title: A radioligand binding assay for antitubulin activity in tumor cells.
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 beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-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 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.
authors.affiliation: Dow Agro Sciences LLC, Indianapolis, IN 46268, USA. dyoung@dow.com
authors.forename: David H
authors.initials: DH
authors.lastname: Young
authors.forename: Fernando M
authors.initials: FM
authors.lastname: Rubio
authors.forename: Paul O
authors.initials: PO
authors.lastname: Danis
mesh_terms: D001549:Benzamides / Q000737:chemistry; D001667:Binding, Competitive / Q000187:drug effects; D016923:Cell Death; D045744:Cell Line, Tumor; D004347:Drug Interactions; D018432:Drug Resistance, Multiple; D006801:Humans; D008870:Microtubules / Q000187:drug effects; D008940:Mitotic Index; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology*; D017239:Paclitaxel / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods*; D014404:Tubulin / Q000378:metabolism*; D050257:Tubulin Modulators / Q000032:analysis* / Q000494:pharmacology; D014747:Vinblastine / Q000494:pharmacology
chemical_list: D001549:Benzamides; D014404:Tubulin; D050257:Tubulin Modulators; D014747:Vinblastine; D017239:Paclitaxel