Lysophosphatidate Reverses G2/M Arrest by Taxol Figure 4. Lysophosphatidate releases MCF-7 cells from Taxol-induced arrest in G2/M and cell death. MCF-7 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 then 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 (where large enough to be shown) for three independent experiments. doi:10.1371/journal.pone.0020608.g004 fraction was decreased significantly by LPA treatment (Fig. 7A). This decrease was balanced by the appearance of Taxol in the soluble compartment of the cells (Fig. 7B) in agreement with the lack effect of LPA on the cellular content of Taxol (Fig. 6A and B). The LPA effect in decreasing the association of Taxol with the polymerized tubulin was reversed by incubation with 40 mM LY294002, the PI 3-kinase inhibitor (Fig. 7A). We confirmed these results in separate experiments where MCF-7 cells were incubated in the presence or absence of LPA or LY294002 for 6 rather than 12 h (results not shown). We were aware that LY294002 has other actions such as the inhibition of casein kinase-2. We, therefore, tested different concentrations of LY294002, Wortmannin (anoth- er PI3K inhibitor) and TBB (4,5,6,7-tetrabromobenzotriazole, a selective casein kinase-2 inhibitor) [38]. LY294002 reversed the LPA-induced displacement of Taxol from polymerized tubulin with a maximum effect at 20 mM (Fig. 7C). Wortmannin also had a similar effect at 50 and 100 nM (Fig. 7D). TBB had no significant effect between 10 and 100 mM (results not shown). We next investigated the role of LPA receptors on the LPA effect on Taxol distribution. Among the LPA receptors, LPA1, LPA2, and LPA3 are the best characterized and have the major role in LPA action [6,39]. Real- time RT-PCR was performed to detect changes in mRNA expression levels for LPA receptors and also ATX in three breast cancer cell lines. We already showed that ATX expression is low in MCF-7 and MDA-MB-231 cells (Samadi et al; Gaetano et al) and ATX mRNA levels are similar in the MDA-MB-468 cells (Fig. 8A). Taxol treatment for 26 h did not significantly change ATX mRNA expression in any of the cell lines. MCF-7 and MDA-MB-468 cells predominantly express LPA2 and MDA-MB-231 mainly express LPA1 receptors [40] and this is reflected in the mRNA levels (Fig. 8B and C). MDA-MB-231 cells expressed relatively more mRNA for LPA3 than MCF-7 or MDA- MB-468 cells (Fig. 8D). Taxol treatment caused a 6-fold increase in LPA3 receptor mRNA in MDA-MB-231 cells and a significant decrease in LPA2 mRNA in MCF-7 cells (Fig. 8B,C,D). PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e20608