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Random Mutagenesis of β-Tubulin Defines a Set of Dispersed Mutations That Confer Paclitaxel Resistance

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ABSTRACT

Purpose

Previous research showed that mutations in β1-tubulin are frequently involved in paclitaxel resistance but the question of whether the mutations are restricted by cell-type specific differences remains obscure.

Methods

To circumvent cellular constraints, we randomly mutagenized β-tubulin cDNA, transfected it into CHO cells, and selected for paclitaxel resistance.

Results

A total of 26 β1-tubulin mutations scattered throughout the sequence were identified and a randomly chosen subset were confirmed to confer paclitaxel resistance using site-directed mutagenesis of β-tubulin cDNA and transfection into wild-type cells. Immunofluorescence microscopy and biochemical fractionation studies indicated that cells expressing mutant tubulin had decreased microtubule polymer and frequently suffered mitotic defects that led to the formation of large multinucleated cells, suggesting a resistance mechanism that involves destabilization of the microtubule network. Consistent with this conclusion, the mutations were predominantly located in regions that are likely to be involved in lateral or longitudinal subunit interactions. Notably, fourteen of the new mutations overlapped previously reported mutations in drug resistant cells or in patients with developmental brain abnormalities.

Conclusions

A random mutagenesis approach allowed isolation of a wider array of drug resistance mutations and demonstrated that similar mutations can cause paclitaxel resistance and human neuronal abnormalities.

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Abbreviations

CHO:

Chinese hamster ovary

cmd:

colcemid

GST:

glutathione S-transferase

HA:

hemagglutinin antigen

ptx:

paclitaxel

tet:

tetracycline

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ACKNOWLEDGMENTS AND DISCLOSURES

These studies were supported by grant CA85935 from the National Institutes of Health to FC.

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Author notes

  1. Changqing Zeng

    Present address: Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100029, China

  2. Malathi Hari

    Present address: Division of Hematology/Oncology, University of Michigan Cancer Center, Ann Arbor, Michigan, 48109, USA

Authors and Affiliations

  1. Department of Integrative Biology and Pharmacology, University of Texas Medical School, 6431 Fannin St., Houston, Texas, 77030, USA

    Shanghua Yin, Changqing Zeng, Malathi Hari & Fernando Cabral

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  1. Shanghua Yin
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  2. Changqing Zeng
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  4. Fernando Cabral
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Corresponding author

Correspondence to Fernando Cabral.

ELECTRONIC SUPPLEMENTARY MATERIAL

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Fig. S1

Paclitaxel prevents the morphological changes caused by mutant tubulin. Wild-type CHO cells (A, B) or the same cells transfected with HAβ1-tubulin containing F85L (C, D) or R320C (E, F) mutations were grown for 3 days without tetracycline but in the presence or absence of 250 nM paclitaxel (Ptx). The cells were stained with antibody DM1A to label the microtubules and with DAPI to stain the nuclei (shown in the insets). Bar = 10 μm (JPEG 130 kb)

High resolution image (TIFF 597 kb)

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Yin, S., Zeng, C., Hari, M. et al. Random Mutagenesis of β-Tubulin Defines a Set of Dispersed Mutations That Confer Paclitaxel Resistance. Pharm Res 29, 2994–3006 (2012). https://doi.org/10.1007/s11095-012-0794-5

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