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Increased Protein Tyrosine Phosphorylation in Apoptotic Neural Cell Death Due to Microtubule Perturbations
Brett A Chromy1, Mary P Lambert1, William L Klein1
1Dept. of Ncurobiology and Physiology, Northwestern University, Evanston, IL 60208.
Abstract:
The microtubule-perturbing drugs colchicine and taxol have been found to induce apoptosis in a CNS neuronal cell line. Apoptosis in drug-treated rat B103 neuroblastoma cells was evident in characteristic morphological changes, internucleosomal DNA fragmentation, and loss of nuclear content. Since colchicine and taxol have opposite actions on microtubule integrity, disruption of the active turnover of the microtubule network appears to be a crucial step for apoptosis to occur. It has been suggested that the basis for apoptosis by these drugs derives from their known block of the cell cycle at G2/M, but this does not appear the sole reason as both colchicine and taxol were able to evoke high levels of apoptosis in cells differentiated by Bt2cAMP or serum withdrawal. Further tests of cellular consequences of microtubule perturbation revealed a specific impact on signal transduction involving protein tyrosine phosphorylation. Immunoprecipitation with antibodies against tyrosine phosphorylated proteins showed a striking increase in the phosphorylation of a Triton-insoluble ~90 kDa protein, roughly concurrent with the onset of internucleosomal DNA fragmentation. Cycloheximide and genistein significantly reduced cell death and blocked appearance of the ~90 kDa tyrosine phosphorylated protein. Data suggest the hypothesis that signal transduction leading to apoptosis can be triggered by anomalous microtubule turnover and that the mechanism involves tyrosine phosphorylation of a ~90 kDa Triton-resistant protein.
Insights
Microtubule disruption by colchicine and taxol induces apoptosis in neuronal cells. This cell death involves tyrosine phosphorylation of a key protein, suggesting a novel signaling pathway for programmed cell death.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Microtubule-targeting drugs like colchicine and taxol can induce apoptosis.
- The exact mechanisms by which these drugs trigger programmed cell death in neurons are not fully understood.
- Previous research suggested cell cycle arrest at G2/M as the primary mechanism.
Purpose of the Study:
- To investigate the role of microtubule disruption in inducing apoptosis in a CNS neuronal cell line.
- To identify the specific molecular signaling pathways involved in drug-induced neuronal apoptosis.
- To explore the potential involvement of protein tyrosine phosphorylation in this process.
Main Methods:
- Treatment of rat B103 neuroblastoma cells with colchicine and taxol.
- Assessment of apoptosis through morphological changes and DNA fragmentation.
- Analysis of protein tyrosine phosphorylation using immunoprecipitation.
- Evaluation of the effects of cycloheximide and genistein on cell death and protein phosphorylation.
Main Results:
- Colchicine and taxol induced characteristic apoptosis in neuronal cells, irrespective of cell cycle arrest.
- Microtubule perturbation led to increased tyrosine phosphorylation of a ~90 kDa Triton-insoluble protein.
- Inhibition of protein synthesis and tyrosine kinase activity reduced both cell death and protein phosphorylation.
Conclusions:
- Disruption of microtubule dynamics, rather than just cell cycle arrest, is a critical trigger for apoptosis in these neuronal cells.
- Tyrosine phosphorylation of a ~90 kDa protein is implicated in the signal transduction pathway leading to microtubule-disruption-induced apoptosis.
- This study proposes a novel mechanism involving aberrant microtubule turnover and tyrosine kinase signaling in neuronal cell death.
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