The primary emphasis of this proposal is to study the regulation of dihydrofolate reductase (Dhfr) gene expression during changes in the proliferative state of the cell. The steady state levels and stability of various mouse Dhfr mRNA species will be analyzed during growth stimulation and quiescence in both the amplified and un-amplified states. Using cell lines with amplified copies of the Dhfr gene has several advantages: i)Dhfr MRNA can easily be detected due to the elevated level of expression in amplified cells and ii)Dhfr expression in the amplified state extends our understanding of its regulation in tumor cells which have used gene amplification as a method of resistance to folate antagonists such as methotrexate. However, Dhfr gene expression in an unamplified cell may be different than in the amplified state. There- fore, Dhfr gene expression will also be studied in the unamplified state. It has previously been difficult to study Dhfr expression in unamplified cells, but the recent development of quantitative PCR to detect mRNA has made this feasible. The sensitivity of the PCR assay will allow the analysis of thiouridine pulsed labeled RNA separated on organomercurial columns, thereby enabling us to study both stable and newly made Dhfr RNA. Secondly, various changes will be made in the primary structure of the Dhfr gene. Yeast genetics will be employed to make changes in the mouse Dhfr gene contained in a YAC clone. These YAC constructs will be transferred to Dhfr-deficient CHO cells and Dhfr expression in normal and tumorigenic cells, and additional insight into the role of Dhfr gene expression in the development of drug resistance in neoplastic cell.
