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Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

Takenouchi T, Yoshioka M, Yamanaka N, Kitani H - Cell Biol Int Rep (2010) (2010)

Bottom Line: This process is also essential for liver morphogenesis in embryonic development.To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes.The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 12, Tsukuba, Ibaraki 3058634, Japan.

ABSTRACT
EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.

No MeSH data available.


Related in: MedlinePlus

Immunocytochemical characterization of RL/DMEM and RL/DF cellsThe RL/DMEM (A) and RL/DF cells (B) were plated in eight-well chamber slides in DMEM- and DF-based medium respectively. The next day, the cells were fixed and stained with specific antibodies against CK18, CK19, OX41, desmin, αSMA, ZO-1 or VE-cadherin. ZO-1′ is a magnified picture of ZO-1. Cell nuclei were non-specifically stained with the anti-ZO-1 antibody. Scale bar = 100 μm, except for ZO-1′ (scale bar = 25 μm).
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Figure 2: Immunocytochemical characterization of RL/DMEM and RL/DF cellsThe RL/DMEM (A) and RL/DF cells (B) were plated in eight-well chamber slides in DMEM- and DF-based medium respectively. The next day, the cells were fixed and stained with specific antibodies against CK18, CK19, OX41, desmin, αSMA, ZO-1 or VE-cadherin. ZO-1′ is a magnified picture of ZO-1. Cell nuclei were non-specifically stained with the anti-ZO-1 antibody. Scale bar = 100 μm, except for ZO-1′ (scale bar = 25 μm).

Mentions: The RL/DMEM cells exhibited an epithelial-like morphology (Figure 2A). However, they were negative for CK18 and CK19, even though this cell line was established from a primary culture of purified parenchymal hepatocytes (Figure 2A). The cell–cell adhesion boundaries of RL/DMEM cells were immunostained with the anti-ZO-1 antibody, suggesting that they still retain some characteristics of epithelial cells (Figure 2A). The RL/DMEM cells were completely negative for OX41, VE-cadherin and desmin, suggesting that they do not have any characteristics of Kupffer cells, sinusoidal endothelial cells or quiescent HSCs. Strikingly, the RL/DMEM cells were strongly positive for αSMA, a marker of hepatic myofibroblasts and activated HSCs (Figure 2A). From these findings, it can be considered that RL/DMEM cells have properties not only of epithelial cells, but also cells of the mesenchymal lineage.


Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

Takenouchi T, Yoshioka M, Yamanaka N, Kitani H - Cell Biol Int Rep (2010) (2010)

Immunocytochemical characterization of RL/DMEM and RL/DF cellsThe RL/DMEM (A) and RL/DF cells (B) were plated in eight-well chamber slides in DMEM- and DF-based medium respectively. The next day, the cells were fixed and stained with specific antibodies against CK18, CK19, OX41, desmin, αSMA, ZO-1 or VE-cadherin. ZO-1′ is a magnified picture of ZO-1. Cell nuclei were non-specifically stained with the anti-ZO-1 antibody. Scale bar = 100 μm, except for ZO-1′ (scale bar = 25 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475435&req=5

Figure 2: Immunocytochemical characterization of RL/DMEM and RL/DF cellsThe RL/DMEM (A) and RL/DF cells (B) were plated in eight-well chamber slides in DMEM- and DF-based medium respectively. The next day, the cells were fixed and stained with specific antibodies against CK18, CK19, OX41, desmin, αSMA, ZO-1 or VE-cadherin. ZO-1′ is a magnified picture of ZO-1. Cell nuclei were non-specifically stained with the anti-ZO-1 antibody. Scale bar = 100 μm, except for ZO-1′ (scale bar = 25 μm).
Mentions: The RL/DMEM cells exhibited an epithelial-like morphology (Figure 2A). However, they were negative for CK18 and CK19, even though this cell line was established from a primary culture of purified parenchymal hepatocytes (Figure 2A). The cell–cell adhesion boundaries of RL/DMEM cells were immunostained with the anti-ZO-1 antibody, suggesting that they still retain some characteristics of epithelial cells (Figure 2A). The RL/DMEM cells were completely negative for OX41, VE-cadherin and desmin, suggesting that they do not have any characteristics of Kupffer cells, sinusoidal endothelial cells or quiescent HSCs. Strikingly, the RL/DMEM cells were strongly positive for αSMA, a marker of hepatic myofibroblasts and activated HSCs (Figure 2A). From these findings, it can be considered that RL/DMEM cells have properties not only of epithelial cells, but also cells of the mesenchymal lineage.

Bottom Line: This process is also essential for liver morphogenesis in embryonic development.To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes.The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 12, Tsukuba, Ibaraki 3058634, Japan.

ABSTRACT
EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.

No MeSH data available.


Related in: MedlinePlus