Spontaneous or carcinogen-mediated amplification of a mutated ras gene promotes neoplastic transformation.

Mouse C3H10T1/2 cells were co-transfected with a plasmid containing the KiSV DNA and a plasmid carrying sequences coding for resistance to mycophenolic acid. Only 30% of the transfected colonies had a transformed phenotype, i.e. highly refractile rounded cells. The remaining colonies had varied morphologies with flat or slightly elongated cells. Analysis of p21 ras protein indicated that higher levels of the protein were expressed in cells with the more transformed phenotype. Tumors formed by a poorly tumorigenic clone were found to have undergone in vivo amplification of the transfected KiSV sequence. Transformed variants of this clone were also isolated in vitro. Treatment with 5-azacytidine resulted in an increase of about 10 fold in the formation of transformed variants. All transformed cells isolated, either spontaneous or 5-azacytidine induced, were tumorigenic in nude mice. The neoplastic conversion of these cells was accompanied by amplification of the transfected K-ras sequences. The data reported here indicate that a chemical agent that modifies DNA structure can enhance the frequency of amplification events in cellular DNA and, by affecting ras copy number, promote cell transformation.