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This version was published on February 1, 2006
Journal of Biomolecular Screening, Vol. 11, No. 1, 82-89 (2006)
DOI: 10.1177/1087057105282300

A Radioligand Binding Assay for Antitubulin Activity in Tumor Cells

David H. Young

Dow Agro Sciences LLC, Indianapolis, IN

Fernando M. Rubio

Eastwoods Consulting, Boylston, MA

Paul O. Danis

Applied Biosystems, Framingham, MA

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 ß-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to ß-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 inwhole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site andwith 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 3HRH-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


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