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14 3 3 Implications in Topoisomerase II Pharmacology

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DNA topoisomerase II (topo II) is a ubiquitous nuclear enzyme that 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 class of chemotherapeutic drugs used widely to treat cancers of the lung, breast, and prostate. The study of topo II biochemistry has revealed several mechanisms by which tumor cells respond to, or evade, the cytotoxic effects of topo Il-directed antitumor drugs. We have developed the hypothesis that topo II activity can be mediated by protein-protein interactions and modulation of these proteins may influence tumor cell response to topo II poisons. Since we originally identified the transcription factors CREB, ATF-2, and c-Jun as topo lI-interactive proteins (TIPs) that stimulate topo LI catalytic activity, others have described TIPs from yeast and Drosophila that are necessary for faithful chromosomal segregation. A human homolog of the yeast TIP, Sgsl, is likely to be the gene that when mutated is responsible for the high tumor frequency in Bloom's syndrome patients and, most recently, the retinoblastoma tumor suppressor gene product Rb has also been shown recently to bind topo II and inhibit its catalytic activity. Therefore, the study of topo II protein-protein interactions has revealed previously unappreciated roles for the enzyme in human neoplasia. This continuation proposal expands on our investigation of topo II protein-protein interactions in the cytotoxic action of topo Il-directed drugs. We have previously identified the epsilon (e) isoform of human 14-3-3 protein as a TIP from screening a human HeLa cell cDNA library with a protein probe comprising a large, C-terminal fragment of the major a isoform of human topo II. 14-3-3 proteins, an unusually highly conserved protein family of distinct gene products found across plants, fungi, and mammals, have been implicated in proto-oncogenic cellular signaling pathways, the G2 DNA damage checkpoint, and in apoptosis regulation. However, mammalian cells have maintained 7 distinct 14-3-3 gene products, perhaps indicating that each isoform possesses unique functions. A very recently described function of some 14-3-3 isoforms is in directing the subcellular compartmentalization of other proteins: either in nuclear export (for cdc25 phosphatase) or, conversely, in nuclear import (for telomerase and the homeobox transcription factor, TLX-2). Using reciprocal affinity chromatography and co-immunoprecipitation methods, we have shown that 14-3-3e, but NOT the G2 arrest protein 14-3-3E, directly binds human topo lla. Functionally, these interactions with 14-3-3e, but NOT 14-3-3a, lead to in vitro inhibition of topo II DNA binding activity and a modest attenuation of etoposide-stabilized DNA damage in purified enzyme and isolated nuclear comet assays. We propose to test a two-part hypothesis that 1) distinct structural determinants within topo lla and 14-3-3 proteins lead to these isoform specific effects and the interactions may be influenced by specific cell cycle and DNA damage dependent phosphorylation events and, 2) that site-directed mutations in these structural motifs (on either topo II or 14-3-3e) or ectopic expression of specific 14-3-3 isoforms or their dominant negative counterparts can influence subcellular compartmentalization of topo lIa in vivo and may play a role in the recently recognized cytoplasmic accumulation of the enzyme and topo Il-drug resistance of various tumor cell lines in plateau phase. Acknowledging that some 14-3-3 isoforms may influence topo II drug efficacy independently of physically interacting with topo II, appropriate controls and alternative paradigms will be employed to distinguish between effects of 14-3-3 on cell cycle distribution or apoptosis induction relative to those directly relating to protein-protein interactions with topo IIa. Since current data suggests that 14-3-3e may protect cells from topo lI-directed antitumor drugs, the long term goal of this work is to identify either structural motifs in either topo II or 14-3-3 that may be targeted by small molecules or peptidomimetics, or kinase inhibitors that disrupt their interaction, to enhance the antitumor efficacy of topo II-directed drugs and/or overcome intrinsic or acquired resistance to these agents.
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