Taxol is a remarkably effective and increasingly widely-used drug for the treatment of cancer. The long-term objective of this work is to understand the antitumor mechanism of taxol. Despite considerable knowledge gained in the past several years about how taxol acts, its precise mechanism of antitumor action is far from understood. Our recent work indicates that the most potent antiproliferative action of taxol in a number of human tumor cell lines appears to be arrest of mitotic progression at the metaphase/anaphase transition. It is hypothesized that the primary mechanism by which taxol inhibits proliferation of sensitive tumor cells is by slowing or blocking cell cycle progression at mitosis through suppression of spindle microtubule dynamics, and that the tubulin isotype composition of a tumor cell may be an important determinant of sensitivity to taxol. To test these hypotheses, the following experimental aims are proposed: 1) To determine the mechanistic relationship between suppression of spindle microtubule dynamics by taxol and inhibition of cell cycle progression from metaphase to anaphase. The dynamics of mitotic spindle microtubules attached to chromosomes will be analyzed by video microscopy using a fluorescent kinetochore marker and by immuno-electron microscopy of tubulin incorporated into spindle microtubules and related to cell cycle blockage. 2) To examine the effects of taxol on the biochemical and ultrastructural interactions of microtubules with key spindle components, including quantitation of the number of microtubules attached to kinetochores. 3) To determine how the tubulin isotype composition of tumor cells contributes to the differential sensitivity of the cells to taxol. The effects of taxol on dynamics of microtubules composed of isotypically-purified abI tubulin combined with other isotypes and of microtubules in pairs of taxol-sensitive and taxol- resistant living cells. By stably transfecting human tumor cells with cDNAs for selected b-tubulin isotypes, the effects of altering levels of tubulin isotype expression on the sensitivity or resistance to taxol will be tested. Our recent work indicates that a common mechanism, i.e., suppression of microtubule dynamics, underlies the actions of many antimitotic drugs. It is important for the clinical use of taxol and for rational design of new drugs to understand precisely how taxol works at the molecular level. Understanding the linkage between microtubule dynamics, mitotic block, and resistance to taxol promises to lead to new drug targets that will improve the efficacy of chemotherapy.
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