A MUTANT YEAST TOPOISOMERASE-II (TOP2G437S) WITH DIFFERENTIAL SENSITIVITY TO ANTICANCER DRUGS IN THE PRESENCE AND ABSENCE OF ATP

To further characterize the mechanistic basis for cellular resistance/ hypersensitivity to anticancer drugs, a yeast genetic system was used to select a mutant type II topoisomerase that conferred cellular resis tance to CP-115,953, amsacrine, etoposide, and ellipticine, The mutant enzyme contained a single point mutation that converted Gly(437) --> Ser (top2G437S), Purified top2G437S displayed wild-type enzymatic acti vity in the absence of drugs but exhibited two properties that were no t predicted by the cellular resistance phenotype, First, in the absenc e of ATP, it was hypersensitive to all of the drugs examined and hyper sensitivity correlated with increased drug affinity. Second, in the pr esence of ATP, top2G437S lost its hypersensitivity and displayed wildt ype drug sensitivity. Since the resistance of yeast harboring top2G437 S could not be explained by alterations in enzyme-drug interactions, p hysiological levels of topoisomerase II were determined. The Gly(437) --> Ser mutation reduced the stability of topoisomerase II and decreas ed the cellular concentration of the enzyme. These findings suggest th at the physiological drug resistance phenotype conferred by top2G437S results primarily from its decreased stability. This study highlights the need to analyze both the biochemistry and the physiology of topois omerase II mutants with altered drug sensitivity in order to define th e mechanistic bridge that links enzyme function to cellular phenotype.