The Journal of Dermatology Vol. 19: 814-817,1992 WS V-4 Alteration of Melanoma Melanogenesis by Phenotypic Modifiers Hiroyuki Takahashi, Takashi Horikoshi, Kazumasa Wakamatsu*, Shosuke Ito* and Peter G. Parsons** Abstract Human melanoma cells (MM96E)were incubated with a phenotypic modifier (L-ethionine) to compare its effects on phenotypic expression with those induced by sodium butyrate and dimethyl sulfoxide. In contrast to the latter agents, L-ethionine (8mM) failed to arrest the cell cycle at the G 1 phase or to inhibit colony formation ability after 48 hr incubation. Tyrosinase. activity changed in parallel with 5-S-cysteinyldopa (5-S-CD) content during treatment with sodium butyrate or dimethyl sulfoxide. Tyrosinase was inhibited in L-ethionine-treated cells, probably because of metabolism of L-ethionine to sulfhydryl compounds; this remains to be clarified. Gamma-glutamyl transpeptidase activity changed inversely with tyrosinase activity after sodium butyrate or dimethyl sulfoxide incubation, whereas L-ethionine did not significantly alter the enzyme activity. In addition, only sodium butyrate induced alkaline phosphatase activity. L- ethionine was less effective than sodium-butyrate or dimethyl sulfoxide in inhibiting expression of the B8G3 melanosomal antigen, as determined by Western blotting. These results suggest that phenotypic modifiers (differentiation inducers) affect melanoma cells in various ways and that melanogenesis therefore reflects only one aspect of differentiation in pigment cells. Introduction Since malignant melanomas are highly resistant to most therapeutic agents; a large number of attempts have been made to overcome this dis- advantage. One such approach has been to in- vestigate how it is possible to induce malignant cells toward a more differentiated state corresponding to normal melanocytes by using chemical reagents. Representative drugs in this category include di- butyryl 3':5'-cyclic monophosphate (dbcAMP) (1), retinoids (2), dimethyl sulfoxide (DMSO) (3), and 12-0-tetradecanoyl phorbol-13-acetate (TPA) (4). Recent reports discussing the potential of sodium butyrate and its derivatives as chemotherapeutic agents have been accumulating (5, 6). Criteria for differentiated states of pigment cells are generally accepted as follows; 1) enhancement of melano- genesis, 2) decrease in cellular growth rate, and 3) morphological changes into normal cells (mela- Department of Dermatology, Sapporo Medical College, Sapporo, *Fujita Health University School of Health Sciences, Toyoake, Aichi, Japan, and **Queensland Instituteof Medical Research, Herston, Australia. R<jprint requests to: H. Takahashi, Departmentof Der- matology, Sapporo Medical College, Minami-1 Nishi-16, Chuo-ku, Sapporo,060,Japan. nocytes) (3, 4). Recently, we reported the effects of 4 phenotypic modifiers (differentiation inducers) on various differentiation markers in human melanoma cells (7). As an extension of this study, we have tested L-ethionine (ET; 2-amino-4-(ethylthio)butyric acid) on cultured human melanoma cells (MM96E) to further study phenotypic alterations, particularly with respect to melanogenesis. Materials and Methods Cell Cultures The human malignant melanoma cell lines used in the present study were as follows: MM96E and L originated from MM96 (9), a former subline pos- sessing a higher tyrosinase activity than MM96L. MM418, established from a primary melanoma lesion, has high tyrosinase activity and melanin content (10) and A2058 has a moderate tyrosinase activity (11). Ail cell lines were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium sup- plimented with 5% fetal calf serum (FCS) (VIV) , penicillin (100 Vlml), streptomycin (100 jlg/ml) , and 3 mM 4-(2-hydroxy-ethyl)-I-piperazine ethane sulfonic acid (HEPES). Medium was changed every 3 days, and cultures were maintained at 37°C in a humidified atmosphere of 5% C0 2/air.