Overexpression of betaIII-tubulin is associated with resistance to tubulin-binding agents (TBA) in a range of tumor types. We previously showed that small interfering RNA silencing of betaIII-tubulin expression hypersensitized non-small cell lung cancer cells to TBAs. To determine whether betaIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of betaIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non-small cell lung cancer cells stably expressing green fluorescent protein-betaI-tubulin. Interphase cells were examined at three vincristine and paclitaxel concentrations that (a) inhibited cell proliferation, (b) induced 5% to 10% mitotic arrest, and (c) induced 30% to 40% mitotic arrest. In the absence of either drug, betaIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC(50)), overall microtubule dynamicity was significantly suppressed in betaIII-tubulin knockdowns (-31.2%) compared with controls (-6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of betaIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (> or =40 nmol/L vincristine; > or =20 nmol/L paclitaxel), betaIII-tubulin knockdown resulted in significantly reduced suppressive effects on microtubule dynamicity with little or no further increase in mitotic arrest, compared with control cells. Importantly, apoptosis was markedly increased by betaIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that betaIII-tubulin knockdown enhances the effectiveness of TBAs through two mechanisms: suppression of microtubule dynamics at low drug concentrations and a mitosis-independent mechanism of cell death at higher drug concentrations.