This work is a continuation of efforts to elucidate the role of DNA topolsomerase II in mediating the anti-tumor effects of a variety of important drugs. Based on the work funded by this grant for the past 10 years as well as the work of others, it is now clear that topolsomerase II is the major intracellular target for a variety of intercalating agents and epipodophyllotoxins. Identification of this target and this biological behavior is now beginning to suggest new strategies for optimizing therapy with topoisomerase inhibitors. Work proposed in the current application will address three major unresolved issues relating to topoisomerase II and DNA? The vpmR cell line is resistant to intercalating agents (1A) and epipodophyllotoxins (EPT) and bears a topoisomerase II which, upon purification, exhibits resistance to these agents as well. In this application, characterization of the vpmR enzyme will be extended by cloning, sequencing, and comparing the cDNA's for wild type and vpmR topoisomerase II. This will allow identification of the mutation sited. The enzyme's essential role as target of EPTs will be confirmed by expressing the wild type gene in the vpmR cell line under an inducible promoter. The transfected cells will then be assayed for sensitivity to VP-16 under induced and non-induced conditions. Once the mutation site of the vpmR enzyme has been identified, monoclonal antibodies to synthetic peptides representing this area of the gene will be generated and tested for neutralization capability with respect to cleavable complex formation and enzyme catalytic activity. 2. How does tumor environment affect regulation of topoisomerase II and resistance to topoisomerase II inhibitors? The basis for the loss of topoisomerase II in mammalian cells exposed to inducers of grips will be defined. In addition, L1210 leukemia cells which become resistant to topoisomerase II inhibitors after exposure to grp inducers without loss of topoisomerase II will be characterized. 3. Why is transient expression of the cleavable complex lethal? the possibility that transient topoisomerase inhibition causes cell death by illegitimate DNA recombination will be explored using an experimental model amenable to both in vivo and in vitro assay. In addition, since G2 phase arrest is an important biological sequela of topoisomerase II inhibition, the basis for this arrest will be explored by studying the expression of recently identified proteins required for movement of cells from G2 to mitosis.
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