Use of an Alpha-Melanocyte-Stimulating Hormone Analogue to Improve Alpha-Melanocyte-Stimulating Hormone Receptor Binding Assay in Human Melanoma

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Abstract
In order to optimize the detection and measurement of .alpha.-melanocyte-stimulating hormone (.alpha.-MSH) receptivity in human melanoma cells, and the authors replaced the natural hormone by [Nle4,D-Phe7]-.alpha.-MSH, a more stable and potent analogue in the receptor binding assay commonly performed with .alpha.-MSH. The following parameters were investigated: temperature, incubation time, number of cells, and ratio of labelled to unlabelled hormone. Optimal conditions for each assay were determined. The results demonstrate that the analogue has identical binding sites to .alpha.-MSH, as similar reciprocal displacements of each labelled (125I) hormone by serial dilutions of unlabelled .alpha.-MSH or [Nle4,D-Phe7]-.alpha.-MSH (10-12 M to 10-6 M) were obtained. To further compare the two hormones, we performed a screening of various human cell lines: ten melanomas and five nonmelanomas. The assay with [Nle4,D-Phe7]-.alpha.-MSH yielded more receptor expression on six of ten melanoma lines against only four of ten with the natural hormone. In conclusion, the use of radiolabelled [Nle4,D-Phe7]-.alpha.-MSH analogue of labelled .alpha.-MSH improved both sensitivity and reproducibility in this receptor binding assay on human melanoma lines.