This study was designed to investigate differentiation of human pancreatic duct carcinoma cells (Capan-1) in vitro. Observations on live cells, and electron microscopic examination, together with enzymological and immunocytochemical methods, have demonstrated that these cells differentiate spontaneously at an early stage. The cells are seen to be joined by apical junctions. High ATPase activity can be detected in the basolateral membranes, and the cells secrete a gastric type mucin (MI) bearing acidic groups. During differentiation in culture, they form domes which are thought to be the morphological expression of trans-epithelial transport of water and electrolytes. This particular structure is transitory, since after 6 days in culture all the cells lose their adhesivity, and form into floating cords. Co-culture of Capan-1 cells and human, nude mice or chick embryo fibroblasts leads to a higher degree of differentiation of epithelial cells, reflected by the earlier appearance of numerous domes. In addition, the anchorage of Capan-1 cells to fibroblasts prevents retraction of the monolayer, and enables the domes to be maintained in the cultures for more than one month. These findings suggest that Capan-1 cells are able to carry out trans-epithelial movement of water and electrolytes. It is suggested that excretion of ions (bicarbonate and/or chloride) is preserved after transformation of pancreatic duct cells. Mucins (MI) and the recently described VIP receptor sites are also thought to play a part in these exchange processes.
Human pancreatic cells of the Capan-1 cell line differentiate in culture. During the exponential growth phase, the cells are undifferentiated, only becoming differentiated during the stationary phase. The formation of domes in this phase is related to the exchange of water and electrolytes. The present study was designed to characterize the localization and expression of alkaline phosphatases (AP) in Capan-1 cells during growth in culture. Biochemical, cytoenzymatic and immunocytochemical methods were employed combined with light and electron microscopic examination. AP essentially of the placental type were expressed progressively during the exponential growth phase, and were seen to be distributed over the surface of the Capan-1 cells. In the stationary phase, the AP became localized on the surface of microvilli. The precipitates of the enzyme reaction highlighted regular four-bodied structures. Biochemical assays showed a progressive increase in activity of this enzyme in cells during both the exponential and stationary growth phases. However, in the stationary phase between days 7 and 8, there was a fall in enzyme activity, with a corresponding increase in this activity in the culture medium. Cytological examination indicated that this fall could be accounted for by loss of AP-positive membranes by vesiculization of apical microvilli and release of microvesicles into the culture medium. Immunoblots showed that Capan-1 cells expressed two types of AP, a placental type (70 kDa) and to a lesser extent a liver type (80 kDa). Expression of the placental type was attributed to a neoplastic derepression of the coding gene, while the liver type was assumed to be a normal gene expression of human duct cells. The placental type AP might thus serve as a marker of transformation, and the liver type as a marker of differentiation.
Human pancreatic cells of the Capan-1 line form domes in culture during the stationary growth stage. The domes are thought to be a result of the transport of water and electrolytes by the Capan-1 cells. In older Capan-1 cultures, the epithelial sheets formed thickenings from several layers of cells of which the outermost ones were joined by tight type junctions. In the intracellular space, deposits of insoluble calcium salts were observed. Culture of Capan-1 cells in the presence of fibroblasts prolonged survival of the cultures with intact domes for more than 80 days. The Capan-1 cells proliferated forming multilayers and closed cavities which we called super-domes. X-ray spectrometry and electron diffraction analysis showed that the abundant deposits inside these cavities consisted of calcium phosphate in an apatite structure. The number of these deposits increased with time in culture, and they appeared to be formed at the sites of contact with an extracellular matrix consisting of cell debris. Deposits were not observed within the culture medium. Cells from domes were stained cytochemically for ATPases and alkaline phosphatases and examined by light and electron microscopy. The Capan-1 cells surrounding the domes were differentiated, polarized cells containing placental type alkaline phosphatases on their apical membranes and Ca2(+)-ATPases on their basolateral membranes. These enzymes were thought to play a role in the accumulation of phosphate and Ca2+ ions in the dome cavities, which then formed crystals in the presence of organic compounds produced by lysis of cells of the deepest layers of the super-domes. The crystals of hydroxyapatite observed in standard Capan-1 cell cultures and those cocultured with fibroblasts were assumed to be a result of transepithelial transport of Ca2+ and phosphate ions by these cells.
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