The overall goal of this proposal is to investigate the relationship between the mutations in the topoisomerase II (topo II) gene and resistance to antineoplastic drugs. topo II is the target for many clinically useful drugs. Mutations in this enzyme are associated with drug for many clinically useful drugs. Mutations in this enzyme are associated with drug resistance in human and hamster cell lines. Despite the facts that these cell lines are of different origins and show different phenotypic patterns, four of the five characterized mutations are located in the region 426-493 of the enzyme. Because of the essential nature of topo II, it is likely that only the mutations interfering with the drug-enzyme interaction without significantly affecting the topo II functions will be viable and confer drug resistance. This strongly suggests that this region interacts directly or indirectly with these drugs although other regions may also be involved in this interaction. Structure-activity analyzes of these drugs do not provide a clear understanding of the drug-enzyme interaction, or to the mechanism of topo II mutation-mediated drug resistance. A systematic analysis of the target of these drugs, the topo II enzyme, will shed some light on this area. The identification of drug-resistant mutations in the topo II provides a starting pint for such an analysis. Some of these mutations confer resistance to a multiple topo II acting drugs while others are only resistant to a single or a few drugs. This suggests that amino acid changes may involve sites in the enzyme affecting interactions with either common or specific structures of these drugs. These drug-resistant mutations induce multiple changes in the protein structure. However, it is not clear that which of these changes is essential for the altered phenotype. In order to have a clear understanding of topo II mutation- mediated drug resistance, the amino acid residues at the known mutation sites will be altered by site-directed mutagenesis to effect a specific structural change while minimally affecting others. The resulting mutant enzymes will be overexpressed and purified. The impact of these specific mutations on the protein structure, and its effects on drug resistance as indicated by the level of drug-induced DNA cleavage (DIDC) will be analyzed. The relationship between the topo II mutations and drug resistance may be elucidated. This may play a role in overcoming the resistance to topo II targeting drugs in cancer chemotherapy.
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