enzyme of melanin pigment formation. The absence of this monophenol monooxygenase in human postnatal retinal pigment epithelium (RPE) is mostly taken for granted. Cultured human RPE cells were fed with isolated rod outer segments (ROS) from cattle and with latex particles. After phagocytosis, RPE cells were tested for tyrosinase with independent methods. Firstly, immunocytochemistry with anti tyrosinase antibodies and, secondly, ultrastructural as well as light microscopic DOPA histochemistry. Finally, mRNA was isolated from human RPE before incubation with ROS, and 5, 20 and 40 h after feeding with ROS. The amount of tyrosinase mRNA was determined quantitatively by Real Time RT-PCR, and the regulation of the tyrosinase protein was investigated by Western Blot of cultured human RPE cells before incubation with ROS, and 5, 20 and 40 h after feeding with ROS. Tyrosinase was definitely found in RPE cells using these methods, but was absent without feeding with ROS. Furthermore, we showed co-expression of rhodopsin and tyrosinase in the RPE cells. Contrary to tyrosinase activity, the mRNA for tyrosinase was clearly present in the cultured RPE cells which had not been exposed to ROS; decreased significantly from 5 h after exposure to ROS and returned to its original non-fed level 40 h after ROS feeding. Compared to classic hypothesis, we provide evidence with two independent methods that expression of tyrosinase and its enzymatic activity are induced by phagocytosis of ROS in human RPE.
PP075 - MELANOCYTE-KERATINOCYTE ADHESION FACILITATES MELANIN TRANSFER
KarolienVanDenBossche1,2, Mireille Van Gele1, Jo Lambert1, Jean Marie Naeyaert1 and Vincent Hearing2
1Department of Dermatology, Ghent University Hospital, Belgium;
2Laboratory of Cell Biology, NCI, NIH, Bethesda, USA
During skin pigmentation melanin is transferred from melanocytes to surrounding keratinocytes. The mechanism by which this transfer process occurs is as yet unknown. It could involve exocytosis of melanosomes, cytophagocytosis of a melanocytic dendrite, intercellular tunnel formation, release of melanosome-containing vesicles or a combination of those mechanisms. One way of addressing this issue is the study of melanocyte-keratinocyte contact in relation to transfer. Different melanoma cell lines were assessed for their adhesive properties to keratinocytes. MNT1 and UACC257 cells adhere to keratinocytes, as opposed to SKMel28 cells. Adhesion can be counteracted by addition of EDTA, suggesting a role for cadherins. This is confirmed by the expression of both E- and P-cadherins at the cell surface of MNT1 and UACC257 cells, and not of SKMel28 cells. When measuring transfer of DiO (as surrogate melanosome membrane marker) after 6 h of co-culture, significantly less dye is transferred from SKMel28 cells as compared to MNT1 and UACC257 cells. Accordingly, transfer is diminished when keratinocytes are treated with UVB, which causes cleavage of E-cadherin, prior to co-culturing. We show that the adhesive properties of melanoma cells to keratinocytes, depending upon E- and P-cadherin expression, relate to melanin transfer.
PP076 - COMBINING EPIDERMAL BLISTER GRAFTS AND EARLY UVB NB AND LASER STIMULATION: A POSSIBLE WAY TO PIGMENT SEGMENTAL VITILIGO AND RESISTANT AREAS LIKE HANDS IN SYMMETRICAL VITILIGO (CASE PRESENTATION)
A Overbeck1 and IC le Poole2.
1Lumiderm, Madrid Spain;
2Department of Pathology/Cardinal Bernardin Cancer Center, Loyola University Chicago, Maywood, IL, USA
Background: Segmental Vitiligo still presents a big problem to repigment as well as hands, fingers and feet in symmetrical Vitiligo.
Aim: To develop an easy to perform, inexpensive and sure method for pigmenting these complicated areas, which still represent a big percentage of patients, which present all criteria for a long lasting or permanent pigmentation except that the site is incompatible with present possibilities of therapy
Result: In a step by step sequence it can be seen, that a blister transfer to dermabrated areas in the face can lead to an almost complete pigmentation with good matching of colors. The author can show, that immediately after the transplant the melanocytes are highly active and expand from minor pigmented patches to fill a 50 cm2 area just by stimulating them early and constantly with UVB-light (excimer laser 308 nm), thus allowing to combine the advantages of a topical surgical treatment with a topical phototherapy. As long as melanocyte culturing is still very expensive this means a economically possible way for Vitiligo patients to cover limited ‘complicated’ areas in stable Vitiligo, segmental and symmetrical.
PP077 - MUTATIONS IN TYROSINASE GENE OF ALGERIAN, CAMEROONIAN, INDIAN AND MOROCCAN PATIENTS WITH OCULOCUTANEOUS ALBINISM TYPE IA(OCA1A)
Robert Aquaron1 and Catherine Badens2.
1Laboratoire de biochimie, Université de la Méditerranée, Faculté de Médecine, Marseille;
2Centre d’Enseignement et de Recherche en Génétique Médicale, Faculté de Médecine,Marseille
In this study, we investigated mutations in tyrosinase gene from six patients with OCA1A, four from North Africa( two Algerian and two Moroccan subjects), one from Cameroon and one Indian from La Réunion. We found in three cases homozygous missense mutations(p.G47D and p.Y327C in two Moroccan subjects and p.G149R in the Indian patient) and one novel homozygous nonsense mutation (p.E250X) in an Algerian patient. Consanguinity occured in these four male subjects. We found only one missense mutation p.H404P in the other Algerian patient. Our Cameroonian patient, which belongs to the Bamileke ethnic group in which a high incidence of OCA2( mutations in P gene) exists, is a compound heterozygote for the previously described p.C89R mutation and for the novel p.261delNfs mutation, a four base-pair deletion(c.781_784 delAACT) which leads to the asparagine 261 deletion and a frameshift with a premature termination codon. Only three cases of OCA1A have been reported in African-Americans. Among the many mutations reported in the literature, there are prevalent mutations according to each ethnic groups like p.G149R in Indians and p.G47D in Jews. Identification of prevalent and/or novel OCA1A alleles will greatly facilitated DNA-based diagnosis, prenatal diagnosis, and carrier screening of OCA1A.
PP078 - ELECTROPHYSIOLOGICAL AND HISTOLOGICAL ANALYSIS OF PHOTORECEPTOR CELLS IN ALBINO TYROSINE HYDROXYLASE TRANSGENIC MICE
Victoria Tovar1, EstherZurita1, Marta Cantero1, Juan Jose Lazcano1, Rima Barhoum2, Pedro de la Villa2 and Lluis Montoliu1
1Department of Molecular and Cellular Biology, Centro Nacional de Biotecnologia (CNB-CSIC), 28049 Madrid (Spain);
2Department of Physiology, University of Alcala, 28871 Madrid, Spain
Albino mammals display a number of retinal-visual abnormalities, including rod-photoreceptor cell deficiency, underdevelopment of central retina and incorrect retinal-brain connections. All these abnormalities result in an impaired visual system, with reduced visual acquity and reduced stereoscopic vision. Oculocutaneous albinism type I (OCAI) is a congenital hypopigmentation syndrome whose molecular basis is a mutation in the tyrosinase gene, resulting in alterations in gene expression or the production of abnormal protein product with absent or suboptimal enzymatic function. Lack or reduction in tyrosinase function appears to be the primary cause of retinal and visual abnormalities recorded in albino animals. Recently, we have shown that the ectopic expression of tyrosine hydroxylase in albino transgenic mice allows the correction of all albino visual abnormalties in the absence of melanin, thus demonstrating the important role of early melanin precursors (i.e. L-DOPA) in the normal development of mammalian retina (Lavado et al. J. Neurochem. 2006, 96(4):1201–11). Here, we have analysed the tyrosine hydroxylase transgenic mice in deeper details, using electrophysiological (electroretinographic recordings, ERG) and histological (detection of photoreceptor cells in sections and whole-mounted retinae) experimental approaches to ascertain whether the observed rescue of the visual system in these mice correlates with proper photoreceptor cell function. A series of standardized ERG protocols have been used in order to differentiate the contribution of cones and rods to the visual function in all animals under study.
PP079 - CLINICAL AND DERMOSCOPIC CHARACTERISTICS OF NEVI IN SPANISH CHILDREN AND THEIR RELATIONSHIP WITH ENVIRONMENTAL AND CONSTITUTIONAL FACTORS
AguileraP1, Romero D1, Guilabert A1, Julià M1, Vicente A2, Gonzalez MA2, Kolm I1, Malvehy J1 and Puig S1
1Dermatology department. Hospital Clínic de Barcelona;
2Hospital Sant Joan de Déu, Barcelona
Childhood and adolescense is an important time of life for the formation and evolution of nevi and a higher number of nevi in early life could anticipate a major risk to develop melanoma. It has been shown that constitutional factors such as hair, eye and skin color as well as environmental factors such as sun exposure and sun protection, are associated with the number of nevi. Data concerning factors associated with melanocytic nevi have been mostly derived from populations in northern Europe, northern America and Australia, and it is unclear whether these data can be extrapolated to populations in other geographic locations and with different prevalent phenotypes. There are no studies defining the most characteristic dermoscopic patterns in children of our population and relating a pattern predominance with constitutional and environmental factors. Our aim was to investigate the number and dermoscopic patterns of melanocytic nevi among Spanish children and to assess constitutional and environmental factors associated with the number of nevi and the dermoscopic pattern predominance. Clinical and dermatoscopic examination were performed in 180 children 1 to 15 years. The mean number of moles was 17’5. Being male, past history of sunburns, facial freckling and family history of breast cancer were independent risk factors for having higher number of nevi. Dominant globular pattern was the most frequent found in children of our population.
PP080 - ATM AND P53 GENE EXPRESSION INVOLVED IN THE PATHOGENESIS OF HUMAN MELANOMA
BoyanoMD1, Alonso S2, Ortega I1, Arroyo J1, Izagirre N2, Gardeazabal J3, Andollo N1, García de Galdeano A1, Díaz-Ramón JL3, Jangi SM1, de la Rúa C2 and Díaz-Pérez JL3.
1Department of Cell Biology and Histology. Faculty of Medicine and Dentistry. University of the Basque Country. 48940 Leioa, Bizkaia, Spain;
2Department of Genetics. Faculty of Science and Technology. University of the Basque Country. 48940 Leioa, Bizkaia, Spain;
3Dermatology Service. Cruces Hospital. 48903 Barakaldo, Bizkaia, Spain
To understand the molecular changes involved in the pathogenesis of melanoma, we compared the gene expression profiles of a series of nevi and primary melanomas. Normal human melanocytes and melanoma cell lines were obtained in our laboratory from biopsies of three independent nevi and four independent primary melanomas. RNA from these specimens were analysed by microarray technology (Affymetrix U133A). After normalization of the data the resulting data table was imported into the Significance Analysis of Microarrays (SAM) software package and Onto-Express. An overexpression of p53 and repression of ATM (Ataxia-Telangectasia- Mutated kinase) genes in melanoma cell lines were the most robust biomarkers identified. These results have been validated by qRT-PCR. We investigated if this observation could be associated to any sequence polymorphism in the coding region of TP53. A mutation at codon 72 responsible for a Pro -> Arg aminoacid change was found in the melanoma cDNAs sequenced. However, a population analysis by ARMS-PCR showed that this is a common polymorphism. This suggests that the increase of TP53 expression is not related to TP53 coding sequence variants. Phosphorylation of ATM and p53 protein has been also studied in the total protein lysates from normal melanocytes and melanoma cell lines and we have found significantly less phosphorylated ATM and p53 protein on melanoma cells than in normal melanocytes. As ATM is an upstream kinase of p53 involved in DNA damage and in the stability of p53 we conclude that ATM might be involved in the pathogenesis of melanoma. Thus, despite of the overexpression of p53 found in melanoma cells, inactivation of ATM inhibits p53 activity suppressing apoptosis and favouring tumorigenesis.
PP081 - ACRAL MELANOMA CELL LINE HAS A HIGH RESISTACE AGAINST APOPTOSIS INDUCED BY HISTONE DEACETYLASE INHIBITOR (HDACI), FK228, DUE TO ITS DOWN-REGULATED BAX EXPRESSION
Ken Futaki1, Yusuke Furukawa2, Mamitaro Ohtsuki3, Hidemi Nakagawa1, Hiroshi Murata4, Minoru Takata4, Toshiaki Saida4 and Genji Imokawa1,5.
1Department of Dermatology, Jikei Medical University, Tokyo;
2Division of Stem Cell Regulation;
3Jichi Medical University, Tochigi;
4Shinshu University School of Medicine, Matsumoto;
5Skin Science Research Institute, Tokyo, Japan
We recently found that acral melanoma cell line (ALM) has a higher resistance against apoptosis induced by HDACi FK288. Despite a significant G1 arrest and apoptosis occurring in the non-ALM after 24 h and 48 h treatment, respectively, with FK228, the same treatment of the ALM induced neither G1 arrest nor apoptosis until 48 h. There was no distinct difference in Cdk2/4 and MITF expression between the untreated ALM and non-ALM. Treatment with FK228 induced a marked depression of MITF followed by Cdk2 attenuation along with up-regulation of p21 in all melanoma cell lines tested. On the other hand, the ALM had a higher and minimal expression of Bcl2 and Bax proteins, respectively. Further analysis revealed that induced % apoptosis correlated not with Bcl2 expression, but with Bax expression in the untreated cells. Consistently, translocation of Bax to mitochondria was distinctly induced by FK228 treatment at 24 h post-incubation in concert with delayed activation of caspase-9. Our results suggest the possibility that acral regions have characteristic tissue circumstances which may affect phenotypic expression of Bax in the developed melanoma cells.
PP082 - THE ROLE OF CDKN2A AND CDK4 IN SPANISH MELANOMA FAMILIES
KolmI1, Cuellar F1, Puig-Butillé JA2, Ogbah Z1, Badenas C2, Milà M2, Martí R1, Malvehy J1 and Puig S1.
1Department of Dermatology;
2Department of Genetics, Hospital Clínic i Provincial de Barcelona, IDIBAPS, Barcelona, Spain
Background: Familial melanoma occurs in 5–10% of all melanoma cases. Two main melanoma genes have been identified, CDKN2A with mutations in 20–60% of families studied and CDK4 with less than 1% of identified mutations.
Objectives: To determine genetic background and describe clinical characteristics in Spanish melanoma families.
Subjects: Seventy-eight Spanish families with at least two well-documented melanoma cases.
Methods: SSCP and sequencing of CDKN2A and CDK4.
Results: Four families with ≥4 cases of MM; 15 families with 3 cases and 59 families with 2 cases of MM. CDKN2A. Mutations were detected in 21% of families (all families with more than 3 cases were carriers of mutations and mutations are more frequent in families with early onset or multiple primary melanoma cases). Mutations in CDK4 were not found. eight different missense mutations and 2 frameshift mutations (358delG and 102-106delG) were identified. The most frequent missense mutation was G101W, which was detected in six families. V59G, D84Y, L65P, R87W, V106V, 34G>T, Q50R were private mutations. A specific surveillance program including dermatological visits with dermoscopy and/or total body photography and digital dermoscopy was offered to the families allowing the early diagnosis of new melanoma cases.
Conclusion: CDKN2A is responsible of genetic susceptibility of a proportion of Spanish familial melanoma. The prevalence of mutations increases with the number of MM cases in a family, early onset of MM and the presence of multiple primary MM cases. A specific surveillance program was useful in the early diagnosis of melanoma either in mutant and wild-type CDKN2A families.
PP083 - MUTATIONAL STATUS OF B-RAF AND N-RAS IN FRESH MELANOMAS
Z. Ogbah1, J.A. Puig-Butille1, C. Badenas1, J. Malvehy1, M.Milà2 and S. Puig1
1Melanoma Unit, Hospital Clínic i Provincial de Barcelona, IDIBAPS, Universitat de Barcelona, Barcelona, Spain
2Genetic Department, Hospital Clínic i Provincial de Barcelona, Barcelona, Spain
The MAPK pathway is involved in fundamental processes as cell growth and proliferation. The B-RAF gene encodes a serine/threonine kinase which couples signaling from activated RAS to downstream MAPK. The most frequent mutation (V600E) affects the kinase activation domain and it is detected in 60–70% of human melanomas and in 5%–33% N-RAS mutations have been reported. The objective of the study was to determine the percentage of B-RAF and N-RAS mutations rate in our panel of tumors. Frozen samples from 78 primary melanomas of which 17 were in situ (55 superficial spreading, 6 nodular, 17 acral lentiginous, 10 lentigo maligna and two unclassified melanoma) and three nevus, were included in the study. SSCP and sequencing were performed for N-RAS and B-RAF. The B-RAF-V600E Mutector Assay Kit was used to corroborate the V600E mutation. B-RAF mutations were detected in 14 tumors (11% SSM, 1% NM, 3% LMM, 0% ALM, 0% unclassified melanoma).No N-RAS mutations were detected. Interestingly, the only common nevus analyzed carried the V600E mutation, while it was not present in the dysplastic or spitzoid nevi. We have identified a 19% of B-RAF mutations in our samples, all of them V600E, and no N-RAS mutations. Our percentage is lower than initially suggested by other authors. These differences may be related to: (i) Proportion of histological type of primary tumors; (ii) type of samples used (frozen vs paraffin embedded); and (iii) mutational analysis method used.
This work further supports the hypothesis that, although MAPK pathway is involved, other genetic pathways may be also implicated in melanoma initiation and proliferation.
PP084 - CHARACTERIZATION OF TUMOR IMMUNITY IN MELANOMA CHEMO-THERMO-IMMUNOTHERAPY (CTI THERAPY) USING NPRCAP-MAGNETITE (NPRCAP/M) NANO-PARTICLES
AkikoSakemoto1, Yasuaki Tamura2, Noriyuki Sato2, Toshiharu Yamashita1, Tomoaki Takada1, Makito Sato1, Masae Ohkura1, Ichiro Ono1, Akira Ito3, Hiroyuki Honda4, Kazumasa Wakamatsu5, Shosuke Ito5 and Kowichi Jimbow1.
Departments of 1Dermatology;
21st Pathology, Sapporo Medical University;
3Department of Chemical Engineering, Kyusyu University;
4Department of Biotechnology, Nagoya University;
5Department of Chemistry, Fujita Health University, Japan
We established chemo-thermo-immunotherapy (CTI therapy) by combining N-propionyl cysteaminylphenol (NPrCAP) with magnetite nano-particles (NPrCAP/M), which were taken by B16 melanoma cells and killed them selectively. Importantly this treatment modality could increase its cytocidal effect by local heat generated through the exposure to alternating magnetic field (AMF) and even rejected secondly-inoculated, re-challenged melanoma on the opposite flank of the first melanoma inoculation. This rejection of melanoma re-challenge test was also seen by NPrCAP/M treatment without AMF, although the degree of its effect was lower than that with AMF. In this study we measured the level of heat shock protein (Hsp) production in the supernatant and the response of CD8+ T cells to dendritic cells pulsed with the supernatant. We found the level of Hsp72 and Hsp90 increased in the supernatant after CTI therapy. CD8+ T cells being responded to dendritic cells were also detected. Thus it is indicated that the in vivorejection of re-challenged melanoma after CTI therapy derives from the release of HSP-peptide complex from necrotic tumor cells which can elicit specific tumor immunity against melanoma cells.
PP085 - MELANOMA EPITHELIAL CELL INTERACTION STIMULATES LOCALIZED MMP2 SECRETION
E.-M. Schnäker, T. Görge, T.A. Luger, M. Steinhoff and S.W. Schneider.
Department of Dermatology, University Hospital Muenster, Muenster, Germany
The presented study focussed on local extracellular Matrix-MetalloProtease (MMPs) activity during invasion of melanoma cells into an epithelial monolayer. In previous studies we showed that human melanoma cells store MMP2 in small cytoplasmatic vesicles which are conveyed along the microtubule network. Disruption of vesicle transport led to diminished protease secretion and attenuated invasion of the melanoma cells (1). We now used this model system to characterize intracellular MMP2 storage as well as extracellular MMP2 distribution during co-culture of melanoma and epithelial cells. Immunofluorescence microscopy revealed that cytoplasmic MMP2 vesicles also contain MT1-MMP. Moreover, we demonstrate that epithelial melanoma interaction induces altered membrane expression of MMP2 and MT1-MMP in melanoma cells. In mono-cultures we found MMP2 bound to the plasma membrane of melanoma cells in irregular distributed spots whereas MT1-MMP and Timp2 were ubiquitously expressed on the cell surface. Co-cultured melanoma cells exhibited a clearly altered MMP distribution: MMP2 and MT1-MMP co-localized at those parts of melanoma cells that were in close contact to epithelial cells, indicating high local proteolytic activity. However, Timp2 distribution remained unaffected. Moreover, atomic force microscopy revealed fusion events of exocytotic vesicles in similar areas of the lamellipodium of melanoma cells. Additional western blot experiments revealed higher membrane associated expression of active MT1-MMP and active MMP2 in co-cultured melanoma cells. Therefore, we conclude that co-culture induces enhanced MT1-MMP and MMP2 secretion in melanoma cells as well as altered MMP surface distribution, representing a host-tumor communication axis.
1. Schnaeker, E.M. et al., Cancer Res. 64, 8924--8931 (2004)
PP086 - MUTATION OF THE TUMOUR SUPPRESSOR P33ING1b IS RARE IN MELANOMA
M. Stark1, J.A. Puig-Butillé2, G. Walker1, C. Badenas2, J. Malvehy2, N. Hayward1 and S. Puig2.
1Queensland Institute of Medical Research, Brisbane, Australia
2Hospital Clínic i Provincial de Barcelona, IDIBAPS, Universitat de Barcelona, Barcelona, Spain
The ING-1 gene (13q34) encodes for p33ING1b, which is involved in the p53-dependent response to DNA damage following ultraviolet radiation exposure. The prevalence of p33ING1b mutations in melanoma samples has been recently reported to be 20% of tumours. The objective of the study is to assess the p33ING1b mutation rate in our large panels of melanomas biopsies and melanoma cell lines. Eighty-three primary melanomas, including 40 superficial spreading, 25 acral lentiginous, seven nodular, seven lentigo maligna, one desmoplastic, two unclassified melanomas, and one malignant blue nevus; and 55 melanoma cell lines were screened for mutations in p33ING1b by PCR and single stranded conformation polymorphism analysis, and direct sequencing. In contrast to previous reports, we found no somatic p33ING1b mutations in our panel of melanomas.We did however find one germline variant, A140V, in a patient with acral lentiginous melanoma, also found a synonymous variant, S173S, in a cutaneous melanoma biopsy. We found that some of the discrepancy between the studies may be due to inadvertent amplification of the ING1 pseudogene (INGX), and/or contamination of some samples with murine Ing1. In conclusion, p33ING1bmutations in melanoma are rare. We have highlighted the importance of allele-specific primer design to avoid pseudogene contamination, and also the necessity to confirm the genetic identity and species of origin of individual cell lines.
Further studies are needed to clarify the possible role of p33ING1b in melanoma tumorigenesis.
PP087 - ORGANIC ANION TRANSPORTING POLYPEPTIDES EXPRESSION IN MELANOCYTIC CELLS AND RELATIONS WITH VASCULOGENIC MIMICRY IN AGGRESSIVE MELANOMA CELLS
Danielle Parriaux1, Alain Vérine1, Dominique Lombardo1 and PatrickVerrando1,2.
1INSERM UMR 777, Glycobiology and cell dysfunctions, Laboratory of Investigations of Skin Diseases, LISD/LIMP (EA3291), University of the Mediterranean, Marseilles, France
Entry transporters of drugs and subsequent signal in melanoma cells have not been truly considered to imbalance resistance of melanoma to chemotherapies that involves the ABC transporter family. This initial study investigated first whether human melanocytic cells expressed members of the superfamily of organic anion transporting polypeptides (OATP/SLCO). We found that normal melanocytes displayed transcripts for six out of the nine known human OATP, with differences in abundance. We analyzed next the occurrence of OATP in two highly- and two poorly-aggressive cell lines derived from cutaneous and metastatic uveal melanoma tumors. While transcripts of two OATP members were detected in either cell lines, aggressive cells expressed specifically four other members and poorly aggressive cells displayed only one specific OATP. Exposure of aggressive cells to OATP substrates (cholate, taurocholate, Rifampicin, Rifamycin), or to either TNF? or Phenobarbital, did not result in main changes in transcript levels, but in modifications of the unique ability of aggressive cells to form tubular structures and patterned networks, termed ‘vasculogenic mimicry’. Focusing on OATP1B3 present only in aggressive cells, we found that these substrates and methotrexate induced some disorganization of the vasculogenic mimicry in a dose-dependent manner. Monoclonal antibody 5F260 against both OATP1B3 and OATP1B1 located these transporters exclusively to tubular structures. We try at present to delineate the molecular mechanisms underlying these OATP-mediated modification of vasculogenic mimicry characteristic of aggressive melanoma cells.
PP088 - FREE RADICAL SITUATION IN MELANOMA CULTURES
JanBorovanský1, Adéla Lipšová1 and Jiří Vachtenheim2.
1Department of Biochemistry & Experimental Oncology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic
2Department of Molecular Biology, Institute of Chest Diseases, Prague, Czech Republic
In our previous study (based on animal melanoma models - B16 and S91 mouse melanomas and MeLiM minipig melanoma) we demonstrated that melanoma progression was not associated with oxidative stress both on organ and serum levels of host organisms. This time we extended our studies by measuring the total antioxidant status (by means of a specific kit - Randox Labs, UK) of media, in which four human melanoma cell lines (HBl, Beu, Malme 3M, SK-Mel-5), mouse melanoma line (B16 F-10) and an osteosarcoma line (U2-OS) had been grown for 72 h, and compared the data with those measured in the corresponding cell free media incubated under the same conditions. In the cells we measured MITF level and also several enzyme activities participating in free radical metabolism - tyrosinase (MBTH assay of Winder&Harris), gamma-glutamyltransferase (GGT100 kit, Pliva-Lachema, Czech Rep.) and glutathione peroxidase (Ransel kit, Randox, UK). U2-OS cells exhibited neither tyrosinase nor the GGT activity; glutathione peroxidase level was generally very low. In the growth media of highly pigmented cells, e.g. in the B16-F10 cultures, the increased values of the total antioxidant status were again surprisingly demonstrated. Although cell cultures in comparison with animal melanoma models represent more simple systems, their free radical situation remains complex.
PP089 - DERMOSCOPY FEATURES OF MELANOMA ASSOCIATED WITH MC1R GENE POLYMORPHISM IN CDKN2A MUTATION SPANISH CARRIERS
FranciscoCuéllar1,2, Celia Badenas1,3, Joan Puig-Butillé1,3, Rosa Martí1,4, Pedro Zaballos1,5, Josep Malvehy1 and Susana Puig1.
1Melanoma Unit, Hospital Clinic Barcelona, Spain;
3Molecular Genetics, Hospital Clinic Barcelona, Spain;
4Dermatology Service, Hospital Universitari Arnau de Vilanova, Lleida, Spain;
5Dermatology Service, Hospital Santa Tecla, Tarragona, Spain
Mutations in CDKN2A gene are melanoma-predisposition alleles with high penetrance and low population frequencies. Variants of melanocortin-1 receptor gene (MC1R) confer much lower melanoma risk but are common in European populations. There are no studies about dermoscopy features of melanoma associated with these genetic changes.
Objective: To describe dermoscopy characteristics of melanoma in patients with MC1R variants in CDKN2A mutation-carrying Spanish patients.
Material and methods: Ten CDKN2A mutation-carrying patients with 11 melanoma lesions were assessed for MC1R genotype and ABCD total dermoscopic score (TDS) was calculated.
Results: None had MC1R wild-type. Five patients had red-hair variants (R151C, R160W, D294H), three of them 2 variants. The ABCD TDS were significantly higher in non-carriers of MC1R red-hair polymorphism compared with carriers of ≥2 MC1R red-hair variants (6.4 ± 0.4 vs. 4.4 ± 0.9 p = 0.009). Differences were significant in colours (1.8 ± 0.3 vs. 1 ± 0.0 p = 0.007) and structures (1.9 ± 0.3 vs. 1.2 ± 0.3 P = 0.018).
Conclusion: CDKN2A mutation Spanish carriers with ≥2 MC1R red-hair polymorphism have lower ABCD TDS that may cause false negatives. High definition image controls and complementary ABCD algorithms could be useful.
PP090 - TRANSPORT BY BLOOD CELLS AND BODY DISTRIBUTION OF AN ALKYLATING PEPTIDE RECOGNIZED BY TUMOR-ASSOCIATED PROTEASES
KarenDierickx, Renato Morandini and Ghanem Ghanem.
LOCE, Bordet Institute, Free University of Brussels, Belgium
Most of conventional anticancer drugs currently in use are based on a differential uptake by tumor cells due to their higher proliferation. Because they also affect normal tissues and produce general toxic side effects, several approaches are being explored to improve drug delivery. This study addresses to one of these mechanisms using tumor-associated proteases as targets for specific drug delivery. Our present work focuses on the alkylating peptide PSF that is able to bind to blood cells (BC) and is efficiently processed by melanoma tumor proteases. The transport of PSF by RBC is supported by the quick clearance of PSF from the blood along with the BC mediated generation of its main metabolite. Binding to artificial membranes (liposomes) was achieved and only competition with melanoma cells or proteolytic enzymes such as Dispase, led to the generation of active metabolites. Body distribution of 14C-PSF in melanoma bearing nude mice showed 1.5 to 2.5 fold tumor to muscle ratios. Interestingly, unusual accumulation was observed in the spleen supporting the high binding to BC, and in the pancreas, validating further the expected protease-mediated drug delivery. Both in vitro and in vivo results support a particular delivery system based on the binding to BC, subsequent transport, followed by a proteolytic release at the targeted area.
PP091 - SIGNIFICANCE OF GLUTATHIONE S-TRANSFERASES M1, T1 AND P1 POLYMORPHISMS IN SWEDISH MELANOMA PATIENTS
Bu H.1, Rosdahl I.1, Holmdahl-Källen K.2, Sun X-F1 and Zhang H.1.
Department of 1Biomedicine and Surgery, Linköping University, SE-581 85 Linköping, Sweden;
2Dermatology Clinic, Kalmar Hospital, SE-391 85 Kalmar, Sweden
Associations between GSTs polymorphisms and melanoma risk and progression were investigated. Polymorphisms of GSTM1, GSTT1 and GSTP1 genes in melanoma patients and tumor-free individuals were examined. Relationships between the polymorphisms and tumor characteristics and pigment phenotypes of the patients were analyzed. DNAs were isolated from mononuclear cells of venous blood. Multiplex PCR and PCR-RFLP techniques were used for the genotyping. There was no significant difference in distributions of the GSTM1 null and GSTT1 null genotypes or in frequency of GSTP1 GG genotype between the melanoma patients and tumor-free controls. In melanoma patients, these polymorphisms were not correlated with early or later onset of melanomas or gender of the patients. Frequency of GSTM1 null genotype was higher in patients with melanoma >2.5 mm than in those with tumors <1.0 mm, and higher frequency was found in nodular melanoma than in the other tumor types. GSTP1 GG genotype was more often found in the patients with brown and mixed eye color or brown and black hair than those with blue and green eyes or blond hair. It is unlikely that polymorphisms of GSTM1, GSTT1 and GSTP1 are general risk factors for melanoma in the Swedish population. GSTM1 null genotype was correlated with Breslow thickness and tumor type, which might serve as an additional biomarker for a rapid tumor progression. GSTP1 GG increases risk for melanoma in the subgroup of individuals with dark eyes or darker hair. The GSTs polymorphisms are not associated with sun exposure-related melanoma.
PP092 - PATHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF UVB-INDUCED EFFECTS ON IRRADIATED NEVI AND SUPPOSED PROTECTIVE ROLE OF SUNSCREENS
CristinaCarrera1, Susana Puig1, Josep Palou1, J. Antón Puig-Butillé2, Zighereda Ogbah2, Mario Lecha1 and Josep Malvehy1.
1Melanoma Unit and Dermatology Department;
2Genetic Service, Hospital Clínic de Barcelona, University of Barcelona, IDIBAPS
Background: Sunscreens have shown a positive impact in the prevention of UVR damage in keratinocytes. However their role in protecting melanocytes against UVR has not been well established. We have previously developed a human in vivo model to demonstrate the UVB-induced changes in nevi, based on this model, we investigated different molecular markers in irradiated nevi and the impact of sunscreens.
Methods: Twenty paraffined samples of 2 MED-UVB irradiated nevi were included. 7 days before excision, 11 of these nevi were irradiated only on a half (physical protection on the other half), while a physical and chemical sunscreen (octocrylene, Parsol 1789, titanium dioxide, Mexoryl SX™, Mexoryl XL™) was applied on a half of the other 9 nevi, before irradiation of the whole lesion. Immunohistochemical stains were performed with HMB45, MART-1, Ki67, p16 and p53.
Results: Histopathological UVB-induced features were parakeratotic scale, and mild lymphocytic perivascular inflammation. Most nevi showed increase size of junctional melanocytes and their dendrites. Molecular changes were: (i) Notable activation of melanocytes, both in the lesions and in adjacent skin (HMB45/MelanA). (ii) Increased number of proliferating cells, mostly lesional keratinocytes (Ki67). (iii) Mild increase expression of p53 in apoptotic irradiated keratinocytes, but not in nevocytes. No differences were observed between physically protected areas and sunscreen protected areas.
Conclusion: In addition to clinical and dermoscopical UVB-induced changes, pathological and molecular effects were demonstrated in irradiated nevi. Molecular differences were found between irradiated halves of nevi vs physically or sunscreen protected areas.
PP093 - THE PROTECTIVE EFFECT OF FERULIC ACID ON UV-INDUCED STRESS IN HUMAN SKIN CELLS
LesterM.Davids1, Susan H. Kidson1, Stephanie Burton2, Fred P.H.T.M.Romijn3, Stan Pavel4, J van Pelt3 and Nico P.M. Smit3.
1Department Human Biology, University of Cape Town, Cape Town, South Africa;
2Department Chemical Engineering, University of Cape Town, Cape Town, South Africa;
3CKCL, Leiden Univerisity Medical Centre, Leiden, The Netherlands;
4Department Dermatology, Ledien University Medical Centre, Leiden, The Netherlands
Ferulic acid (FA) is a hydroxycinnamic acid present in all plants and is found in high levels in olives and maize. Its biological role as an antioxidant and UV absorber is well recognized. Since UV-A induces elevated intracellular reactive oxygen species (ROS) which in turn leads to apoptosis and cell death, our aim was to assess whether cultured human melanocytes and keratinocytes treated with FA alone and/or in combination with vitamins C and E in vitro would (i) be protected against the effect of UV-induced ROS, (ii) would be protected against UV-induced apoptosis and (iii) up or down-regulate cellular gene expression. Primary cultures of melanocytes and keratinocytes (passages 4–9) were treated with 800 μM FA alone or in combination with 35 μM vitamin E and 100 μM vitamin C for 18 h, exposed to 225 kJ/m2 UV-A and analysed 24 h later. ROS were measured using dihydrorhodamine dye and FACS analysis. Apoptosis was measured by counting apoptotic cells using the Hoechst nuclei stain and using annexin V/propidium iodide ratios. Pre-treating both melanocytes and keratinocytes with 800 μM FA alone for 18 h resulted in a reduction of UV-induced ROS and apoptotic cells. A 2-fold reduction was evident with cells treated with the FA-vitamin combination. Gene expression analysis of the FA-vitamin combination showed more than 1.5x upregulation by several genes including caspase-8, glutathione peroxidase and NF-kappa B in response to UV and a downregulation/suppression of caspase 10, Bcl-2 and TNF. Overall, pre-treatment of skin cells with the antioxidant ferulic acid significantly protects human skin cells from UV-induced damage and induces a variety of gene expression patterns.
PP094 - UVB-INDUCED G2/M ARREST INVOLVES CDC2 SEQUESTRATION BY GADD45A IN NUCLEAR SPECKLES OF MELANOCYTES
Caroline Fayolle1, Julie Pourchet1, Rémy Pedeux2, Alain Puisieux1, Claude Caron de Fromentel1, Jean-François Dore1 and Thibault Voletzel1.
1INSERM U590 «Oncogenèse et Progression Tumorale», Université Lyon 1, Centre Léon Bérard, 28 rue Laënnec, 69373 LYON cedex 08, France
2Present Address: INSERM U578, Institut Albert Bonniot, 38706 La Tronche, France
Exposure to solar UVB radiation is involved in the development of cutaneous melanoma in Human. We previously showed that human melanocytes and melanoma cells respond to UVB radiation via a p53-independent pathway involving GADD45A activation. Here, we investigated the role of Gadd45a in UVB-induced G2/M arrest. We determined that Gadd45a and its partner p21Waf1 co-localize in nuclear bodies called Nuclear Speckles. We further observed that UVB-induced G2/M arrest is associated with Cdc2 accumulation in these Nuclear Speckles. Knock-down of Gadd45a expression by RNA interference prevents both UVB-induced Cdc2 accumulation in Nuclear Speckles and G2/M arrest. Our results demonstrate that UVB-induced G2/M arrest of melanoma cells is Gadd45a-dependent. Furthermore, we show that Cdc2 sequestration by Gadd45a occurs in Nuclear Speckles, suggesting a new role for these nuclear bodies, so far only linked to RNA maturation.
PP095 - AN IN VITRO STUDY OF MELANOCYTE MIGRATION IN A CO-CULTURE SYSTEM
DheshnieGovender1, Lester M. Davids1, Dorothy C. Bennet2, Zalfa Abdel-Malek3 and Susan H. Kidson1.
1University of Cape Town, Faculty of Health Sciences, Department of Human Biology, Observatory, Cape Town, South Africa;
2Centre for Molecular and Metabolic Signaling, Division of Basic Medical Sciences (Anatomy), St.George's, University of London, Cranmer Terrace, London SW17 ORE, UK;
3University of Cincinnati College of Medicine Albert Sabin Way Cincinnati, Ohio 45267-0592, USA
Vitiligo is a depigmenting disorder, with unknown etiology, making it a difficult task to find effective treatment. Repigmentation of lesional areas, following treatment often occurs perifollicularly and then spreads. The theory is that melanocytes from the stem cell niche are stimulated to migrate or proliferate out and enter the interfollicular epidermis. Although there is evidence of melanocyte migration in the murine hair follicle this process of migration into the interfollicular epidermis has not been demonstrated in humans. The aim of this study was to determine whether melanocytes were capable of migrating through a monolayer of keratinocytes. Using immortalized human melanocytes (Hermes 4a) and immortalized human keratinocytes (HaCats), cells were plated onto coverslips as two isolated populations separated by a space of 2 mm. The behavior of the cells was assessed in terms of migration of melanocytes to keratinocytes and cell-cell interactions. Melanocyte specific markers, tyrosinase and tyrosinase-related protein 2, and vital dyes were used to monitor these cells. The results show that when keratinocytes form an epidermal sheet they have a “barrier effect” on melanocyte migration, whereas when keratinocytes are arranged in clumps, this effect is lost, and melanocytes are able to move over and through the keratinocytes. These preliminary results may suggest that in vivo cell-cell interactions formed between the keratinocytes may yield the structure impermeable to melanocytes and therefore implies that melanocytes migrate via an alternate route, possibly through the dermis or along the basement membrane.
PP096 - EXPRESSION AND REGULATION OF NRF1-3 IN HUMAN SKIN CELLS
AgathaKokot, Meinhard Schiller, Dieter Metze, Thomas Luger, and Markus Böhm.
Department. of Dermatology and Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, University of Münster, Münster, Germany
Nrf1-3 are members of the Cap’ n’ collar family of transcription factors and play a crucial role in regulation of protective genes and therefore in the cellular stress response. Surprisingly little is known about Nrf1-3 in human skin which is constantly exposed to various stressors, especially ultraviolet (UV) light. RT-PCR analysis revealed constitutive expression of all Nrf members at the RNA level in the majority of epidermal and dermal human cell types with highest amounts in epidermal melanocytes. Western blotting confirmed expression of Nrf1 and two at protein level in all tested cell types. Immunohistochemistry of healthy skin disclosed the presence of Nrf1 in nuclei of epidermal cells, dermal fibroblasts and distinct epithelia of adnexal structures while Nrf2 was found only in the latter within the cytoplasm. Functional studies using various stressors including UVB, H202 and chemical oxidants revealed time-dependent and subtype-specific regulation of Nrf1-3 in epidermal melanocytes, keratinocytes and dermal fibroblasts. Moreover, exposure to oxidants resulted in a time-dependent nuclear translocation of Nrf2 and 3 but not Nrf1 as shown by immunofluorescence. Our data present a first insight into the expression and regulation of Nrf members in human skin cells and will be the base for further studies of Nrf function in skin cells under physiologic and pathophysiologic conditions.
PP097 - MOLECULAR RESPONSES OF NORMAL HUMAN CAUCASIAN MELANOCYTES IN CULTURE EXPOSED TO SIMULATED SOLAR UV: COULD MELANIN AND ITS PRECURSORS BEHAVE AS ENDOGENEOUS PHOTOSENSITIZERS?
Laurent Marrot, Jean-Philippe Belaïdi, Christophe Jones, Philippe Perez and Jean-Roch Meunier.
L’OREAL Recherche, Phototoxicity, Aulnay sous bois, France
Melanocytes have a central role in the response of skin to sunlight exposure. They are directly involved in UV-induced pigmentation as a defence mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The aim of this work was to analyze the behaviour of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to UV radiation from a solar simulator (SSUV: 300–400 nm). Even at relatively high doses (12 kJ/m2 UVB and 110 kJ/m2 UVA), cell death was limited. Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2/M phase, and this correlated well with a high induction level of the gene GADD45, 4 h post-exposure. Among the genes involved in DNA repair, gene XPC was the most inducible since its expressionincreased more than two fold 15 h, whereas expression of P48, also involved in the nucleotide excision repair pathway,was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UVA radiation only (320–400 nm) and stimulation of melanogenesis prior to irradiation further increased HO1 induction. Such a behavior confirmed previous results showing that photoinduced DNA breakage by UVA (detected using the comet assay) was also enhanced when melanogenesis was triggered. This work provides new data about the stress reponse of human melanocytes exposed to UV and underlines the probable involvement of sunlight in melanoma initiation.
PP098 - CLINICAL IMPROVEMENT BY MATRICARIA CHAMOMILLA EXTRACT ON FACIAL LENTIGO SENILIS
TakahiroMatsuki1, Sayuri Sato1, Makoto Kawashima2 and Genji Imokawa3
1Department of Dermatology, Sanno Hospital, Tokyo, Japan;
2Tokyo Women's Medical University, Tokyo, Japan;
3Skin Science Research Institute, Tokyo, Japan
We have recently reported that up-regulation of melanin synthesis within melanocytes in the lesional epidermis of lentigo senilis is mediated via stimulated endothelin linkage. We have also demonstrated that Matricaria Chamomilla extract can act as antagonist against endothelin B receptor and has an ability to reduce UVB-induced pigmentation which is triggered by a stimulated endothelin linkage. Thus, it is intriguing to determine whether topical application with M. Chamomilla extract has a potential to reduce the pigmentation in lentigo senilis. In this report, we performed clinical study using lentigo senilis patients (n = 33) for 2–6 months. One to three pigment spots on the face of lentigo senilis patients were selected for the application site and each pigment spot was evaluated by dermatologists as well as by color difference meter. Clinical evaluation revealed that the treatment for 2-6 months resulted in 15–48% and 40–70% of distribution in marked improvement and slight improvement, respectively, and there was a significant difference in delta L value evaluated by color difference meter among before, 1 and 2 months after treatment. In two cases, the lentigo senilis completely disappeared with a marked increase in delta L value (8.0 and 7.0) after 6 months of treatment. These clinical results suggest that M. chamomilla extract is a useful tool for improving endothelin-associated hyperpigmentary disorders.
PP099 - ELUCIDATION OF THE ENIGMATIC ROLE OF MELANOCYTES WITHIN THE SEBACEOUS GLAND
KaiTolk1, Thomas Luger1, Christos C. Zouboulis2 and Markus Böhm1.
1Department. of Dermatology and Ludwig Boltzmann Institute for Cell Biology and Immunobiology of the Skin, University of Münster, Münster, Germany;
2Departments. of Dermatology and Immunology, Dessau Medical Center, Dessau, Germany
Since many years it is known but largely ignored that melanocytes are present in adnexal structures of the skin including the sebaceous gland. To shed light into the enigmatic role of melanocytes within sebaceous glands we first confirmed the presence of scattered non-pigmented melanocytes in sebaceous glands of normal human skin by immunofluorescence using an antibody against premelanosomal antigens. Next, normal human melanocytes (NHM) and SZ95 sebocytes, the latter an immortalized sebaceous gland cell line derived from human facial sebaceous glands, were co-cultured in vitro. NHM communicated with SZ95 sebocytes via dendrite formation albeit long-term culture of both cell types was not successful. Conditioned medium (CM) from NHM profoundly inhibited lipid formation of SZ95 sebocytes induced by linoleic acid. Moreover, apoptosis of SZ95 sebocytes induced by serum deprivation was significantly suppressed by CM from NHM. Coincubation with an antibody against alpha-melanocyte-stimulating hormone (alpha-MSH) abrogated the suppressive effect of CM from NHM on apoptosis of SZ95 sebocytes induced by serum starvation. Exogenous alpha-MSH mimicked the effect of CM from NHM on apoptosis of SZ95 sebocytes induced by serum starvation. These data provide a first glimpse into the function of melanocytes within sebaceous glands, i. e. via secretion of soluble mediators melanocytes may act as regulators of differentiation and survival of sebocytes.
P100 - ABSENCE OF MC1R DOES NOT IMPAIR THE INFLAMMATORY RESPONSE TO UV RADIATION IN ADULT MOUSE SKIN.
AgnieszkaWolnicka-Glubisz1,2, Edward De Fabo2 and Frances Noonan2.
1Department of Biophysics, Faculty of Biotechnology, Jagiellonian University, Krakow, Poland;
2Department of Environmental and Occupational Health, School of Public Health and Health Services, The George Washington University Medical Center, Washington, DC, USA
We have previously shown a dose and time dependent infiltration of Ly6G+ neutrophils into adult mouse skin 24-48 h after UVB, but not UVA irradiation. Here we have investigated the effect of Mc1r on this response using FACS analysis and immunohistochemistry. Ly6G positive cells as 31% ± 5.4 total cells was similar in FVB, C57BL/6-c (albino), C57BL/6-Mc1re/e (yellow), and C57BL/6 (black) mice 48 h after a dose of 8.4 kJ/m2 from F40sunlamps. The effect was dose dependent. Also there were no differences between mice in UV-induced edema. DNA damage as determined by thymine dimer staining was similar in all groups. We conclude that the absence of Mc1r does not impair the inflammatory response to UV radiation in adult mouse skin.