Analyses of mixed melanogenesis in tyrosinase cDNA-transfected human amelanotic melanoma cells

In the pigment cell the synthesis of tyrosinase and the formation of premelanosomes are independent, yet coordinated, processes. However, the interrelationship between the two processes has not been elucidated previously. In this study, an expression plasmid for human tyrosinase cDNA was constructed and transfected into a human amelanotic melanoma cell line, G-361. Stable transfected cells (G-CMHT-3) were obtained with high tyrosinase activity and distinct melanization occurred. As for the type of melanin, both pheo- and eumelanin contents increased in G-CMHT-3 cells. Interestingly, catalase activity as one of the other melanogenic enzymes was decreased in G-CMHT-3 cells. The decrease of catalase activity was considered to play a role in melanin-polymer formation, resulting in the increase of both pheo- and eumelanin contents. Under electron microscopic observation, dopa-oxidase – positive Golgi-associated endoplasmic reticulum of lysosome, coated vesicles, and premelanosomes were observed in pigmented G-CMHT-3 cells, and the expressed tyrosinase was considered to be well translocated to these organelles. In addition, the number of premelanosomes (stages I-III) as well as melanosomes (stage IV) increased in G-CMHT-3 cells compared to those in G-361 cells. It is also noted that G-CMHT-3 cells showed more normal phenotype premelanosomes with occasional transitional premelanosomes exhibiting partial melanin polymer formation within their concentric whorl-like internal membranes. Further- more, the number of eumelanosomes in G-CMHT-3 cells was much larger than that in G-361 cells. These results suggest that the tyrosinase introduced by its cDNA transfection induced active and structurally non-aberrant premelanosome formation resulting in the upregulated pheo- and eumelanogenesis.