Taxol, a complex diterpene isolated from extracts of the bark of the western yew Taxus brevifolia, is a compound that shows great promise as a cancer chemotherapeutic agent. Taxol has recently been found to have exciting potential for the treatment of advanced ovarian carcinoma, and the drug may be effective against other solid tumors including melanoma and lung, gastric, breast, colon, and cervical carcinomas. The main target of taxol in cells appears to be microtubules, but the mechanism appears to differ significantly from the mechanisms of other drugs that affect microtubules. Taxol inhibits the progression of several tumor cells at metaphase of mitosis, but the precise molecular mechanism of action and the reasons for its effectiveness against certain solid tumors are not understood. Based upon work in our laboratory, we hypothesize that inhibition of tubulin addition and loss dynamics at the ends of specific mitotic spindle microtubule populations is responsible for inhibition of cells at mitosis, and possibly, for the cytotoxic effects of taxol. With this hypothesis in mind, we will determine the relationship between inhibition of mitosis by taxol and cytotoxicity caused by taxol in HeLa cells and in a number of other human tumor cell lines. We will use video microscopy and immunofluorescence microscopy to analyze mitotic blockage, multinucleation, and microtubule organization and a microculture colorimetric assay to assess cytotoxicity. To elucidate how taxol inhibits mitosis, we will analyze the effects of taxol on the dynamics of tubulin exchange at the ends of spindle microtubules in HeLa cells in relation to inhibition of spindle function. Studies will involve labelling spindle microtubule subsets in living cells by microinjecting biotin-labelled HeLa cell tubulin and visualizing the dynamics of the microtubules in control and taxol-blocked spindles by immunofluorescence light microscopy and by immunoelectron microscopy. We will investigate the contribution to mitotic inhibition of taxol-induced microtubule bundling and altered interactions of microtubules with other cell proteins. Finally, by computer-enhanced video microscopy, we will characterize quantitatively the effects of taxol on the dynamics of tubulin exchange at microtubule ends in vitro.
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