Mechanisms of Taxol-induced Cell Death Are Concentration Dependent1

Although the ability of Taxol to stabilize cellular microtubules is well accepted, the mechanisms by which Taxol induces growth arrest and cell death remain unclear. Recent evidence indicates that Taxol alters specific intracellular signal transduction events, such as the activation of Raf-1 kinase, that may be essential for drug-induced apoptosis. To determine whether Raf-1 kinase activation occurs at different concentrations of Taxol and in response to disruption of the normal microtubule cytoskeleton, A549 cells were treated with different concentrations of Taxol after which Raf-1 activation and the microtubule cytoskeleton were analyzed. Raf-1 activation was observed at Taxol concentrations of 9 DM and greater. However, disruption of the normal microtubule cytoskeleton was seen at lower Taxol concentrations (1-7 MM),indicating that this process begins in the absence of Raf-1 activation. Raf-1 activation correlated with the induction of a G2-M block. Depletion of Raf-1 resulted in the accu mulation of cells in the ( ; ,-M phase of the cell cycle, suggesting that Raf-1 may play an important role in the passage through mitosis. Supporting this idea, Raf-1 was activated in mitotic cells. Low concentrations of Taxol induced cell death in the absence of Raf-1 activation, indicating that Taxol-induced cell death is not dependent on Raf-1 activation. At concen trations of drug lower than the critical concentration required for Raf-1 activation, p53 and p21WAF~'were induced independently of Raf-1. These studies suggest that Taxol-mediated cell death may result from two dif ferent mechanisms. At low Taxol concentrations (<9 MM),cell death may occur after an aberrant mitosis by a Raf-1 independent pathway, whereas at higher Taxol concentrations (a'< n\n cell death may be the result of a terminal mitotic arrest occurring by a Raf-1-dependent pathway.