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Microtubule Disruption Induced in Vivo by Alkylation of ß-Tubulin by 1-Aryl-3- (2-Chloroethyl)Ureas, a Novel Class of Soft Alkylating Agents¹

Biotechnology Unit, Biomaterial Institute of Quebec, Centre Hospitalier Universitaire de Quebec, Pavillon St-François d’Assise, Laval University, Quebec City, Quebec, Canada, G1L 3L5

We have previously reported that 4-tert-butyl-[3-(2-chloroethyl)ureido] benzene (4-tBCEU), a potent cytotoxic agent, modulates the synthesis of tubulins, suggesting that its cytotoxicity may be mediated through an antimicrotubule mechanism. Indeed, 4-tBCEU and its 4-iso-propyl (4-iso-propyl [3-(2-chloroethyl)ureido] benzene) and 4-sec-butyl (4-sec-butyl [3-(2-chloroethyl)ureido] benzene) homologues induced disruption of the cytoskeleton and arrest of the cell cycle in G₂ transition and mitosis. To better understand the mechanisms responsible for microtubule disruption by 1-aryl-3-(2-chloroethyl)ureas (CEU), we first examined their cytotoxicity on Chinese hamster ovary cells resistant to vinblastine and colchicine due to the expression of mutated tubulins (CHO-VV 3–2). These cells showed resistance to CEU, e.g., 4-tBCEU having an IC₅₀ of 21.3 ± 1.1 µM as compared with an IC₅₀ of 11.6 ± 0.7 µM for wild-type cells, suggesting a direct effect of the drugs on tubulins. Western blot analysis confirmed the disruption of microtubules and evidenced the formation of an additional immunoreactive ß-tubulin with an apparent lower molecular weight on SDS polyacrylamide gel. Incubation of MDA-MB-231 cells with [urea-¹⁴C]-4-tBCEU revealed the presence of a radioactive protein that coincided with the additional ß-tubulin band, indicating that CEU could covalently bind to the ß-tubulin. The 4-tBCEU-binding site on ß-tubulin was identified by competition of the CEU with colchicine, vinblastine, and iodoacetamide, a specific alkylating agent of sulfhydryl groups of cysteine residues. Colchicine, but not vinblastine, prevented the formation of the additional ß-tubulin band, suggesting that 4-tBCEU alkylates either Cys239 or Cys354 residues near the colchicine-binding site.

To determine the cysteine residue alkylated by 4-tBCEU, we incubated the radiolabeled drug with human neuroblastoma cells (SK-N-SH) that overexpress the ßIII-tubulin, an isoform where Cys239 is replaced by a serine residue. The results clearly showed that ßIII-tubulin is not alkylated by [urea-¹⁴C]-4-tBCEU, suggesting that cysteine 239 residue is essential for the reactivity of 4-tBCEU with ß-tubulin. Taken together, these findings indicate that the mechanism of cytotoxicity of CEU involves microtubule depolymerization through alkylation of ß-tubulin.

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