Comparison of tyrosinase levels in amelanotic and melanotic melanoma cell cultures by a competitive enzyme‐linked immunoadsorbent assay and by immunotitration analysis

Abstract

Melanogenesis in mammalian pigment cells is regulated by changes in the activity of tyrosinase, the rate-limiting enzyme for melanin synthesis. Because recent evidence suggests that this enzyme may exist in pigment cells in both active and inactive stages, a competitive enzyme-linked immunoadsorbent assay (ELISA) was developed to compare tyrosinase levels in amelanotic and melanotic melanoma cell clones. The melanotic cell line used for this study, MEL-11A, had basal tyrosinase levels approximately 40 times that of the amelanotic cell line, AM-7. Both cell lines responded to melanocyte-stimulating hormone by demonstrating large increases in tyrosinase activity. For competitive ELISA analysis of tyrosinase levels in these two clones, microtiter plates were coated with purified tyrosinase, and trypsinized cell extracts were tested for their ability to compete with bound tyrosinase for antibody binding. Although tyrosinase activity in the amelanotic clone was 1/40 that of the melanotic clone, immunoreactive tyrosinase levels in AM-7 cells were found to be approximately one-half that present in the melanotic clone. Additional evidence for the presence of an inactive (or at least, catalytically less active) enzyme in AM-7 cells was obtained from immunotitration analysis of tyrosinase in cell extracts from both cell lines. These results suggest that at least some amelanotic melanoma cells may contain significant levels of catalytically inactive tyrosinase molecules and that the level of pigmentation in mammalian melanocytes may be regulated by a tyrosinase activation process.

Analysis of Mammalian Pigmentation at the Molecular Level

“…In addition, the expression of the products of the c and b loci are not dramatically increased in response to stimulation of differentiation (such as by MSH), although there are increases in the order of two-to threefold. These latter patterns, however, are in keeping with evidence that tyrosinase synthesis is in many ways constitutive, and that rapid and significant variations in melanogenic activity result more from activation of latent enzyme than from increases in enzyme synthesis [Fuller et al, 1987[Fuller et al, ,1988Jimenez et al, 1988;Wong and Pawelek, 19751. The sum ofthese studies emphasizes the importance of other ongoing work on the regulation of melanin production by post-tyrosinase factors, inhibitors, and other control mechanisms [Barber et al, 1984;Korner and Pawelek, 1980;Lamoreux et al, 1986;Pawelek et al, 19801. Thus the time has arrived for a critical reevaluation of all studies performed on the various pigment mutations at the biochemical, enzymatic level.…”

Expression of melanocyte stimulating hormone receptors correlates with mammalian pigmentation, and can be modulated by interferons

“…Similar conclusio~ its inhibitor is critical to the output of melanin bY the have been reached based on kinetic and immunologic melanocYte; e.g.9 in our studies On the effects Of the analyses of JBMS wearing et al, 1988; Jimenez et al, inhibitor on purified enzyme (Table 3) it can be seen that 1988), B16 and S91 (Pawelek, 1976; while only a slight inhibition of tyrosine hYdroxYlase Fuller et al, 1987Fuller et al, , 1988 melanoma cells, where in-activity occurs at the concentrations employed (10 to creases in the synthesis of tyrosinase and/or the adiva-30%'0), this has a profound effect on the produdion of tion of latent tyrosinase were insufficient to account for melanin in the same reaction mixture (50 to 70%). the dramatic increases in melanin Production elicited by MSH.…”

Microphthalamia-associated transcription factor: a critical regulator of pigment cell development and survival

“…Alpha-melanocyte stimulating hormone (a-MSH) is one such peptide hormone that has been suggested to modulate the pigment response. Studies have shown a role of a-MSH in promoting pigment synthesis (Fuller et al, 1987;Aroca et al, 1993). a-MSH binds to the melanocortin 1 receptor (MC1r), which is a member of the seventransmembrane receptor family that activates adenyl cyclase via G-protein signaling.…”