Genes CDH1 CDH3 DSP OCLN KRT5 KRT14 CDH2 CDH11 FN1 VIM FBN1 FGF1 FGFR1 SPARC TWIST2 WNT5A BMP4 FZD7 ZEB1 Epithelial cell markers Mesenchymal cell markers Known regulators of EMT 228 2138 29 27 2538 232 33 74 6 4 3 3 9 6 3 4 3 3 3 3E-6 2E-10 6E-5 5E-3 4E-11 2E-13 1E-10 1E-14 7E-3 13E-3 8E-15 2E-6 1E-12 2E-2 9E-5 1E-4 1E-3 1E-3 10E-12 Kim JB [62] Jarrard DF [63] Savagner P [64] Medici D [65] Kogan I [20] Kogan I [20] Lee JM [2] Sarrio´ D [67] Yang Z [68] Billottet C [69] Kiemer AK [23] Billottet C [69] Acevedo VD [66] Gotoh N [70] Yang J [71] Dissanayake SK [72] Theriault BL [73] Vincan E [74] Eger A [75] Table 1. Known markers of epithelial and mesenchymal cells were changed in EPT1 cells. Fold change EPT1/EP156T p-value References doi:10.1371/journal.pone.0003368.t001 Cytogenetic analysis of EP156T and EPT1 Abnormal karyotypes are typically observed in transformed prostate epithelial cells [36–38]. By G-banding we found no pronounced chromosomal alterations occurring between EP156T and EPT1 cells (Figure S2). Most likely a clonal selection has occurred as the EPT1 cells did not show any of the additional marker chromosomes. Both the EP156T cells and the progeny EPT1 cells harboured a marker chromosome which by FISH (fluorescent in situ hybridization) analysis was shown to be a derivative chromosome 20 (der(20), Supplementary Figure 2), Whether gain of der(20) occurred prior to immortalization or as a consequence of EP156T immortalization is unknown. However, a gain of chromosome 20 was found in both non-malignant and malignant cells immortalised by hTERT [39]. As this gain is not a common finding in prostate cancer [40–42] this could result from the immortalization process. Discussion This work is the first report of an EMT model based on primary, immortalized prostate epithelial cells. The derivation of EPT1 from EP156T was clearly verified by the similar expression levels of hTERT, the puromycin resistance and the very similar karyotypes of both cell lines, including a common derivative chromosome, der(20). Several ways to induce EMT in prostate cancer cells have been described, including overexpression of CAV1 and ID1 or MMP14 in LNCaP cells [13,14], EGF treatment of DU145 cells [15], depletion of PDEF [16] or BMP7 treatment of PC3 cells [18]. However, some of these studies cannot easily be reconciled, such as EMT and Transformation both EMT and MET induction of PC3 cells [16–19]. Long term passages of these cell lines in different laboratories may cause them to differ significantly from the original patient cells, and new models closer to prostate tissue are desirable. EMT is not easily observed in histological examinations of cancer tissue sections, even by experienced pathologists [2,43]. This has led to the idea that EMT may be transient during cancer progression [44,45] or occur only in a subpopulation of the tumour cells, such as cells at the invasive front [5,8,46–48] or cancer stem cells [11,49,50]. The successful establishment of an EMT model based on primary prostate cells with many traits of the prostate basal cell phenotype is important in light of a recent report on the significance of EMT in prostate cancer[12]. EMT has been considered an event following malignant transformation to endow cancer cells invasive and metastatic competence [6,8,9]. The present work shows that primary prostate epithelial cells underwent complete EMT, while most of the transformation assays remained negative, including serum and growth factor independent growth, as well as anchorage independent growth and chromosome instability (Figure 4). At the same time, loss of contact inhibition combined with similar differential gene expression patterns as in prostate cancer cell lines indicated that EPT1 cells are in the early steps towards transformation. Regarding the temporal relationship between transformation and EMT, one recent report has also observed that EMT emerged at a very early stage of transformation (early HF1 cells) [10]. However, EMT in that system was far from full completion, lacking mesenchymal characteristics in both early and later transformation stages, and the downregulation of E-cadherin was significant neither in early (0.960.1) nor in later (0.560.2) stages of transformation, and N-cadherin was not found upregulated. To our knowledge, EPT1 cells represent the first model in which EMT has finished completely at very early stages of malignant transformation. It has previously been shown that ectopic expression of hTERT may be sufficient to immortalize cells in vitro, but not sufficient to achieve, anchorage-independent cell growth or tumorigenesis [25]. Full malignant transformation is a stepwise process and can be achieved by additional expression of oncogenes such as the SV40 early region plus activated RAS [25,51]. EMT was induced in the hTERT immortalized primary prostate epithelial EP156T cells without exogenous introduction of oncogenes and without transformation. Furthermore, karyotype analysis revealed that the EPT1 cells were diploid and apparently contained much less karyotypic abnormalities than previously described for the prostate cancer cell lines LNCaP, PC3 or DU145 [52,53]. This work shows that EMT itself appears as a coordinated gene expression program that may be switched on in either non-transformed or transformed cells. It is possible that EMT may contribute towards malignant transformation in non-transformed cells, and that in transformed cells EMT may contribute to a more aggressive phenotype. Genomic features of transformation such as loss of heterozygocity or chromosomal rearrangements cannot easily be reversed. In this respect EMT regulation may provide an important target for cancer therapy. Work is ongoing to further transform EPT1 cells by additional stimulation or oncogene introduction. This model is therefore valuable for understanding the mechanistic relationship between EMT and transformation. EPT1 cells lost contact inhibition accompanied by EMT during the selection of cells insensitive to full confluence. EP156T cells ceased growing once cells abutted on each other in contrast to EPT1 cells whose protrusions passed across neighbouring cells, eventually leading to heaps of cells. It is still unknown what happened first or emerged simultaneously in EPT1 cells. Actually, PLoS ONE | www.plosone.org 6 October 2008 | Volume 3 | Issue 10 | e3368