Microscopic visualization of melanocyte/melanoma melanotropin receptors.

Abstract

The objectives of this research were to determine whether melanotropin receptors are characteristic (constant) membrane markers of melanocytes, and, more importantly human melanoma cells. Methodologies were developed to visualize these receptors by light, scanning electron (SEM) and fluorescence microscopy. Multiple copies (up to a hundred) of [Nle⁴, D-Phe⁷] α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to macromolecular carriers, either latex beads (microspheres and macrospheres) or another inert substrate, polyvinyl alcohol (PVA). Multiple copies (up to a hundred) of a fluorophore were also attached to the PVA. Incubation in the presence of the macromolecular conjugates of the analog resulted in binding of human normal epidermal melanocytes and melanoma cells (but not other cells) to both small and large beads and to the fluorescent conjugate. Binding of the microspheres or the fluorescent conjugate to the cells exhibited a unique cluster pattern (capping) suggesting a receptor internalization related phenomenon. Only a single aggregate of beads was present on each cell suggesting a focal site of endocytosis. Most importantly, every cell of every melanoma cell line possessed receptors as visualized by fluorescence microscopy. Since the cells were not synchronized, binding apparently took place during all phases of the cell cycle. Therefore, receptor expression is not cell-cycle dependent. Specificity of binding of the macromolecular conjugates was demonstrated by several studies: (1) binding of melanocytes/melanoma cells to the conjugates was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog, (2) the macromolecular substrates (latex beads, PVA) lacking bound ligand did not bind to the melanoma cells or melanocytes, (3) another peptide ligand (e.g., substance-P) attached to the substrates failed to bind to the cells, and (4) cells of nonmelanocyte origin (e.g., mammary cancer cells, lung cancer cells, fibroblasts) did not bind to the macromolecular conjugates. Thus, cell specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes and melanoma cells. Fluorescent melanotropin conjugates should be useful in determining whether all human melanoma (primary and metastatic) tumors possess such receptors. These receptors may provide targets for melanotropic peptides for the identification, localization, and chemotherapy of melanoma.