Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories.
ABSTRACT Caco-2 cells, widely used to study carrier mediated uptake and efflux mechanisms, are known to have different properties when cultured under different conditions. In this study, Caco-2 cells from 10 different laboratories were compared in terms of mRNA expression levels of 72 drug and nutrient transporters, and 17 other target genes, including drug metabolising enzymes, using real-time PCR. The rank order of the top five expressed genes was: HPT1>GLUT3>GLUT5>GST1A>OATP-B. Rank correlation showed that for most of the samples, the gene ranking was not significantly different. Functionality of transporters and the permeability of passive transport markers metoprolol (transcellular) and atenolol (paracellular) were also compared. MDR1 and PepT1 function was investigated using talinolol and Gly-Sar transport, respectively. Sulfobromophthalein (BSP) was used as a marker for MRP2 and OATP-B functionality. Atenolol permeability was more variable across laboratories than metoprolol permeability. Talinolol efflux was observed by all the laboratories, whereas only five laboratories observed significant apical uptake of Gly-Sar. Three laboratories observed significant efflux of BSP. MDR1 expression significantly correlated to the efflux ratio and net active efflux of talinolol. PepT1 mRNA levels showed significant correlation to the uptake ratio and net active uptake of Gly-Sar. MRP2 and OATP-B showed no correlation to BSP transport parameters. Heterogeneity in transporter activity may thus be due to differences in transporter expression as shown for PepT1 and MDR1 which in turn is determined by the culture conditions. Absolute expression of genes was variable indicating that small differences in culture conditions have a significant impact on gene expression, although the overall expression patterns were similar.
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ABSTRACT: The intestine-specific caudal-related homeobox transcription factor CDX2 seems to play a key role in intestinal development and differentiation. Inactivation of one Cdx2 allele predisposes mice to develop colon polyps, and loss of CDX2 expression is a feature of some poorly differentiated colon carcinomas in humans. Conversely, aberrant CDX2 expression is often seen in intestinal metaplasias in the stomach and esophagus and in some gastric carcinomas. To better understand CDX2 function, we sought to define CDX2-regulated genes. HT-29 colon cancer cells with minimal endogenous CDX2 expression were engineered to express exogenous CDX2, and gene expression changes relative to control cells were assessed using high-density oligonucleotide arrays. The gene for liver intestine (LI)-cadherin (cadherin 17) was strongly induced by CDX2 in HT-29. In other colorectal cancer lines, endogenous CDX2 and LI-cadherin expression were well correlated. Activation of a ligand-regulated form of CDX2 rapidly induced LI-cadherin gene expression, even in the presence of protein synthesis inhibitor. Analysis of the 5'-flanking region of the LI-cadherin gene defined 2 CDX2 responsive elements, and chromatin immunoprecipitation assays indicate CDX2 binds to the elements. In primary colorectal cancers and intestinal metaplasias in the stomach, CDX2 and LI-cadherin expression were tightly correlated. CDX2 regulates LI-cadherin gene expression in normal, metaplastic, and neoplastic tissues of the gastrointestinal tract via binding to elements in the 5'-flanking region of the gene. Given the well-established roles of cadherins in morphogenesis and differentiation, LI-cadherin may be a key factor mediating CDX2 function in intestinal cell fate determination.Gastroenterology 11/2002; 123(5):1565-77. · 12.82 Impact Factor
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ABSTRACT: The transport of specific molecules across lipid membranes is an essential function of all living organisms and a large number of specific transporters have evolved to carry out this function. The largest transporter gene family is the ATP-binding cassette (ABC) transporter superfamily. These proteins translocate a wide variety of substrates including sugars, amino acids, metal ions, peptides, and proteins, and a large number of hydrophobic compounds and metabolites across extra- and intracellular membranes. ABC genes are essential for many processes in the cell, and mutations in these genes cause or contribute to several human genetic disorders including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response. Characterization of eukaryotic genomes has allowed the complete identification of all the ABC genes in the yeast Saccharomyces cerevisiae, Drosophila, and C. elegans genomes. To date, there are 48 characterized human ABC genes. The genes can be divided into seven distinct subfamilies, based on organization of domains and amino acid homology. Many ABC genes play a role in the maintenance of the lipid bilayer and in the transport of fatty acids and sterols within the body. Here, we review the current knowledge of the human ABC genes, their role in inherited disease, and understanding of the topology of these genes within the membrane.The Journal of Lipid Research 08/2001; 42(7):1007-17. · 4.39 Impact Factor
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ABSTRACT: The adhesive function of classical cadherins depends on the association with cytoplasmic proteins, termed catenins, which serve as a link between cadherins and the actin cytoskeleton. LI-cadherin, a structurally different member of the cadherin family, mediates Ca2+-dependent cell-cell adhesion, although its markedly short cytoplasmic domain exhibits no homology to this highly conserved region of classical cadherins. We now examined whether the adhesive function of LI-cadherin depends on the interaction with catenins, the actin cytoskeleton or other cytoplasmic components. In contrast to classical cadherins, LI-cadherin, when expressed in mouse L cells, was neither associated with catenins nor did it induce an upregulation of beta-catenin. Consistent with these findings, LI-cadherin was not resistant to detergent extraction and did not induce a reorganization of the actin cytoskeleton. However, LI-cadherin was still able to mediate Ca2+-dependent cell-cell adhesion. To analyze whether this function requires any interaction with proteins other than catenins, a glycosyl phosphatidylinositol-anchored form of LI-cadherin (LI-cadherin(GPI)) was constructed and expressed in Drosophila S2 cells. The mutant protein was able to induce Ca2+-dependent, homophilic cell-cell adhesion, and its adhesive properties were indistinguishable from those of wild type LI-cadherin. These findings indicate that the adhesive function of LI-cadherin is independent of any interaction with cytoplasmic components, and consequently should not be sensitive to regulatory mechanisms affecting the binding of classical cadherins to catenins and to the cytoskeleton. Thus, we postulate that the adhesive function of LI-cadherin is complementary to that of coexpressed classical cadherins ensuring cell-cell contacts even under conditions that downregulate the function of classical cadherins.The Journal of Cell Biology 04/1997; 136(5):1109-21. · 10.82 Impact Factor