Lysophosphatidate Reverses G2/M Arrest by Taxol Figure 5. Lysophosphatidate releases MDA-MB-468 cells from Taxol-induced arrest in G2/M and cell death. MDA-MB-468 cells were preincubated with 10% delipidated serum and 50 nM Taxol for 26 h. Taxol was then maintained or removed in the presence of 10% delipidated serum and 5 mM LPA was added as indicated (Protocol C). Cells were stained with DAPI, anti-phospho-histone and with anti-tubulin at the times indicated. The Panels show the percentage of: A) cells in mitosis, B) percentage of cells in mitosis that had abnormal spindles, C) mononucleated cells, D) multinucleated cells and E) dead and dying cells. Total number of cells (100%) includes mononucleated, multinucleated, dead and dying and mitotic cells. Time zero shows 26 h pretreatment with Taxol. Results are means 6 SD for three independent experiments. doi:10.1371/journal.pone.0020608.g005 To investigate the role of these LPA receptors in the displacement of Taxol from polymerized tubulin, we applied VPC51299, an LPA1/3, antagonist and studied Taxol localization in both cytosolic and polymerized tubulin compartments. In MCF-7 cells, 1 mM VPC51299 failed to change the LPA-induced displacement Taxol from polymerized tubulin (Fig 9A). However, in MDA-MB-231 cells, VPC51299 attenuated the action of LPA (Fig 9B). Discussion The present work demonstrates that the effect of LPA in decreasing the Taxol-induced accumulation of breast cancer cells in G2/M [7] does not depend on an LPA-induced export of Taxol from the cells, or increased Taxol metabolism. Neither does LPA decrease the Taxol-induced accumulation of MCF-7 cells in mitosis by delaying entry into G2/M or by inducing selective death of the cells arrested in mitosis. Instead, LPA produced the surprising effect of reversing Taxol action in producing abnormal mitotic spindles. This effect was dependent on PI3K and the displacement of Taxol from its association with the tubulin polymer fraction. We also showed that the LPA-induced release from mitotic arrest protected the cells from the consequent Taxol- induced cell death [41,42,43]. We designed our experiments to investigate this latter phenomenon by preincubating cells for 26 h with delipidated serum and Taxol to produce mitotic arrest. The delipidated serum provides protein growth factors required for cell cycle progression, but it lacks lipid growth factors including LPA [7]. This enabled us to study the effects of Taxol removal or LPA addition on the structure of spindles in the cells and the progression of cells from G2/M into G1. Although removal of Taxol after 26 h caused a decrease in Taxol within the cells and allowed more cells to progress to G1, this effect was not as potent as the addition of LPA. LPA did not increase the expulsion of Taxol from the cells, but it did cause a redistribution of Taxol within the cells by displacing it from the polymerized tubulin fraction. We, therefore, conclude that LPA has a specific effect in facilitating normal spindle structure in the presence of Taxol. This LPA effect will decrease Taxol-induced mitotic arrest and also enable cancer cells to escape PLoS ONE | www.plosone.org 7 May 2011 | Volume 6 | Issue 5 | e20608