ASSESSMENT OF MICROTUBULE STABILIZERS BY SEMIAUTOMATED IN-VITRO MICROTUBULE PROTEIN POLYMERIZATION AND MITOTIC BLOCK ASSAYS

Paclitaxel(Taxol) a clinically active anticancer agent, exerts its cyt otoxicity by inducing tubulin polymerization, leading to cellular mito tic block. In contrast, other antimitotic drugs, such as colchicine, : podophyllotoxin, and vinblastine, act by depolymerizing microtubules. We report here (a) a semiautomated assay which measures the tubulin-po lymerizing activity of paclitaxel analogs and (b) a cellular assay to measure the potential of these compounds to block cells in mitosis. Th e microtubule-polymerizing assay measured the turbidity of bovine brai n microtubule protein (MTP) polymerized by the test compound in a 96-w ell plate. We maximized the sensitivity of this assay by conducting th e polymerization reaction at 20 degrees C, at which temperature the ba seline reaction, i.e. the basic ability of the untreated MTP control t o polymerize, was minimal. At 20 degrees C, the effect of 0.05 mu g/ml of paclitaxel on MTP could be detected, whereas at 37 degrees C, > 1 mu g/ml of paclitaxel was required to detect a significant effect rela tive to untreated MTP. We describe the analysis of 1:he complex curves of MTP polymerization with varying concentrations of test compounds. The polymerization of microtubules leads to cells being blocked in mit osis. This mitotic blocking effect in intact cells was determined usin g a cell settling chamber which allowed eight samples to be deposited on a slide. This method required a smaller number of cells (10(3) - 10 (5)), maintained cell morphology, and allowed for rapid screening of s amples. The activity of several new paclitaxel analogs is reported.