Microtubule-Disrupting Chemotherapeutics Result in Enhanced Proteasome-Mediated Degradation and Disappearance of Tubulin in Neural Cells

Abstract

We sought to examine the effects of microtubule-targeting agents (MTA) on neural cells to better understand the problem of neurotoxicity, their principal side effect, and to possibly develop a model of clinical toxicity. Studies showed that microtubule-depolymerizing agents (MDA) not only disassembled microtubules in neural HCN2 cells but also led to rapid disappearance of tubulin, and that this was specific for MDAs. Tubulin levels decreased to 20% as early as 8 hours after adding vincristine, and to 1% to 30% (mean, 9.8 ± 7.6%; median of 7%) after 100 nmol/L vincristine for 24 hours. This disappearance was reversible. An increase in both glu-terminated and acetylated tubulin, markers of stable tubulin, preceded reaccumulation of soluble tubulin, suggesting a priority for stabilizing tubulin first as microtubules before replenishing the soluble pool. Similar results were shown with other MDAs. Furthermore, microtubule reassembly did not arise from a central focus but instead appeared to involve dispersed nucleation, as evidenced by the appearance of small, stable microtubule stubs throughout the cytoplasm. In contrast, experiments with four nonneural "normal" cell lines and four cancer cell lines resulted in microtubule destabilization but only modest tubulin degradation. Evidence for proteasome-mediated degradation was obtained by demonstrating that adding a proteasome inhibitor before vincristine prevented tubulin disappearance. In summary, MDAs lead to rapid disappearance of tubulin in neural but not in other normal or cancer cells. These results underscore the fine control that occurs in neural cells and may further our understanding of neurotoxicity following MDAs.

Evaluation of tubulin polymerization and chronic inhibition of proteasome as citotoxicity mechanisms in bortezomib-induced peripheral neuropathy

“…33 Based on the reported evidence of changes in tubulin dynamics obtained in different cellular systems and on the relevance of tubulin interference in the pathogenesis of toxic peripheral neuropathies, 21,34,35 we focused on α-tubulin polymerization as a possible mechanism of neurotoxicity in BiPN. Despite the fact that the severity of nerve damage was more marked in the caudal nerve than the sciatic nerve, we selected the latter tissue to assay proteasome inhibition.…”

“…16,17 Pathogenic hypotheses can be reliably investigated in vivo using well-characterized animal models reproducing chronic BiPN as well as neuronal systems in vitro. [18][19][20][21] In this context, we have studied for the first time, using parallel in vivo and in vitro experiments, the possible correlation existing between proteasome inhibition and α-tubulin polymerization in order to understand the pathogenic basis of BiPN onset.…”

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

“…Blots were incubated in antibodies discussed in SI Materials and Methods. This process was followed by IRDye infrared secondary antibodies (LiCor) and expression quantitated using the Odyssey Infrared Imager (LiCor) (35). The percentage of cytoplasmic retention is indicated and calculated as [C/(C+N)] × 100%.…”

“…Cells were harvested at 4°C, in 20 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA, 1% TX-100, with protease (aprotinin, Sigma, and cOmplete Mini, EDTA-free, Roche) and phosphatase inhibitors (PhosSTOP, Roche), frozen, and sonicated before SDS/PAGE. Western blots were probed with antibodies for γ-H2AX, total H2AX, or GAPDH, followed by IRDye infrared secondary antibodies (LiCor) and expression quantitated using the Odyssey Infrared Imager (LiCor) (35).…”

“…A549 cells were fixed in either ice-cold 100% methanol for 8 min at −20°C, or in 4% (vol/vol) paraformaldehyde for 30 min at 22°C, followed by 100% methanol for 8 min at 20°C, or in 4% (vol/vol) paraformaldehyde for 15 min at 22°C, followed by 0.3% TX-100 for 60 min. Cells were processed for immunofluorescence microscopy with modifications of previously described methods (35,38) and the antibodies used are described in SI Materials and Methods.…”