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Abstract
Using DIC and confocal microscopy, changes in morphology, migratory characteristics and adherence junctions (AJs) were analyzed in the mammary carcinoma cell line MCF-7-SNAI1 after activation of the EMT transcription factor SNAI1. Western Blot analysis showed that after removal of tetracycline from the cell culture medium expression of SNAI1 reached its peak in 24 hours and then plateaued for 7 days. During the 7 days the cells continued to express E-cadherin; however, tangential AJs typical for cells with stable cell-cell adhesion, changed into radial AJs. The radial AJs continued to accumulate E-cadherin during 24‑72 hours after tetracycline removal. As a result of SNAI1 activation, the cells underwent epithelial-mesenchymal transition (EMT) and became migratory. On a two-dimensional substrate, cells exhibited both individual and collective migration. As the tetracycline washout period progressed, the fraction of the cells capable of migrating through migration chamber membranes increased; on the contrary, cells’ ability to invade an epithelial monolayer decreased. These results demonstrate that retaining a hybrid epithelial/mesenchymal phenotype and accumulation of E-cadherin in AJs during early stages of EMT do not impede disruption of stable cell-cell adhesion and cells’ acquisition of migratory activity.
Highlights
Snail1 is a 29 kDa transcription factor belonging to the Snail superfamily
As the tetracycline washout period progressed, the fraction of the cells capable of migrating through migration chamber membranes increased; on the contrary, cells’ ability to invade an epithelial monolayer decreased. These results demonstrate that retaining a hybrid epithelial/mesenchymal phenotype and accumulation of E-cadherin in adherence junctions (AJs) during early stages of epithelial-mesenchymal transition (EMT) do not impede disruption of stable cell-cell adhesion and cells’ acquisition of migratory activity
MCF-7 cells stably expressing a tetracyclineregulated SNAI1 (MCF-7-SNAI1) were subjected to Western Blot analysis to determine the kinetics of expression of EMT transcription factor SNAI1 as well as the AJ protein E-cadherin after removal of tetracycline (Tet) which triggers SNAI1 expression
Summary
Snail (human SNAI1) is a 29 kDa transcription factor belonging to the Snail superfamily. Snail works as a transcription repressor for E-cadherin, occludin and claudins [2,3,4]. Snail induces expression of vimentin and fibronectin [6]. Snail together with Twist induce Zeb expression [6, 7]. It is considered that repression of E-cadherin expression induced by Snail weakens cell-cell adhesion during epithelial-mesenchymal transition (EMT) [2, 3], a process that promotes invasion and metastasis. During EMT, epithelial cells lose epithelial markers (E-cadherin, occludin, cytokeratins etc.) and begin to express mesenchymal markers (vimentin, fibronectin etc.). They lose basal-apical polarity and acquire migratory capabilities [8]. The goal of the present study was using DIC and confocal microscopy, to study changes in cell phenotype resulting from expression of the exogenous EMT transcription factor SNAI1 in the mammary tumor cell line MCF-7-SNAI1
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