Andrea Quaroni

Chapter 20 Establishment and Characterization of Intestinal Epithelial Cell Cultures

Publisher Summary The chapter discusses various methods for establishment and characterization of intestinal epithelial cell cultures. Suspensions of isolated intestinal epithelial cells, minimally contaminated by mesenchymal and plasma elements, are relatively easy to obtain and represent an obvious source of cells for primary culture. The chapter discusses the establishment of intestinal epithelial cell lines (IEC cells). The general approach used is to maintain whole fragments of tissue floating near the surface of the culture medium (containing 100–200 μg/ml collagenase) while slowly releasing cells, which are then found, attached to the surface of the dish. During the first 2 months in culture, and until pure epithelioid colonies are selected with cloning cylinders, the presence of a small amount of collagenase (20–50 μg/ml) is required in the culture medium. For characterization, the IEC cells have the typical morphologic appearance of epithelioid cells in culture. They have a large, centrally located nucleus, and grow as tightly packed colonies of polygonal cells. Most aspects of the regulation of IEC cell proliferation are still undefined. Preliminary results suggest that these cells possess a number of intriguing regulatory properties.

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans‐Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90–98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.

Study of intestinal cell differentiation with monoclonal antibodies to intestinal cell surface components

Monoclonal antibodies that react with antigens of the plasma membrane of rat intestinal villus and crypt cells have been prepared by fusion of mouse myeloma (NSI) cells with spleen cells of mice immunized with various intestinal cellular fractions, including the luminal membrane of adult villus and crypt cells, and of newborn rat intestinal cells. The antigenic targets of most antibodies have been identified. They include major protein components of the brush border (luminal) membrane of adult villus cells (sucrase-isomaltase, maltase, lactase, aminopeptidase N, alkaline phosphatase) and newly identified protein antigens specific for intestinal epithelial cells. Of 25 independently derived monoclonal antibodies prepared, 18 reacted exclusively with the brush border membrane of the villus cells, confirming its unique protein composition. Antibodies specifically staining the crypt cells, the newly differentiated epithelial cells present in the lower half of the villi, the top villus cells, and both villus and crypt cells were also obtained and characterized. These antibodies have been used to study the expression of cell- and tissue-specific functions during differentiation and development of the intestinal epithelium. Contrary to results obtained with polyclonal antisera, no inactive forms of the brush border enzymes have been detected in the crypt cells. The identification of cell surface components expressed at different levels of the villi, and in both undifferentiated and differentiated intestinal cells, suggests that cell differentiation in the intestinal epithelium is a continuous and gradual process involving both transcriptional and translational regulation of different sets of genes.

Mosaic pattern of lactase expression by villous enterocytes in human adult-type hypolactasia

Abstract Immunohistological analysis of the expression of lactase protein in adults with hypolactasia has been carried out using monoclonal antibodies. Eight different antibodies that recognize at least three distinct epitopes on the lactase protein each gave the same result. Strong brush border staining was observed in all the lactase-persistent adults. No staining at all was detected in 9 of the hypolactasic subjects. In the remaining 12 individuals a mosaic pattern of expression was observed: small patches of enterocytes stained strongly, whereas the surrounding areas showed no staining at all. Sucraseisomaltase, in contrast, showed no such mosaicism in these or in any of the other individuals. The mosaicism observed in the 12 hypolactasic individuals suggests that the differentiation of the columnar cells along the villus is not homogeneous. Furthermore, the existence of two patterns of expression of the lactase protein in the lactase-deficient individuals (i.e., absence of protein and mosaicism), if characteristic of the entire length of the intestine of the individuals tested, would suggest the existence of two phenotypes of adult-type hypolactasia in the population studied.

Establishment and characterization of a new human hepatocellular carcinoma cell line

SummaryA human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α1-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.

Involvement of p21(WAF1/Cip1) and p27(Kip1) in intestinal epithelial cell differentiation

Using the conditionally immortalized human cell line tsFHI, we have investigated the role of cyclin-dependent kinase inhibitors (CKIs) in intestinal epithelial cell differentiation. Expression of cyclins, cyclin-dependent kinases (Cdk), and CKIs was examined under conditions promoting growth, growth arrest, or expression of differentiated traits. Formation of complexes among cell cycle regulatory proteins and their kinase activities were also investigated. The tsFHI cells express three CKIs: p16, p21, and p27. With differentiation, p21 and p27 were strongly induced, but with different kinetics: the p21 increase was rapid but transient and the p27 increase was delayed but sustained. Our results suggest that the function of p16 is primarily to inhibit cyclin D-associated kinases, making tsFHI cells dependent on cyclin E-Cdk2 for pRb phosphorylation and G1/S progression. Furthermore, they indicate that p21 is the main CKI involved in irreversible growth arrest during the early stages of cell differentiation in association with D-type cyclins, cyclin E, and Cdk2, whereas p27 may induce or stabilize expression of differentiated traits acting independently of cyclin-Cdk function.

Glucocorticoids have pleiotropic effects on small intestinal crypt cells

Glucocorticoids have long been known to accelerate maturation of the intestinal tract, but the molecular mechanisms that account for their physiological function in the epithelium remain poorly characterized. Using rat intestinal epithelial cell lines (IEC-6, IEC-17, and IEC-18) as models, we have characterized glucocorticoid receptors in crypt cells and documented striking morphological, ultrastructural, and functional alterations induced by these hormones in intestinal cells. They include arrest of growth, formation of tight junctions, appearance of long, slender microvilli, reorganization of the endoplasmic reticulum and trans-Golgi network, and downregulation of the cell cycle regulatory proteins cyclin-dependent kinase 6 and p27Kip1. These effects are consistent with the activation or modulation of multiple genes important in the physiological function of absorptive villous cells but are probably not directly involved in the induction of cell differentiation.Glucocorticoids have long been known to accelerate maturation of the intestinal tract, but the molecular mechanisms that account for their physiological function in the epithelium remain poorly characterized. Using rat intestinal epithelial cell lines (IEC-6, IEC-17, and IEC-18) as models, we have characterized glucocorticoid receptors in crypt cells and documented striking morphological, ultrastructural, and functional alterations induced by these hormones in intestinal cells. They include arrest of growth, formation of tight junctions, appearance of long, slender microvilli, reorganization of the endoplasmic reticulum and trans-Golgi network, and downregulation of the cell cycle regulatory proteins cyclin-dependent kinase 6 and p27(Kip1). These effects are consistent with the activation or modulation of multiple genes important in the physiological function of absorptive villous cells but are probably not directly involved in the induction of cell differentiation.

Luminal substrate "brake" on mucosal maltase-glucoamylase activity regulates total rate of starch digestion to glucose.

Background: Starches are the major source of dietary glucose in weaned children and adults. However, small intestine α-glucogenesis by starch digestion is poorly understood due to substrate structural and chemical complexity, as well as the multiplicity of participating enzymes. Our objective was dissection of luminal and mucosal α-glucosidase activities participating in digestion of the soluble starch product maltodextrin (MDx). Patients and Methods: Immunoprecipitated assays were performed on biopsy specimens and isolated enterocytes with MDx substrate. Results: Mucosal sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM) contributed 85% of total in vitro α-glucogenesis. Recombinant human pancreatic α-amylase alone contributed <15% of in vitro α-glucogenesis; however, α-amylase strongly amplified the mucosal α-glucogenic activities by preprocessing of starch to short glucose oligomer substrates. At low glucose oligomer concentrations, MGAM was 10 times more active than SI, but at higher concentrations it experienced substrate inhibition whereas SI was not affected. The in vitro results indicated that MGAM activity is inhibited by α-amylase digested starch product “brake” and contributes only 20% of mucosal α-glucogenic activity. SI contributes most of the α-glucogenic activity at higher oligomer substrate concentrations. Conclusions: MGAM primes and SI activity sustains and constrains prandial α-glucogenesis from starch oligomers at approximately 5% of the uninhibited rate. This coupled mucosal mechanism may contribute to highly efficient glucogenesis from low-starch diets and play a role in meeting the high requirement for glucose during childrens brain maturation. The brake could play a constraining role on rates of glucose production from higher-starch diets consumed by an older population at risk for degenerative metabolic disorders.

Nivalenol and deoxynivalenol affect rat intestinal epithelial cells: a concentration related study.

The integrity of the gastrointestinal tract represents a crucial first level defence against ingested toxins. Among them, Nivalenol is a trichotecenes mycotoxin frequently found on cereals and processed grains; when it contaminates human food and animal feed it is often associated with another widespread contaminant, Deoxynivalenol. Following their ingestion, intestinal epithelial cells are exposed to concentrations of these trichothecenes high enough to cause mycotoxicosis. In this study we have investigated the effects of Nivalenol and Deoxynivalenol on intestinal cells in an in vitro model system utilizing the non-tumorigenic rat intestinal epithelial cell line IEC-6. Both Nivalenol and Deoxynivalenol (5–80 µM) significantly affected IEC-6 viability through a pro-apoptotic process which mainly involved the following steps: (i) Bax induction; (ii) Bcl-2 inhibition, and (iii) caspase-3 activation. Moreover, treatment with Nivalenol produced a significant cell cycle arrest of IEC-6 cells, primarily at the G0/G1 interphase and in the S phase, with a concomitant reduction in the fraction of cells in G2. Interestingly, when administered at lower concentrations (0.1–2.5 µM), both Nivalenol and Deoxynivalenol affected epithelial cell migration (restitution), representing the initial step in gastrointestinal wound healing in the gut. This reduced motility was associated with significant remodelling of the actin cytoskeleton, and changes in expression of connexin-43 and focal adhesion kinase. The concentration range of Nivalenol or Deoxynivalenol we have tested is comparable with the mean estimated daily intake of consumers eating contaminated food. Thus, our results further highlight the risks associated with intake of even low levels of these toxins.

Activated H-ras transforms rat intestinal epithelial cells with expression of α-TGF

Abstract We describe malignant transformation of cultured rat intestinal epithelial cells (IEC-18) by transfection with the activated human H-ras gene cloned from the EJ bladder carcinoma. Transformed cells showed a marked morphological change, expressed high levels of the transfected H-ras gene, were able to grow in agar and expressed antigenic markers identical with parental IEC-18 cells. When injected into syngeneic rats these cells formed rapidly growing tumors expressing the same intestinal-specific antigenic markers as the injected cells. Parallel to the high expression of H-ras mRNA in the transformants we document overexpression of rat α-TGF mRNA.