JSID Abstracts/Journal of Dermatological Science 10 (1995) 61-102 87

145 THE SIGNIFICANCE AND EXPRESSION OF KEFtATlN PEPTIDES IN CULTURED MALIGNANT MELANOMA CELLS. Y. Katagata,* Y. Hozumi and S. Kondo. Department of Dermatology, Yamagata University School of Medicine, Yamagata, Japan.

Melanoma has become malignant of melanocytes and nevus cells which derived from a neural crest. Only few studies have so far been made at keratin expression in melanoma cells. Moreover, the physiological significance of keratin peptides was almost unknown in the processing of tumorigenesis. So, we first elucidated keratin peptides in several cultured malignant melanoma cells using 2D- PAGE. To extract tie keratin peptide, cultured cells were treated with 2 kind of solutions(l; aqueous solution, II; high salt solution), separately. In the fraction treated with I, many kind of ketatin pep- tides were extracted, however, only several keratin peptides were detected in the fraction treated with II which has been used by other investigators. In addition, we would like to discuss also about keratin monoclonal antibodies and mRNA levels.

146 u -MSH/ACTH OF KERATINOCYTES AND MSH RECEPTOR ON MElANOCYTES ARE UP-REGULATED BY ULTRAVIOLET B IRRADIATION. Y. Funasaka*. A. Cbakrabortv. A. Ohashi. e M. N Department of Dsrnlatology. Kobe University Medical School, Kobe, Japan.

Cultured keratmocyle has been shown to produce and secrete both u -MSH and ACTH. In addition, sdmulaliin of n -MSH receptor system &as been proved to increase proliferation and rnelanogenesis of normal human melanocytes. We asked whether a -MSlVAiZTH-MSH receptor system *s wolved in Ultraviolet B ( UVB ) induced melanogenesis using rwrnal human keralinocyies and melanocytes. We also examined the effect of UVB-induced cyiokines ( IL1 rz j3, TNF a, endothefin 1) and quenchers of H202 ( NAC) on this svstem. To evaluate u -MSH/ACTH oroducdon and secretion we performed the radioimmunoassay (RIA). Th’e MSH receptor regulation &as studied bvthe bindino assav usino 12511abelled Nle4DPhe7 o-MSH and Northern blotting u&g mel&oco&n 1 receptor ( MC1 -R ) cDNA as a probe UVB upregulated u -MSH/ACTH production and secretion and (I -MSH receptor bonding activity with increased MC1 -R expression. This activation was inhibited by NAC. ILl. and ET1 upregulated either or both of this system. TNF (I inhlbited MSH-R activity These results suggest that UVB modulates both directly and indirectly LI MSHIACTH-MSH receptor system and direct UVB effect involves active oxygen spiecies

147 IMMUNOREACTIVITY OF a-MELANCCME STIMULATING HORMONE AND0 -ENDORPHIN I N CUTANEOUS MELANOCYTIC LES0NS. M. Naaahama’. Department of Dermatology, Kobe University Medical school, Kobe, Japan

a -MSH s+imulalion has been shown lo give melastalic ability via up- rsouietion of CDKNP l ol@MTSl I in rnxrse melanoma 816 cell lime. We h&e recently d&x& D -MSH production and MSH receptor ( melanocortin 1 receptor gene ) expression in cultured normal human nmfanocy&andmelenomac%lk Todarify apossiblerdeo! CI- MSHiMSH receptor in melanoma development and progression, we immunohiklochemkally analyzed (I -MSH expression using polyclonal antibody. To determine whether MSH immunoreaciivity reflect on bindung of secmtti MSH fmm other tissues with MSH R on meknoma calls or melanoma celk produce MSH. we furlher examined 6 erdorphin ~mmunoreaclivily which is one of the proc@omelanocortin (POMC ) gene derwatives like o -MSli bul wilh 110 effinily to MCI -A. None d 20 cases of ~lmmon and dysplsstic nevi showed no detectable MSH staining in nevus celk buf 40 % cases were positive in B-etirphin immunoreacitiy. In melanomas, 16 out of 39 cases showed a -MSH and 29 showed 6. endorphin irnmwroreaclivily and melanoma cells of ALM and m&static lesions were strongly posidve. These results suggest that POMC gene derivatives are involved in melanoma progression possibly in autocrine manner

148 MONOCLONAL ANTI-C-KIT ANTIBODY (ACK2) INDUCED APOPTOSIS IN CULTURED NEURAL CREST CELLS. m Y.Kawa. M.Ohkura. H.Ono#. Y.Kubotaand M.a Dep. of Dermatoloav, XMarianna Univ. School of Medicine. Kawasaki. Jaoan. #Dep. of B%Iogy, Keio Univ. Yokohama, Japan.

We studied the role of stem cell factor (SCF) in the c-Kit expression and melanogenesis of cultured mouse neural crest cells and reported that c-Kit positive melanoblasts appeared in the SCF-added medium when the neural tubes of 9.5.day-old mice embryos were cultivated for several days. In this study, using this culture system, we studied whether ACK2 can induce apoptosis in melanoblasts. Apoptolic cells of the cultured neural crest cells were detected by using an Apop Tag’” kit. There was a significantly higher number of apoptotic cells in cultivated cells to which we added ACK2 on the 7th day, as compared to the control group. Moreover, apoptotic cells were also confirmed by electron microscopic study. Nishikawa et al reported that melanoblasts disappeared from murine skin during embryonic life with ACK2 injections. However, its precise mechanisms remain unknown. Our in vitro study shows that ACK2 induced apoptosis in c-Kit positive melanoblasts.

149 POSSIBLE INVOLVEMENT OF PHOSPHOLIPASE 4 IN THE

INDUCTION OF MELANOCYTE MOVEMENT BY MELANOCYTE MITOGENS. T. Horikawa*‘. I.G. MorelI?, D.A. Norris”, I.B. Travers’,

150 DTNAMICS OF MI-D-1 ANTIBODIES IN PATIENTS UITH MELANOMA ALONG WITH B-IFN TREATHMT. S. Kako’ , H. Nagai, K. Hayashibe and M. Ichihashi. Dapartaant of Darn!atolagy. Koba University School of Msdi i Koba. Japan.

f-fR&ne encoding human melanoma-associated antigen immunogenic in patients with lnelanona vas characterized to be limitedly expressed in melanoma cells and to be potentially useful for active specific immunotherapy. This study aimed at detecting the dynamics of titer of anti-D-1 antibodies in patients with melanoma receiving D-l racomhinant peptide and fi -IFN, based on tha following observation; 1) there are 2 groups in patients of high and low titer of anti-D-1 antibodies before at.3 even after receiving D-l paptida alone. 2) D-l mRNA could be increased in cultured human malanona cells after treatment with

B-IFN. Thus, such a ncdulation of malarmma calls in viva by B-IFN might affect host innune response to melanoma. The titer

of antibodies to D-l was measured by double determinants immunoassay and observed with clinical course of patients rasaivad. Enhancamant by B-IFN of antibody-titer seam to be observed in patients belong to high titer group before immunization. This suggests D-l expression on cell surface nay linked with specific HLA molacules.