Preferential binding of E7010 to murine beta 3-tubulin and decreased beta 3-tubulin in E7010-resistant cell lines.

Abstract

N-[2-[(4-Hydroxyphenyl)amino]-3-pyridyl]-4-methoxybenzenesulfonamide+ ++ (E7010) is a novel sulfonamide antimitotic agent, which is active against mouse and human tumors. E7010 binds to beta-tubulin and inhibits polymerization of microtubules. In order to clarify the mechanisms of E7010-resistance, two murine leukemic P388 subclones resistant to E7010, 0.5r-D and 4.0r-M, were characterized. The two clones showed approximately 10- and 100-fold resistance to E7010-induced growth-inhibitory effects, respectively, compared with the parental cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. These cell lines showed no cross-resistance to other anticancer agents such as taxanes, vinca alkaloids, mitomycin C, cisplatin and irinotecan hydrochloride (CPT-11). Increased alpha- and beta-tubulin protein and mRNA levels were observed in 0.5r-D and 4.0r-M cells as compared with the parental cells. We examined the isotype-specific expression of beta-tubulin in these E7010-resistant cells by a competitive reverse transcription-polymerase chain reaction method. Although a 50% increase in beta 5 isotype mRNA levels was observed in 4.0r-M cells, the levels of beta 3 isotype message in the two resistant clones were approximately 50% less than the parental cells. To elucidate the binding properties of E7010 with beta-tubulin isotypes, we prepared isotype-specific fusion proteins of beta-tubulins. Direct photoaffinity labeling of the isotype-specific fusion proteins with [14C]E7010 revealed that E7010 preferentially binds to the beta 3 isotype rather than beta 2, beta 4, and beta 5 isotype proteins. Therefore, altered expression of beta-tubulin isotypes, especially beta 3 isotype, to which E7010 binds with high affinity, may account for the decreased sensitivity of these resistant clones to E7010.

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