Effects of taxol on the polymerization and posttranslational modification of class III beta-tubulin in P19 embryonal carcinoma cells.

Abstract

Undifferentiated P19 embryonal carcinoma cells and P19 cells induced to differentiate along a neuronal pathway by 10(-6) M retinoic acid were treated with taxol to examine the effects of this microtubule-stabilizing drug on the subcellular sorting of class III beta-tubulin and on neurite outgrowth. P19 cells were grown on cover slips and then treated with taxol at concentrations of 10(-6) to 10(-9) M for 24 h. The microtubule cytoskeleton was examined after double-immunofluorescence labelling with a monoclonal antibody to alpha-tubulin (YOL 1/34) and a monoclonal neuron-specific class III beta-tubulin antibody (TuJ1). Treatment of undifferentiated P19 cells with concentrations of taxol greater than 4 x 10(-8) M caused microtubule bundling and multiple aster formation and promoted polymerization of the low levels of class III beta-tubulin found in these cells. In neurons, at 2 x 10(-8) M taxol, bundling of microtubules at the base of the neurite was apparent. At taxol concentrations greater than 1 x 10(-7) M, enhanced assembly of class III beta-tubulin was apparent, although long neurites were not observed. Using isoelectric focusing followed by western blotting, we detected an additional isoform of class III beta-tubulin after treatment with 10(-6) M taxol. The results indicate taxol treatment alters the normal subcellular sorting of tubulin isotypes, promotes the polymerization and posttranslational modification of class III beta-tubulin, and interferes with neurite outgrowth.