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137 Cadherin molecules expressed in human melanoma cell line
JSID Abstracts
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EXPRESSION AND THE SIGNIFICANCE OF KERATIN IN MEIANOCYTE. Y. Katagata andS. Kondo. Dept. of Dermatol., Yamaaata Universiw. School of Medicine. Yamaaata. Japan. Melanin is synt&sized in melanosomk and may play a role of defence of our body. To present of intermediate filaments in melanocyte reported already only electron microscopically, however, their facts and functions are not clarified up until now. [MaterialsandMethods] Meianocytes were obtained at a semiconfluent condition using Morinaga-normal human cultured melanocyte kit. They were washed and sonicated 3 time-s,respectively, with an aqueoussolution[lO mMTris-HCl(pH 7.2)/10 mM EDTA/PMSF(Of mg /ml)]. Resulting the final residue was treated with 50 pl of 10 M urea/S% b-ME/5% NP40 for 6 h at 3pC. The extract was analyzed by 2DPAGE and immune reaction with anti-keratin monoclonal antibodies. [Results and Discussion] No evidence of keratin is in melanocyte, so far. Wedetectedsix kinds of keratin subunit (Kl, K5, K8, KlO, K14, K16) in tbe above melanocytes. We suggest that keratin may play a role of structure and maintenance in melanocyte and a transfer of melanosomeinto keratincwtes.
ISGUTIONANDCBABACTENIzATION iIF HUNAN N8L4NONA C8LLLINES DePICI8NTIN GusGANGLIGSIDE EXPRBSSION.Junji Nakano*“*. B.K. KohawNaj’. Chidori Asagami’and Kenneth0. Lloyd’. ‘Imunology Program,Neaorial Slcen-Kettering Cancer Center, NW York, Ner York, U.S.A., 2Departmeotof Deraatology. Ywchi University School of Nadicine, Uhe. Japan. In order to investigate the function of GD3ganglioside we have selected Gn.-deficient hw nelaoom cell lines. This was done br treating &NBL-28 cells with anti-CD3 antit& (B24) and rabbit coapleaant and subsequentsub-cloning of the surviving cells. This resulted in the derivation of.t.wo cell lines deficient in the cell surface expression of Goa. Both cell lines (designated SK-HBL-28-Nlsod SN-NIU-28-NZ)had no detectable cell surface expression of Gusas analrzed by a mixed haem&utination assay with mAbB24. No Goawas detectable in either cell line by glycolipid isolation, TU: (thin layer chromtography) and resorcinol-BCl spray. Horover. TLC-i-staining with aAb N84 sh& the presenceof los aaunts of GD~in both WI and 112 (I/40 of the aunt. in the parent cell line in Nl and l/500 in N2). The mutant call lines had slaer growth rates in vitro sod did not grow in nu/nu mice. These results indicate that Goaganglioside play an important role in proliferation and growth of malcells.
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ANALYSIS OF THE ROLE OF SMALL GTP BINDING PROTElN RAE IN INTRACELLULAR MEIANOSOME TRANSPORT. K. ARAKI#, T. HORIKAWA, K. NAKAGAWA, Y. FUNASAKA AND M. ICHIHASHI. #Deparbneni of Dermatobgy. Kobe University Mediil School,KobeJJapan. Melanoaomes are produced and fully developed h pigment calls. It is known that m&womas are transfered into keratinocytes,frcrn the tip of dendritic processes of m&anocytes. But the mechwww of hkomlkrlmr melenosome transports are still un&ar. Recently, acw-mu!ated evidences revealed that Rab protein pla s a key m!e of in-la! vesicle transporl h secretive cells, suei as synaptic ne”rons. In this studywe investgated whet&r a Rab pmtein is associated with rnebwam h pigment calls. Meianosoine enr!ched fracticn was cblained fmm cullwed El6 nose melanmw c&s by sucrose density gradient cantrii method. We kind Rab3A h the melanosonw enriched fr&%M b Westernblot analysis. We also da&ted ab hilin-3A. a Iargei molawla for Rab3A ,in the mekmmorns emiohsd 5 actbn.The results hdical~~~~4 possibly plays an mportant role h hlmchlar transport h pigment cells.
CADHERIN MOLECULES EXPRESSEDIN HUMAN MELANOMA CELL LINE N. Matsuvoshi. T. Tanaka. K.-L Toda. S. hnamura. Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Cell-cell adhesion moleculesare supposed to play an important role on metastasis. No-1 melanocytes express E-cadhetin, but most melanomacells do not expres it in spite of their strong cell-cell adhesiveness. We studied the expressionof cadherin moleculesin a humanmelanomacell line (MeWo)by RT-I’CR, and found the several fragFents of cadherin n~lecules including human N-cadhedn and protocadlwin 1PC42). Ftibermore several new cadhetin molecules werealso identified. Theseresults indicate that melanonw express various kinds of cadherin molecules, and the expression of the cadherinsmay be crucial to the melanomacell-cell adhesiveness.
135 INACTIVE NELANOCVTES IN THE OUTER ROOT SHEATH OF THE HAIR FOLLICLE AND THE SEBACEOUS GLANO. T.Horikaua’ , J.G.Morelli* , D.A.Norris* , R.Ichihashl’ . ’ Department of Dermatology, Koke Dnlversity school of Medicine, Kobe, Japan. Depertaent of Dermatology, University of Colorado, Colorado, U.S.A. Outer root sheath melanocytes(ORS-HCs) are known to be DOPA negative. Activation, proliferation and migration of ORS-MCs are believed to play a critical role in repigmenting phase of vitiligo. We found that ORS-WCs react with antipte8elanosomal antibodies NKI/beteb and A4Fll. However, ORS-t4Cs did not react with antibodies to l elanosome related cvtoplasmic anticran. or n elanosome related glycoproteins, tyrosinase, .TRP1. and TRP-2. He also found these inactive elanocytes in sebacereous glands and ducts. The RS-HCs became DOPA positive after cultivation i;i using TPA, bFGF and ET-l. We suggest that these inactive nelanocytes may be activated by certain factors and becoae DOPA positive.
138 TRP-2 CAN PREVENT CELL DEATH INDUCED BY EXPRESSION OF TYROSINASE IN NON-PIGMENT CELLS. mmd Y.MisLical. Miibiom rnditule for Dermatological Research, Kobe 657, Japaa Ia melaain-polymer formath, thtre are two pathway fro-m $fPAchron$DC). Oae is DIiICA-melaaie pathway abkb 1 m&am-monomer DIIICA coverted from DC by DOPAcbrome tautomerase(TRP-2) aad DHICA-metaaia ls produced from DIIICA by DHICA-oxidase (TRP-1). hatkr ia DHI-melanin pmthwmy is vim moetber mamow DHl spontaneously coaverted from DC. The expression of tyrosinase alone in flbroblasts after geae trasfectioa induces cell death in these cells. Previously it bas beea reported that TRP-1 caa reduce tbls type of cell death We found TRP-2 caa reduce more effectively this type of cell death Dexamethaxoaeinducible tyroslnase cDNA expression vector was ceastructed and tranfected into the flbroblasts withlwltbout TRP-2 cDNA pre-transfected. It bar been found that cell denth was induced io tbe cells without TRP-2 exprtssioa la coatrast to TRP-2 expressing cells. Tbls result suggested that tbe DOPAcluome tautomerase activity of TRP-2 converted DOPAcbrome to DHICA having lower cytotoxity.
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EXPRESSION AND THE SIGNIFICANCE OF KERATIN IN MEIANOCYTE. Y. Katagata andS. Kondo. Dept. of Dermatol., Yamaaata Universiw. School of Medicine. Yamaaata. Japan. Melanin is synt&sized in melanosomk and may play a role of defence of our body. To present of intermediate filaments in melanocyte reported already only electron microscopically, however, their facts and functions are not clarified up until now. [MaterialsandMethods] Meianocytes were obtained at a semiconfluent condition using Morinaga-normal human cultured melanocyte kit. They were washed and sonicated 3 time-s,respectively, with an aqueoussolution[lO mMTris-HCl(pH 7.2)/10 mM EDTA/PMSF(Of mg /ml)]. Resulting the final residue was treated with 50 pl of 10 M urea/S% b-ME/5% NP40 for 6 h at 3pC. The extract was analyzed by 2DPAGE and immune reaction with anti-keratin monoclonal antibodies. [Results and Discussion] No evidence of keratin is in melanocyte, so far. Wedetectedsix kinds of keratin subunit (Kl, K5, K8, KlO, K14, K16) in tbe above melanocytes. We suggest that keratin may play a role of structure and maintenance in melanocyte and a transfer of melanosomeinto keratincwtes.
ISGUTIONANDCBABACTENIzATION iIF HUNAN N8L4NONA C8LLLINES DePICI8NTIN GusGANGLIGSIDE EXPRBSSION.Junji Nakano*“*. B.K. KohawNaj’. Chidori Asagami’and Kenneth0. Lloyd’. ‘Imunology Program,Neaorial Slcen-Kettering Cancer Center, NW York, Ner York, U.S.A., 2Departmeotof Deraatology. Ywchi University School of Nadicine, Uhe. Japan. In order to investigate the function of GD3ganglioside we have selected Gn.-deficient hw nelaoom cell lines. This was done br treating &NBL-28 cells with anti-CD3 antit& (B24) and rabbit coapleaant and subsequentsub-cloning of the surviving cells. This resulted in the derivation of.t.wo cell lines deficient in the cell surface expression of Goa. Both cell lines (designated SK-HBL-28-Nlsod SN-NIU-28-NZ)had no detectable cell surface expression of Gusas analrzed by a mixed haem&utination assay with mAbB24. No Goawas detectable in either cell line by glycolipid isolation, TU: (thin layer chromtography) and resorcinol-BCl spray. Horover. TLC-i-staining with aAb N84 sh& the presenceof los aaunts of GD~in both WI and 112 (I/40 of the aunt. in the parent cell line in Nl and l/500 in N2). The mutant call lines had slaer growth rates in vitro sod did not grow in nu/nu mice. These results indicate that Goaganglioside play an important role in proliferation and growth of malcells.
134
137
ANALYSIS OF THE ROLE OF SMALL GTP BINDING PROTElN RAE IN INTRACELLULAR MEIANOSOME TRANSPORT. K. ARAKI#, T. HORIKAWA, K. NAKAGAWA, Y. FUNASAKA AND M. ICHIHASHI. #Deparbneni of Dermatobgy. Kobe University Mediil School,KobeJJapan. Melanoaomes are produced and fully developed h pigment calls. It is known that m&womas are transfered into keratinocytes,frcrn the tip of dendritic processes of m&anocytes. But the mechwww of hkomlkrlmr melenosome transports are still un&ar. Recently, acw-mu!ated evidences revealed that Rab protein pla s a key m!e of in-la! vesicle transporl h secretive cells, suei as synaptic ne”rons. In this studywe investgated whet&r a Rab pmtein is associated with rnebwam h pigment calls. Meianosoine enr!ched fracticn was cblained fmm cullwed El6 nose melanmw c&s by sucrose density gradient cantrii method. We kind Rab3A h the melanosonw enriched fr&%M b Westernblot analysis. We also da&ted ab hilin-3A. a Iargei molawla for Rab3A ,in the mekmmorns emiohsd 5 actbn.The results hdical~~~~4 possibly plays an mportant role h hlmchlar transport h pigment cells.
CADHERIN MOLECULES EXPRESSEDIN HUMAN MELANOMA CELL LINE N. Matsuvoshi. T. Tanaka. K.-L Toda. S. hnamura. Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Cell-cell adhesion moleculesare supposed to play an important role on metastasis. No-1 melanocytes express E-cadhetin, but most melanomacells do not expres it in spite of their strong cell-cell adhesiveness. We studied the expressionof cadherin moleculesin a humanmelanomacell line (MeWo)by RT-I’CR, and found the several fragFents of cadherin n~lecules including human N-cadhedn and protocadlwin 1PC42). Ftibermore several new cadhetin molecules werealso identified. Theseresults indicate that melanonw express various kinds of cadherin molecules, and the expression of the cadherinsmay be crucial to the melanomacell-cell adhesiveness.
135 INACTIVE NELANOCVTES IN THE OUTER ROOT SHEATH OF THE HAIR FOLLICLE AND THE SEBACEOUS GLANO. T.Horikaua’ , J.G.Morelli* , D.A.Norris* , R.Ichihashl’ . ’ Department of Dermatology, Koke Dnlversity school of Medicine, Kobe, Japan. Depertaent of Dermatology, University of Colorado, Colorado, U.S.A. Outer root sheath melanocytes(ORS-HCs) are known to be DOPA negative. Activation, proliferation and migration of ORS-MCs are believed to play a critical role in repigmenting phase of vitiligo. We found that ORS-WCs react with antipte8elanosomal antibodies NKI/beteb and A4Fll. However, ORS-t4Cs did not react with antibodies to l elanosome related cvtoplasmic anticran. or n elanosome related glycoproteins, tyrosinase, .TRP1. and TRP-2. He also found these inactive elanocytes in sebacereous glands and ducts. The RS-HCs became DOPA positive after cultivation i;i using TPA, bFGF and ET-l. We suggest that these inactive nelanocytes may be activated by certain factors and becoae DOPA positive.
138 TRP-2 CAN PREVENT CELL DEATH INDUCED BY EXPRESSION OF TYROSINASE IN NON-PIGMENT CELLS. mmd Y.MisLical. Miibiom rnditule for Dermatological Research, Kobe 657, Japaa Ia melaain-polymer formath, thtre are two pathway fro-m $fPAchron$DC). Oae is DIiICA-melaaie pathway abkb 1 m&am-monomer DIIICA coverted from DC by DOPAcbrome tautomerase(TRP-2) aad DHICA-metaaia ls produced from DIIICA by DHICA-oxidase (TRP-1). hatkr ia DHI-melanin pmthwmy is vim moetber mamow DHl spontaneously coaverted from DC. The expression of tyrosinase alone in flbroblasts after geae trasfectioa induces cell death in these cells. Previously it bas beea reported that TRP-1 caa reduce tbls type of cell death We found TRP-2 caa reduce more effectively this type of cell death Dexamethaxoaeinducible tyroslnase cDNA expression vector was ceastructed and tranfected into the flbroblasts withlwltbout TRP-2 cDNA pre-transfected. It bar been found that cell denth was induced io tbe cells without TRP-2 exprtssioa la coatrast to TRP-2 expressing cells. Tbls result suggested that tbe DOPAcluome tautomerase activity of TRP-2 converted DOPAcbrome to DHICA having lower cytotoxity.