Chou S C, Yang C F, Kimler V A, Taylor J D, Tchen T T
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Cell Differ Dev. 1989 Nov;28(2):105-17. doi: 10.1016/0922-3371(89)90047-6.
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.
我们之前报道过一种黑色素化细胞系的分离，该细胞系可经历可逆的去分化和再分化过程。一种色素沉着严重的细胞系，命名为P15，最初是通过鱼血清诱导一些GEM 81细胞黑色素化而分离得到的，在鱼血清培养基中克隆并连续传代，在胎牛血清培养基中培养几个月后色素明显减少，在相同培养基中再培养一年后完全无色素。向这种去分化细胞系（命名为P15D）的培养基中添加鱼血清，一周内可诱导色素沉着。这种重新诱导色素沉着的细胞系，命名为P15DI，在胎牛血清培养基中培养一个月后会失去色素。我们在此报告，P15的去分化发生在两个阶段。去除鱼血清一周后，培养物中酪氨酸酶的比活性下降约70%，并在这个降低的水平至少维持一个月。一年后，酪氨酸酶的比活性降至几乎检测不到的水平，培养物完全无色素（P15D）。电子显微镜研究表明，P15D细胞没有黑素小体，可能没有用于形成黑素小体的大囊泡，但有一些多巴阳性的反式高尔基体网络（TGN）。向P15培养物的生长培养基中添加鱼血清会导致酪氨酸酶比活性稳步增加，一天后即可检测到。一天内多巴阳性TGN的数量也会增加。三天后首次出现黑素小体，一周后数量增多。去除鱼血清后，这些重新诱导的细胞（P15D1）酪氨酸酶的比活性迅速下降，八天后达到P15D细胞中所见的基础水平。我们还报告，一种命名为p75（分子量约75,000）的蛋白质，先前显示与源自金鱼的两种黑色素化细胞类型中的黑素小体相关，存在于所有黑色素化细胞系中，包括P15和P15DI，但不存在于未黑色素化的细胞系中，包括P15D。
