The long-term objectives of our research are to acquire a thorough understanding of the mechanisms of action and of resistance to Taxol, an antitumor agent that is known to be efficacious in the treatment of human cancer. An intense interest in drug development is now concentrated on the epothilones and discodermolide, natural products whose chemical structures are distinct from Taxol but whose mechanisms of action and resistance have similarities, but are definitely not identical, to those of Taxol. Our plan is to dissect the similarities and differences between these three microtubule stabilizing drugs. The epothilones and/or discodermolide may have superior clinical activity compared to Taxol, particularly in tumors resistant to taxanes. An overriding theme of this grant application is the role that low doses of Taxol, the epothilones and discodermolide play in the killing of cancer cells. This emphasis on low drug dosage is a new focus for our laboratory that developed as it became clear that Taxol was responsible for cell death at low doses of drug that did not block cells in mitosis. There are undoubtedly diverse pathways by which low doses of Taxol kill cancer cells. The availability of preclinical data is essential and must contribute to the decision to move drugs into clinical trials.
The specific aims of this proposal are to: 1. Determine the mechanism(s) by which cells treated with low concentrations of Taxol escape the spindle checkpoint. HeLa cells will be transfected with MAD2, a checkpoint component, to determine if its overexpression can rescue from cell death, those cells that escape from the mitotic checkpoint. The relationship between overexpression of the protein CENP-E, a component of the mitotic checkpoint, and drug resistance will also be investigated. 2. Examine the contribution of p53-dependent apoptosis to cytotoxicity caused by low concentrations of Taxol. Determine if aberrant mitosis leads to p53-dependent apoptosis. DNA microarrays will be used to survey and compare gene expression profiles of A549 cells treated with different concentrations of microtubule stabilizing drugs. 3. Investigate how the expression level and the phosphorylation status of the microtubule regulatory proteins stathmin and MAP4 in cancer cells relate to differential sensitivity to microtubule-stabilizing agents, and how these agents affect the expression profile of these proteins.
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