Lysophosphatidate Reverses G2/M Arrest by Taxol Figure 3. Morphology of Taxol-treated MCF-7 cells. MCF-7 cells were preincubated with 10% delipidated serum and 50 nM Taxol for 26 h. Cells were then stained with DAPI, anti-phospho-histone and with anti-tubulin. The appearance of the cell in mitosis (assessed by DAPI and P-histone staining) is shown in Panel A. Panel B shows cells that did not stain for P-histone and which were identified as being mononucleated, multinucleated or dead. Panel C shows examples of the appearance of cells that were identified as having abnormal spindles. doi:10.1371/journal.pone.0020608.g003 load the cells with Taxol as in the experiments described in Fig. 4. These cells were incubated in 10% delipidated serum, in which endogenous LPA and other bioactive lipids were removed [7]. Cells were then incubated in the presence or absence of 5 mM LPA for a further 12 h. At the end of the 26 h incubation, 7068% of the total Taxol was in the cells and incubation for a further 12 h increased (p,0.05) the proportion of Taxol inside the cells to 8065% (Fig. 6A). Removal of Taxol during the final 12 h incubation was accompanied by the loss (p,0.05) of 3268% of the [3H]Taxol from the cells into the medium (Fig. 6B). LPA did not significantly affect these Taxol distributions in either protocol. We, therefore, rejected our first hypothesis. We also analyzed the [3H]Taxol under all conditions by thin layer chromatography [37]. There was no significant formation of 39-p-hydroxytaxol and 6a-hydroxytaxol, the major Taxol metab- olites, either in the absence or presence of LPA (results not shown). We also determined whether the LPA-induced escape of the breast cancer cells from mitotic arrest can be explained by the release of Taxol from its association with the polymerized tubulin fraction. MCF-7 cells were pretreated for 26 h with 50 nM 3 [ H]Taxol to load the cells with Taxol and produce mitotic arrest as in Fig. 4. The medium was maintained, 5 mM LPA was added as indicated and the cells were then incubated for and additional 12 h. The amount of [3H]Taxol associated with the polymerized tubulin PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20608