ALTERED TOPOISOMERASE IN ATYPICAL MULTIDRUG RESISTANCE

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Multiple drug resistance (MDR) refers to the fact that tumor cells can display a broad cross-resistance to a variety of "natural product" compounds of widely differing structure and mechanism of action. One type of MDR (Pgp-MDR) has been characterized by decreased drug accumulation and overexpression of the gene coding for a large membrane glycoprotein, termed, Pgp. We have developed progressively-VM-26-resistant human leukemic cell lines (CEM/Vm-1 and CEM/VM-1-5) with an "atypical" pattern of cross-resistance and cellular pharmacology. These cells retain sensitivity to the Vinca alkaloids, are not impaired in their transport of drugs, and do not overexpress the pgp gene. There is now considerable evidence that "atypical-MDR" (at-MDR) is due to an alteration in the activity of the essential nuclear enzyme, topoisomerase II, an unique and important target for many anticancer agents. Whether this alteration is a consequence of a mutation in the gene coding for topoisomerase II or in a gene that modulates it is not clear. The long-term objective of the proposed work is to determine the molecular basis of at-MDR. To accomplish this goal, five specific aims will be addressed. The first aim is to purify and characterize the topoisomerase II protein from at-MDR cells. A second aim is to characterize the topoisomerase II mRNA and its regulation in the at-MDR cells using a topoisomerase II-specific nucleotide probe. The third aim is to identify specific mutations in the at-MDR topoisomerase II mRNA using nuclease mapping techniques and cDNA sequence determinations. The fourth aim is to confirm that any identified alterations in the at-MDR topoisomerase II are indeed responsible for the expression of the at-MDR phenotype. Finally, if the topoisomerase II mRNA and enzyme is unaltered in the resistant cells, the fifth aim is to characterize by biochemical and molecular procedures the nature of the altered modulator of topoisomerase II. Characterization of mutations in the at-MDR cells that are responsible for the alterations in topoisomerase II activity will yield information about the interaction of clinically important drugs with the critical enzyme, may provide information pertinent to the mechanism of action of topoisomerase II, and may provide insights into the design of new drugs and therapeutic modalities to overcome this form of resistance.