Mechanisms of drug resistance inv-src transformed rat fibroblasts.

Abstract

The central question of my dissertation is does cellular transformation increase the occurrence of gene amplification. I used a model system in which cellular transformation could be controlled by shifting the temperature. Rat-1 was the parental cell line and was not transformed at any temperature. The LA24 cell line carries an avian sarcoma virus with a temperature sensitive mutation in the v-src gene. These cells are transformed at 35°C, but become nontransformed at 40°C. Measurements of cell transformation included (1) growth on plastic, (2) growth in soft agar and (3) v-src RNA and protein expression. My first experiments were to examine the frequency and mechanisms of MTX and colchicine resistant variants. The frequency in generating MTX resistant clones was the same in transformed and nontransformed LA24 cells. However, dihydrofolate reductase gene amplification was the preferred mechanism of MTX resistance in transformed LA24 clones. The frequency in generating colchicine resistant clones was no different in transformed and nontransformed LA24 cells. P-glycoprotein gene amplification was also the preferred mechanism of colchicine resistance in transformed LA24 clones. To directly study the mutation rate of colchicine resistant variants on transformed and nontransformed LA24 cells, Luria-Delbruck fluctuation analysis was performed. Colchicine resistant variants were derived from a single cell that were grown to a population size of 10⁵ cells; they were then selected with colchicine. The data showed no increase in the mutation rate of transformed LA24 cells compared to untransformed cells upon selection in colchicine. In summary, I have shown that v-src transformation in Rat-1 fibroblasts causes a high frequency of P-glycoprotein gene amplification when selected with colchicine, and a high frequency of dihydrofolate reductase gene amplification when selected with MTX. Even though gene amplification is enhanced by v-src transformation, the frequency of generating colchicine and MTX variants is not related to cellular transformation state.