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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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Related in: MedlinePlus

A818-6 hollow sphere development as documented by phase–contrast microscopy. (a) A818-6 cells grown as a monolayer culture under standard cell culture conditions. (b) Signet ring–like cells (arrows) displaying a preform of hollow sphere development observed 2 d after seeding the cells on solid agarose. (c) Mature hollow spheres after 14 d in culture. Bars, 10 μm.
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Figure 1: A818-6 hollow sphere development as documented by phase–contrast microscopy. (a) A818-6 cells grown as a monolayer culture under standard cell culture conditions. (b) Signet ring–like cells (arrows) displaying a preform of hollow sphere development observed 2 d after seeding the cells on solid agarose. (c) Mature hollow spheres after 14 d in culture. Bars, 10 μm.

Mentions: Under standard cell culture conditions, A818-6 cells grew as a monolayer culture (Fig. 1 a) with a doubling time of 69 h. When adherence of these cells to culture substratum was prevented by coating tissue culture plates with solid agarose, hollow sphere development was observed within a time frame of 6–12 d after the initial seeding. The development of hollow spheres can be subdivided into two phases: (a) initiation and (b) maturation. The phase of initiation proceeded from the day of seeding to days 6–8 of the development. Signet ring–like cells with an expanded vacuole (Fig. 1 b, white arrows) appeared. 6–8 d after seeding, hollow spheres were the predominant component of the culture. Other remaining structures were identified as compact spheroids and signet ring–like cells. The maturation phase commenced after hollow spheres were retransferred into noncoated standard cell culture flasks. The hollow spheres became more homogenous in size and shape over the next 4 d (Fig. 1 c). In comparison to the early stages, only a few apoptotic cells were visible inside the hollow spheres. In contrast to other remaining cell clusters, hollow spheres did not attach to the surface of tissue culture grade flasks. Reattachment of the hollow spheres to the surface of the dish was only seen after mechanical disruption and was associated with regrowth as a monolayer culture (data not shown).


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)

A818-6 hollow sphere development as documented by phase–contrast microscopy. (a) A818-6 cells grown as a monolayer culture under standard cell culture conditions. (b) Signet ring–like cells (arrows) displaying a preform of hollow sphere development observed 2 d after seeding the cells on solid agarose. (c) Mature hollow spheres after 14 d in culture. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: A818-6 hollow sphere development as documented by phase–contrast microscopy. (a) A818-6 cells grown as a monolayer culture under standard cell culture conditions. (b) Signet ring–like cells (arrows) displaying a preform of hollow sphere development observed 2 d after seeding the cells on solid agarose. (c) Mature hollow spheres after 14 d in culture. Bars, 10 μm.
Mentions: Under standard cell culture conditions, A818-6 cells grew as a monolayer culture (Fig. 1 a) with a doubling time of 69 h. When adherence of these cells to culture substratum was prevented by coating tissue culture plates with solid agarose, hollow sphere development was observed within a time frame of 6–12 d after the initial seeding. The development of hollow spheres can be subdivided into two phases: (a) initiation and (b) maturation. The phase of initiation proceeded from the day of seeding to days 6–8 of the development. Signet ring–like cells with an expanded vacuole (Fig. 1 b, white arrows) appeared. 6–8 d after seeding, hollow spheres were the predominant component of the culture. Other remaining structures were identified as compact spheroids and signet ring–like cells. The maturation phase commenced after hollow spheres were retransferred into noncoated standard cell culture flasks. The hollow spheres became more homogenous in size and shape over the next 4 d (Fig. 1 c). In comparison to the early stages, only a few apoptotic cells were visible inside the hollow spheres. In contrast to other remaining cell clusters, hollow spheres did not attach to the surface of tissue culture grade flasks. Reattachment of the hollow spheres to the surface of the dish was only seen after mechanical disruption and was associated with regrowth as a monolayer culture (data not shown).

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