Most descriptions of transformed epithelial cells in vitro focus on cell lines that underwent EMT. We, however, decided to study transformed epithelial cell lines that had a slightly changed morphology. We chose an IAR epithelial line transformed in vitro with dimethylnitrosamine (IAR-6-1). As previously reported [35], the IAR-6-1 line was tumorigenic in syngenic rats. In comparison with IAR-2 cells, the morphology of IAR-6-1 cells was minimally changed. Single IAR-6-1 cells were polygonal or discoid. In confluent culture, IAR-6-1 cells formed a monolayer similar to the IAR-2 monolayer (Figure 1E and 1F). Western blot analysis showed that transformed IAR-6-1 epithelial cells retained expression of epithelial E-cadherin and did not express mesen- chymal N-cadherin (Figure S1). Differences between nontrans- formed IAR-2 cells and transformed IAR-6-1 cells became evident when they were fluorescently stained for the actin cytoskeleton and E-cadherin. In contrast with nontransformed IAR-2 cells, IAR-6-1 cells had only randomly oriented actin bundles in the cytoplasm; the marginal actin bundles had disappeared (Figure 1F). We also revealed the dramatic rearrangement of E-cadherin-based AJs in IAR-6-1 cells that were organized into strands arranged perpendicularly or at different angles to the cell edge (Figure 1G). We designated AJs of transformed epithelial cells ‘radial AJs’. Double fluorescence staining showed that AJs in transformed IAR- 6-1 cells were colocalized with short straight actin bundles (Figure 1G and 1H). In dense cultures, some AJs were oriented along the cell-cell borders. We also established 14 clones of IAR-2 cells transformed with Ras and compared their morphology. Cells of nine Ras- transformed clones (IAR1162 clones) underwent EMT: they lost the epithelial phenotype and acquired fibroblastic-like morphol- ogy. As Western blotting showed, IAR1162 cells did not expressed epithelial E-cadherin and did express mesenchymal N-cadherin (data not shown). We investigated, however, the other five clones of Ras-transformed IAR-2 cells (IAR1170-D6, -D11, -F9, -H5, and -D12), which in comparison with nontransformed IAR-2 cells had slightly changed morphology. Ras-transformed IAR1170 cell lines were tumorigenic in nude mice (Figure S2). All five clones had similar morphology. The shapes of IAR1170 cells were near- discoid or polygonal. In confluent culture, IAR-1170 cells formed monolayers. As an example, we give images of IAR1170-H5 clone (Figure 1I and 1J). Western blot analysis showed that cells of IAR1170 clones retained E-cadherin and also expressed N- cadherin (Figure S1). A characteristic feature of the actin cytoskeleton in IAR1170 cells was the disappearance of the marginal actin bundle (Figure1J). In IAR1170 cells, we also revealed the reorganization of E-cadherin-based AJs. The continuous adhesion belt that was typical of IAR-2 cells was destroyed. AJs stained for E-cadherin were seen as punctate clusters at the cell-cell boundaries. The circumferential actin belts were disorganized (Figure 1K and 1L). Thus, we described the early stages of the morphological transformation of epithelial cells, when cells retained expression of E-cadherin, had discoid or polygonal morphology, and formed monolayers. Neoplastic transformation, however, led to the disappearance of the marginal actin bundle and to the dramatic rearrangement of E-cadherin-based AJs. Alterations of cell–cell interactions and promotion of motile behavior of epithelial cells caused by neoplastic transformation As neoplastic transformation leads to disappearance of the marginal actin bundle and to rearrangements of AJs in epithelial cells, we decided to test whether these alterations contribute to cell-cell interactions of transformed IAR-6-1 and IAR1170 cells. Adhesive Interactions To compare cell-cell interactions in cultures of nontransformed and transformed cells, sparse cultures of IAR-2, IAR-6-1, and IAR1170 cells were observed with time-lapse videomicroscopy. Nontransformed IAR-2 cells in sparse cultures formed islands. Time-lapse imaging showed that cell-cell contacts in these islands were stable (Figure 2A and Video S1) and were disrupted only during mitosis. Contacts between neighboring islands or single IAR-2 cells led to the establishment of strong cell-cell adhesion and development of a continuous monolayer for 2–3 days. In cultures of transformed IAR-6-1 and IAR1170 cells, we revealed significant alterations in cell-cell interactions. In contrast with nontransformed IAR-2 cells, cell-cell contacts in cultures of transformed IAR-6-1 and IAR1170 cells were unstable and often broke (Figure 2B and 2C, Video S2, and Video S3). Transformed cells in contact extended lamellipodia at free edges. During 6–7 h of observation, the same cell could establish and break contacts with different neighboring cells and could move in different directions. This behavior of transformed epithelial cells may promote their migratory activity. To explore the effects of changed motile behavior of transformed IAR-6-1 and IAR1170 cells on their migration, the ability of cells to migrate through polycarbonate membrane inserts with 8-mm pores in Bio-Coat migration chambers was examined. Migration assay showed that the number of migrating IAR-6-1 and IAR1170 cells was higher than that among nontransformed IAR-2 cells (Figure 3). Thus, disappearance of the marginal actin bundle and rearrangement of E-cadherin-based AJs is accompa- nied by reduction in cell-cell adhesion and promotion of migratory activity of transformed cells. Analysis of the dynamics of cell–cell contact formation in a narrow wound For detailed analysis of cell behavior during formation of contact with neighboring cells, nontransformed IAR-2 cells and transformed descendants were imaged in a narrow wound (,100 mm) in a monolayer. As IAR1170 expressed both E- and N-cadherin and there are findings that N-cadherin promotes motility when expressed by epithelial cells [24], for detailed studies we focused on IAR-6-1 cells, which expressed E-cadherin and did not express mesenchymal N-cadherin. We made narrow wounds by scraping cell monolayers with a sterile needle. After 3–4 h of incubation, cells from adjacent monolayers began to form new cell-cell contacts in the area of colliding lamellipodia. In the case of nontransformed IAR-2 cells, there was lateral expansion of initial stable contact between opposing cells (Figure 4A and Video S4). During the first 5–10 min after the establishment of a stable contact, cells formed lamellipodia both in the zone of contact and at the free edges. Within the next 15–20 min, dramatic cessation of protrusive activity along the entire contact (contact paralysis) developed. It began simultaneously along the entire length of the contact. The cells formed small lamellipodia only at the edges of the contact. Contact paralysis did not extend to the free edges of contacting cells. We observed, however, a decrease of protrusive activity at the free edges of contacting cells. We analyzed protrusive activity using kymographs (Figure 4B). Quantitative analysis of kymographs showed that both phases of the protrusion- retraction cycle, especially protrusions, were affected during contact paralysis. The rate of lamellipodial protrusion at the site of the contact was strongly diminished by 15–20 min after the establishment of the contact. The rate of lamellipodial retraction at the site of the contact was also reduced. Analysis of kymographs also showed statistically significant decrease of the mean rate of lamellipodial protrusion at the free edges of contacting cells. (Figure S3). PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e8027