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Tubulin-Targeted Drug Action: Functional Significance of Class II and Class IVb β-Tubulin in Vinca Alkaloid Sensitivity

¹ Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia and ² University of New South Wales, Sydney, New South Wales, Australia

Requests for reprints: Maria Kavallaris, Children's Cancer Institute Australia for Medical Research, High Street (P. O. Box 81), Randwick, New South Wales 2031, Australia. Phone: 61-2-9382-1823; Fax: 61-2-9382-1850; E-mail: m.kavallaris{at}ccia.unsw.edu.au.

Key Words: β-tubulin • drug resistance • non–small cell lung cancer • tubulin-binding agents • microtubules

Aberrant expression of β-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 β-tubulin isotypes in cellular sensitivity to TBAs, and to gain insights into the functional role of βII- and βIVb-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 βII- or βIVb-tubulin hypersensitized the lung cancer cell lines to Vinca alkaloids, with the effects more pronounced following βIVb-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 βII- and βIVb-tubulin knockdown cells to undergo G₂-M cell cycle block following 5 nmol/L vincristine treatment, with the βIVb knockdown cells being more sensitive than the βII-tubulin knockdown cells compared with control. In contrast to βII-tubulin knockdown, βIVb-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, βIVb-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 βII- or βIVb-tubulins have functionally distinct roles and expression of these isotypes may serve as strong predictors of Vinca alkaloid response and resistance. [Cancer Res 2008;68(23):9817–24]