Microtubule Dynamics, Mitotic Arrest, and Apoptosis: Drug-Induced Differential Effects of βIII-Tubulin

Abstract

Overexpression of βIII-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 βIII-tubulin expression hypersensitized non-small cell lung cancer cells to TBAs. To determine whether βIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of βIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non-small cell lung cancer cells stably expressing green fluorescent protein-βI-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, βIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC 50 ), overall microtubule dynamicity was significantly suppressed in βIII-tubulin knockdowns (−31.2%) compared with controls (−6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of βIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (≥40 nmol/L vincristine; ≥20 nmol/L paclitaxel), βIII-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 βIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that βIII-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. Mol Cancer Ther; 9(5); 1339-48. ©2010 AACR.

Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins

“…Given the importance of MTs in cell division and the widely accepted concept that cancer cells divide more rapidly than normal cells, it has been generally assumed that MTAs mediate cytotoxicity by interfering with mitosis (1, 2). Elegant in vitro and preclinical data have demonstrated time and again that MTAs lead to mitotic arrest and in turn cell death (5,(7)(8)(9)(10)(11)(12). Arrest in mitosis as the mechanism that leads to cell death is possible in these preclinical models because their doubling times range from a few hours to at most a few days, and even brief drug exposures are likely to encounter a substantial fraction of cells traversing through mitosis.…”

Class III β-Tubulin Expression Predicts Prostate Tumor Aggressiveness and Patient Response to Docetaxel-Based Chemotherapy

“…Because the primary effect of taxane is to bind microtubules, thereby enhancing the microtubule polymerization and decreasing microtubule dynamicity, it has been suggested that βIII-tubulin-containing microtubules are more prone to overcome the suppressive effects of taxanes on microtubule dynamics. Although it should be noted that all the above studies (12,13,42) have focused on the assembly of purified microtubules in cell-free systems and therefore remain controversial given contradictory reports obtained in intact cells, and hence more biologically relevant, that βIII-tubulin did not intrinsically affect microtubule dynamic, at least in some instances (38,43). Interestingly, in these last studies, the effects of paclitaxel on microtubule dynamics were altered, suggesting a role for βIII-tubulin in drugmicrotubule interactions.…”

TUBB3/βIII-Tubulin Acts through the PTEN/AKT Signaling Axis to Promote Tumorigenesis and Anoikis Resistance in Non–Small Cell Lung Cancer

“…H460 and A549 NSCLC cells were obtained from ATTC and grown as described (8)(9)(10). Upon receipt from ATCC, cell master stocks were prepared and cells for experiments were passaged for less than 6 months.…”

“…Clinical studies show that high bIII-tubulin expression correlates with chemoresistance and poor survival in different tumor types, including breast, ovarian, gastric, and NSCLC (5,6). Previously, we demonstrated a functional role for bIIItubulin in regulating chemosensitivity in NSCLC using RNA interference to silence bIII-tubulin expression in NSCLC cells (7)(8)(9)(10). Knockdown of bIII-tubulin increased sensitivity via an increase in apoptosis to chemotherapeutic agents both in vitro and in vivo (7)(8)(9)(10).…”

“…Previously, we demonstrated a functional role for bIIItubulin in regulating chemosensitivity in NSCLC using RNA interference to silence bIII-tubulin expression in NSCLC cells (7)(8)(9)(10). Knockdown of bIII-tubulin increased sensitivity via an increase in apoptosis to chemotherapeutic agents both in vitro and in vivo (7)(8)(9)(10). However, despite evidence to link bIII-tubulin expression to chemotherapy drug sensitivity, its roles in the tumorigenic and metastatic potential of NSCLC are unknown.…”