Tyrosinase activities of cell-free extracts and living cells of cultured melanoma cells

Melanogenesis in the course of monolayer culture of a stably melanotic clonal line C 2M, derived from a mouse melanoma B 16, was investigated. Tyrosinase activity per cell of cell-free extracts was highest when the extract was prepared from cells in the mid-exponential phase of growth, when it was more than 6 times the activity of that prepared from a fully grown culture or a culture in the very early phase. On the other hand, the enzyme activity per cell of living cells in culture was highest in the early phase of culture and decreased rapidly to a level of less than one tenth of the maximum activity, in the stationary phase. The upper limit of population density of cultured melanoma cells permissive for melanin synthesis (2 to 3 × 10 5 cells/cm 2) was much higher than that of normal (nonneoplastic) melanocytes, which had been reported to produce melanin only under conditions of clonal growth. The relative efficiency of tyrosinase activity in situ, expressed by the ratio of tyrosinase activity in culture to that of cell-free extract, decreased rapidly in the exponential phase of growth. This decrease correlates to the cell density in the culture, and little if at all to the division rate, and suggests a suppressing mechanism of melanin synthesis working at the enzyme level.