Human melanocytes and melanoma show a paradoxical migration response pattern dependent on RAS, cPLA2 and lipoxygenase, but not on cyclooxygenase

SlOl

ESDR I JSID I SID Abstracts

0601

0604

MYOSIN-V GO-LOCALIZATION WITH TYROS~NASE RELATED PROTEIN-I LABELED MELANOSOMES IN HUMAN MELANOCYTES. H.R ndaloh Bvers a d Nicole L. Jalbert. Department of Dermatology, Bostoh School zf Medicine, Boston, MA, USA. The dilute gene, whose mutation in mice produces a grey rather than black coat, encodes myosin-V. Affected melanocytes are unable to transport melanosomes to the cell periphery and myosin-V co-localiies with murine melanoma melanosomes indicating an important role for myosin-V in melanosomal transport. We investigated the relationship of myosin-V to melanosomes in human melanocytes in vitro by confocal microscopy, double fluorescent labeling of antibodies to myosin-V and tyrosinase Western related protein-l (TRP-1). and image analysis techniques. immunoblotting for myosin-V using solubilized human melanocytes confined the expected band at a relative mobility of 190 kDa. Time-lapse studies on melanocytes in vitro confirmed resolution of single melanosomes. Confocal phase and double fluorescent microscopy demonstrated co-localization of melanosomes, myosin-V and TRP-1. Melanosomes in the cell periphery showed intense myosin-V and TRP-1 staining. Pigment (melanin) density within 10 Km2 areas of cell cytoplasm of multiple cells or of 0.14 pm2 melanosomal sized areas within individual cells directly correlated with myosin-V fluorescence intensity (pcO.001). providing stattstical confirmation of myosin-V melanosome co-localization. These findings support the role of myosin-V in the peripheral cytoplasmic transport of human melanosomes.

MALIGNANT MELANOMA CELLS IN ZONES OF PROLIFERATION EXPRESS HlGHER TELOYERASE ACTIVITY THAN CENTRAL CELLS OF THE TUMOR Alexander Glaess?. Ania-Katrin Bosserhofl *, Reinhard Buettnef. Michael LandthaleFand Wiihelm St&? Department of DermatoloQJ and Institute of Pathology’. University of RegensburQ 93042 Regensburg, Germany Telomerase prevents the shortening of &lomeraS by elonQatinQ telomeric TTAGGGsequences and is suppressed in normal human somatic cells but reactivated durfna tumor progression. It is known that most malignant melanomas express telomeras; activity. To investigate the mle of telomerase activity within different zones malignant melanomas were microdissected and telomeraSe activity was deteded using the TRAP-ELISA-Assay (Boehringer, Mannheim, Genany). For quantifying the level of tetomerase aclivily in frozen tissue samples the Sk Mel 28 cell line was used es an Internal standard. By application of the micmdissection technique, equal amounts of lumor cells were obtained, controlled by measurements of protein concentration and counting cells in single cell suspensions. In 9 melanomas, telomerase activity was compared either between the invasive and the intraepidermal component or behveen the peripheral and the central areas of the tumor. Invasive cells displayed 31-39% higher levels of telOmeraSe adivity Compared to intraepidermal cells. In comparison to sreas located centrally the peripheral tumor areas showed 21.68% higher levels in 5 of 8 cases. In one case an equal level of Momerase actiwty could be detected, Our results indicate that malignant melanomas express different levels of telomerase activity within the same tumor. i.e. hiah telomerase activitv in zones of omliferatian and lower activity I” centrally br sup&icially located areas of the tumor: Therefore, telomerase may play a crucial role in the tumor eroaression and orollferation of malignant melanomas.

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HUh4ANMFLANOCYTESANDMEJ_4NOMASHOWAPAR.4DOXICAL MIGRATION RESPONSE PATTERN DEPENDENT ON R4S. cPL42 AND !_lPOXYGWASE, ElUf NOT ON CYCLOOXYGENASE. H. Yaai. K.H. Pfenninger. M. F&a. D. A. Noms Dept. of Dermatology and Dept. of Cellular and Structural Bid:, Univ. of Colorado School of Medicine, Denver, Colorado, USA. We have previously shown thai mrgration of human melsnccytes and melanoma cell hnes is dependent on growth factor stimulation of cytoplasmic phosphohpase A2 (cFLA2) mediated through activation of the ms signaling pathway. In tlus project we describe a highly distinctive biphasic movemenl response in melanccytes and melanoma m wbxh maximal sumulauon inhibits rather than enhancing nugration. Normal human melaaocylcs and low mvasive-potential melanomas show stmng chemotacllc responses in Boyden chamber assaysto growth factors such a~ IGF-I, ET1andbFGF. Thrcmbin also strongly induces migration in these ceils aSSoCiati with increasedcPLA2activity. I” crmtmst the highly mmtatic melanana cell hae WM 1617 has lugh sponcane~us mrgration associated with high cPLA2 activity, but addition of small amounts of ET-1 or thmmhn decreases cell mipra(ion while causing further increases in cPLA2. Low mvaPive-potential melanoma cells transfected wth activating ras muratlons also showed high baseline migration whxh was inbibHed by further growth factor stimulation. The increases in nugranon induced by growth factors in low invasive potenhal melanoma, and the mhrbition of migration in invasive melanomas was Mocked by CDC, a selective 12.lipoxygenase Inhibitor. No effect on migration was found with mdomethacm. a selectivecyclooxygenase Inhibitor. We describe a biphasic movement response m melsnocytes and melanomas, mediated by ras-dependent cPIA2 actwation and hpoxygenase. The mhihitory portion of the response to added growth factors is mcst promment in advanced melanomas. Better understanding of this biphasic response, and modulation with hpoxygenase itibitors may influence progression of melanoma.

0603

0606

PURIFIED IMMUNOGLOBULIN G FROM NON-SEGMENTAL-TYPE VITILIGO lNDUCES MELANOCYTOTOXICITY VIA A DIVERSE Yu. Yu_’u SPECTRUM OF IMMUNE MECHANISMS. H&&t v Department of Dermatology, Kaohsiung Medical College, Kaohsiung, Taiwan. Functional melanocytes in patients with vitiligo vulgaris disappear from involved skin by a mechanism(s) that has not yet been identified. An immunological hypothesis is currently proposed as a possible patbogenesis of non-segmental-type vitiligo. IgG antimelanocyte antibodies were reported to induce melanocyte damage in vitro by a complement-mediated mechanism and antibody-dependent cellular cytotoxicity. For determining the role of antimelaoocyte autoantibodies in the damage of melanocytes, IgG was purified from the sera of patient@-IgG) and normal individuals(N-IgG). After incubation of purified IgGs with cultured melsnocytes, the results revealed: (1) the binding ability of V-I&i 10 cultured melanocytes (MC) was higher than that of N-IgG; (2) V-IgG induced IL-8 release from MC, but not IL-l& IL-lp and IFNq; (3) V-IgG sGmulated HLA-DR expression on MC, but not HLA-DP and HIA-DQ; (4) ICAM- expression on MC was induced by V-&G. These results suggest that V-IgG plays important roles in melanocytic cytotoxicity via a diverse spectrum of immune mechanisms.

INFLUENCE OF ENDOTHELIN 1 TO EXI’RFSSION OF FOCAL ADHESION KINASE (pl25FAK) AND CELL MIGRATION IN CULTUZED MFLANOCYTE. MOhara. S. MA, and T. Akasaka. Dept. of Dermatology, Iwate Medical University, Schc~J of Medicine, Morioka, Japan 020. Among several factors affecting melanccyte molphobiology, endothelin 1 is believed to a mediator of melamxyte dendricity, melanogenesis and proliferation. We studied innuence of endothelin 1 to p125FAK expression, distribution and cell migration in cultured melanocyte. MeIanocytes cultured in four categories of endothelin 1 omcentration on fibmnsctin coated cover-slips express p125FAK containing plaques, which are distributed in theperiphery of dendrites and localized to actio stress fiber termination sites, using double immunofluorescence staininp. Higher mean migration rate time-lapse image analysis and longer dendrites were expressed in melanocytes cultured in higher end&h&n 1 concentration. Higher mean p125FAK plaques / cell exhibited significantly higher mean migration rates. These results indicate that endothelin 1 induce the increased expression of p125FAK and the functional relation of p125FAK expression to high migration on ECM components swsts that endothelin 1 modulates cytoskeletal functmn of cell motility in melsnocytes.