The levels of topoisomerase Ha (topo Ha) are elevated in many tumors, contributing to increased sensitivity to a number of clinical anti-cancer drugs. Clearly, the regulation of this protein is of critical importance. We have studied the regulation of topo Ha expression with the use of time lapse and fluorescence imaging techniques which allow cell cycle analyses without cell synchronization. A portion of the topo Ha promoter was identified which is remarkably dependent upon and responsive to Ras activity. Nevertheless, topo IIalpha protein levels in cycling cells were not altered by this oncogene. Only a small population of slowly cycling NJH3T3 cells increased topo Ha levels following injection of Ras protein. Interestingly, these slowly cycling cells containing Ras activity were highly sensitive to the anti-topo II drug etoposide. This proposal is designed to extend our studies of topo IIalpha regulation by testing models to explain these results. First, we will determine why topo IIalpha protein levels are not altered by Ras activity in cycling cells, despite a ras responsive region in the promoter. Is the difference between promoter activity and protein expression due to alterations in topo Ha protein or mRNA stability? Is it possible that other regions of the topo IIalpha promoter suppress the ras responsive region in cycling cells? These experiments will utilize traditional biochemical procedures, as well as our new imaging techniques. Next, we will analyze slowly cycling cells. These cells behave similarly to many tumor cells. While they retain proliferative capacity, they are temporarily located in a prolonged Gi-phase. Experiments involving time lapse analysis, studies of signaling molecules, and analysis of promoter activity will be performed in these cells to determine how topo Ha regulation differs in these cells compared to cycling NIH3T3 cells. Finally, we will perform experiments to demonstrate that these slowly cycling NlH3T3 cells serve as a culture model for tumor cells. It should be emphasized that most cells of a tumor are not cycling at any give time, but must, nevertheless, be sensitive to an anti-tumor treatment. Perhaps our studies of these slowly cycling cells will yield information of value in understanding these temporarily non-cycling tumor cells and their sensitivity to drug treatment.
