EFFECT OF 5-AZACYTIDINE ON DNA METHYLATION AND THE MALIGNANT PROPERTIES OF B16-MELANOMA CELLS

Abstract

The role of DNA methylation in the expression of the metastatic phenotype in B16 murine melanoma cells in syngeneic C57BL/6 mice has been investigated. B16 cultures were incubated in vitro for either 6 or 18 h with the DNA hypomethylating agents, 5-azacytidine (5-Aza-CR) or 5-fluoro-2''-deoxycytidien (FCdR). At various times (1-13 days) following treatment, tumor cells were tested for their ability to form metastatic deposits when injected at different doses either i.v. (experimental metastasis) or s.c. in the footpad (spontaneous metastasis). Both 5-Aza-CR (0.5-15 .mu.M) and FCdR (0.3-30 .mu.M) caused a dose-dependent increase in the ability of B16 cells to form experimental pulmonary metastases. Increased capacity to form experimental pulmonary metasases was evident 24 h following treatment with 5-Aza-CR and 13 days following treatment with FCdR. The enhanced metastatic burden involved both an increase in the median number of lung colonies and a substantial increase in the size of individual lesions. 5-Aza-CR or FCdR treatment of B16 cell populations did not influence either the tumorigenicity or their ability to form spontaneous metastases. Parallel in vitro experiments using high-performance liquid chromatography analysis of cellular DNA demonstrated that under conditions in which 5-Aza-CR and FCdR enhanced formation of experimental metastases by B16 cells, there were readily detectable alterations in the 5-methylcytosine levels in DNA extracted from drug-treated cultures. These data suggest that drug-induced alterations in DNA methylation can affect biochemical pathway(s) whose expression is associated with the successful organ colonization by circulating tumor cells.