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Receptors for alpha-melanocyte-stimulating hormone (alpha-MSH) on human malignant melanoma cell lines were investigated with a specific binding assay and characterized with structural analogues of alpha-MSH and adrenocorticotropic hormone and by photoaffinity cross-linking of the hormone-receptor complex. Specific binding of high-performance liquid chromatography-purified, monoiodinated alpha-MSH in the presence of 1 mM 1,10-phenanthroline as protease inhibitor was highest after a 2-h incubation at 37 degrees C. The nonspecific binding was less than 20% and dissociation of the ligand-receptor complex was relatively slow. Ten out of 12 human cell lines showed specific binding sites for alpha-MSH with Kp values ranging from 0.195 to 2.87 nM and the sites/cell being approximately 400 to approximately 1600. Virtually identical results were obtained in an assay where the cells remained attached to the culture dishes during the entire experiment. The study of hormone analogues with the D10 cell line showed that oxidized alpha-MSH had an approximately 40-fold lower affinity than alpha-MSH whereas [Nle4,D-Phe7]-alpha-MSH displayed a threefold and the adrenocorticotropic hormone fragments (1-17) and (1-24) a 20- and 8-fold higher affinity. Cross-linking of the alpha-MSH-receptor complex of three cell lines using monoiodinated [Nle4,D-Phe7,Trp(2-nitro-4-azidophenylsulfenyl)9]-alpha-MSH as photoaffinity label revealed a major Mr 45,000 protein band on sodium dodecyl sulfate-polyacrylamide gels, analogous to the MSH receptor of mouse B16 melanoma cells.

The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.

Treating human melanoma lines with dibutyryl adenosine 3':5'-cyclic monophosphate (dbc AMP) resulted in morphologic changes associated with the altered expression of cell surface antigens. After treatment, cells developed long cellular projections characteristic of mature melanocytes and showed the presence of an increased number of Stage II premelanosomes. In addition, induction of melanin synthesis, detected as brown perinuclear pigmentation, was observed. The AMP further drastically reduced the growth rate of the five melanoma cell lines that were tested. The influence of dbc AMP was completely reversible 3 days after the agent was removed from the culture medium. The antigenic phenotype of the melanoma lines was compared before and after dbc AMP treatment. This was done with four monoclonal antibodies directed against major histocompatibility complex (MHC) Class I and II antigens and 11 monoclonal antibodies defining eight different melanoma-associated antigenic systems. Treatment with dbc AMP reduced the expression of human leukocyte antigen (HLA)-ABC antigens and beta-2-microglobulin in five of five melanoma lines. In the two HLA-DR-positive cell lines dbc AMP reduced the expression of this antigen in one line and enhanced it in the other. No induction of HLA-DR or HLA-DC antigens was observed in the Class II negative cell lines. Furthermore, dbc-AMP modulated the expression of the majority of the melanoma antigenic systems tested. The expression of a 90-kilodalton (KD) antigen, which has been found to be upregulated by interferon-gamma, was markedly decreased in all the five cell lines. A similar decrease in the expression of the high molecular weight proteoglycan-associated antigen (220-240 KD) was observed. The reduced expression of Class I and II MHC antigens as well as the altered expression of the melanoma-associated antigens studied were shown to be reversible after dbc AMP was removed. Our results collectively show that the monoclonal antibody-defined melanoma-associated molecules are linked to differentiation. They could provide useful tools for monitoring the maturation of melanomas in vivo induced by chemical agents or natural components favoring differentiation.