Reestablishment of Rabbit Gallbladder Epithelial Cells in Collagen Gel Culture and their Alterations by Cytochalasin B and Transforming Growth Factor Beta-1

Yoshida, Kazuharu; Kawakami, Kazuyoshi; Kawamura, Yasuhito; Saito, Katsuhiko; Nakanuma, Y

doi:10.1016/s0344-0338(96)80119-1

Cited by 6 publications

(13 citation statements)

References 18 publications

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“…Normal rabbit gallbladder epithelial cells primarily cultured in the collagen gel matrix form monolayered spherical cystic structures. These cells can completely reestablish polarity, showing microvilli on the apical surface, basal lamina on the basal site, and a completed junctional complex at the upper lateral site, and they tightly adhere to neighboring cells (Yoshida et al 1996;Nakanuma et al 1997;. Since the spherical cystic structures formed by GB-d1 cells in SC were microscopically and electron-microscopically similar to those formed by normal rabbit gallbladder cells, it can be speculated that GB-d1 cells in SC behave like moderately differentiated or well-differentiated cancer cells.…”

Section: Discussionmentioning

confidence: 99%

The role of E-cadherin in the differentiation of gallbladder cancer cells

Mukai

Miyazaki

Yakushiji

2001

Cell and Tissue Research

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Cell adhesion molecules are essential for development and maintenance of epithelial architecture. To clarify the role of these molecules in the morphology of gallbladder cancers, four human gallbladder cancer cell lines (GB-d1, KMG-C, GBK-1, and G-415) were examined in vitro. They showed noticeably different morphologies in our standard gel cultures (SC). GB-dl and KMG-C formed cystic and spheroid structures, respectively, which seemed to represent well-differentiated and moderately differentiated cancers, respectively. GBK-1 and G-415 showed branching and "pseudoglandular" structures, respectively, both of which seemed to indicate original dedifferentiated cancers. In floating gel culture (FC), only GB-d1 showed a highly increased tendency toward cyst formation. Expression of E-cadherin and alpha-catenin in the gallbladder cancer cell lines was investigated by Western-blotting analysis. Expression was detected in GB-d1 and KMG-C, but not in GBK-1 and G-415 cells. Furthermore, E-cadherin expression in GB-dl was 1.82 times greater in FC than in SC, while E-cadherin expression levels of KMG-C did not change. Neither GB-d1 nor KMG-C showed any difference in a-catenin expression between SC and FC. Immunostaining of GB-d1 revealed that these proteins were localized to the cell membrane. In contrast, heterogeneous localization of these proteins was detected in the spheroid structures of KMG-C, in both SC and FC. Electronmicroscopic examination revealed that reestablishment of the junctional complex occurred only in GB-d1 cells cultured in FC. The formation of cystic structures in GB-d1 was completely inhibited by an antibody against human E-cadherin. Both expression of E-cadherin and its membranous localization are required for well-differentiated-type morphogenesis in gallbladder cancer cells.

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Section: Discussionmentioning

confidence: 99%

The role of E-cadherin in the differentiation of gallbladder cancer cells

Mukai

Miyazaki

Yakushiji

2001

Cell and Tissue Research

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“…Epimorphin was added to monolayer-cultured gallbladder epithelial cells at a concentration of 100 µg/ml for 7 days, starting at day 1 of culture, and was added directly to the collagen gel matrix of collagen gel cultured gallbladder epithelial cells at a concentration of 100 µg/ml. The concentrations of EGF, HGF, epimorphin, and TGF-β1 were based on other in vitro studies (Sugihara et al 1993;Halttunen et al 1996;Yoshida et al 1996;Koshida and Hirai 1997;Toda et al 1997;Li et al 1998;Nishimura et al 1998). …”

Section: Primary Culture Of Rgb Epithelial Cells In Collagen Gel Matrixmentioning

confidence: 99%

Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels

Mori

Miyazaki

2000

Cell and Tissue Research

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Although peptide growth factors play an important role in the morphogenesis of gallbladder, little is known about how they effect the morphogenesis of gallbladder epithelial cells. Rabbit gallbladder epithelial cells (RGEC) were isolated and cultured in monolayer or collagen gels. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), epimorphin, transforming growth factor-beta 1 (TGF-beta 1), and fibroblast-conditioned medium (FCM) were added to the cultured cells to clarify the effects of these peptides and FCM on morphogenesis of RGEC. RGEC suspended in collagen gels form spherical cysts with morphologic polarity. EGF, HGF, epimorphin, and FCM promoted cyst maturation by accelerating the proliferation and aggregation of clear, polarized vesicles. In contrast, TGF-beta 1 markedly inhibited DNA synthesis in both monolayer and collagen gel cultures and promoted formation of branching structures in collagen gels. Furthermore, in the presence of EGF, TGF-beta 1 induced a drastic change in morphogenesis, with the formation of branching networks that showed cell-cell contact only at sites where branches touched. RGEC-forming multicellular cysts did not express vimentin but expressed significant amounts of cytokeratin and regained junctional complexes. In contrast, TGF-beta 1-treated cells strongly expressed vimentin along with branching structures and showed decreases in cytokeratin expression and junctional complexes. Thus, TGF-beta 1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular changes, with loss of both epithelial polarization and junctional complexes. These results suggest that the morphogenetic program of RGEC is likely to be determined by the interaction of these peptides and the timing of their presence.

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“…2B). These lectins are known to bind to the specific carbohydrate residues of glycoproteins (Terada and Nakanuma, 1994;Yoshida et al, 1996). Therefore, it seems reasonable to propose that such carbohydrate residues which are detectable by lectin histochemistry Fig.…”

Section: Other Phenotypic Markers Of Gallbladdermentioning

confidence: 99%

“…Loss and restoration of epithelial cell polarity are evaluable using this model (Yoshida et al, 1996).…”

Section: Three-dimensional Cell Culturementioning

confidence: 99%

“…Yet there are a number of growth factors, like transforming growth factor ␤1 (TGF-␤1) and hepatocyte growth factor (HGF), and of adhesion molecules like ␣2␤1, ␣3␤1, and ␣6␤4 integrins, that act as morphogens (Berdichevsky et al, 1994;Yoshida et al, 1996). For example, the addition of TGF-␤1, which is able to influence the proliferation and maturation of epithelial cells, caused the epithelial cells in the multicellular cysts cultured in the collagen gel to become taller and more multilayered (Fig.8A).…”

Section: Modifications Of Monolayer and Three-dimensional Cell Culturmentioning

confidence: 99%

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Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium

Nakanuma

Kawakami

Kawamura

et al. 1997

Microsc. Res. Tech.

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Several models for preparing and isolating human and animal gallbladder epithelial cells, including low‐grade gallbladder carcinoma cells, as well as proposed systems for culturing these isolated epithelial cells are reviewed here. Several reports concerning tissue culture of the gallbladder are also reviewed. The cell culture systems are divided into monolayer cell culture on collagen‐coated or uncoated culture dishes or other culture substrate and three‐dimensional cell culture in collagen gel. To prepare and isolate gallbladder epithelial cells, digestion of the gallbladder mucosa, abrasion of the mucosal epithelial cells, and excision of epithelial outgrowth of mucosal explants are applied. In monolayer cell culture, most of the specific biological features of isolated and cultured cells characteristic to the gallbladder are gradually lost after several passages, though quantitative and objective analyses of the pathophysiology of cultured cells and their secretory substances can be performed. Tissue culture using explants of the gallbladder has mainly been used for physiological studies of the gallbladder, such as investigating the transport of water and electrolytes. In this tissue culture system, quantitative assessment is difficult, though the original and specific biological and histological characteristics of the gallbladder are retained. Three‐dimensional collagen gel culture could be an ideal model combining monolayer cell culture and tissue culture systems, and create controllable conditions or environments when several biologically active substances, such as growth factors, proinflammatory cytokines and adhesion molecules, are added to the culture medium. Advantages and shortcomings of individual cultivation models are discussed, and selecting the culture model most appropriate to the purpose of the study will facilitate investigations of the biology and pathogenetic mechanisms of gallbladder diseases such as cholelithiasis. Microsc. Res. Tech. 39:71–84, 1997. © 1997 Wiley‐Liss, Inc.

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Reestablishment of Rabbit Gallbladder Epithelial Cells in Collagen Gel Culture and their Alterations by Cytochalasin B and Transforming Growth Factor Beta-1

Cited by 6 publications

References 18 publications

The role of E-cadherin in the differentiation of gallbladder cancer cells

The role of E-cadherin in the differentiation of gallbladder cancer cells

Factors affecting morphogenesis of rabbit gallbladder epithelial cells cultured in collagen gels

Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium

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