Melanotropic peptide receptors: membrane markers of human melanoma cells

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Abstract
The objectives of this research were to determine whether melanotropin receptors are characteristic (constant) membrane markers of human melanoma cells. Methodologies were developed to visualize these receptors by fluorescence microscopy. Multiple copies (10-20) of both [Nle4,D-Phe7]alpha-MSH, a superpotent analog of alpha-melanocyte stimulating hormone (alpha-MSH), and a fluorophore, were conjugated to polyvinyl alcohol (PVA). Incubation in the presence of the multivalent macromolecular conjugate (FITC-PVA-MSH) resulted in binding of human epidermal melanocytes and keratinocytes and human melanoma cells (both melanotic and amelanotic) to the fluorescent conjugate. Binding of the conjugate to the cells exhibited a unique cluster pattern (capping) suggesting a receptor internalization related phenomenon. Most importantly, every cell of every melanoma cell line, melanotic or amelanotic, possessed receptors as visualized by fluorescence microscopy. Since the cells were not synchronized, some binding apparently took place during all phases of the cell cycle. Therefore, receptor expression appears not to be cell-cycle dependent. Specificity of binding of FITC-PVA-MSH was demonstrated by several studies. (i) Binding of the conjugate to melanoma cells could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; [Nle4,D-Phe7]alpha-MSH. (ii) The macromolecular conjugate lacking bound ligand (FITC-PVA) did not bind to the melanoma cells. (iii) Another peptide, a substance-P analog, attached to the substrate (FITC-PVA-SP) failed to bind to the cells. (iv) With the exception of keratinocytes, other cells of nonmelanocyte origin (e.g., fibroblasts, spleen, liver, kidney cells, and mammary cancer cells, lung cancer cells) did not bind to the conjugate. Thus, cell-specific melanotropin receptors appear to be characteristic cell surface markers of epidermal melanocytes, keratinocytes, and melanoma cells. In several human melanoma cell lines these receptors appeared to be functional since [Nle4,D-Phe7]alpha-MSH stimulated tyrosinase activity. Fluorescent melanotropin conjugates might prove useful in determining whether all human melanoma (primary and metastatic) tumors possess such receptors. These receptors might then provide targets for melanotropic peptides for the identification, localization, and chemotherapy of melanoma.