Selective Aberration and Pigment Loss in Melanosomes of Malignant Melanoma Cells in Vitro by Glycosylation Inhibitors: Premelanosomes as Glycoprotein

We have found that glucosamine (1 mg/ml) or tunicamycin (0.2–0.4 μg/ml), specific inhibitors of lipid carrier-dependent glycosylation of protein, when added to cultured B-16 melanoma cells produce a marked loss of pigmentation, accompanied by distinctive, biochemical as well as ultrastructural aberrations in their melanogenic compartments. Electron microscopic analysis shows that these newly induced unpigmented cells form uniquely altered melanosomes containing little or no melanin, although their population is not substantially reduced. Within the melanogenic compartments, selective aberration of melanosomes is seen, that is, deformity, bulging, and segregation of their interior membrane, as well as the intramelanosomal formation of irregularly concentric lamellar structure. No apparent structural deformity of Golgi apparatus, Golgi-associated endoplasmic reticulum of lysosome (GERL), and coated vesicles has been observed. Further, no substantial alteration is seen in mitochondria or other cellular structures. Quantitative analysis of altered and nonaltered melanosomes has revealed that the ratio of altered premelanosomes to the total number increases to 44% in glucosamine-treated cells and to 99.5% in tunicamycin-treated cells, compared to 13% in the control.Electron microscopic dopa reaction has also revealed that these altered melanosomes seem to exhibit a weakly positive or a negative dopa reaction in both glucosamine- and tunicainycin-treated melanoma cells although a number of dopa-positive altered melanosomes are still seen. However, GERL and coated vesicles show no apparent decrease in dopa reaction. It may be concluded that glycoprotein synthesis is integral to the formation of normal melanosomes and to their specific melanizing function, which could be impaired by inhibition of the synthesis of asparagin-linked mannose-containing sugar chains. This results in retrogressive changes in the premelanosomal tyrosinase during its maturation process