Taxol binds to cellular microtubules.

Abstract

ABSlRACT Taxol is a low molecular weight plant derivative which enhances microtubule assembly in vitro and has the unique ability to promote the formation of discrete microtubule bundles in cells. Tritium-labeled taxol binds directly to microtubules in vitro with a stoichiometry approaching one (Parness, J., and S. 8. Horwitz, 1981, J. Cell Biol. 91:479-487). We now report studies in cells on the binding of [aH]taxol and the formation of microtubule bundles.[aH]Taxol binds to the macrophagelike cell line, J774.2, in a specific and saturable manner. Scatchard analysis of the specific binding data demonstrates a single set of high affinity binding sites. Maximal binding occurs at drug concentrations which produce maximal growth inhibition. Conditions which depolymerize microtubles in intact and extracted cells as determined by tubulin immunofluorescence inhibit the binding of [aH]taxol. This strongly suggests that taxol binds specifically to cellular microtubules. Extraction with 0.1% Nonidet P-40 or depletion of cellular ATP by treatment with 10 mM NaNa prevents the characteristic taxol-induced bundle formation. The binding of [aH]taxol, however, is retained under these conditions. Thus, there must be specific cellular mechanisms which are required for bundle formation, in addition to the direct binding of taxol to cytoplasmic microtubules.Taxol, a novel diterpenoid, was originally isolated from the stem bark of the western yew, Taxus brevifolia, and has been found also in the leaves, stems and roots of a variety of other Taxus species (35,63). The drug is a complex ester, shown to be a taxane derivative containing a rare oxetan ring, and is the first compound of this type to have antileukemic and tumor inhibitory properties. Early work in our laboratory demonstrated that the drug inhibits replication of HeLa cells (47). Studies with P-388 cells taken from taxol-treated mice identified taxol as a mitotic spindle poison (22).Studies in vitro have shown that in marked contrast to other antimitotic drugs, taxol enhances both the rate and yield of microtubule assembly. The critical concentration of microtubule protein required for assembly is reduced, and the microtubules formed are stable to depolymerization by calcium or cold (47). Taxol also assembles tubulin under conditions in which polymerization would not normally occur. These include the absence of microtubule-associated proteins, 36S tubulincontaining ring structures, exogenously added guanosine 5' triphosphate, or organic buffer (27, 50). Taxol induces microtubule assembly even at low temperatures (24,47,60). Microtubules assembled to steady state and then incubated with taxol become resistant to depolymerization by calcium, suggesting that there is a taxoi-binding site on the microtubule. Maximal effects in vitro are seen at taxol concentrations stoichiometric with the tubulin dimer concentration (49). Recent work with [3H]taxol confirms that the drug does bind to the microtubule, and that such binding occurs with a stoichiometry approaching one (41).HeLa ...

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