Natural products have historically been the source of most of the microtubule (MT)-targeting small molecules whose properties have allowed them to become useful drugs. That remains true of most but not all of the compounds that we have used in this study. These include the clinically established MT-active drugs colchicine, combretastatin, vincristine, taxol, and others. Almost all such agents were developed first in pre-clinical research that included in vitro studies of the effect of the compounds on polymerization of tubulin to microtubules as well as the effect of such compounds on cell behavior, especially examining the ability of the compounds to disrupt mitosis through effects on the MT arrays that comprise the mitotic spindle. Indeed the ability to cause mitotic arrest in rapidly growing cell cultures in the laboratory is often considered to be an assay of the principal mechanism of these drugs. Nonetheless we have argued that mitosis is not the central target of these drugs in patient tumors. Human tumors grow very slowly compared to laboratory cultures and mitosis is rare, and therefore is not an abundant target. Microtubules are abundant in non-mitotic as well as mitotic cells, however, and hence we have explored the nature of the microtubule functions that these drugs do target. One of those is intracellular transport, which occurs constantly, throughout the cell cycle, and which is required for many central parts of cell metabolism. This traffic is directional and requires the array of organized microtubules to coordinate the process. One aspect of this is the requirement of directional transport to move enzymes from the cytoplasm to the nucleus in response to DNA damage. This occurs on microtubules, and is disrupted by anti-microtubule drugs. We discovered that this interference with DNA repair processes underlies the well-verified potentiation by microtubule-targeting drugs of the anti-cancer activity of DNA-damaging chemotherapy. This insight may allow better scheduling of therapy and rational development of new therapeutic drug combinations. Additionally we have found that tubulin and microtubules have important roles in the regulation of cell oxidative metabolism through interaction with mitochondria. It has been often reported that microtubule-targeting drugs cause release of bursts of reactive oxygen species. These oxidative bursts result in production of carbonyl residues on cellular proteins. In order to study thus better, we have developed a new fluorescent reporter molecule which reacts covalently with carbonyl residues in cellular macromolecules. This allows the quantitation of oxidative events, the imaging of the distribution of these events in the cell, and potentially the isolation of the oxidized macromolecules (through pull-down experiments).

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U.S. National Inst/Child Hlth/Human Dev
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