- Email: [email protected]
Expression of agouti signal protein in murine skin characterized by immunostaining with an agouti antibody(αPEP16)
ESDR I JSID I SID Abstracts
0583 EXPRESSION OF AGOUTI SIGNAL PROTEIN IN MURINE SKIN CHARACTERIZED BY IMMUNOSTAINING WlTH AN AGOUTI ANTlBODY(txPEP16). Naoko Matsunaaa, Chie Sakai, Vincent J. Hearing. Laboratory of Cell Biology, National hstiNtes of Health, MD, USA Switching between production of two types of melanins, eumelanin and pheomelanin, in follicular melanocytes elicits a temporary shift from eu- to pheomelanogenesis, which is responsible for the wild-type agouti hair color pattern. This switch is controlled by the agouti locus (which encodes agouti signal protein, ASP) from 4 - to 6 days postnatal. ASP is thought to be produced by dermal papillae cells and is a paracrine factor that regulates the production of pigment by follicular melanocytes. PEP16 is a rabbit polyclonal antiserum generated against a synthetic peptide which corresponds to the carboxy terminus of ASP. The specificity of the aPEP reactivity was antibody was measured by ELISA and its specific confirmed with ASP by western blot analysis. In this study we observed the expression of ASP in newborn (3, 6, 9 day) non-agouti black, agouti and lethal yellow mouse skin specimens by immunostaining with aPEP16. High expression is observed in 6 day agouti and 6 day and 9 day lethal yellow mouse skin. These results suggest that in the lethal yellow mouse, ASP expression increases day by day and that in the agouti mouse, ASP expression is observed during pheomelanogenesis.
0586 Department of Dermatology, CD95 ligand (CD95L)
Kinki University
School of Medicine,
Osaka, Japan.
potently induces apoptosis by activating CD95 on target cells. Recently, it has been reported that melanoma cells in viva express the significant amount of CD95L. thereby being able to kill lesionally infiltrating CD95bearing immune competent cells. Therefore, we were interested to investigate at which stage the melanoma CD95L expression is turned on. In this study, we employed the immunohistochemical technique using an antibody directed against CD9SL. Skin biopsy of 49 lesions from 46 patients were assessed and included benign and dysplastic neavi, melanomas in situ, stage I melanomas categorized either to horizontal growth phase (HGP) or to vertical growth phase (VGP), advance invasive melanomas (Clark’s level 4 or 5) and lymphnode metastases. CD95L was expressed in all VGP tested, whereas neither HGP, benign neavi, nor dysplastic neavi positively reacted with the antibody. Furtbemmre, all advanced invasive melanomas as well as lymphnode m&stases of cutaneous origins gave raise to positive immunofluorescence. To analyse the link between the positivity and the size of hunors, the data were reanalysed on the basis of Breslow’s thickness, indicating that the expression was observed only when they were thicker than 0.76 mm. Moreover, by employing Clark’s criteria, it was appeared that CD95L was expressed on melanoma cells only when those were invading to dermis (Clark’s level 2 to 5). Taken together, our present study indicates the significant correlation between the hanorigenicity and the expression of CD95L, and thereby gives raise to the possibility that CD95L may be an useful prognostic marker for melanomas.
0584
0587
P”NCTIONAL MELANOCYTES ARE PRESENT IN WAITE EPIDERMIS IN VITILIGO OF LONG DURATION. Ansela Shoulder. Desmond J. Tobin Nelle N Swanson. Kann U. Schsllreuter. Clinical and Experimental Dermatology, Department of Biowdical Sciences, University of Bradford, Bradford, England, ‘Dept of Dermatology, Mayo Clime,
FAS EXPRESSION AND FUNCIlON IN HUMAN MELANOCYTES. NEVUS CELLS AND MELANOMA C&L LINES: LOSS OF EXFRESSI,ON $D,OR FUl$Tl?; W 7 OF 13 MELANOMA CELL LINES. J?, Wehrll. R. I.-H. Deparnaentof Dermatology and DHURDV. Geneva University Hospital, *Ludwig Inst. for Cancer Research. Lausanne, and tlnstitute. of Biochemistry. University of Lausanne, Switzerland. Fas (APO-1,CD951is a widely expresseddeath receptorthat belongs ta the umor necmsis factor receptorsuperfamilyand is a potent mediatorof apoptotic cell death. Crosslinking of Fas by its ligand (FasL) is one of the two major wagons used by cytotoxic T-cells for killing their targets. Melanomas are immunogenic and induce the pmducticm of specific Cl’Ls. but .w usually able to escape immune destruction. We have recently shown that Fas expression is downregulated in cutanews m&mxydc lesions with malignant progression. and have now analyzed the expression and function of Fas in similar human cell types in vitro. FACS analysis using a specific anti-human Fas monoclonal antibody revealed mcdemte to strong Fas expression in primary melanocytes, a nevus cell line and 8 out of 13 melanoma cell lines. In 5 of 13 (38%) melanoma cell lines however, Fas expression was low 10 undetectable. ‘The function of the Fwmediated death signalling cascade was assessed in the same cell types by measuring the percentage of cell death following expasun to recombinant .wluble FasL (sFasL). Using this functional assay, news cells and all but two of the Fas expressing melanoma cell lines (6 of 8) wen sensitive to SF.& showing that F&s expression correlates with functional Fas signaling in all except two of the melanoma cell lines. Accordingly. the 5 melanoma cells expressing little or no Fas were resistant to sFa&mediated death. Taken logether, our results show lhat Fas expression is lost in a subset of melanoma cell lines as wmparcd to primary mclanccylcs or news cells. and that loss of Fas expression in melanoma cell lines conelates with resistance to Fwmediated cell death. In addition. they show that resistance to Fas-mediated cell death can occur in melanoma cell lines despite conserved Fas expression,as two of the 13 melanoma cell lines analyzed were resistant to sFa.sL despite Fas expression. Down regulation of Fas expression and/or loss of Fas signaling may provide an additional means for melanomas 10 escape tumor specific cell-mediated immune resp0nsc.s
Minnesota, USA.
0585
0588
THE SCF/KIT PATHWAY PLAYS A CRITICAL ROLE IN THE CONTROL OF NORMAL HUMAN MELANOCYTE HOMEOSTASIS. James M. Grxhnik,*P James A. Burch,’ James Burchette,’ and CbristopherR.Shea*.l Departments of *Medicine (Division of Dermatology), ‘Cell Biology, and ‘Pathology, Duke University Medical Center, Durham, North Carolina, USA During development, the interaction of stem cell factor (SCF) with its receptor, KIT, is critical for the survival of melanwcytes. Limited in viva human studies have
SUPRANUCLEAR MELANIN CAPS REDUCE ULTRAVIOLETINDUCED DNA DAMAGE IN HUMAN EPIDERMIS. Nobuhiio Kobavashi. Akemi Nakagawa. Tsutomu Muramatsu. Yukio Yamashina, Toshihiko Shirai and Toshio Mori.* Department of Dermatology and *Radioisotope Center, Nara Medical University, Kashihara, Nara, Japan. Melanin can form sopranoclear caps in human epidermis. This suggests that intracellular melanin reduces UV transmission to the underlying cell nucleus and inhibits the formation of UV-induced DNA damage. The purpose of this study was to determine the photoprotective effect of epidetmal melanin. We irradiated normal human skin explants with UVB and determined the formation of cyclobutane pyrimidine dimers and (6-4)photoproducts in individual epidermal cells by indirect immunofluorescence and by laser cytometry using monoclonal antibodies specific for either cyclobutane dimers or (6.4)photoproducts. We found that epidermal cells with supranuclear melanin caps had significantly less DNA damage (both types) than epidermal cells without supranuclear melanin caps. Moreover, the protection factor for the two types of photolesions correlated with melanin concentration in epidemml cells. These results indicate that melanin reduces UV-induced photoproduct type DNA damage in normal human epidermis in a concentration-dependent manner.
suggested a possible activating role of SCF on adult human melanocytes. In order to study the impact of this pathway on normal melanocyte homeostasis, human skin xenografts were treated with serial injections of recombinant human SCF or a KITinhibitory antibody (K44.2). On histologic evaluation, SCF injection increased, while KIT inhibition decreased, the number, size, and dendricity of melanocytes. Immunohistochemical expression of melanacyte diffcrcntmtion antigens, including tyroslnase-related-protein:i and gplOOipmell7, was markedly increased by treatment with SCF, and decreased by K44.2 treatment. The number of Ki67-positwe melanocytes was increased in the SCF-treated tissue, suggesting a direct proliferative effect of SCF; conversely, treatment with K44.2 resulted in melanocyte loss, which did not appear reversible with prolonged treatment. These findings demonstrate that the SCFiKIT pathway remains critical in adult human skin, and that philrmacologic modulatmn of this single pathway can control cutaneous melanocyte homeostasis.
0583 EXPRESSION OF AGOUTI SIGNAL PROTEIN IN MURINE SKIN CHARACTERIZED BY IMMUNOSTAINING WlTH AN AGOUTI ANTlBODY(txPEP16). Naoko Matsunaaa, Chie Sakai, Vincent J. Hearing. Laboratory of Cell Biology, National hstiNtes of Health, MD, USA Switching between production of two types of melanins, eumelanin and pheomelanin, in follicular melanocytes elicits a temporary shift from eu- to pheomelanogenesis, which is responsible for the wild-type agouti hair color pattern. This switch is controlled by the agouti locus (which encodes agouti signal protein, ASP) from 4 - to 6 days postnatal. ASP is thought to be produced by dermal papillae cells and is a paracrine factor that regulates the production of pigment by follicular melanocytes. PEP16 is a rabbit polyclonal antiserum generated against a synthetic peptide which corresponds to the carboxy terminus of ASP. The specificity of the aPEP reactivity was antibody was measured by ELISA and its specific confirmed with ASP by western blot analysis. In this study we observed the expression of ASP in newborn (3, 6, 9 day) non-agouti black, agouti and lethal yellow mouse skin specimens by immunostaining with aPEP16. High expression is observed in 6 day agouti and 6 day and 9 day lethal yellow mouse skin. These results suggest that in the lethal yellow mouse, ASP expression increases day by day and that in the agouti mouse, ASP expression is observed during pheomelanogenesis.
0586 Department of Dermatology, CD95 ligand (CD95L)
Kinki University
School of Medicine,
Osaka, Japan.
potently induces apoptosis by activating CD95 on target cells. Recently, it has been reported that melanoma cells in viva express the significant amount of CD95L. thereby being able to kill lesionally infiltrating CD95bearing immune competent cells. Therefore, we were interested to investigate at which stage the melanoma CD95L expression is turned on. In this study, we employed the immunohistochemical technique using an antibody directed against CD9SL. Skin biopsy of 49 lesions from 46 patients were assessed and included benign and dysplastic neavi, melanomas in situ, stage I melanomas categorized either to horizontal growth phase (HGP) or to vertical growth phase (VGP), advance invasive melanomas (Clark’s level 4 or 5) and lymphnode metastases. CD95L was expressed in all VGP tested, whereas neither HGP, benign neavi, nor dysplastic neavi positively reacted with the antibody. Furtbemmre, all advanced invasive melanomas as well as lymphnode m&stases of cutaneous origins gave raise to positive immunofluorescence. To analyse the link between the positivity and the size of hunors, the data were reanalysed on the basis of Breslow’s thickness, indicating that the expression was observed only when they were thicker than 0.76 mm. Moreover, by employing Clark’s criteria, it was appeared that CD95L was expressed on melanoma cells only when those were invading to dermis (Clark’s level 2 to 5). Taken together, our present study indicates the significant correlation between the hanorigenicity and the expression of CD95L, and thereby gives raise to the possibility that CD95L may be an useful prognostic marker for melanomas.
0584
0587
P”NCTIONAL MELANOCYTES ARE PRESENT IN WAITE EPIDERMIS IN VITILIGO OF LONG DURATION. Ansela Shoulder. Desmond J. Tobin Nelle N Swanson. Kann U. Schsllreuter. Clinical and Experimental Dermatology, Department of Biowdical Sciences, University of Bradford, Bradford, England, ‘Dept of Dermatology, Mayo Clime,
FAS EXPRESSION AND FUNCIlON IN HUMAN MELANOCYTES. NEVUS CELLS AND MELANOMA C&L LINES: LOSS OF EXFRESSI,ON $D,OR FUl$Tl?; W 7 OF 13 MELANOMA CELL LINES. J?, Wehrll. R. I.-H. Deparnaentof Dermatology and DHURDV. Geneva University Hospital, *Ludwig Inst. for Cancer Research. Lausanne, and tlnstitute. of Biochemistry. University of Lausanne, Switzerland. Fas (APO-1,CD951is a widely expresseddeath receptorthat belongs ta the umor necmsis factor receptorsuperfamilyand is a potent mediatorof apoptotic cell death. Crosslinking of Fas by its ligand (FasL) is one of the two major wagons used by cytotoxic T-cells for killing their targets. Melanomas are immunogenic and induce the pmducticm of specific Cl’Ls. but .w usually able to escape immune destruction. We have recently shown that Fas expression is downregulated in cutanews m&mxydc lesions with malignant progression. and have now analyzed the expression and function of Fas in similar human cell types in vitro. FACS analysis using a specific anti-human Fas monoclonal antibody revealed mcdemte to strong Fas expression in primary melanocytes, a nevus cell line and 8 out of 13 melanoma cell lines. In 5 of 13 (38%) melanoma cell lines however, Fas expression was low 10 undetectable. ‘The function of the Fwmediated death signalling cascade was assessed in the same cell types by measuring the percentage of cell death following expasun to recombinant .wluble FasL (sFasL). Using this functional assay, news cells and all but two of the Fas expressing melanoma cell lines (6 of 8) wen sensitive to SF.& showing that F&s expression correlates with functional Fas signaling in all except two of the melanoma cell lines. Accordingly. the 5 melanoma cells expressing little or no Fas were resistant to sFa&mediated death. Taken logether, our results show lhat Fas expression is lost in a subset of melanoma cell lines as wmparcd to primary mclanccylcs or news cells. and that loss of Fas expression in melanoma cell lines conelates with resistance to Fwmediated cell death. In addition. they show that resistance to Fas-mediated cell death can occur in melanoma cell lines despite conserved Fas expression,as two of the 13 melanoma cell lines analyzed were resistant to sFa.sL despite Fas expression. Down regulation of Fas expression and/or loss of Fas signaling may provide an additional means for melanomas 10 escape tumor specific cell-mediated immune resp0nsc.s
Minnesota, USA.
0585
0588
THE SCF/KIT PATHWAY PLAYS A CRITICAL ROLE IN THE CONTROL OF NORMAL HUMAN MELANOCYTE HOMEOSTASIS. James M. Grxhnik,*P James A. Burch,’ James Burchette,’ and CbristopherR.Shea*.l Departments of *Medicine (Division of Dermatology), ‘Cell Biology, and ‘Pathology, Duke University Medical Center, Durham, North Carolina, USA During development, the interaction of stem cell factor (SCF) with its receptor, KIT, is critical for the survival of melanwcytes. Limited in viva human studies have
SUPRANUCLEAR MELANIN CAPS REDUCE ULTRAVIOLETINDUCED DNA DAMAGE IN HUMAN EPIDERMIS. Nobuhiio Kobavashi. Akemi Nakagawa. Tsutomu Muramatsu. Yukio Yamashina, Toshihiko Shirai and Toshio Mori.* Department of Dermatology and *Radioisotope Center, Nara Medical University, Kashihara, Nara, Japan. Melanin can form sopranoclear caps in human epidermis. This suggests that intracellular melanin reduces UV transmission to the underlying cell nucleus and inhibits the formation of UV-induced DNA damage. The purpose of this study was to determine the photoprotective effect of epidetmal melanin. We irradiated normal human skin explants with UVB and determined the formation of cyclobutane pyrimidine dimers and (6-4)photoproducts in individual epidermal cells by indirect immunofluorescence and by laser cytometry using monoclonal antibodies specific for either cyclobutane dimers or (6.4)photoproducts. We found that epidermal cells with supranuclear melanin caps had significantly less DNA damage (both types) than epidermal cells without supranuclear melanin caps. Moreover, the protection factor for the two types of photolesions correlated with melanin concentration in epidemml cells. These results indicate that melanin reduces UV-induced photoproduct type DNA damage in normal human epidermis in a concentration-dependent manner.
suggested a possible activating role of SCF on adult human melanocytes. In order to study the impact of this pathway on normal melanocyte homeostasis, human skin xenografts were treated with serial injections of recombinant human SCF or a KITinhibitory antibody (K44.2). On histologic evaluation, SCF injection increased, while KIT inhibition decreased, the number, size, and dendricity of melanocytes. Immunohistochemical expression of melanacyte diffcrcntmtion antigens, including tyroslnase-related-protein:i and gplOOipmell7, was markedly increased by treatment with SCF, and decreased by K44.2 treatment. The number of Ki67-positwe melanocytes was increased in the SCF-treated tissue, suggesting a direct proliferative effect of SCF; conversely, treatment with K44.2 resulted in melanocyte loss, which did not appear reversible with prolonged treatment. These findings demonstrate that the SCFiKIT pathway remains critical in adult human skin, and that philrmacologic modulatmn of this single pathway can control cutaneous melanocyte homeostasis.