STUDIES ON TRITIUM-LABELLED - TAXOL BINDING TO MICROTUBULES

Abstract

Reports from our laboratory have demonstrated that an experimental anti-tumor agent, taxol, enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules to depolymerization in vitro and in cells. To directly assess the interaction of taxol with tubulin and microtubules, we prepared tritium-labeled drug. Drug binding was correlated with microtubule morphology by electron microscopy. Microtubules assembled in the presence of ('3)H-taxol bind drug specifically with an apparent binding constant, K('app), = 8.7 x 10('-7) M and binding saturates with a calculated maximal binding ratio, B(,max), = 0.6 moles taxol bound/mole tubulin dimer. ('3)H-Taxol also binds and assembles phosphocellulose purified tubulin, suggesting that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimer concentration. Unlike ('3)H-colchicine, ('3)H-taxol does not bind to microtubule protein under conditions which do not support the assembly of microtubules even in the presence of taxol. Under assembly conditions,podophyllotoxin and vinblastine inhibit the binding of ('3)H-taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. ('3)H-Taxol binds to assembled microtubules with approximately the same stoichiometry as is found in microtubules that are assembled in the presence of drug. Unlabeled taxol competitively displaces ('3)H-taxol from microtubules, while podophyllotoxin, vinblastine and CaCl(,2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound ('3)H-taxol in the pellet remains constant. We conclude that taxol binds specifically to a polymerized form of tubulin with a stoichiometry approaching unity, thereby stabilizing microtubules to depolymerization.;Structure-activity relationships of natural and semisynthetic taxanes in vitro and in cell culture are reported. Cytotoxicity and in vitro activity require both an intact taxane ring and ester side chain at position C-13. Addition of acetyl moieties at positions 2' and 7 results in loss of in vitro activity but not cytotoxicity.