14-3-3 IMPLICATIONS IN TOPOISOMERASE II PHARMACOLOGY

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DESCRIPTION: DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme which catalyzes the interconversion of the various tertiary structures of DNA. This enzyme is absolutely essential to cellular proliferation since it decatenates physically interlocked DNA prior to mitosis. Topo II is also a clinically relevant target for a widely useful class of chemotherapeutic drugs. The study of topo II biochemistry has revealed several mechanisms by which tumor cells respond to, or evade, the cytotoxic effects of the topo-II-directed anticancer drugs. In the past three years, the Principal Investigator has been developing the hypothesis that topo II activity can be mediated by protein-protein interactions and modulation of these proteins may also affect tumor cell response to topo II poisons. Others have described topo II interactive proteins (TIPs) from yeast and Drosophila that are necessary for faithful chromosomal segregation. A human homolog of the yeast TIP, Sgs1, is likely to be the causative gene that is altered in Bloom's syndrome. Therefore, study of topo II protein interactions has revealed unappreciated roles for the enzyme in human neoplasia. The Principal Investigator's studies propose to focus instead on the role of topo II protein interactions in the cytotoxic action of topo-II-directed drugs. Toward this aim, a human HeLa cell cDNA expression library has been screened for proteins that interact with the C-terminus of the major alpha form of human topo II. One of the TIPs has been identified as the epsilon isoform of human 14-3-3 protein. 14-3-3 proteins, an unusually highly conserved protein family found in plants, fungi, and mammals, have been implicated repeatedly in numerous protooncogenic and oncogenic cellular signaling pathways. 14-3-3 proteins also appear to sequester the apoptotic death agonist, Bad, in IL-3 protection of T cells. The Principal Investigator therefore proposes 1) to test whether the 14-3-3/topo II interaction is authentic and occurs in intact cells and 2) to investigate the biochemical and pharmacological consequences of this interaction in tumor cells overexpressing 14-3-3 protein. The high level of conservation of 14-3-3 proteins, their implication in cellular growth signaling and now apoptosis, suggest that 14-3-3/topo II interactions are likely to be biologically significant. Most importantly, these interactions may also modulate tumor cell susceptibility to topo-II-directed antitumor drugs.