Structural and functional analysis of amplified DNA in a tumorigenic mouse cell line: Organization of amplified genes and identification of a novel gene with oncogenic potential

Amplification of oncogenes in tumor cells is a commonly observed phenomenon. Presumably, overexpression of an amplified oncogene abrogates normal regulation of cellular proliferation, thereby conveying a selective growth advantage to cells. I have been studying a tumorigenic mouse cell line (3T3DM) that stably maintains amplified DNA in the form of double minutes (DMs) and has amplified no known oncogene. Two genes amplified in these cells have been previously isolated. The goals of my work were (1) to determine the structure of amplified DNA in 3T3DM cells; (2) to isolate additional amplified and overexpressed genes; and (3) to test each gene identified for oncogenic potential. The approach for a structural analysis was based on locating CpG islands within the amplified domain. This provided valuable probes for long-range mapping and for identifying amplified genes. A model for the DMs, showing them to be circular structures consisting of two identical, imperfect inverted repeat units, was constructed. This model is consistent with mechanisms of DNA amplification derived from studies of acquired drug resistance. Also, analysis of amplified CpG islands led to the isolation of a third gene and indicated that it is likely that no additional expressed genes occur within the amplified domain. To test the oncogenic potential of the three amplified genes, the ability of each to tumorigenically transform NIH3T3 and Rat-2 cells was assessed. Use of a protocol for stepwise amplification of transfected sequences permitted overexpression of each gene. One gene, mdm-2, consistently demonstrated oncogenic potential in multiple, independent trials. Further analysis has shown that mdm-2 is highly conserved, widely expressed and most likely encodes a zinc finger DNA binding protein. These results suggest that mdm-2 probably functions as a transcriptional activator in pathways of cellular proliferation. Overexpression of mdm-2 may abrogate mechanisms that regulate such pathways and activate its oncogenic potential