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Paclitaxel-dependent mutants have severely reduced microtubule assembly and reduced tubulin synthesis
Steven B Barlow1, Manuel L Gonzalez-Garay, Fernando Cabral
1Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas 77225, USA.
Abstract:
A subset of mutant cell lines selected for resistance to the antitumor drug paclitaxel are unable to progress normally through mitosis unless the drug is present in the growth medium. Without paclitaxel the cells form defective spindles, undergo aberrant mitoses, fail to complete cell division and eventually die. Analysis of these drug-dependent cells revealed a low amount of microtubule polymer and less tubulin production than wild-type cells. Ribonuclease protection experiments indicated that the decreased tubulin protein was due to decreased tubulin mRNA. Enhancing microtubule assembly by treating the cells with paclitaxel, restored tubulin to levels comparable with those of paclitaxel-treated wild-type cells, which demonstrated that the drug-dependent cells do not have a permanent impairment in their capacity to synthesize tubulin. Paclitaxel-resistant (but not dependent) cells have a smaller reduction in microtubule polymer with little or no decrease in tubulin production, whereas colcemidresistant cells have increased microtubule assembly but also exhibit little or no change in tubulin production. Finally, a mutant cell line producing an unstable beta-tubulin protein has normal growth as well as normal synthesis and polymerization of tubulin, despite an approximately 30% decrease in steady state tubulin content. These studies establish a lower limit of tubulin assembly needed for cell survival and indicate that tubulin assembly must fall below this point to trigger a significant decrease in tubulin synthesis.
Insights
Drug-dependent cells require paclitaxel for normal mitosis due to low tubulin levels. This study reveals a critical threshold for tubulin assembly essential for cell survival and division.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mitosis is crucial for cell division and is regulated by microtubule dynamics.
- Paclitaxel is an anti-cancer drug that stabilizes microtubules, arresting cells in mitosis.
- Mutant cell lines resistant to paclitaxel exhibit altered responses to the drug.
Purpose of the Study:
- To investigate the molecular mechanisms underlying drug-dependent cell survival in mitosis.
- To determine the relationship between tubulin synthesis, microtubule assembly, and cell viability.
- To establish the critical threshold of tubulin assembly required for normal cell cycle progression.
Main Methods:
- Analysis of mutant cell lines selected for paclitaxel resistance.
- Measurement of microtubule polymer and tubulin production.
- Ribonuclease protection assays to quantify tubulin mRNA levels.
- Assessment of cell division and survival under varying drug concentrations.
Main Results:
- Drug-dependent cells exhibit reduced microtubule polymer and tubulin synthesis, requiring paclitaxel for mitotic progression.
- Paclitaxel treatment restores tubulin levels in dependent cells, indicating no permanent synthesis defect.
- Mutant cells with unstable beta-tubulin maintain normal growth despite reduced tubulin content, suggesting a critical assembly threshold.
Conclusions:
- Cell survival is dependent on maintaining a minimum level of tubulin assembly.
- A significant decrease in tubulin assembly below this threshold triggers reduced tubulin synthesis.
- These findings elucidate the intricate regulation of tubulin dynamics and its role in mitotic fidelity.
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