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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

Expression of CK18 and αSMA in RL/DF cells in DF- and DMEM-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DF cells were routinely passaged in DF-based medium [DF in (A)]. The cells were also cultured for 11 days after replacement of the medium with a DMEM-based medium [DF→DMEM in (A)]. These cells were then returned to DF-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides under the conditions indicated, then fixed, and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.
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Figure 4: Expression of CK18 and αSMA in RL/DF cells in DF- and DMEM-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DF cells were routinely passaged in DF-based medium [DF in (A)]. The cells were also cultured for 11 days after replacement of the medium with a DMEM-based medium [DF→DMEM in (A)]. These cells were then returned to DF-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides under the conditions indicated, then fixed, and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.

Mentions: Similarly, we examined the ability of RL/DF cells to exhibit phenotypic conversion between epithelial and mesenchymal phenotypes. The monolayer formed by RL/DF cells was cobblestone-like in morphology, but shrank slightly as observed in RL/DMEM cells cultured in DF-based medium (Figure 4A, upper panels). When the RL/DF cells were transferred to DMEM-based medium and maintained for 11 days in culture, the αSMA expression increased in some, but not all, cells, whereas the expression level of CK18 did not change (Figure 4A, lower panels). When the medium was returned to a DF-based medium, the αSMA-positive cells disappeared (Figure 4B). These findings suggest that a reversible epithelial–mesenchymal phenotypic conversion occurs in at least some populations of RL/DF cells.


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)

Expression of CK18 and αSMA in RL/DF cells in DF- and DMEM-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DF cells were routinely passaged in DF-based medium [DF in (A)]. The cells were also cultured for 11 days after replacement of the medium with a DMEM-based medium [DF→DMEM in (A)]. These cells were then returned to DF-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides under the conditions indicated, then fixed, and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Expression of CK18 and αSMA in RL/DF cells in DF- and DMEM-based mediumThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RL/DF cells were routinely passaged in DF-based medium [DF in (A)]. The cells were also cultured for 11 days after replacement of the medium with a DMEM-based medium [DF→DMEM in (A)]. These cells were then returned to DF-based medium to determine their plasticity (B). The cells were plated in eight-well chamber slides under the conditions indicated, then fixed, and stained with specific antibodies against CK18 or αSMA. Scale bar = 100 μm.
Mentions: Similarly, we examined the ability of RL/DF cells to exhibit phenotypic conversion between epithelial and mesenchymal phenotypes. The monolayer formed by RL/DF cells was cobblestone-like in morphology, but shrank slightly as observed in RL/DMEM cells cultured in DF-based medium (Figure 4A, upper panels). When the RL/DF cells were transferred to DMEM-based medium and maintained for 11 days in culture, the αSMA expression increased in some, but not all, cells, whereas the expression level of CK18 did not change (Figure 4A, lower panels). When the medium was returned to a DF-based medium, the αSMA-positive cells disappeared (Figure 4B). These findings suggest that a reversible epithelial–mesenchymal phenotypic conversion occurs in at least some populations of RL/DF cells.

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