Tubulin-targeted drug action: functional significance of class ii and class IVb beta-tubulin in vinca alkaloid sensitivity.

Aberrant expression of beta-tubulin isotypes is frequently described in tumor tissues and tubulin-binding agent (TBA)-resistant cell lines. There is limited understanding of the role of specific beta-tubulin isotypes in cellular sensitivity to TBAs, and to gain insights into the functional role of betaII- and betaIVb-tubulin, we examined these isotypes in lung cancer cell lines NCI-H460 (H460) and Calu-6. Drug-treated clonogenic assays revealed that small interfering RNA-mediated knockdown of either betaII- or betaIVb-tubulin hypersensitized the lung cancer cell lines to Vinca alkaloids, with the effects more pronounced following betaIVb-tubulin knockdown. In contrast, there was no change in paclitaxel sensitivity following knockdown of either isotype. Cell cycle analysis revealed a greater propensity for the betaII- and betaIVb-tubulin knockdown cells to undergo G2-M cell cycle block following 5 nmol/L vincristine treatment, with the betaIVb knockdown cells being more sensitive than the betaII-tubulin knockdown cells compared with control. In contrast to betaII-tubulin knockdown, betaIVb-tubulin knockdown cells showed a significant increase in the sub-G1 population (cell death) following treatment with both 5 and 40 nmol/L of vincristine compared with controls. Importantly, betaIVb-tubulin knockdown in H460 cells caused a significant dose-dependent increase in Annexin V staining in response to vincristine but not paclitaxel. Therefore, increased sensitivity to induction of apoptosis is one mechanism underlying the Vinca alkaloid hypersensitivity. This study provides direct evidence that betaII- or betaIVb-tubulins have functionally distinct roles and expression of these isotypes may serve as strong predictors of Vinca alkaloid response and resistance.