EP156T is a primary prostate cell line immortalized by retroviral hTERT expression and retains many features of the prostate basal cell phenotype [20]. In an attempt to select for cells insensitive to contact inhibition [21,22], EMT was observed in EP156T cells accompanied by loss of contact inhibition, and these changed new cells were named EPT1. The progeny EPT1 cells exhibited a distinct morphology and increased migration and invasion compared with the parental EP156T cells, and gene expression analysis revealed a striking number of gene expression changes previously found as markers of EMT [2,9,10,23,24]. Downregulation of E-cadherin (CDH1) and upregulation of N-cadherin (CDH2) - the so called cadherin- switch - were most evident in EPT1 cells. It appeared that entire gene modules involved in cell-to-cell and cell-to-matrix interac- tions changed their transcription patterns in a coordinated manner in EPT1 cells. Transformation assays revealed, however, that EPT1 cells still were serum and growth factor dependent and had not acquired ability to anchorage indepen- dent cell growth. This model therefore allows a study of EMT in primary prostate cells and separated from full malignant transformation. Results EP156T cells lost contact inhibition after long term culture at high confluence The EP156T cell line is a primary prostate epithelial cell line immortalized by hTERT and has been previously characterized [20]. EP156T cells exhibit a significant pattern of authentic prostate-specific features with the capacity to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. It has previously been shown that ectopic expression of hTERT may be sufficient to immortalize cells in vitro, but not sufficient to achieve transfor- mation [25]. This is consistent with the behaviour of EP156T cells, which exhibited properties of primary prostate basal cells [20]. EP156T cells grow in a monolayer and cease proliferation upon reaching confluence, a phenomenon referred to as cell contact inhibition. Loss of contact inhibition is one feature of malignant transformation [26]. One approach of transformation is to select cells that are insensitive to contact inhibition [22]. In order to transform EP156T cells to prostate cancer cells, the cells were kept growing at passage 43 at high confluence to select cells that survived contact inhibition. Cell death increased once they reached full confluence. Following 12 weeks thereafter with very slow proliferation, cells started to grow faster and kept on dividing and piled up despite high cell density. The changed cells were named EPT1. The growth curves of EPT1 and EP156T cells clearly displayed different behavior when cells were grown at high confluence. Many of the EP156T cells died while EPT1 cells continued proliferation (Figure 1A). Actually, EPT1 cells can grow on top of each other and pile up even under low cell density as shown in the DAPI nuclear staining in Figure 1B. Loss of cell contact inhibition may emerge before anchorage- independent cell growth and tumour formation in animals during the transformation process [27,28]. The ability of EPT1 cells to override contact inhibition suggested development in the direction of transformation. EMT emerged during the change from EP156T to EPT1 Apart from their loss of contact inhibition, EPT1 cells changed significantly in morphological features compared with EP156T cells. EP156T cells have a very clear and round boundary with EMT and Transformation individual cells abutting on each other in a uniform array. There are regularly spaced cell to cell junctions and adhesions between neighbouring cells (Figure 2A). Very differently, EPT1 cells have a much longer and irregularly scattered cell shape, character- istically varying in composition and density like mesenchymal cells (Figure 2A) and corresponding to known features of epithelial to mesenchymal transition [2]. The morphological traits of EMT were consistent with key differences between EP156T and EPT1 cells regarding E-cadherin and N-cadherin gene expression as shown in Figure 2B–D and detailed below. hTERT was expressed at similar levels in both EP156T and EPT1 and the exogenous nature was supported by very high puromycin resistance of these cells, which is consistent with hTERT expression from a puromycin resistance retroviral vector (data not shown) [20]. EPT1 cells were trypsinized and propagated in standard EP156T medium with FCS increased to 5%. Increased migration and invasion of EPT1 cells EMT is characterized by increased motility and gain of invasiveness. To determine whether EPT1 cells had become more active in migration and invasion than the EP156T cells, we evaluated the serum-induced migration of cells using transwell chamber assays. As shown in Figure 3A, EPT1 migrated through the pores more than 2.5 fold faster than EP156T cells. The invasive ability of EPT1 and EP156T cells was examined by the serum-induced invasion through the transwell extracellular matrix layer. As shown in Figure 3B, EPT1 cells exhibited 3 fold increase in invasion compared to EP156T cells after 24 h incubation. The faster migration of EPT1 compared with EP156T cells was also confirmed by the wound healing assay for 24 h (Figure 3C). EP156T cells moved as a sheet en block as described for epithelial cells, whereas EPT1 cells migrated considerably more dynamically and moved individually and sometimes left a part of the trailing region behind as described for mesenchymal cells [2]. All these results indicated that the EPT1 cells had acquired much higher ability to migrate and invade during the transition to mesenchy- mal-like cells in comparison with EP156T cells. EPT1 cells display striking gene expression switch patterns characteristic of EMT To identify gene markers involved in EMT from EP156T to EPT1 cells, we profiled the gene expression of both cell lines using the Agilent Human Whole Genome Oligo Microarray, which contained 44 k probes. There were 965 genes downregulated and 893 genes upregulated more than 3 fold in EPT1 cells compared with EP156T cells. A number of gene expression changes associated with EMT varied significantly between EPT1 and EP156T (Table 1). Loss of E-cadherin (CDH1), the prototypic epithelial adhesion molecule in adherens junctions, and gain of N- cadherin (CDH2) are among the main hallmarks of EMT [2]. CDH1 was downregulated 27 fold and CDH2 upregulated 33 fold in EPT1 cells. The cadherin switch was verified by both real-time qPCR (mRNA level) and Western-blotting (protein level) (Figure 2B–C). Immunofluorescence staining showed very clearly the disappearance of E-cadherin and gain of N-cadherin in the membrane of EPT1 cells (Figure 2D). The epithelial markers including cytokeratin 14 (KRT14), KRT5 and p63 that charac- terized EP156T [20], were all downregulated more than 100 fold in EPT1 cells. In contrast, many mesenchymal markers were upregulated in EPT1 cells, including cadherin 11 (CDH11), vimentin (VIM) and fibronectin (FN). Apart from these EMT markers, several transcription factors that are known to regulate PLoS ONE | www.plosone.org 2 October 2008 | Volume 3 | Issue 10 | e3368