Abstract

Microtubule binders are cell cycle-specific agents with preferential cytotoxicity toward mitotic cells. We have characterized vincristine-selected human leukemia cells to establish whether development of vincristine resistance was accompanied by changes in cell cycle kinetics and distribution. Our results indicate that vincristine resistance is accompanied by delayed G₂ transit and prolonged early mitosis in both the absence and the presence of the microtubule binder nocodazole. The altered G₂/M regulation is accompanied by resistance to short-term (12 h) but not continuous nocodazole exposure in agreement with the transient nature of the observed cell cycle alterations. Western blot analysis indicates that vincristine-selection is accompanied by down-regulation of topoisomerase IIα without detectable alterations of the other mitotic regulators studied, including Cdk1, p21, 14-3-3σ, and 14-3-3ϵ. This was associated with at least 7-fold less chromosome-associated topoisomerase IIα, decreased catalytic activity, and cross-resistance to topoisomerase II inhibitors. Characterization of isogenic cell lines expressing different levels of topoisomerase II proteins shows that cellular levels of topoisomerase IIα, but not the closely related topoisomerase IIβ, directly influence the cell cycle kinetics in G₂ and early mitosis as well as the resistance to nocodazole. These results underline the importance of topoisomerase IIα in late G₂ and early M phases and provide evidence for an as-yet-unsuspected interaction between topoisomerase II and microtubule-directed agents.