The aim of this project is to the test hypothesis that transformed cells have higher rates of genome instability than non-transformed cells. The proposed studies will examine a model system in which cellular transformation is altered by shifting the temperature of incubation. LA24 is a rat cell line which has been infected with an avian sarcoma virus containing a temperature sensitive mutation of the v-src gene. At the permissive temperature, cell morphology is transformed, while at the restrictive temperature, cell morphology is normal. We propose to study the frequency of gene amplification in LA24 cells that are transformed and non- transformed by selecting for methotrexate and colchicine resistant clones. A common mechanism for methotrexate and colchicine resistance is amplification of the target gene. We will calculate the frequency of methotrexate- and colchicine-resistant mutants by Luria-Delbruck fluctuation analysis in transformed and non-transformed cells. Individual clones produced by fluctuation analysis will be expanded and will be analyzed by molecular biologic techniques to see if gene amplification or altered RNA expression for either the dihydrofolate reductase gene or p-glycoprotein genes occur in resistant clones. DNA synthesis inhibitors will be used to perturb exponentially growing cultures of transformed and non-transformed cells to see if enhancement of gene amplification by DNA synthesis inhibition occurs more frequently in transformed cells. These studies will allow us to test whether transformed cells have higher rates of genome instability and whether genome instability plays an important role in acquired drug resistance.