EMT and Transformation Figure 2. EPT1 cells have undergone EMT. A. Representative light microscopic images show morphological changes of EPT1 cells derived from EP156T cells at both low density (a, d) and high density cultures (b, c, e, f). EPT1 cells are fusiform and much longer than EP156T cells. Overlapping growth of EPT1 cells is also shown (d, e, f). B. Real-time qPCR showing the cadherin switch of mRNA levels in EPT1 compared to EP156T using TaqMan assays for E-cadherin (Hs00170423_m1) and N-cadherin (Hs00169953_m1). C. Western blots show the cadherin switch of protein levels. Both primary antibodies against E-cadherin and N-cadherin (BD Transduction Labs.) were diluted 1:2500. The anti-actin mouse Mab was diluted 1:250. The HRP- conjugated anti-Ig was diluted 1:8000. Molecular weights (kD) were estimated based upon the ECL DualVue Marker in wells labelled M. D. Indirect immunofluorescence images (206) show decreased E-cadherin and increased N-cadherin in EPT1 cells (right panels). The primary antibody against E- cadherin was diluted 1:50 and against N-cadherin (BD Transduction Labs.) 1:25 and the FITC-conjugated secondary antibody 1:250. doi:10.1371/journal.pone.0003368.g002 members of gap junction protein beta family exhibited dramat- ically reduced expression in EPT1 cells (Table 2). Hemidesmo- somes and focal adhesions are required for epithelial cells to attach to the underlying basement membrane. Most components of the hemidesmosomes were downregulated in EPT1 cells compared with the parental cells, especially dystonin and keratins. Compo- nents of the focal adhesions were also changed in EPT1 cells (Table 2). These observations, together with the consistently changed EMT markers, indicated that the regulation of EMT was orchestrated not only in cell phenotype transition, but also in entire modules of cell junctions. The complete changes of cell junctions make EPT1 an ideal model to study the complex regulatatory networks of EMT. EPT1 cells display gene expression patterns in common with prostate cancer cell lines EMT has been frequently observed in transformed cell lines. We asked if EPT1 cells have similar gene expression profiles as prostate cancer cells represented by PC3 and DU145. Differen- tially expressed genes between EP156T and EPT1 and between EP156T and prostate cancer cell lines were compared. In total 1858 genes differed more than 3 fold between EPT1 and EP156T cells. As shown in Figure S1, most of the genes altered after EMT in EPT1 cells were also altered when comparing the PC3 and DU145 to the EP156T cells. More than 70% of the downregulated genes in EPT1 overlapped with genes underexpressed in PC3 cells compared to the EP156T cells. Interestingly, in PC3 cells many epithelial markers including E-cadherin and the cytokeratins KRT4 and KRT5 were also significantly downregulated, and the mesenchymal markers including N-cadherin were also upregu- lated. This is consistent with a previous report that PC3 cells are mesenchymal-like cells that can be induced to mesenchymal to epithelial transition (MET) by overexpression of secreted Frizzled- related protein 3 (sFRP3) [19]. Both the loss of contact inhibition of EPT1 and the similar gene expression changes of EPT1 and prostate cancer cells versus EP156T cells indicated a development towards transformation of EPT1 cells. EPT1 cells are not transformed although they have undergone EMT The observation that changed genes in EPT1 share patterns with prostate cancer cell lines made us ask whether EPT1 cells were transformed. An ability to survive and proliferate under serum-free condition is one of the well-known features of cancer cells in vitro [32]. We examined the proliferation of EPT1 cells in medium with different concentrations of fetal calf serum (FCS). As shown in Figure 4A, both EP156T and EPT1 cells grew much slower in 1% FCS than in 5% FCS and completely stopped growing and died in medium without FCS after 48h incubation, while prostate DU145 cancer cells still grew well in FCS-free medium although slower than in complete medium. The long PLoS ONE | www.plosone.org 4 October 2008 | Volume 3 | Issue 10 | e3368