Tissue-specific patterns of 5-methylcytosine in vertebrate DNAs are thought to play significant roles in the control of gene expression in normal and transformed cells. We propose to use the cancer chemotherapeutic agent 5-azacytidine (5-aza-Cr) to perturb these patterns, so that their importance in the stability of cellular phenotypes can be better understood. In particular, we are interested in the relationships between DNA methylation, cellular differentiation and oncogenic transformation. The 5-Aza-Cr will be tested for its ability to induce the formation of epidermal cells from C3H/10T1/2C18 mouse mesenchymal precursors and muscle cells from diploid human fibroblasts. Myogenic lines will be isolated from treated 10T1/2 cells and characterized with respect to chromosomal and 5-methylcytosine contents. Isolated DNA from myogenic lines will be tested for its ability to induce the formation of muscle cells in untreated recipient cells, to test the hypothesis that the emergence of mesenchymal phenotypes is under the control of one or a few regulatory genes. The mechanism of inhibition of DNA methylation by 5-az-Cr and the number of cell divisions required after exposure for new gene expression will also be investigated. Hemimethylated duplex DNA from treated cultures will be used to establish and in vitro methylation assay to isolate and characterize vertebrate DNA methyltransferases. The abilities of diverse chemical carcinogens to inhibit DNA methyltransferases in vitro and in living cells will be investigated. These experiments will determine whether chemical carcinogens can induce heritable changes in cellular phenotype by altering DNA methylation patterns.
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