S. phenotype (epithelial-to-mesenchymal changeover, or EMT) is a hallmark tumor dissemination and progression in many carcinomas (14, 15); however, ovarian carcinomas exhibit an early gain of epithelial phenotype or mesenchymal-to-epithelial transition. This gain of an epithelial gene profile aids in disseminating cells in multicellular aggregate (spheroid) formation and anoikis resistance (16). Later in progression, the cells revert to a mesenchymal phenotype (EMT), and it is proposed that this phenotypic plasticity contributes to decreased response to conventional therapeutics (17,C20). Furthermore, recent work has demonstrated a role for the mesenchymal phenotype in mesothelial cell invasion at metastatic sites in the peritoneal cavity (21). Understanding the mechanism(s) regulating this phenotypic plasticity may enable reversal of acquired resistance to chemotherapeutic agents. Acquisition of mesenchymal phenotype via EMT can be initiated by extracellular matrix context, disruption of adhesion, and soluble signaling factors (22, 23). Of these factors, lysophosphatidic acid (LPA) has emerged as a potentially important mediator of EMT in ovarian carcinoma due in part to its high concentration in the ovarian cancer microenvironment (up to Dyphylline 80 m) (24, 25). LPA modulates mitogenic activity, motility, escape from anoikis, and survival effects through a class of heterotrimeric G-protein-coupled receptors, LPA1 to LPA5 (26, 27). One mechanism by which LPA modulates motility and facilitates the gain of a mesenchymal phenotype in EOC is disruption of E-cadherin-based cell-cell adhesions (28,C31). Of particular interest is the fate of adherens junction-associated and/or cytoplasmic -catenin following disruption of cell-cell adhesions. In intact epithelial tissues, -catenin displays junctional localization and is associated with the cytoplasmic domain of E-cadherin. Junction disruption can induce pathways that subsequently target -catenin for proteosome-mediated degradation or for nuclear translocation and transcriptional regulation (32). As previous work has demonstrated the intersection of cytoplasmic and nuclear Dyphylline -catenin pools (33), this study LRAT antibody investigated the consequences of LPA-induced disruption of E-cadherin-mediated cell junctions on subcellular -catenin localization and Tcf/Lef/-catenin transcriptional activity. These results provide additional support for the role of ligand-independent -catenin activity in serous epithelial Dyphylline ovarian carcinomas. Experimental Procedures Cell Culture OVCA429 and OVCA433 cell lines were generously provided by Dr. Robert Bast, Jr. (M.D. Anderson Cancer Center, Houston, TX), and were maintained in MEM (Gibco Invitrogen), 10% fetal bovine serum (Gibco Invitrogen), penicillin/streptomycin (Gibco Invitrogen), amphotericin B (Cellgro by Mediatech), nonessential amino acids (Cellgro by Mediatech, Herndon, VA), and sodium pyruvate (Cellgro by Mediatech) at 37 C in 5% CO2. Multicellular aggregates were formed in 96-well plates coated with 50 l of 0.5% agarose in serum-free media by seeding 5000 cells per well in serum-free media and incubating overnight at 37 C in 5% CO2. Aggregate formation was confirmed by light microscopic visualization. Lysophosphatidic Acid LPA was purchased from Cayman Dyphylline Chemical (Ann Arbor, MI) and was prepared for use by dehydrating the lyophilized lipid under a tissue culture hood, on ice, overnight. LPA was resuspended in 1% BSA Dyphylline in PBS at a final concentration of 2 mm, allowed to dissolve on a rotator at 4 C overnight, and then aliquoted. Aliquots in use were stored at ?20 C and at ?80 C for long term storage. Where indicated, cells were pretreated for 15 min prior.