Various factors that modulate the differentiation of malignant cells are known to affect their experimental metastatic potential (EMP), or lung colonization after intravenous injection into syngeneic animals. However, some results and conclusions on the relation between cell differentiation and metastasis have appeared to conflict. We have reanalysed this by measurement of EMP of B16 melanoma sublines after culture with agents or conditions that acted on differentiation through various intracellular pathways. All tested agents did affect the EMP. EMP was usually positively correlated with differentiation under diverse conditions, but exceptions showed that there is no direct causal connection. Nor could all findings be explained in terms of cell proliferation or expression of major histocompatibility antigens. Some data helped to explain disparities between previous reports. Specific novel findings included the following. The stimulation of EMP by melanocyte-stimulating hormone (MSH) as well as all other tested effects of MSH were prevented by extended exposure to 12-O-tetradecanoyl phorbol acetate (TPA), suggesting a requirement for protein kinase C activity as well as G-protein coupling in MSH action. Cells grown with cholera toxin were always more differentiated than untreated cells, but the EMP could be either markedly increased or markedly decreased by cholera toxin under different conditions. The basic culture medium apparently determined this striking reversal. The EMP was also significantly affected by the extracellular pH.

We have established two new immortal lines of mouse melanocytes, melan-b and melan-c, from mice homozygous for the brown (b) and albino (c) mutations respectively. Both lines were derived through differentiation in vitro of embryonic epidermal melanoblasts. The brown melanocytes are visibly brown by light microscopy, and centrifuged cell suspensions form brown pellets. The albino melanocytes form white pellets and contain abundant unpigmented premelanosomes as shown by transmission electron microscopy. Like normal, non-immortal melanocytes and like the immortal black melanocyte line melan-a, both lines show little or no growth in a standard, serum-supplemented medium, but proliferate well in the presence of 12-o-tetradecanoyl phorbol-13-acetate (TPA). Sustained growth of the albino cells also requires either keratinocyte feeder cells or 2-mercaptoethanol (2-ME). The modal chromosome numbers are 39 for melan-b and 40 (diploid) for melan-c. Neither line is tumorigenic in nude mice. Heterokaryons between the two lines can be constructed and form wild-type, black pigment. Melanocyte lines can now be reproducibly generated from mice of different strains, and provide tools for molecular studies of germline coat-colour mutations. These two lines provide elegant means to study the developmentally controlled expression of the two complementary genes, B and C, with black melanin pigment as a readily detectable natural marker.

Summary Murine and human melanoma cells differ relatively reliably from non-tumorigenic melanocytes in certain biological properties. When cultured at low pH, melanocytes tend to be pigmented and melanoma cells unpigmented. The growth of virtually all metastatic melanoma cells is inhibited by phorbol esters such as TPA (12-0-tetradecanoyl phorbol-13-acetate), which stimulate melanocyte growth. Melanocytes fail to grow in suspension culture or produce tumours when implanted in animals, while many melanoma lines can do both. Here we studied which of these properties were dominant in hybrid cells formed by fusion of drug-resistant murine B16-FIORR melanoma cells to melanocytes of the albino and brown lines, melan-c and melan-b. The albino melanocytes are unpigmented but well-differentiated, the brown melanocytes produce pale brown pigment and the melanoma cells are unpigmented under the conditions used. All hybrid colonies observed produced black pigment, except some melan-b/melanoma hybrids when growing sparsely with TPA. Thus pigmentation was generally dominant. 14/15 hybrid lines showed stimulation of proliferation by TPA, as do melanocytes. Most hybrid lines showed no or reduced capacity for growth in suspension, though some grew better in suspension when TPA was present. There was marked suppression of the tumorigenicity of the parental melanoma cells in 4/8 hybrids examined, and tumorigenicity was reduced in the others, despite considerable chromosome loss by the passage level tested. Thus most properties of the non-tumorigenic pigment cells were dominant, as often observed for other cell lineages, and providing further evidence for gene loss in the genesis of malignant melanoma.