A radioreceptor assay for alpha-MSH is described which is based on cultured mouse B16 melanoma cells and bioactive monoiodinated [Nle4]-alpha-MSH tracer. The assay was used (1) to study the binding characteristics of alpha-MSH to B16 cells, (2) to determine the relative binding activity of MSH peptides, and (3) to measure MSH in tissue extracts. The association of alpha-MSH to B16 cells reached a stable plateau after 3 h at 15 degrees C. At 25 degrees or 37 degrees C, the binding was transient and at 0-1 degree C, the association was very slow. The hormone-receptor complex was relatively stable between 0 degrees and 15 degrees C whereas a 50% dissociation was reached after 90 min at 25 degrees C and after 35 min at 37 degrees C. The mean KD for alpha-MSH of four saturation experiments was 1.3 nM and the number of receptors 9570 per cell. 1,10-Phenanthroline had a stabilizing effect in the binding assay when used at a 0.3 mM concentration. From the MSH peptides tested in the binding assay, some showed similar potencies in three bioassays (tyrosinase, melanin and Anolis skin), whereas others displayed considerably lower bioassay values than expected from the binding data. This shows that binding and bioactivity can be dissociated in some of the MSH peptides. The biological activity of MSH from the neurointermediate lobe of the rat pituitary as measured by its binding to B16 cells corresponds fairly well with RIA results; in the anterior lobe, alpha-MSH values are overestimated because of the large amount of ACTH present.

We analyzed mechanisms responsible for organ-specific metastasis by using two melanoma sublines derived from the same mouse tumor, of which one colonizes the lungs (Flo) and the other colonizes the liver (L8) after intravenous injection. Both lines were obtained by selective growth in lung or liver after injection of tumor cells into a tail vein or portal vein. Contrary to common concepts, the cells of the liver-colonizing melanoma line do not accumulate preferentially in the liver after intravenous administration in vivo. However, the selective survival and proliferation of these melanoma cells in the target organ (liver) may be explained by the unexpected observation that they can be specifically stimulated to proliferate in the presence of hepatocytes, whereas the cells of the lung-colonizing line cannot. Growth promotion under coculture conditions in vitro was monitored both by thymidine incorporation into DNA and by increase in cell numbers. The proliferative stimulus is not mediated by an easily diffusible factor but rather depends upon direct contact between liver cells and those tumor cells that metastasize to that particular organ.It is common clinical experience that a malignant primary cancer, although shedding viable cells throughout the body, will only give rise to gross metastases in certain specific tissues (1,2) and that the specific nature of these secondary sites depends upon the histological origin of the primary tumor. Apparently, the host tissue must be able to reciprocate this recognition in some way, if only passively (3).Some progress in explaining the general mechanisms of metastasis has been made recently through the recognition of autocrine motility factors that induce pseudopodia formation and hence exploratory cell migration (4). Earlier advances were made with variant and mutant cells that had different degrees of metastatic ability correlating with cell-surface alterations (5). Later, variants of metastasizing mouse melanoma cells were selected with preference for the liver (6). Fusion of nonmetastatic tumor cells with reticuloendothelial cells can also give rise to metastatic hybrids having distinct site specificities (7).This phenomenon has some interesting analogies with other homing and tissue interaction systems such as the inhibitory regulation of a melanoma by embryonic skin (8), the growth stimulation of neuroblastoma cells by neonatal sympathetic ganglia (9), fetal fat tissue and its supportive interaction with numerous fetal epithelia (10), and the stimulation by human melanoma and carcinomas of fibroblasts to produce hyaluronate (11).We show here that a specific interaction occurs between adult hepatocytes and liver-metastasizing melanoma cells resulting in the stimulation of the proliferation of the tumor cells. MATERIALS AND METHODSIn Vivo Experiments. Organ specificity. An organ-specific mouse melanoma of the F1 line of B16 melanoma cells (from I. J. Fidler, Houston) was isolated by administration into the portal vein, recovery and growth of liver colonies in ...

MSH receptors and their binding characteristics of [125I]-labelled derivatives of alpha-MSH have been studied extensively on various mouse and human melanoma cell lines in culture. The aim of this study was to determine the binding characteristics of alpha-MSH radioligands to MSH receptors occurring in experimental mouse and human melanoma tumours as well as in human melanoma biopsies. For this reason, solid tumours were grown on experimental animals by inoculation of murine B16-F1 and human D10 and HBL melanoma cells. After excision and cryosectioning of the tumours, frozen tissue sections were incubated with [(125I)Tyr2]-alpha-MSH or [(125I)Tyr2,Nle4,D-Phe7]-alpha-MSH and specific alpha-MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentrations of unlabelled alpha-MSH during incubation with tracer led to a dose-dependent displacement of the radioligand. Quantitative analysis of the autoradiograms produced dissociation constants which were comparable with those obtained with cell binding assays: KD = 1.87 and 1.31 nmol/l for B16 tumours and cells, respectively; 0.32 and 0.33 nmol/l for D10, and 2.24 and 1.36 nmol/l for HBL tumours and cells, respectively. This indicates similar binding properties of alpha-MSH radioligands to both cultured melanoma cells and tissue sections of melanoma tumours from experimental animals. Similar binding characteristics were also observed with human melanoma tissue sections originating from biopsies of melanoma patients.

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Pharmacological concentrations of iodide (> 1 x 10(-6) mol/l) are known to inhibit thyroid follicular cell growth in vitro. However, the inhibitory effect varies widely, depending on experimental conditions, and usually does not exceed 50%. We demonstrate that iodide (10(-4) mol/l) inhibits the growth of FRTL-5 cells in different passages by 11-67%. When five subclones of FRTL-5 cells were compared to the wild type, iodide-induced growth inhibition varied between 25% and 46%. The individual degree of inhibition of each clone was reproducible in two subsequent passages, suggesting that it is a stable constitutive trait. When FRTL-5 cells were grown first in three-dimensional clusters and then transplanted onto nude mice with high endogenous thyrotropin secretion, iodide at a serum concentration of less than 5.7 x 10(-7) mol/l nearly completely blocked cell replication in the transplants but not in the mice's own thyroid. Five cell lines, prepared from autonomously growing hyperthyroid feline multinodular goiters, were nearly completely resistant to the growth-inhibitory effect of iodide. These observations suggest that the sensitivity towards the growth-inhibiting effect of iodide is a highly variable, stable trait of each thyrocyte, even in cloned cell populations. Some FRTL-5 cells and, even more so, cells prepared from autonomously growing nodular feline goiters are resistant constitutively to the growth-inhibiting effect of iodide.(ABSTRACT TRUNCATED AT 250 WORDS)