The proportions in which two eumelanin monomers, namely 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI), compose the eumelanin polymer are believed to determine properties of the pigment including its color. These proportions are, however, not well elucidated for naturally occurring eumelanins, largely because of methodological difficulties. In this study we estimate the content of DHICA-derived units in mammalian eumelanins using a combination of two analytical techniques: 1) quantitation of DHICA-derived eumelanin by measuring the yield of pyrrole-2,3,5-tricarboxylic acid (PTCA index) and 2) spectrophotometrical quantitation of total (DHI + DHICA) eumelanin at 350 nm (A350 index). The ratio of PTCA/A350 measured for melanins synthesized from DHI and DHICA mixed in various molar proportions correlates well with the content of DHICA in synthetic polymers. Using this relationship as a standard curve we estimated the proportion of DHICA-derived units in mammalian eumelanins from hair and melanoma cells and found it to be much higher in rodent pigments (58.8%-98.3%; two species, mouse and hamster were examined) as compared to human eumelanins (19.2%-41.8%; one Caucasian and one Oriental individual were examined). No relationship between proportion of DHICA-derived units in eumelanin and hair color is found. The latter seems to be determined predominantly by the ratio of pheo- to eumelanin synthesis.
The melanogenic gene-transfected cell system serves as a useful tool for the study of the symphonic relation between melanin synthesis and intracellular organelles such as melanosomes in melanocytes. We constructed melanin-producing mouse fibroblasts by transfection of human tyrosinase cDNA to investigate the intracellular changes caused by tyrosinase expression. DHICA-oxidase (5,6-dihydroxyindole-2-carboxylic acid oxidase) activity without TRP-1 (Tyrosinase Related Protein-1) expression in the cells suggested that human tyrosinase also possesses a DHICA-oxidase activities different from mouse tyrosinase. Electron microscopic observation indicated that melanin-deposit organelles have some lysosomal features. These properties of melanin-deposit organelles in tyrosinase expressing fibroblasts provide one evidence for the hypothesis that melanosome is the specialized lysosome in melanocytes.
DOPAchrome tautomerase (DCT) is known to control the ratio of DHICA/DHI formed within the melanocyte, but physiologic significance of this activity is not yet fully elucidated. In this study the two melanin monomers are shown to inhibit with different efficacy the initial, tyrosinase-controlled, melanogenic reaction, namely conversion of L-tyrosine to DOPAchrome (2-carboxy-2,3-dihydroindole-5,6-quinone). This is demonstrated in the test tube assay system whereby formation of DOPAchrome is catalyzed by i) isolated premelanosomes (PMS), ii) tyrosinase-rich PMS glycoproteins, or iii) tyrosinase purified from fibroblasts transfected with human tyrosinase gene. Both DHI and DHICA suppress the conversion of L-tyrosine to DOPAchrome when added to reaction mixture but the inhibitory effect is far more strongly pronounced by DHI. DHI inhibits both activities of tyrosinase--tyrosine-hydroxylation and DOPA-oxidation--more strongly than DHICA. The different extent of inhibition is shown to reflect i) the ability of the two monomers to compete with tyrosinase substrates for the enzyme's active center and ii) the rate of interaction between melanin monomers and DOPAquinone. Consequently, we demonstrate that the tyrosinase-catalyzed DOPAchrome formation can be modulated by the ratio of DHICA/DHI among melanin monomers with the increased proportion of DHICA resulting in more efficient DOPAchrome formation. These results raise the possibility that DOPAchrome tautomerase plays a role in positive control of the tyrosinase-catalyzed early phase of melanogenesis.
Pigmented hamster melanoma tumors growing in situ contain two subpopulations of melanoma cells that have different electrophoretic mobilities (EPM). A mild neuraminidase treatment, which removes sialic acid residues from the cell surface glycoproteins, reduces the EPM of both groups of melanoma cells yielding an electrophoretically uniform population. This shows that the differences in the EPM between the subpopulations of pigmented melanoma cells stem from the different content of sialic acid residues on the cell surface. The relationship between the different EPM melanoma cell subpopulations was, therefore, examined during tumor growth, development, and formation of metastases. The relative content of cells having high electrophoretic mobility, the "fast moving" cells, increases as the tumors grow larger. However, tumors of the same diameter contain nearly the same fraction of "fast moving" cells despite their age. The proportion of the "fast moving" cells is significantly higher in the central part than in the outermost layer of pigmented melanoma tumors. These data suggest that the development of "fast moving" cells is promoted by some size-dependent changes in the intratumor environment. In vivo selection of melanoma cells for their ability to colonize lungs renders tumors that reveal elevated metastatic potential and contain a significantly higher fraction of cells possessing high electrophoretic mobility than the parent tumor. Moreover, the metastatic nodules contain a remarkably elevated fraction of the "fast moving" cells. The reported correlation between the "fast moving" cell fraction and the metastatic potential suggests that the relative content of cells having high electrophoretic mobility may determine the metastaticity of pigmented hamster melanoma.
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