A Radioligand Binding Assay for Antitubulin Activity in Tumor Cells

Rohm and Haas Company, Spring House, PA.

^* To whom correspondence should be addressed. E-mail: dyoung{at}dow.com.

	Abstract

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the -subunit of tubulin in the colchicine binding region. Binding of ³H-RH-5854 to -tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. ³H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of ³H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of ³H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the ³H-RH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.

Key Words: tubulin, benzamide, RH-5854, cell-based binding assay, tumor cells

First published on November 28, 2005, doi:10.1177/1087057105282300

Journal of Biomolecular Screening 2006;11:82.

A more recent version of this article appeared on February 1, 2006


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