Taxol and the Vinca alkaloids are highly effective cancer chemotherapeutic drugs. Their primary targets in tumor cells appear to be dynamic spindle microtubules required for proper segregation of chromosomes to the daughter cells at mitosis. Despite considerable knowledge gained recently about how taxol and Vinca alkaloids act to inhibit mitotic progression by suppression of spindle microtubule dynamics, their precise mechanisms of action and the reasons that tumor cells are resistant or sensitive to the drugs are far from understood. It is hypothesized that a) the tubulin isotype composition of the microtubules in tumor cells, the composition of microtubule-associated mitotic regulatory proteins, and mutated forms of tubulin may all be important determinants of sensitivity to antimitotic drugs, and b) that microtubule-targeted antitumor drugs inhibit proliferation of, and kill, tumor cells in several ways, not only by suppressing spindle microtubule dynamics and disrupting mitotic spindle function, but by binding to tubulin components of the centrosomes (spindle poles), which could lead to aberrant spindle function and aberrant cytokinesis. To test these hypotheses, the following experimental aims are proposed: 1) To determine to what extent the beta-tubulin isotype composition, tubulin mutations, and regulatory microtubule-associated proteins (MCAK, stathmin, and survivin) affect the resistance of cells to these drugs. 2) To elucidate the mechanistic relationship between altered expression of mitotic regulatory proteins (using RNAi), suppression of mitotic spindle microtubule and centromere dynamics, inhibition of tumor cell proliferation and cytokinesis, induction of aneuploidy and cell killing by microtubule-targeted drugs, and 3) To elucidate the interactions between microtubule-targeted drugs and centrosomal tubulins and their effects on tumor cell proliferation and killing. The binding of radiolabeled taxol or vinblastine to purified centrosomal tubulins, the effects of antimitotic drugs on the organization of gamma-, delta-, and epsilon-tubulins by light, electron and confocal microscopy of fixed and living cells, on the nucleation of microtubules at centrosomes, and on centriolar migration prior to cytokinesis will be determined. The results will elucidate mechanisms and predict possible clinical efficacy of combination therapies directed at modulating mitotic microtubule regulatory proteins in combination with taxanes and Vinca alkaloids.
Showing the most recent 10 out of 56 publications
