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Effect of 5-Azacytidine on DNA Methylation and the Malignant Properties of B16 Melanoma Cells

Department of Cell Biology, Smith Kline and French Laboratories, Philadelphia, Pennsylvania 19101 [D. L. T., T. K., F. M., R. G., G. P.], and Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 [G. P.]

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'-deoxycytidine (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 µM) and FCdR (0.3–30 µM) caused a dose-dependent increase in the ability of B16 cells to form experimental pulmonary metastases. Increased capacity to form experimental pulmonary metastases 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.

¹ To whom requests for reprints should be addressed, at Department of Cell Biology (L-109), Smith Kline and French Laboratories, 1500 Spring Garden Street, Philadelphia, PA 19101.

Received 10/ 5/84. Revised 8/16/85. Accepted 8/22/85.