The aim of this proposal is to define the pharmacologic, biochemical, and molecular determinants of the resistance of a line of human leukemia cells called HL-60/AMSA to topoisomerase (topo) II-reactive DNA intercalating agents. Purified HL-60/AMSA topo II is resistant to the inhibiting actions of the intercalator m-AMSA. HL-60/AMSA cells and topo II are also resistant to a number of other topo II-reactive DNA intercalators. However HL-60/AMSA cells and topo II are not resistant to the nonintercalating topo II-DNA-drug interaction that eventually produce cytolysis are intact in HL- 60/AMSA, but the initial interaction is clearly different for intercalators and nonintercalators, and altered in the case of the HL-60/AMSA topo II- intercalator-DNA interaction. We will use assays devised by Osheroff and purified HL-60 and HL-60/AMSA topo II to identify the precise steps in the topo II topoisomerization cycle affected by intercalators and etoposide with particular focus on the steps affected differently for the two drug classes. This will identify differences between the drug effects on these enzymes that could be critical biochemical determinants of HL-60/AMSA's drug resistance. We will amplify and clone the HL-60 and HL-60/AMSA topo II genes. We will sequence these genes looking for mutations in the HL-60/AMSA sequence. Such mutations are likely as rearrangements in the HL-60/AMSA topo II gene have been found and HL-60 contains only one topo II allele so that a change in this single allele could have produced the resistance. To prove that the mutation or mutations produce the drug resistance we will transfect the topo II gene into mammalian and yeast systems in which cellular and biochemical pharmacologic studies can test whether the topo II genes confer m-AMSA-sensitivity or resistance on the transfectants. This will identify the molecular determinants of HL-60/AMSA's drug resistance. The manner in which HL-60/AMSA arose, repeated intermittent treatment with a DNA-reactive drug, is analagous to the way human tumor resistance may arise in the clinic. Defining the precise biochemical and molecular determinants of topo II-reactive drug resistance will give insight into the process of induced drug resistance, identify the key sites of drug cytolytic action, and suggest ways to design new agents to overcome drug resistance.
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