Cells. The origin and properties ofthe murine B16 melanoma cell lines, B16-Fl (low lung metastasis) and B16-FlO (high lung metastasis), have been described (7,8). Clones were isolated from these lines by replica plating as described (16), and subclones were isolated from cloned lines by the same method. Aliquots of all clones and subclones, together with the parent cell populations from which they were derived, were stored at liquid nitrogen temperature for use as reference stocks. The isolation and properties of cell variants resistant to concentrations of ouabain (Ouar), trifluorothymidine (TFTr), and diaminopurine (DAPr) toxic to wild-type B16 cells will be described elsewhere. All cultures were grown in Dulbecco's modified Eagle's minimal essential medium/10% -.fetal bovine serum (GIBCO) without antibiotics at 37°C in humidified 5% COJ/ 95% air as described (8)
The metastatic properties of tumor cell clones isolated from individual lesions of B16 melanoma metastatic to lung have been examined at different stages in the evolution of metastasis. Clonal analysis of metastatic lesions produced by B16 melanoma populations containing clones with identifiable, stable drug-resistance markers revealed that the majority (>80%) of experimental metastases produced by intravenous injection oftumor cells are of unicellular origin. During the early stages of their growth (<25 days after initial tumor cell arrest), the majority of metastatic lesions contain cells with indistinguishable metastatic phenotypes (intralesional clonal homogeneity) although different clonally homogeneous lesions from the same host contain tumor cells with different metastatic phenotypes (interlesional clonal heterogeneity). Progressive growth of metastatic lesions is accompanied by emergence, within originally clonally homogeneous lesions, of variant tumor cells with altered metastatic properties (intralesional clonal heterogeneity). By 40-45 days after initial arrest of injected tumor cells in the lung, 90% of the metastatic lesions are populated by cells with heterogeneous metastatic phenotypes.Studies in several laboratories have shown that malignant tumors contain subpopulations of cells that differ widely in their metastatic abilities (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). This cellular heterogeneity is believed to result from the formation of variant subpopulations of tumor cells with altered metastatic properties during progressive tumor growth (15). The factors that influence the genesis and regulation of cellular diversity within malignant tumors are poorly understood. Recent studies (16)(17)(18)(19) suggest that the rate at which tumor cell variants with altered metastatic properties are generated in vitro is influenced by the extent of subpopulation diversity within the overall cell population. We have shown that the rate of formation of metastatic variants is significantly higher in populations containing a limited number of tumor cell subpopulations than in highly heterogeneous, polyclonal populations containing multiple cellular subpopulations (16,17). We have also shown that the majority oflung metastatic lesions produced by the murine B16 melanoma arise from the single cells (17). Formation of such lesions thus represents a situation in which subpopulation diversity is restricted. It was therefore considered of interest to determine whether this situation would stimulate rapid formation of new tumor cell variants to generate phenotypically diverse subpopulations oftumor cells within individual metastases. Cells. The origin and properties of the B16 melanoma B16-F10 subline have been described (14,20). Drug-resistant (Dr) variants were selected from the B16-FlO subline by treatment with the chemical mutagen N-methyl-N'-nitro-N-nitrosoguanidine as described (16,17). Variants were selected for resistance to trifluorothymidine (2 ,ug/ml; TFT%), diaminopurine (47 p.M; DAPr), vin...
Metastasis is a complex process whereby tumour cells from a primary neoplastic growth disseminate throughout the body and establish secondary tumour foci in distant organs. Biochemical traits associated with, or essential for, the expression of the metastatic phenotype have not yet been identified. In the course of examining stimulation of the B16 murine melanoma adenylate cyclase by melanocyte-stimulating hormone (MSH) and by the diterpene forskolin, we noted that tumour cell clones isolated from common parent cell populations differed widely in their responses to these agonists. We report here that the accumulation of cyclic AMP induced by MSH or forskolin shows a strong positive correlation with the ability of B16 melanoma clones to form pulmonary tumour colonies when injected intravenously (i.v.) into syngeneic mice ('experimental metastasis'). In parallel in vitro analyses of cyclic AMP metabolism and in vivo assays of experimental metastasis using replicate cell preparations, highly metastatic tumour cell clones consistently show greater than a 30-fold increase in cellular cyclic AMP when exposed to MSH or forskolin. By contrast, clones with limited metastatic abilities respond to the same agonists with only a two- to threefold increase in cellular cyclic AMP. These data suggest that cyclic AMP metabolism is linked with biochemical pathways that are responsible for the formation of experimental metastasis by the B16 melanoma.
The regulation of adenylate cyclase in murine melanoma tumor cell clones with different metastatic capacities has been studied in intact cells and isolated membrane preparations. Analysis of the responses of intact cells from a number of B16 melanoma clones revealed that treatment with melanocyte-stimulating hormone (MSH) or the diterpene, forskolin, produced significantly greater accumulation of intracellular cyclic adenosine 3',5' monophosphate (cAMP) in strongly metastatic clones than in weakly metastatic tumor cell clones. In contrast, in isolated membranes from the same panel of clones, the extent of activation by forskolin but not by MSH correlated with metastatic capacity. Sodium fluoride and 5'-guanyl-beta-gamma-imidodiphosphate [Gpp(NH)p] also stimulated adenylate cyclase in isolated membranes but the extent of activation did not correlate with the metastatic behavior of the donor cells. A combination of forskolin and Gpp(NH)p proved to be a sensitive prospective indicator for identifying differences in the metastatic capabilities of individual B16 melanoma clones. Adenylate cyclase in membrane preparations from strongly metastatic B16 clones displayed synergistic activation but stimulation of the enzyme from weakly metastatic clones was less than additive. To test the generality of these findings, similar investigations were performed on B16-BL6 melanoma cells, a highly invasive subline of the B16 melanoma, and the K1735, an ultraviolet-light-induced murine melanoma arising in a different mouse strain (C3H). Consistent with their high metastatic potential, clones derived from the B16-BL6 melanoma displayed elevated levels of hormonally-stimulated adenylate cyclase, thereby confirming, for this tumor system, a close association between hormonal responsiveness and metastatic capacity. In contrast, K1735 melanoma cell clones exhibited significant interclonal variation in adenylate cyclase activity and metastatic performance, but no consistent relationship between the two traits was detected. Differences in the regulation and/or the intrinsic catalytic capacity of adenylate cyclase may account, at least in part, for the variation in hormonal responsiveness observed among B16 clones with distinct metastatic properties and suggest that cAMP-dependent molecular processes may be required for the expression of B16 melanoma experimental metastatic potential.(ABSTRACT TRUNCATED AT 400 WORDS)
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