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 β‐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 α‐ and β‐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 β‐tubulin in these E7010‐resistant cells by a competitive reverse transcription‐polymerase chain reaction method. Although a 50% increase in β5 isotype mRNA levels was observed in 4.0r‐M cells, the levels of β3 isotype message in the two resistant clones were approximately 50% less than the parental cells. To elucidate the binding properties of E7010 with β‐tubulin isotypes, we prepared isotype‐specific fusion proteins of β‐tubulins. Direct photoaffinity labelling of the isotype‐specific fusion proteins with [14C]E7010 revealed that E7010 preferentially binds to the β3 isotype rather than β2, β4, and β5 isotype proteins. Therefore, altered expression of β‐tubulin isotypes, especially β3 isotype, to which E7010 binds with high affinity, may account for the decreased sensitivity of these resistant clones to E7010.