Colon Adenocarcinoma Cell Line Caco-2 Research Articles

Immunohistochemical Localization of Insulin-Degrading Enzyme Along the Rat Intestine, in the Human Colon Adenocarcinoma Cell Line (Caco-2), and in Human Ileum

Insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin in insulin target cells. Knowledge of the existence of this enzyme in the intestine will be beneficial to the achievement of clinical oral efficacy of insulin. A comparative study was conducted with rat intestine, human colon adenocarcinoma (Caco-2) cells, and human ileum. Confocal microscopy analysis using the anti-IDE antibody showed that IDE was localized in the mucosal cells of rat and human intestines, as well as in Caco-2 cells. Immunostaining of this enzyme was homogeneous throughout the cell excluding nucleus, indicating a typical cytosolic distribution in rat and human enterocytes and in Caco-2 cells.

Characterization of the cDNA and Pattern of Expression of a New Gene Over‐Expressed in Human Hepatomas and Colonic Tumors

A cDNA clone of 6.449 kb ch-TOG (for colonic and hepatic tumor over-expressed gene) initially selected from various human libraries and completed by 5' rapid amplification of cDNA ends (RACE) PCR is described. The original cDNA clone was extracted from an expression library constructed from a human tumoral brain. This library was screened with an antibody raised against the cytochrome P450tu that was shown to be over-expressed in chemically induced mouse hepatic tumors. Using this cDNA as a probe, a full-length cDNA was characterized. Its nucleotide sequence shows no significant similarity with any of the gene sequences collected in the various DNA data bases. The translation of the larger open reading frame leads to a putative protein of 1972 amino acids (molecular mass = 218453 Da). Hybridization analyses on Southern blot and on metaphase chromosomes indicate that this gene is present as a single copy in the genome and is localized on the short arm of chromosome 11. ch-TOG transcripts are present in several human tissues. Over-expression of ch-TOG in neoplastic liver and colon compared with the corresponding normal corresponding tissues is demonstrated. The level of the expression of ch-TOG transcripts was also studied in the various differentiation stages of the human colonic adenocarcinoma cell line Caco-2.

Apical polarity in human colon carcinoma cell lines

The non-polar human adenocarcinoma cells (HT29) when grown as monolayers or aggregates, have no tight junctions and no brush border. When these cells are treated with forskolin (15–100 μM) or cholera toxin (1 nM) intercellular lumina appear between the cells and about 30% of the cells facing the medium or the lumina are fully covered with a brush border. Aggregates embedded in collagen type I and treated with forskolin form a brush border only on cells facing the intercellular lumina. Monolayers of the polar human colon adenocarcinoma cell line Caco-2 express spontaneously tight junctions and a brush border in all the cells. When grown in aggregates the inner cells lose their polarity and only the cells facing the medium are polar. This polarity was not found when the aggregates were embedded in collagen gels. Aggregates embedded in collagen and treated with forskolin form bubble-like structures with a single layer of polar cells facing a central lumen. The data indicate that cell polarity is probably controlled by both internal factors such as cAMP and external factors such as cell-cell and cell-substratum molecules.

Expression of IGF-II and IGF binding proteins in differentiating human intestinal Caco-2 cells.

The mitogenic and metabolic effects of insulin-like growth factor-II (IGF-II) can be modulated by six distinct IGF binding proteins (IGFBPs). As a first step toward understanding the role of IGFs and their binding proteins in intestinal epithelial cell differentiation, the expression of IGF-II and IGFBPs was characterized in the human colon adenocarcinoma Caco-2 cell line. Northern blot analysis revealed two IGF-II transcripts of 5.4 and 4.5 kb, and ribonuclease protection assays indicated that IGF-II mRNA levels are regulated during Caco-2 differentiation. A specific radioimmunoassay detected IGF-II in serum-free conditioned medium, the level of which was three- to fivefold higher in proliferating cells than in differentiated cells. Immunoprecipitation and ligand blot analyses of conditioned medium demonstrated that IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6 are synthesized by Caco-2 cells, with IGFBP-2 and IGFBP-4 being the major IGFBPs secreted, and that the levels of IGFBP-2 and IGFBP-6 decreased as differentiation proceeded. These results indicate that the expression of IGF-II, IGFBP-2, and IGFBP-6 is regulated in a differentiation-dependent manner in Caco-2 cells.

Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells

The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes α- d-mannosidase, β-hexosaminidase, β-glucuronidase, and β-galactosidase increased ∼ 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors (MPRs), the insulin-like growth factor II/cation-independent MPR (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not change significantly during Caco-2 differentiation. In addition, quantitative Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout differentiation, the level of expression of the MPRs was sufficient to target the increased levels of lysosomal enzymes to the lysosome. Unlike the expression of lysosomal enzymes, Western blot analysis demonstrated an ∼ 40% and ∼ 30% decrease, respectively, in the steady-state levels of free and conjugated ubiquitin during Caco-2 differentiation. Taken together, these results show that the ubiquitin-dependent proteolytic pathway is regulated differently than the lysosomal degradative pathway during Caco-2 differentiation.

Phorbol Myristate Acetate Selectively Stimulates Apical Endocytosis via Protein Kinase C in Polarized MDCK Cells

We have examined the regulation of endocytosis in polarized Madin-Darby canine kidney (MDCK) cells. Using quantitative electron microscopy and biochemical measurements, we found that incubation with the tumor promoter phorbol 12-myristate 13-acetate (TPA) stimulated endocytosis of horseradish peroxidase (HRP) and ricin four- to fivefold at the apical side in MDCK cells, whereas the uptake at the basolateral membrane was unaffected. The use of several protein kinase inhibitors and TPA analogues indicated that the stimulation of apical endocytosis was mediated via protein kinase C independently of protein kinase A. This stimulation occurred even when the clathrin-dependent pathway was inhibited by acidification of the cytosol, suggesting that the TPA-stimulated uptake was associated with a clathrin-independent mechanism. Moreover, we found that TPA also stimulated recycling of ricin to the apical domain. Ultrastructural analysis of MDCK cells preincubated with TPA revealed that neither the morphology nor the size of the endosomes was altered compared to control cells. Using morphometry, no marked change in the apical plasma membrane area was detected after incubation with TPA. These data indicate that the TPA-stimulated endocytosis involved neither ruffling nor formation of macropinosomes in MDCK cells. Finally, we found that TPA also selectively stimulated apical endocytosis in the human colon adenocarcinoma cell line (Caco-2). The data suggest that protein kinase C is involved in a strong stimulation of apical endocytosis and might participate in the regulation of membrane trafficking between the apical plasma membrane and apical endosomes in polarized epithelial cells.

Insulin-degrading enzyme in a human colon adenocarcinoma cell line (Caco-2).

The activity of insulin-degrading enzyme (IDE), a thiol metalloprotease degrading insulin in many insulin target cells, was determined in human colon adenocarcinoma (Caco-2) cells. Insulin-degrading activity was localized in the cytosol of Caco-2 cells, accounting for 88% of total activity. Western blots and immunoprecipitation showed that IDE was present in the cytosol of Caco-2 cells and contributed to more than 93% cytosolic insulin-degrading activity. Cytosolic insulin degradation was strongly inhibited by IDE inhibitors, including N-ethylmaleimide, 1,10-phenanthroline, p-chloromericuribenzoate, and EDTA, but was not significantly or not as extensively inhibited by strong inhibitors of proteasome, i.e., chymostatin, soybean trypsin inhibitor, leupeptin, and Dip-F. These results suggest that IDE is present in Caco-2 cells, that Caco-2 IDE has properties similar to those of its counterparts in insulin-target tissues, and that it significantly contributes to intracellular insulin degradation.

Increased tumorigenicity after differentiation of colon cancer cell line: Absence of association with mucin synthesis

Background/Aims: Postconfluence differentiation followed by a decrease in mucin synthesis has been described for the colonic adenocarcinoma cell line Caco-2. Because the onset of differentiation usually heralds the end of proliferation, we expected Caco-2 would be more tumorigenic in the exponential phase. Thus, we compared tumorigenicity, clonigenicity, and mucin synthesis in the exponential and stationary growth phases. Methods: We estimated mucin synthesis in Caco-2 cells by measuring the amount of high-molecular-weight [3H]glucosamine-labeled glycoprotein released into the culture medium and in the cytosolic fraction. Colony forming efficiency in soft agar and tumorigenicity in nude mice were assessed. Results: Cells in the exponential phase synthesized significantly more mucin compared with stationary phase cells. Pretreatment with benzyl-GalNAc reduced production in both phases but did not change clonigenicity. Colonics did not grow from cells seeded in the exponential phase. The average weight of xenografts raised from postconfluence cells was twice that of xenografts raised from cells in the exponential phase. Conclusions: Neither exponential cell growth nor increased mucin production predicts clonigenicity and tumorigenicity. It is speculated that a stable process takes place in Caco-2 cells after confluence which makes them more clonigenic in vitro and enhances tumor growth in vivo.

Induction of lactase biosynthesis in the human intestinal epithelial cell line Caco-2.

The human colonic adenocarcinoma cell line Caco-2 forms monolayers of differentiated enterocyte-like cells when cultured on permeable supports. After confluency, Caco-2 cells express a number of brush-border enzymes including lactase-phlorizin hydrolase, sucrase-isomaltase and dipeptidylpeptidase IV. We have studied, with particular emphasis on lactase-phlorizin hydrolase, the modulation of biosynthesis of these enzymes by stimulating second messenger systems. Forskolin induced lactase-phlorizin hydrolase synthesis approximately fourfold within 7 h, suppressed sucrase-isomaltase synthesis, and had little effect on dipeptidylpeptidase IV. Dibutyryl-cAMP, 8-bromo-cAMP and vasoactive intestinal peptide also increased lactase-phlorizin hydrolase biosynthesis, indicating c-AMP dependent regulation. The induction of lactase-phlorizin hydrolase biosynthesis could be inhibited by actinomycin D and was preceded by a fourfold increase in lactase-phlorizin hydrolase mRNA levels, suggesting transcriptional control. Phorbol 12-myristate 13-acetate had an inhibitory effect on brush-border enzyme synthesis, in particular on sucrase-isomaltase, and blocked the forskolin-induced biosynthesis of lactase-phlorizin hydrolase. Lactase-phlorizin hydrolase synthesis was also inducible by hydrocortisone, but maximal induction required at least 3 days during which time sucrase-isomaltase synthesis diminished. The results indicate opposite regulation of lactase-phlorizin hydrolase and sucrase-isomaltase via cAMP and corticosteroids, and suggest that the Caco-2 cell line can serve as a model system to study aspects of the humoral regulation of human intestinal brush-border enzymes in cell culture.

Differential cytokine regulation of complement C3, C4, and factor B synthesis in human intestinal epithelial cell line, Caco-2.

In the intestinal tract, the local synthesis of C3 and components of both the classical (C4) and alternative (factor B) C activation pathway has previously been demonstrated in vivo. However, the cellular source of this local C synthesis has not been identified. In this study, we demonstrated the syntheses of C3, C4, and factor B in the human colonic adenocarcinoma cell line Caco-2, which is regarded as a good experimental model of normal human intestinal epithelial cells. The results of metabolic labeling experiments indicated that the intra- and extracellular molecular sizes and subunit structures of Caco-2-derived C3, C4, and factor B were compatible with previously reported values for these components in other cells. The functional activities of C3 and C4 in the supernatants were also demonstrated by hemolytic titration assay. Furthermore, C syntheses in this line were independently upregulated by several human cytokines: C3 synthesis was dose-dependently enhanced by the addition of IL-1 beta or TNF-alpha; C4 synthesis was enhanced by the addition of IL-6 or IFN-gamma in the same manner; and the addition of IL-1 beta or IL-6 also induced a dose-dependent increase in factor B synthesis. These enhancing effects were confirmed to be specific for individual cytokines by experiments using anti-human cytokine antibodies. It is likely that intestinal epithelial cells are local production sites of C3, C4, and factor B, and that local C syntheses in the intestine are independently regulated by several cytokines, derived from monocytes/macrophages and T cells resident in the mucosal microenvironment.

Human Caco-2 cells transfected with c-Ha-Ras as a model for endocrine differentiation in the large intestine

Abstract Endocrine cells occur in approximately 30% of all colorectal adenocarcinomas, and this feature appears to correlate with a relatively poor prognosis. To study the factors regulating endocrine differentiation in colorectal cancer, which may bear resemblance to the regulation of endocrine differentiation in normal intestinal mucosa, models in which differentiation can be manipulated are essential. However, endocrine features in colorectal cancer cell lines are scarce and are almost exclusively observed in xenografts, presumably as a result of differentiation induction by stromal components. We attempted to demonstrate endocrine differentiation in the colonic adenocarcinoma cell line Caco-2, which is frequently used as a model for enterocytic differentiation. In vitro endocrine tumor cells were not encountered. In vivo studies were cumbersome, because of the low take rate of Caco-2 cells. We did manage to establish nude mouse xenografts of Caco-2 cells by inoculating cells in collagen gel and by suppressing natural killer cell activity. In an attempt to induce a better take rate and to investigate the effect of Ras oncoprotein overexpression on endocrine differentiation, Caco-2 cells were transfected with a point-mutated c-Ha-Ras gene. The cell line Caco-2 EJ6, generated from these experiments, could be xenografted in nude mice with a high take rate, yielding a moderately well differentiated adenocarcinoma, morphologically identical to the tumors derived from untransfected Caco-2 cells. The xenografts displayed goblet cell, enterocytic, Paneth cell and endocrine differentiation. In vitro endocrine differentiation was observed neither under standard conditions nor with extracellular matrix components as differentiation inducers. We conclude that the Caco-2 cell line and its c-Ha-Ras transfected subline Caco-2 EJ6 in vivo display endocrine differentiation. Ras overexpression does not enhance endocrine differentiation. Due to its favorable growth properties in vivo, Caco-2 EJ6 is a suitable model for studies on endocrine differentiation in colorectal cancer.

Expression of human intestinal dipeptide transporter in Xenopus laevis oocytes

The human colon adenocarcinoma cell line Caco-2 retains the H +/dipeptide cotransporter. To identify the structure of the human dipeptide transporter, we have examined the expression of the transporter in Xenopus laevis oocytes injected with Caco-2 poly(A) +RNA, by monitoring the uptake of bestatin, a dipeptide-like anticancer agent. The bestatin uptake in the poly(A) +RNA-injected oocytes was inhibited by excess glycyl- l-leucine, and showed pH dependence (optimal pH of 5.5–6.0). These observations suggest that the human intestinal dipeptide transporter can be expressed functionally in Xenopus oocytes.

Isolation and characterization of the human sucrase-isomaltase gene and demonstration of intestine-specific transcriptional elements.

The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5'-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5'-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.

Normal inflammatory bowel disease mucosa conceals alterations in natural killer cell activity.

Non-major histocompatibility complex-restricted cytotoxicity or natural killer (NK) activity could be detected in all intestinal lamina propria mononuclear cell preparations of histologically normal mucosa from 57 patients with gastrointestinal disease. Similar levels of NK activity were detected among the different disease groups. Within the inflammatory bowel disease patient group, however, Crohn's disease patients showed a threefold higher level of NK activity than detected in ulcerative colitis patients. Cytotoxicity levels in Crohn's disease patients were also higher than in the control carcinoma patients, whereas ulcerative colitis patients had considerably lower cytotoxicity levels than the carcinoma patients. Thus, unaffected normal inflammatory bowel disease mucosa conceals alterations in NK activity which might occur before the inflammation. The colon adenocarcinoma cell line Caco-2 was found to be a representative target for detecting individual differences in NK activity of lamina propria mononuclear cells compared with standard K-562 targets. The latter can be of relevance when studying mucosal immunoregulatory mechanisms in intestinal disease.

Vitamin D-regulated calcium transport in Caco-2 cells: unique in vitro model

The human colon adenocarcinoma cell line Caco-2 is the only intestinal cell line to differentiate spontaneously in culture exhibiting structural and biochemical characteristics of mature enterocytes and to possess a vitamin D receptor in the fully differentiated state. Transepithelial calcium transport was characterized in differentiated Caco-2 cells grown on permeable filters supports to assess the potential utility of this cell line as an in vitro model to study 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced calcium transport. Calcium transport was increased in a dose-dependent manner by 1,25(OH)2D3. Total calcium transport at different calcium concentrations could be fitted to a modified Michaelis-Menten equation containing a linear transport component. The maximum rate of saturable calcium transport was increased by 4.3-fold (P less than 0.005) in cells treated with 10(-8) M 1,25(OH)2D3. This treatment also increased the apparent buffer calcium concentration that results in half-maximal velocity from 0.4 to 1.3 mM but had no significant effect on nonsaturable calcium transport. Caco-2 cells grown on permeable filter supports provide a unique in vitro human cell culture model to study the mechanism of vitamin D-regulated transepithelial intestinal calcium transport.

Endocytosis, recycling, and lysosomal delivery of brush border hydrolases in cultured human intestinal epithelial cells (Caco-2).

Lysosomes of intestinal epithelial cells in vivo and in culture display strong immunoreactivity with monoclonal antibodies against various brush border enzymes as visualized by immunoelectron microscopy. Novel subcellular fractionation procedures were developed to study, by the pulse-chase technique and by internalization assays, the pathway along which two microvillar hydrolases, sucrase-isomaltase and dipeptidylpeptidase IV, are transported to lysosomes in the differentiated colon adenocarcinoma cell line Caco-2. 7-9% of metabolically labeled sucrase-isomaltase of dipeptidylpeptidase IV were present in lysosomes after 7-8 h of chase as intact complex-glycosylated molecules. Appearance of these enzymes in lysosomes was biphasic. Endocytosis studies with radioiodinated antienzyme monoclonal antibodies (monovalent antigen-binding fragments) and by means of cell surface iodination revealed only slow transport of the enzymes to lysosomes at a low level. However, both enzymes were internalized with different efficiencies and recycled to the cell surface via endosomes. These results suggest that in Caco-2 cells a significant amount of newly synthesized sucrase-isomaltase and dipeptidylpeptidase IV is directly imported into lysosomes bypassing the brush border membrane.

Time-dependent activity and expression of glutathione S-transferases in the human colon adenocarcinoma cell line Caco-2.

The human colon carcinoma cell line Caco-2 was examined for glutathione S-transferase (GST) composition and activity. Freshly seeded cells and cells until 4 days after confluency contain only the placental (Pi) form of glutathione transferase. Cells in culture for longer periods start to express class-Alpha GST isoenzymes. Confluent cells in culture for 20 days or longer contain up to 90% class-Alpha GST. Class-Mu GSTs are not detectable. GST activity gradually increases from 564 +/- 28 to 5381 +/- 165 nmol/min per mg of protein at day 0 and 32 after confluency respectively. With regard to GST composition, Caco-2 cells in culture for longer periods most resemble small-intestinal cells, whereas short-time cultures have characteristics of colonic cells. This cell line is very well suited for the study of both the in vitro properties and the expression of class Alpha and Pi GSTs.

Dissection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface.

Novel subcellular fractionation procedures and pulse-chase techniques were used to study the intracellular transport of the microvillar membrane hydrolases sucrase-isomaltase and dipeptidylpeptidase IV in the differentiated colon adenocarcinoma cell line Caco-2. The overall rate of transport to the cell surface was two fold faster for dipeptidylpeptidase IV than for sucrase-isomaltase, while no significant differences were observed in transport rates from the site of complex glycosylation to the brush border. The delayed arrival of sucrase-isomaltase in the compartment where complex glycosylation occurs was only in part due to exit from the endoplasmic reticulum. A major slow-down could be ascribed to maturation in and transit of this enzyme through the Golgi apparatus. These results suggest that the observed asynchronism is due to more than one rate-limiting step along the rough endoplasmic reticulum to trans-Golgi pathway.

Development of vasoactive intestinal peptide-responsive adenylate cyclase during enterocytic differentiation of Caco-2 cells in culture. Evidence for an increased receptor level.

The purpose of this work was to study vasoactive intestinal peptide (VIP) receptors and the adenylate cyclase response to VIP upon enterocytic differentiation of the human colon adenocarcinoma cell line Caco-2 in culture. The VIP-stimulated enzyme activity is very low, e.g. 20% above basal activity in undifferentiated cells (day 5) and is enhanced markedly at confluency reaching a maximum, e.g. 270%, above basal activity in fully differentiated cells (day 30). VIP potency is also slightly enhanced, the EC50 of VIP ranging from 0.31 nM at day 5 to 0.07 nM at day 30. Modifications of the adenylate cyclase system are not responsible for the development of VIP response. Indeed, forskolin-stimulated adenylate cyclase activity is unchanged during differentiation supporting no alteration of the enzyme catalytic subunit. The same holds true for NaF and guanosine 5'-(beta, gamma-imido)trisphosphate, indicating a constant activity of the guanine nucleotide regulatory unit which mediates hormonal stimulation of adenylate cyclase (Ns). This is further supported by the similar extent of cholera toxin-catalyzed [32P]ADP-ribosylation of the Ns protein that is observed during differentiation. In sharp contrast, a dramatic increase of VIP receptor concentration is observed ranging from 32 fmol/mg of protein at day 5 to 414 fmol/mg of protein at day 30. This is confirmed by affinity cross-linking experiments showing an increased specific incorporation of 125I-VIP in a major 66,000-dalton component during differentiation. A slight increase in receptor affinity is also observed during differentiation with Kd ranging from 0.39 nM at day 5 to 0.08 nM at day 30. These data indicate that one population of VIP receptors accumulates during Caco-2 cell differentiation, representing the crucial event in the development of adenylate cyclase response to the peptide.

Common characteristics for Na+-dependent sugar transport in Caco-2 cells and human fetal colon.

The recent demonstration that the human colon adenocarcinoma cell line Caco-2 was susceptible to spontaneous enterocytic differentiation led us to consider the question as to whether Caco-2 cells would exhibit sodium-coupled transport of sugars. This problem was investigated using isotopic tracer flux measurements of the nonmetabolizable sugar analog alpha-methylglucoside (AMG). AMG accumulation in confluent monolayers was inhibited to the same extent by sodium replacement, 200 microM phlorizin, 1 mM phloretin, and 25 mM D-glucose, but was not inhibited further in the presence of both phlorizin and phloretin. Kinetic studies were compatible with the presence of both a simple diffusive process and a single, Na+-dependent, phlorizin- and phloretin-sensitive AMG transport system. These results also ruled out any interaction between AMG and a Na+-independent, phloretin-sensitive, facilitated diffusion pathway. The brush-border membrane localization of the Na+-dependent system was inferred from the observations that its functional differentiation was synchronous with the development of brush-border membrane enzyme activities and that phlorizin and phloretin addition 1 hr after initiating sugar transport produced immediate inhibition of AMG uptake as compared to ouabain. Finally, it was shown that brush-border membrane vesicles isolated from the human fetal colonic mucosa do possess a Na+-dependent transport pathway(s) for D-glucose which was inhibited by AMG and both phlorizin and phloretin. Caco-2 cells thus appear as a valuable cell culture model to study the mechanisms involved in the differentiation and regulation of intestinal transport functions.