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Regulation of Melanin Synthesis of B16 Mouse Melanoma Cells by 1,25-Dihydroxyvitamin D₃ and Retinoic Acid¹

Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 [J. H., T. K.], and Department of Biochemistry, School of Dentistry, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142 [E. A., T. S.], Japan

Melanin synthesis of B16 mouse melanoma cells was found to be stimulated dose and time dependently by 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the hormonal form of vitamin D₃. The stimulation of melanogenesis resulted from an increase in the activity of tyrosinase, a key enzyme in melanin synthesis. The minimum dose required for this stimulation was as low as 0.05 ng/ml, or 0.12 nM, a physiological level of plasma 1,25(OH)₂D₃. The stimulation by 1,25(OH)₂D₃ was specific; other derivatives of vitamin D₃ caused no stimulation at a concentration of 500 ng/ml. When the cells were plated on agar plates, the proportion of dark or black colonies was not increased by the exposure to 1,25(OH)₂D₃. Furthermore, this compound did not induce melanin synthesis of an amelanotic variant. Thus, its stimulatory effect seemed to be due to stimulation of melanin synthesis of melanotic cells, rather than to conversion of amelanotic clones to melanotic ones. 1,25(OH)₂D₃ did not induce intracellular cyclic adenosine 3':5'-monophosphate, while cholera toxin induced cyclic adenosine 3':5'-monophosphate and stimulated melanin synthesis and tyrosinase activity much more than did 1,25(OH)₂D₃, suggesting that 1,25(OH)₂D₃ stimulates melanin synthesis by a cyclic adenosine 3':5'-monophosphate-independent mechanism.

B16 melanoma cells contained specific receptors for 1,25(OH)₂D₃. Scatchard plot analysis revealed two types of receptor; the high-affinity receptor had a K_d of 18.3 pM and an N_max of 10.6 fmol/mg of protein. The specificity of receptor binding was demonstrated by studies showing that, for 50% displacement of 1,25(OH)₂D₃ binding, other derivatives were required at 500 times higher concentrations or more.

In contrast to 1,25(OH)₂D₃, retinoic acid inhibited melanin synthesis and tyrosinase activity of B16 melanoma cells dose and time dependently. On simultaneous treatment, 1,25(OH)₂D₃ and retinoic acid caused mutual interference, and a balance between their respective stimulating and inhibitory effects was obtained at a molar ratio of 10:1; i.e., with 10 nM 1,25(OH)₂D₃ and 1 nM retinoic acid.

¹ Supported in part by a Grant for Cancer Research from the Ministry of Education, Science and Culture of Japan.

² To whom requests for reprints should be addressed.

Received 7/31/84. Revised 11/29/84. Accepted 12/19/84.

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