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Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro.

Lehnert L, Lerch MM, Hirai Y, Kruse ML, Schmiegel W, Kalthoff H - J. Cell Biol. (2001)

Bottom Line: A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells.Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres.A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, Department of General and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany.

ABSTRACT
Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.

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Confocal laser scanning analysis of coculture experiments of A818-6 cells with Kif-5 fibroblasts transduced with the gene coding for the EGFP. After 6 d of hollow sphere development, A818-6 cells were stained for the mucin-1 antigen. Mucin-1 displayed a staining of apical membranes of A818-6 hollow sphere cells (a). Fibroblasts are depicted by EGFP-derived fluorescence (b). (c) A differential interference contrast image of the analyzed hollow sphere with fibroblasts closely associated with the basal side of hollow sphere cells (c and d, yellow arrow) accompanied by a putative sequestered, nonfluorescing single A818-6 cell (c and d, black arrow). Bars, 10 μm.
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Figure 6: Confocal laser scanning analysis of coculture experiments of A818-6 cells with Kif-5 fibroblasts transduced with the gene coding for the EGFP. After 6 d of hollow sphere development, A818-6 cells were stained for the mucin-1 antigen. Mucin-1 displayed a staining of apical membranes of A818-6 hollow sphere cells (a). Fibroblasts are depicted by EGFP-derived fluorescence (b). (c) A differential interference contrast image of the analyzed hollow sphere with fibroblasts closely associated with the basal side of hollow sphere cells (c and d, yellow arrow) accompanied by a putative sequestered, nonfluorescing single A818-6 cell (c and d, black arrow). Bars, 10 μm.

Mentions: Fibroblast cocultures with A818-6 cells were set up using foreskin-derived KIF-5 fibroblasts. When A818-6 cells were seeded onto a confluent fibroblast monolayer culture, invasive growth and the development of plaques of A818-6 tumor cells were seen (Fig. 5 a). When premature A818-6 hollow spheres (Fig. 5 b) were seeded onto a confluent fibroblast monolayer culture, these hollow spheres increased in size by increasing the number of cells per hollow sphere and developed a homogenous shape within 48 h (Fig. 5 c). In a second experiment, A818-6 monolayer cells were mixed with fibroblasts and subsequently seeded onto agarose-coated dishes. This procedure was also found to enhance A818-6 hollow sphere development. Hollow sphere development was seen 2 d after initial coculture of A818-6 cells with KIF-5 fibroblasts; it was normally observed after 6–8 d in cultures without fibroblasts (Fig. 5 d). The same experiment was repeated with EGFP-transduced fibroblasts and revealed a localization of fibroblasts in the lumen of hollow spheres closely associated to the basal side of A818-6 cells (Fig. 6, a–d). Fibroblast-conditioned medium was not able to replace the direct presence of fibroblasts in the same manner (data not shown). Analysis of the cell cycle–associated proteins cyclin B and p27/Kip after coculture revealed a downregulation of p27/Kip and a weak upregulation of cyclin B in A818-6 hollow spheres (Fig. 4 B, lane 3). This is in accordance with an observed stimulatory effect of fibroblasts on A818-6 hollow sphere development (Fig. 5b and Fig. c).


Autocrine stimulation of human pancreatic duct-like development by soluble isoforms of epimorphin in vitro.

Lehnert L, Lerch MM, Hirai Y, Kruse ML, Schmiegel W, Kalthoff H - J. Cell Biol. (2001)

Confocal laser scanning analysis of coculture experiments of A818-6 cells with Kif-5 fibroblasts transduced with the gene coding for the EGFP. After 6 d of hollow sphere development, A818-6 cells were stained for the mucin-1 antigen. Mucin-1 displayed a staining of apical membranes of A818-6 hollow sphere cells (a). Fibroblasts are depicted by EGFP-derived fluorescence (b). (c) A differential interference contrast image of the analyzed hollow sphere with fibroblasts closely associated with the basal side of hollow sphere cells (c and d, yellow arrow) accompanied by a putative sequestered, nonfluorescing single A818-6 cell (c and d, black arrow). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198814&req=5

Figure 6: Confocal laser scanning analysis of coculture experiments of A818-6 cells with Kif-5 fibroblasts transduced with the gene coding for the EGFP. After 6 d of hollow sphere development, A818-6 cells were stained for the mucin-1 antigen. Mucin-1 displayed a staining of apical membranes of A818-6 hollow sphere cells (a). Fibroblasts are depicted by EGFP-derived fluorescence (b). (c) A differential interference contrast image of the analyzed hollow sphere with fibroblasts closely associated with the basal side of hollow sphere cells (c and d, yellow arrow) accompanied by a putative sequestered, nonfluorescing single A818-6 cell (c and d, black arrow). Bars, 10 μm.
Mentions: Fibroblast cocultures with A818-6 cells were set up using foreskin-derived KIF-5 fibroblasts. When A818-6 cells were seeded onto a confluent fibroblast monolayer culture, invasive growth and the development of plaques of A818-6 tumor cells were seen (Fig. 5 a). When premature A818-6 hollow spheres (Fig. 5 b) were seeded onto a confluent fibroblast monolayer culture, these hollow spheres increased in size by increasing the number of cells per hollow sphere and developed a homogenous shape within 48 h (Fig. 5 c). In a second experiment, A818-6 monolayer cells were mixed with fibroblasts and subsequently seeded onto agarose-coated dishes. This procedure was also found to enhance A818-6 hollow sphere development. Hollow sphere development was seen 2 d after initial coculture of A818-6 cells with KIF-5 fibroblasts; it was normally observed after 6–8 d in cultures without fibroblasts (Fig. 5 d). The same experiment was repeated with EGFP-transduced fibroblasts and revealed a localization of fibroblasts in the lumen of hollow spheres closely associated to the basal side of A818-6 cells (Fig. 6, a–d). Fibroblast-conditioned medium was not able to replace the direct presence of fibroblasts in the same manner (data not shown). Analysis of the cell cycle–associated proteins cyclin B and p27/Kip after coculture revealed a downregulation of p27/Kip and a weak upregulation of cyclin B in A818-6 hollow spheres (Fig. 4 B, lane 3). This is in accordance with an observed stimulatory effect of fibroblasts on A818-6 hollow sphere development (Fig. 5b and Fig. c).

Bottom Line: A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells.Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres.A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology, Department of General and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany.

ABSTRACT
Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5-6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.

Show MeSH
Related in: MedlinePlus