Topoisomerase II (topo II) is a ubiquitous enzyme that is required for many essential cell functions such as DNA replication, recombination and repair and chromosome segregation. Acting as a dimer, topoisomerase II passes a double stranded DNA segment through a transient double strand break in a second DNA strand to modify the topology of the DNA molecule. Topo II is also a major structural protein in the nuclear scaffold. There are two topo II genes: one expressing a 170 kDa topo IIalpha form, and the other coding for a 180 kDa topo IIbeta form. The crucial role of topo II in DNA replication and repair would indicate that the topo II genes are highly and specifically regulated. Several classes of antitumor drugs inhibit topoisomerase II through the stabilization of cleavable DNA-enzyme complexes. Cells that are selected for resistant to a single topo II- interacting drug often exhibit cross-resistance to many topo II inhibitors resulting in an altered-topoisomerase multidrug resistant phenotype (at- MDR). The first major objective of this project is to identify and characterize mutations in the topo II enzyme that result in the at-MDR phenotype. A first step in meeting this objective will be to confirm the direct correlation of an Arg to Gln substitution at a conserved position in a consensus ATP binding motif of topo IIalpha with the at-MDR phenotype exhibited by a VM-26-resistant CEM cell line. Mutations at other nucleotide binding sites in both topo IIalpha and topo IIbeta will be introduced by in vitro mutagenesis of a topo II-expression vector in order to further define the sites and structures involved in the development of a drug-resistant topo II enzyme. A panel of at-MDR cell lines and banked DNA from clinical samples will be screened to determine if similar topo II alterations are present. The other major objective is to characterize the regulatory controls for the expression of the two topoisomerase II genes in order to further understand the specific and/or overlapping functions of the two forms of topoisomerase II. The promoter regions of topo IIalpha and beta will be isolated and characterized to identify promoter sites and cis-acting elements. Trans-acting factors that effect regulation of topoisomerase II in association with cell cycle, proliferation status or transformation state will be identified and characterized. Analysis 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 this critical enzyme, and may provide information pertinent to the mechanism of action and regulation of topoisomerase II.
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