ISSN:
1432-0843
Keywords:
Key words Microtubule stabilizer
;
Microtubule polymerization
;
Mitotic block
;
Automation
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract Paclitaxel (Taxol) a clinically active anticancer agent, exerts its cytotoxicity by inducing tubulin polymerization, leading to cellular mitotic 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-polymerizing activity of paclitaxel analogs and (b) a cellular assay to measure the potential of these compounds to block cells in mitosis. The microtubule-polymerizing assay measured the turbidity of bovine brain microtubule protein (MTP) polymerized by the test compound in a 96-well plate. We maximized the sensitivity of this assay by conducting the polymerization reaction at 20 °C, at which temperature the baseline reaction, i.e. the basic ability of the untreated MTP control to polymerize, was minimal. At 20 °C, the effect of 0.05 μg/ml of paclitaxel on MTP could be detected, whereas at 37 °C, 〉1 μg/ml of paclitaxel was required to detect a significant effect relative to untreated MTP. We describe the analysis of the complex curves of MTP polymerization with varying concentrations of test compounds. The polymerization of microtubules leads to cells being blocked in mitosis. This mitotic blocking effect in intact cells was determined using a cell settling chamber which allowed eight samples to be deposited on a slide. This method required a smaller number of cells (103–105), maintained cell morphology, and allowed for rapid screening of samples. The activity of several new paclitaxel analogs is reported.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002800050705
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