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Cell-based screen for identification of inhibitors of tubulin polymerization
J M Kokoshka1, C M Ireland, L R Barrows
1University of Utah, Department of Pharmacology and Toxicology, Salt Lake City 84112, USA.
Abstract:
This assay is based on morphological changes of rat glioma cells treated with db-cAMP. The db-cAMP treatment induces a tubulin-dependent change causing the cells to acquire a spherical shape. Pretreatment with tubulin inhibitors brings about the disintegration of tubulin polymer and/or prevents its polymerization. Cells with inhibited tubulin fail to respond to db-cAMP treatment. Cells treated with inhibitors of tubulin polymerization are then separated from the spherical cells by aspiration. A semiautomated scanning procedure evaluates the final culture density and yields graphical data.
Insights
This study shows that blocking tubulin polymerization prevents rat glioma cells from changing shape in response to db-cAMP. This tubulin-dependent assay identifies inhibitors of tubulin polymerization.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- Glioma cells exhibit morphological changes in response to specific stimuli.
- Tubulin polymerization is a critical cellular process involved in cell structure and division.
- Cyclic AMP (cAMP) signaling pathways can influence cell morphology.
Purpose of the Study:
- To develop and validate an assay for identifying inhibitors of tubulin polymerization.
- To investigate the role of tubulin in db-cAMP-induced morphological changes in rat glioma cells.
Main Methods:
- Rat glioma cells were treated with dibutyryl cyclic AMP (db-cAMP) to induce morphological changes.
- Cells were pretreated with various tubulin inhibitors to assess their impact on db-cAMP response.
- Tubulin polymerization inhibition was confirmed by the failure of cells to adopt a spherical shape.
- Spherical cells were separated from non-responsive cells via aspiration.
- A semiautomated scanning procedure quantified final culture density and generated graphical data.
Main Results:
- db-cAMP treatment induced a tubulin-dependent morphological change in glioma cells, causing them to become spherical.
- Pretreatment with tubulin polymerization inhibitors blocked the db-cAMP-induced morphological change.
- Inhibition of tubulin polymerization prevented cells from responding to db-cAMP.
- The assay successfully differentiated between responsive and non-responsive cells.
Conclusions:
- The described assay effectively utilizes db-cAMP-induced morphological changes to identify tubulin polymerization inhibitors.
- Tubulin polymerization is essential for the observed morphological plasticity of rat glioma cells.
- This method offers a semiautomated approach for evaluating compounds that affect tubulin dynamics.