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A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance
M L Gonzalez-Garay1, L Chang, K Blade
1Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77030, USA.
Abstract:
Analysis of beta-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged beta-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin beta-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.
Insights
Mutations in beta-tubulin
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Paclitaxel resistance in cancer treatment is a significant challenge.
- Beta-tubulin is a key component of microtubules and a target for paclitaxel.
- Understanding mutations in beta-tubulin is crucial for developing new therapeutic strategies.
Purpose of the Study:
- To investigate the role of specific beta-tubulin mutations in paclitaxel resistance.
- To identify critical amino acid residues within beta-tubulin involved in drug binding and microtubule stability.
- To elucidate the functional consequences of these mutations on microtubule dynamics and cell proliferation.
Main Methods:
- Analysis of beta-tubulin alleles from paclitaxel-resistant Chinese hamster ovary (CHO) cell lines.
- Site-directed mutagenesis to introduce specific Leu-215, Leu-217, and Leu-228 substitutions into beta-tubulin cDNA.
- Transfection of mutant beta-tubulin into wild-type CHO cells using tetracycline-regulated expression plasmids.
- Assessment of paclitaxel resistance, microtubule assembly, cell cycle progression, and microtubule acetylation levels.
Main Results:
- Identification of a mutation cluster affecting Leu-215, Leu-217, and Leu-228 in paclitaxel-resistant CHO cells.
- Introduction of L215H, L217R, and L228F mutations conferred paclitaxel resistance at low to moderate expression levels.
- High-level expression of mutant beta-tubulin led to microtubule assembly disruption, mitotic arrest, and impaired proliferation.
- Mutant beta-tubulin expression correlated with reduced microtubule acetylation, indicating decreased microtubule stability.
Conclusions:
- The leucine cluster at residues 215, 217, and 228 is a critical structural motif for microtubule assembly and stability.
- Specific substitutions within this leucine cluster can confer paclitaxel resistance.
- The level of mutant beta-tubulin expression influences cellular response, with high levels causing toxicity due to microtubule dysfunction.