The role of DNA methylation in the generation of tumor cell variants with altered growth behavior has been investigated. Cultures of the clonally heterogeneous B16 melanoma cell line and a clonal population (B16-CL) derived from it were treated with the DNA hypomethylating agent, 5-azacytidine (5-Aza-CR). The tumorigenic and metastatic properties of (sub)clones isolated from these cultures before and after drug treatment were assayed by injection via multiple routes into syngeneic C57BL/6 mice using a range of cell doses. The rate of tumor growth was monitored following intrafootpad (i.f.p.) injection and the tumor incidence was calculated from the frequency of tumor formation at i.f.p. and supraclavicular subcutaneous (s.c.) sites. Formation of both spontaneous (i.f.p., s.c. inoculations) and experimental (intravenous (i.v.) inoculation) metastatic potential was also investigated. The most consistent effect of 5-Aza-CR was the introduction of heterogeneity with respect to the tumorigenic phenotype. The effect of 5-Aza-CR treatment on metastatic behavior was variable. The majority of tumor cell variants that arose following 5-Aza-CR treatment displayed decreased malignant potential and reduced DNA methylation levels relative to untreated control cells, but the correlation was not absolute. The decreases in DNA methylation levels induced by 5-Aza-CR were unstable and began to rebound within 1 week of drug treatment. The results of the current study indicate that although 5-Aza-CR can introduce significant shifts in the malignant properties of treated cells, the direction and magnitude of the induced alterations are not predictable and are influenced by a variety of experimental parameters including the starting tumor cell population, route of tumor cell inoculation, and the drug treatment protocol. In addition, because DNA methylation levels can rebound rapidly (days) it is difficult to correlate changes in this parameter with the observed alterations in malignancy, which can only be assessed in long-term biological assays (weeks).

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Experimental metastasis correlates with cyclic AMP accumulation in B16 melanoma clones.

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.

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