Reversible dedifferentiation and redifferentiation of a melanized cell line from a goldfish tumor

We reported previously the isolation of a melanized cell line that can undergo reversible dedifferentiation and redifferentiation. A heavily pigmented cell line, designated as P15, originally isolated by fish serum-induced melanization of some GEM 81 cells, cloned and serially passaged in fish serum medium, became noticeably less pigmented after several months in fetal calf serum medium and completely unpigmented after another year in the same medium. Addition of fish serum to the medium of this dedifferentiated cell line, designated P15D, induced pigmentation within a week. This re-induced pigmented cell line, designated as P15DI, became unpigmented when cultured in fetal calf serum medium for one month. We report here that the dedifferentiation of P15 occurs in two stages. One week after withdrawal of fish serum, the specific activity of tyrosinase of the culture dropped by approximately 70% and remained at this reduced level for at least one month. After one year, the specific activity of tyrosinase had dropped to a barely detectable level and the culture became completely unpigmented (P15D). Electron microscopic studies showed that the P15D cells have no melanosomes, probably no large vesicles for melanosome formation, but some dopa-positive trans-Golgi network (TGN). Addition of fish serum to the growth medium of P15 cultures led to a steady increase in the specific activity of tyrosinase, detectable after one day. There was also an increase in the amount of dopa-positive TGN within one day. Melanosomes first appeared after three days and became numerous after one week. Upon removal of fish serum, these re-induced cells (P15D1) underwent a rapid decrease in the specific activity of tyrosinase, reaching, after eight days, the basal level seen in P15D cells. We also report that a protein designated as p75 (Mr approximately 75,000), previously shown to be associated with melanosomes in two melanized cell types of goldfish origin, is present in all melanized cell lines, including P15 and P15DI but absent in unmelanized cell lines, including P15D.