The long-term objective of the proposed study is to elucidate the factors responsible for overexpression of topoisomerase I in tumors with emphasis on the role of phosphorylation in the regulation of this important antitumor drug target. The preliminary studies show that the phosphorylation state of topoisomerase I varies over the cell cycle and changes with cellular growth status or RNA synthetic activity. These studies indicate the importance of phosphorylation in the regulation of this drug target.
The specific aims are to: 1) analyze the regulation of topoisomerase I by phosphorylation in tumorigenic and nontumorigenic cell lines; 2) characterize the phosphorylation of topoisomerase I; 3) analyze the expression of topoisomerase I in colon and upper GI tumors and matched normal mucosa with the aim of identifying correlates of topoisomerase I overexpression. Phosphopeptide maps of topoisomerase I will be generated to identify phosphorylations that change: over the cell cycle; when transcription is inhibited; as growth conditions are varied; when the phosphorylation of topoisomerase I is stimulated or inhibited by activators or inhibitors of components of signal transduction pathways; in tumorigenic as compared to nontumorigenic cell lines. Important phosphopeptides will be isolated and sequenced in order to determine if they correspond to consensus phosphorylation sites for specific protein kinases. Colon and upper GI tumors and matched normal mucosa, and tumor cell lines have defined alterations in tumorigenic genes will be assayed for topoisomerase I expression using northern and western blotting to determine whether steady state mRNA levels and specific tumorigenic alterations may be correlates of topoisomerase I overexpression.
