Tubulin from paclitaxel-resistant cells as a probe for novel antimicrotubule agents

Abstract

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 resistance to paclitaxel. Methods: The assay measured the promotion of microtubule polymerization when target agents were added to solutions containing tubulin purified from cultured cells. Tubulin was prepared from PTX-sensitive 1A9 ovarian carcinoma cells and from a PTX-resistant clone. Polymerization was monitored spectrophotometrically and validated by electron microscopy. Results: Exposure of tubulin isolated from PTX-sensitive 1A9 ovarian carcinoma cells to substoichiometric PTX resulted in polymerization equivalent to that observed with brain tubulin. In contrast, tubulin from a PTX-resistant 1A9 clone failed to polymerize under identical conditions. If a C-2-modified analog of PTX (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substituted for PTX in the same experiment, the tubulins from both sensitive and resistant cells polymerized as well as brain tubulin. As predicted from these results, the PTX analog was nearly as cytotoxic to the PTX-resistant cells as it was to the parental cells: the relative resistance of the resistant cells compared to the parental is only 3–5-fold for the PTX analog versus 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 paclitaxel resistance.