half-life about 3 min in mice [19,20]. This half life depends on the balance of ATX activity in producing LPA [19] and the ecto- activities of lipid phosphate phosphatases (LPPs), which degrade extracellular LPA [6,20,21]. Increased ATX expression is strongly associated with tumor growth, invasion, angiogenesis and metastasis [22,23,24]. Recent work in vivo supports the importance of ATX and LPA in tumor development. Increased expression of ATX, LPA1, LPA2 or LPA3 receptors in mice increased the frequency of invasive, estrogen receptor-positive and metastatic breast cancer [25]. ATX activity is required for lysophosphatidylcholine to stimulate cancer cell migration [19,26,27] and to antagonize Taxol-induced cell death [7]. ATX action also antagonizes carboplatin-induced apoptosis in ovarian cancer cells [28]. We proposed that inhibiting ATX activity or expression, and thereby LPA formation, could provide an important supplement for chemotherapy or surgery [7,26]. The present work was performed to identify how LPA production by ATX decreases the Taxol-induced accumulation of cells in G2/M, an event that precedes apoptosis [7]. This work is a necessary initial step in elucidating the signaling pathways used by LPA to cause Taxol resistance. We now show that this LPA action does not depend on increased expulsion of Taxol from cancer cells, increased Taxol metabolism, a delay in the entry into G2/M or selective killing of cells in G2/M. Surprisingly, LPA has a remarkable effect of enabling cells that had been arrested in G2/M by Taxol to normalize spindle formation, divide and thus escape from cell death. This LPA action depends on the activation of PI 3-kinase, which causes Taxol to be displaced from the tubulin polymer fraction. Results LPA decreases the Taxol-induced accumulation of cells in G2/ M by increasing the escape from mitotic arrest. We selected MCF-7 cells for this study based on our previous work [7] and the widespread use of these cells to study apoptosis. To investigate how LPA antagonizes the Taxol-induced arrest in G2/M and subsequent cell death [7], we determined if LPA affects cell cycle progression or induces selective killing of cells arrested in mitosis by Taxol. MCF-7 cells were treated with bromodeoxyuridine (BrdU) for 1 h to label cells in S-phase (Protocol A; Fig. 1). Progression through the cell cycle of the labeled cells was measured by FACS analysis for 12 h after treatment with 10% delipidated serum, in which LPA and other bioactive lipids were removed [7] and 50 nM Taxol was added in the presence or absence of 5 mM LPA. These concentrations gave optimum cell killing and efficient rescue, respectively [7]. This Taxol concentration is similar to that achieved during chemo- therapy [29] and the LPA concentration is in physiological/ pathological range [17]. Sixty percent of the labeled cells were still in S-phase at the end of the labeling period, as expected. Untreated cells started to exit S-phase immediately thereafter, and approximately 60% of these labeled cells had progressed into the G2/M phase at 2 h after labeling (Fig. 1A,B). Labeled cells started to exit mitosis, and enter into the G1 phase after 4 h from labeling and they continued to accumulate in the G1 peak during 12 h after labeling. LPA alone slowed the progression of BrdU-labeled cells out of S-phase. Fewer cells accumulated in G2/M until 8 h after labeling. However, there was no significant change in the progression of BrdU-labeled cells through mitosis and into the following G1 phase (Fig. 1A,B,C). The percentage of cells in the G1 peak increased in parallel in LPA treated and control cells from 4 to 12 h after labeling. Lysophosphatidate Reverses G2/M Arrest by Taxol Taxol slightly delayed the transit of labeled cells out of S-phase, resulting in a lower decrease of cells in S-phase for the first 4 h of treatment. It also inhibited progression through mitosis, as expected [30]. The percentage of labeled cells in the G2/M peak increased continuously from the time of Taxol addition up to 12 h later (up to 70614% at 12 h), whereas there was no significant progression of labeled cells through mitosis into the following G1 (Fig. 1A,D). Addition of LPA to the Taxol treatment restored the passage of the cells through S-phase to the levels observed in the absence of Taxol. Cells treated with both Taxol and LPA started to exit S-phase immediately after labeling. Approximately 60% of labeled cells had progressed into the G2/M phase at 2 h after labeling. Labeled cells started to exit mitosis and enter into the G1 phase after 4 h from labeling. They continued accumulating in the G1 peak to 12 h after labeling, much like the untreated cells did. In fact, the percentage of cells in the G1 peak increased as fast in Taxol and LPA treated cells as it did in control cells from 4 to 12 h after labeling (Fig. 1A,E). LPA, therefore, enabled the Taxol- treated cells to start exiting G2/M after 4 h. There was consequently a marked increase in accumulation of cells in the G1 peak from 665% at 4 h to 4969% after 12 h incubation (p,0.05) (Fig. 1A,E). BrdU-labeled cells, therefore, progressed through the cell cycle and accumulated in the G1 peak. This evidence indicates that LPA releases the cells from G2/M arrest in the presence of Taxol. To investigate further this apparent progression through mitosis in the presence of Taxol and LPA, we pretreated MCF-7 cells with 50 nM Taxol in the presence of 10% delipidated serum for 24 h to establish a G2/M arrest. We then labeled cells that were still cycling through S-phase with BrdU for 2 h. The cells were then incubated further with Taxol in the presence or absence of LPA (Protocol B; Fig. 2). Maintaining Taxol increased (p,0.05) the percentage of BrdU-labeled cells in the G2/M peak from 5068% at the end of the labeling period to a maximum of 6368% at 4 h later, as expected (Fig. 2A,B). Although the mitotic block was not absolute and the percentage of labeled cells in the G2/M peak decreased after 4 h; only 40%68% of labeled cells had re-entered into the G1 peak at 12 h after the labeling. Moreover, these cells did not progress significantly into the following S-phase. The percentage of labeled cells detected in the S-phase peak decreased during the first 4 h and remained at this low level to the end of the experiment at 12 h. Addition of LPA to the Taxol-treated cells decreased (p,0.05) the percentage of labeled cells in the G2/M peak from 4 h until 12 h after time of addition (Fig. 2A,C). After 12 h only 1965% remained in G2/M. Consistent with a release from the G2/M block, LPA consistently induced the expected concomitant increase in the percentage of labeled cells detected in the G1 peak from 1764% at the time of addition to 6867% at 12 h later (p,0.01) (Fig. 2A,B,C). These results show that LPA does not produce a decrease in the percentage of cells in G2/M by inducing selective killing of these cells. Instead, it induces their release from the Taxol-induced G2/M arrest. The cells released from mitotic arrest underwent mitosis and started another cell cycle, entering the following G1 phase in less than 4 h, and the following S phase after 8 h (Fig. 2A,C). We next verified these highly unexpected conclusions by studying the effects of Taxol and LPA on cell death and the morphology of the nucleus and spindles in MCF-7 compared to MDA-MB-468 cells. MCF-7 cells do not express caspase-3 [31] whereas MDA-MB-468 cells express relatively high caspase-3 activity [32]. We incubated both cell lines for 26 h with 10% delipidated serum and 50 nM Taxol and then used microscopy to study their fates in the presence or absence of LPA (Protocol C). We also removed Taxol from the incubations for the last 12 h to PLoS ONE | www.plosone.org 2 May 2011 | Volume 6 | Issue 5 | e20608