TUBULIN FROM PACLITAXEL-RESISTANT CELLS AS A PROBE FOR NOVEL ANTIMICROTUBULE AGENTS

Purpose: Treatment with paclitaxel (PTX) can lead to the appearance of drug resistance with accompanying changes in tubulin. The purpose of this study was to develop an assay for microtubule-active agents that are able to circumvent changes in tubulin that result in acquired resi stance to paclitaxel. Methods: The assay measured the promotion of mic rotubule polymerization when target agents were added to solutions con taining tubulin purified from cultured cells. Tubulin was prepared fro m PTX-sensitive 1A9 ovarian carcinoma cells and from a PTX-resistant c lone. Polymerization was monitored spectrophotometrically and validate d by electron microscopy. Results: Exposure of tubulin isolated from P TX-sensitive 1A9 ovarian carcinoma cells to substoichiometric PTX resu lted in polymerization equivalent to that observed with brain tubulin. In contrast, tubulin from a PTX-resistant 1A9 clone failed to polymer ize under identical conditions. If a C-2-modified analog of PTX (2-deb enzoyl-2-(m-azidobenzoyl)paclitaxel) was substituted for PTX in the sa me experiment. the tubulins from both sensitive and resistant cells po lymerized as well as brain tubulin. As predicted from these results. t he PTX analog was nearly as cytotoxic to the PTX-resistant cells as it was to the parental cells: the relative resistance of the resistant c ells compared to the parental is only 3-5-fold for the PTX analog vers us 25-30-fold for PTX. Conclusion: Polymerization of purified tubulin from the paclitaxel-resistant cells provided an assay for agents able to circumvent the tubulin alterations that result in acquired paclitax el resistance.