Characterization of the WIDR: a human colon carcinoma cell line.

We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Nucleo-cytoplasmic cycling of the vitamin D receptor in the enterocyte-like cell line, Caco-2

We examined the effects of 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) on the distribution and mobility of the vitamin D receptor (VDR) in the enterocyte-like Caco-2 cell. Confocal microscopy showed that a green fluorescent protein-vitamin D receptor (GFP-VDR) fusion protein is predominantly nuclear (58%) and it does not associate with the apical or basolateral membrane of proliferating or polarized, differentiated cells. In contrast to the previously studied cell types, neither endogenous VDR nor GFP-VDR levels accumulate in the nucleus following 1,25(OH)(2)D(3) treatment (100 nM, 30 min). However, in nuclear photobleaching experiments nuclear GFP-VDR import was significantly increased by 1,25(OH)(2)D(3) during both an early (0-5 min) and later (30-35 min) period (20% per 5 min). Compared to the natural ligand, nuclear import of GFP-VDR was 60% lower in cells treated with the 1,25(OH)(2)D(3) analog, 1-alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228, 5 min, 100 nM). Downstream events like ligand-induced association of VDR with chromatin at 1 h and the accumulation of CYP24 mRNA were significantly lower in Ro-26-9228 treated cells compared to 1,25(OH)(2)D(3) (60 and 95% lower, respectively). Collectively our data are consistent with a role for ligand-induced nuclear VDR import in receptor activation. In addition, ligand-dependent VDR nuclear import appears to be balanced by export, thus accounting for the lack of nuclear VDR accumulation even when VDR import is significantly elevated.     

Characterization of WiDr: A human colon carcinoma cell line

Summary We describe the establishment and characterization of WiDr, a cell line derived from a human colon carcinoma. It produces carcinoembryonic antigen in culture, and has a doubling time of 15 hr with plating efficiency of 51%. The HLA antigenic profile and the allozyme genetic signature (composed of eight gene-enzyme systems) of WiDr cells are different from those of HeLa cells. Furthermore, WiDr cells possess three marker chromosomes, again distinct from the HeLa marker chromosomes. Finally, it is highly tumorigenic in four different xenogeneic animal models. Based on these studies, WiDr represents a useful model cell line for tumor cell biology investigations.     

Analysis of proteomic changes induced upon cellular differentiation of the human intestinal cell line Caco-2

The human intestinal cell line Caco-2 is a well-established model system to study cellular differentiation of human enterocytes of intestinal origin, because these cells have the capability to differentiate spontaneously into polarized cells with morphological and biochemical features of small intestinal enterocytes. Therefore, the cells are widely used as an in vitro model for the human intestinal barrier. In this study, a proteomic approach was used to identify the molecular marker of intestinal cellular differentiation. The proteome of proliferating Caco-2 cells was compared with that of fully differentiated cells. Two-dimensional gel analysis yielded 53 proteins that were differently regulated during the differentiation process. Pathway analysis conducted with those 34 proteins that were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed subsets of proteins with common molecular and cellular function. It was shown that proteins involved in xenobiotic and drug metabolism as well as in lipid metabolism were upregulated upon cellular differentiation. In parallel, proteins associated with proliferation, cell growth and cancer were downregulated, reflecting the loss of the tumorigenic phenotype of the cells. Thus, the proteomic approach in combination with a literature-based pathway analysis yielded valuable information about the differentiation process of Caco-2 cells on the molecular level that contributes to the understanding of the development of colon cancer or inflammatory diseases such as ulcerative colitis--diseases associated with an imbalanced differentiation process of intestinal cells.     

Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco-2: modulation by forskolin

The human colon cancer line Caco-2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco-2 cells with an ED50 of 7 X 10(-6)M. Three different treatment protocols with FK (10(-5)M) were applied: 1) the cells were treated during all the time in culture (20 days); 2) the treatment was started after confluency; 3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 or 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: a) an inhibition of the post-confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; b) an inhibition of the post-confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.     

Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line.

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiate in vitro by replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60-90%) of CEA release.     

Dome formation in the human colon carcinoma cell line Caco-2 in culture. Influence of ouabain and permeable supports

We studied formation of domes in cell monolayers of the human colon carcinoma cell line Caco-2 which has been shown to exhibit signs of enterocytic differentiation and transport properties. After a 24 hr incubation with 4 X 10(-8) M ouabain, the number of domes seen on Caco-2 cell monolayers grown on plastic dishes was not significantly altered. After a 90 min preincubation with ouabain, 86rubidium uptake by Caco-2 cells was inhibited by ouabain, indicating that the cells have an ouabain-sensitive Na+, K+-ATPase, while dome formation was unaffected by ouabain. Domes were observed in Caco-2 cell monolayers grown on Nuclepore filters when the pore size was 0.015 micron but not when it was 0.030 micron. Our results suggest that dome formation in the Caco-2 cell line could be independent of Na+, K+-ATPase activity and might be due to accumulation of molecules having an effective hydrodynamic radius comprised between 0.015 and 0.030 micron.     

Characterization of galactosyl glycerolipids in the HT29 human colon carcinoma cell line.

Glycoglycerolipids constitute a family of glycolipids with apparently very restricted expression in human tissues. They have previously been detected only in the testis and the nervous system. In the present study, two glycoglycerolipids were isolated from the HT29 human colon carcinoma cell line. The glycoglycerolipids were structurally characterized as a monogalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl)-sn-glycerol) and a digalactosylglycerolipid (1-O-alkyl-2-O-acyl-3-O-(beta-galactosyl(1-4)alpha-galactosyl)-sn-glycerol) using NMR and mass spectrometry. This digalactosylglycerolipid has not previously been structurally characterized. When HT29 cells were allowed to differentiate into more enterocyte-like cells by culture in glucose-free medium, expression of both of these glycoglycerolipids was greatly diminished. The presence of glycoglycerolipids in a human colon carcinoma cell line indicates that expression of this family of glycolipids may not be as restricted as previously thought. Instead this class of glycolipids may serve as differentiation antigens in various normal tissues and in tumor development. The Galalpha1-4Gal epitope was previously identified as a receptor for bacterial adhesins and toxins. The finding that this epitope is also linked to a glycerolipid moiety opens up new possible roles for this carbohydrate receptor in intracellular signaling.     

Aldosterone binding in the human colon carcinoma cell line HT29: correlation with cell differentiation

The purpose of the present study is to investigate aldosterone binding to human colon carcinoma HT29 cells. These cells grow as undifferentiated epithelial cells when cultured under standard conditions (Dulbecco's MEM; 10% FCS). Modification of the culture medium induced two types of differentiation: (1) enterocytic differentiation when glucose (25 mM) is replaced by galactose (5 mM) (2) mucus secreting cells in FCS free medium. Aldosterone specific binding was detected in the cytosolic fraction of enterocyte-differentiated cells. This binding corresponded to a single class of sites with affinity, specificity and anion-exchange chromatographic behaviour similar to those observed in the epithelial crypts of human colon. These putative aldosterone receptors were also detected in mucus secreting cells that are derived from the enterocyte-differentiated cells. Enterocytic differentiation appears thus to be a necessary step for the "induction" of aldosterone receptors in HT29 cells.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na+-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na+-independent pathways, possibly mediated by an organic anion transporter, and Na+-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na+-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na+-dependent transport of succinate by NaDC1.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na⁺-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na⁺-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na⁺-independent pathways, possibly mediated by an organic anion transporter, and Na⁺-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na⁺-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na⁺-dependent transport of succinate by NaDC1.     

Attachment of Lactobacillus casei strain GG to human colon carcinoma cell line Caco-2: comparison with other dairy strains

The degree of adhesion of Lactobacillus casei strain GG to human Caco-2 cell line was investigated. Assessment of adhesion was compared to the adhesion of enterotoxigenic human Escherichia coli strain H 10407 and enterotoxigenic bovine E. coli strain B44 (non-adhesive). Freeze-dried Lactobacillus GG or samples from dairy products had medium to strong binding to the Caco-2 cell line. Lactobacillus acidophilus (NCFB 1748) and L. bulgaricus showed no adhesion to the cell line while four tested Bifidobacterium strains had no or very weak adhesion to the Caco-2 cell line.     

Enterocytic differentiation of the human Caco-2 cell line correlates with alterations in integrin signaling

We previously reported that the enterocytic differentiation of human colonic Caco-2 cells correlated with down-regulation of fibronectin (FN) and laminin (LN), two extracellular matrix components interacting with cell surface integrin receptors. We now investigated whether Caco-2 cell differentiation was associated with alterations in integrin signaling with special interest in the expression and activity of focal adhesion kinase (FAK) and mitogen-activated protein (MAP) kinase. The differentiation of Caco-2 cells was associated with: 1) down-regulation of beta1 integrin expression at the mRNA and protein levels; 2) increased FAK expression together with decreased FAK autophosphorylation; 3) decreased FAK's ability to associate with PI3-kinase and pp60c-src; and 4) increased MAP kinase expression along with decreased MAP activity. In addition, we show that FAK and MAP kinase belong to distinct integrin signaling pathways and that both pathways remain functional during Caco-2 cell differentiation since the coating of differentiating cells on FN and LN but not on polylysine increased the tyrosine phosphorylation of FAK and of its endogenous substrate paxillin, and stimulated MAP kinase activity. In conclusion, our results provide evidence that FAK and MAP kinase, two signaling molecules activated independently by beta1 integrins in Caco-2 cells, undergo alterations of both expression and activity during the enterocytic differentiation of this cell line.     

Phosphodiesterase in human colon carcinoma cell line CaCo-2 in culture

In a series of in vitro experiments we characterised the relationship between DNA distribution in the G1, S and G2/M phases of cell cycle and PDE and GST activity in CaCo-2 cells. The DNA distribution in CaCo-2 cells, was assessed by flow cytometry, with fluorescent dyes at different time points of culture. The exponential increase in cell number continued until day 10 when there was cell saturation. The effect of medium replacement on PDE activity was assayed in the first 10 h after medium replacement. The 6^th hour is the time at which PDE activity was found to be highest. We have assayed the PDE enzyme with cGMP and cAMP as substrates. Only cAMP was consumed from this enzyme. We found a very close correlation between the DNA distribution in the various phases of the cell cycle and the PDE activity. PDE activity was very high during the active replication phase, whereas GST activity was high after confluency.     

Castanospermine: a potent inhibitor of sucrase from the human enterocyte-like cell line Caco-2

The addition of castanospermine (5-50 microM) to a culture medium of Caco-2 cells results in a specific suppression of sucrase activity without modification of the biosynthesis of the enzyme. This effect is due to a direct inhibiting effect of castanospermine on Caco-2 sucrase activity. This inhibition is time-dependent (half-maximum efficiency at 10 min for 100 nM), enhanced by preincubation (suggesting a strong interaction with the enzyme), dose-dependent (ED50 at 4 nM after 1 h preincubation period) and of the fully non-competitive type. The calculated Ki (2.6 nM) suggests that castanospermine is the most potent inhibitor of sucrase so far reported.