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Pascale Anderle

Publications and source records attributed to Pascale Anderle.

13 recordsLinked to original sources
PubMed2005

Genetic variants of the human dipeptide transporter PEPT1.

We tested whether genetic polymorphisms affect activity of the dipeptide transporter PEPT1, which mediates bioavailability of peptidomimetic drugs. All 23 exons and adjoining intronic sections of PEPT1 (SLC15A1) were sequenced in 247 individuals of various ethnic origins (Coriell collection). Of 38 single nucleotide polymorphisms (SNPs), 21 occurred in intronic and noncoding regions and 17 in exonic coding region, of which nine were nonsynonymous. Eight nonsynonymous variants were cloned into expression vectors and functionally characterized after transient transfection into Cos7 and Chinese hamster ovary cells. None of the variants had altered transport activity for various ligands, supporting previous results, except for the new, low-frequency PEPT1-F28Y. This variant displayed significantly reduced cephalexin uptake attributable to increased K(m). Altered pH dependence of substrate transport suggested a role for F28Y in H(+)-driven translocation. Haplotype analysis revealed significant differences among ethnic populations. To search for cis-acting polymorphisms affecting transcription and mRNA processing, we measured allelic PEPT1 mRNA expression in human intestinal biopsy samples using a frequent-marker SNP in exon 17. Of 24 heterozygous samples, significant differences in allelic mRNA levels of 20 to 30% were observed in seven tissues. However, the small difference suggests that cis-acting regulatory factors have only limited effects on transporter activity. We also measured the relative formation of a splice variant (PEPT1-RF). PEPT1-RF mRNA levels ranged from 2 to 44% of total PEPT1-related mRNA, with potential consequences for drug absorption. Together with previous results, this study reveals a relatively low level of genetic variability in polymorphisms affecting both protein function and gene regulation.

Animals↗
PubMed2005

Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes.

BACKGROUND: The purpose of this work was to characterize the expression of drug and nutrient carriers along the anterior-posterior and crypt-villus axes of the intestinal epithelium and to study the validity of utilizing whole gut tissue rather than purified epithelial cells to examine regional variations in gene expression. RESULTS: We have characterized the mRNA expression profiles of 76 % of all currently known transporters along the anterior-posterior axis of the gut. This is the first study to describe the expression profiles of the majority of all known transporters in the intestine. The expression profiles of transporters, as defined according to the Gene Ontology consortium, were measured in whole tissue of the murine duodenum, jejunum, ileum and colon using high-density microarrays. For nine transporters (Abca1, Abcc1, Abcc3, Abcg8, Slc10a2, Slc28a2, Slc2a1, Slc34a2 and Slc5a8), the mRNA profiles were further measured by RT-PCR in laser micro-dissected crypt and villus epithelial cells corresponding to the aforementioned intestinal regions. With respect to differentially regulated transporters, the colon had a distinct expression profile from small intestinal segments. The majority (59 % for p cutoff < or = 0.05) of transporter mRNA levels were constant across the intestinal sections studied. For the transporter subclass "carrier activity", which contains the majority of known carriers for biologically active compounds, a significant change (p < or = 0.05) along the anterior-posterior axis was observed. CONCLUSION: All nine transporters examined in laser-dissected material demonstrated good replication of the region-specific profiles revealed by microarray. Furthermore, we suggest that the distribution characteristics of Slc5a8 along the intestinal tract render it a suitable candidate carrier for monocarboxylate drugs in the posterior portion of the intestine. Our findings also predict that there is a significant difference in the absorption of carrier-mediated compounds in the different intestinal segments. The most pronounced differences can be expected between the adjoining segments ileum and colon, but the differences between the other adjoining segments are not negligible. Finally, for the examined genes, profiles measured in whole intestinal tissue extracts are representative of epithelial cell-only gene expression.

Analysis of Variance↗
PubMed2005

Novel markers of the human follicle-associated epithelium identified by genomic profiling and microdissection.

BACKGROUND & AIMS: Regulation of gene expression in the follicle-associated epithelium (FAE) over Peyer's patches is largely unknown. CCL20, a chemokine that recruits immature dendritic cells, is one of the few FAE-specific markers described so far. Lymphotoxin beta (LTalpha1beta2) expressed on the membrane of immune cells triggers CCL20 expression in enterocytes. In this study, we measured expression profiles of LTalpha1beta2-treated intestinal epithelial cells and selected CCL20 -coregulated genes to identify new FAE markers. METHODS: Genomic profiles of T84 and Caco-2 cell lines treated with either LTalpha1beta2, flagellin, or tumor necrosis factor alpha were measured using the Affymetrix GeneChip U133A. Clustering analysis was used to select CCL20 -coregulated genes, and laser dissection microscopy and real-time polymerase chain reaction on human biopsy specimens was used to assess the expression of the selected markers. RESULTS: Applying a 2-way analysis of variance, we identified regulated genes upon the different treatments. A subset of genes involved in inflammation and related to the nuclear factor kappaB pathway was coregulated with CCL20 . Among these genes, the antiapoptotic factor TNFAIP3 was highly expressed in the FAE. CCL23 , which was not coregulated in vitro with CCL20 , was also specifically expressed in the FAE. CONCLUSIONS: We have identified 2 novel human FAE specifically expressed genes. Most of the CCL20 -coregulated genes did not show FAE-specific expression, suggesting that other signaling pathways are critical to modulate FAE-specific gene expression.

Biomarkers↗
PubMed2004

Transporter and ion channel gene expression after Caco-2 cell differentiation using 2 different microarray technologies.

mRNA expression profiles had previously been measured in Caco-2 cells (human colonic carcinoma cells) using either custom-designed spotted oligonucleotide arrays or Affymetrix GeneChip oligonucleotide arrays. The Caco-2 cells used were from different clones and were examined under slightly different culture conditions commonly encountered when Caco-2 cells are used as a model tissue for studying intestinal transport and metabolism in different laboratories. In this study, we compared gene expression profiles of Caco-2 cells generated with different arrays to assess the validity of conclusions derived from the 2 independent studies, with a focus on changes in transporter and ion channel mRNA expression levels on Caco-2 cell differentiation. Significant changes in expression levels upon differentiation were observed with 78 genes, with probes common to both arrays. Of these, 18 genes were upregulated and 36 genes were downregulated. The 2 arrays yielded discrepant results for 24 genes, showing significant changes upon differentiation. The results from the 2 arrays correlated well for genes expressed above average levels (r = 0.75, P < 0.01, n = 25) and poorly for genes expressed at low levels (r = 0.08, P > 0.05, n = 25). Overall correlation across the 2 platforms was r = 0.45 (P < 0.01) for the 78 genes, with similar results from both arrays. Despite differences in experimental conditions and array technology, similar results were obtained for most genes.

Caco-2 Cells↗
PubMed2004

Membrane transporters and channels: role of the transportome in cancer chemosensitivity and chemoresistance.

Membrane transporters and channels (collectively the transportome) govern cellular influx and efflux of ions, nutrients, and drugs. We used oligonucleotide arrays to analyze gene expression of the transportome in 60 human cancer cell lines used by the National Cancer Institute for drug screening. Correlating gene expression with the potencies of 119 standard anticancer drugs identified known drug-transporter interactions and suggested novel ones. Folate, nucleoside, and amino acid transporters positively correlated with chemosensitivity to their respective drug substrates. We validated the positive correlation between SLC29A1 (nucleoside transporter ENT1) expression and potency of nucleoside analogues, azacytidine and inosine-glycodialdehyde. Application of an inhibitor of SLC29A1, nitrobenzylmercaptopurine ribonucleoside, significantly reduced the potency of these two drugs, indicating that SLC29A1 plays a role in cellular uptake. Three ABC efflux transporters (ABCB1, ABCC3, and ABCB5) showed significant negative correlations with multiple drugs, suggesting a mechanism of drug resistance. ABCB1 expression correlated negatively with potencies of 19 known ABCB1 substrates and with Baker's antifol and geldanamycin. Use of RNA interference reduced ABCB1 mRNA levels and concomitantly increased sensitivity to these two drugs, as expected for ABCB1 substrates. Similarly, specific silencing of ABCB5 by small interfering RNA increased sensitivity to several drugs in melanoma cells, implicating ABCB5 as a novel chemoresistance factor. Ion exchangers, ion channels, and subunits of proton and sodium pumps variably correlated with drug potency. This study identifies numerous potential drug-transporter relationships and supports a prominent role for membrane transport in determining chemosensitivity. Measurement of transporter gene expression may prove useful in predicting anticancer drug response.

ATP-Binding Cassette Transporters↗
PubMed2004

Regional variations in ABC transporter expression along the mouse intestinal tract.

The ATP-binding cassette (ABC) family of proteins comprise a group of membrane transporters involved in the transport of a wide variety of compounds, such as xenobiotics, vitamins, lipids, amino acids, and carbohydrates. Determining their regional expression patterns along the intestinal tract will further characterize their transport functions in the gut. The mRNA expression levels of murine ABC transporters in the duodenum, jejunum, ileum, and colon were examined using the Affymetrix MuU74v2 GeneChip set. Eight ABC transporters (Abcb2, Abcb3, Abcb9, Abcc3, Abcc6, Abcd1, Abcg5, and Abcg8) displayed significant differential gene expression along the intestinal tract, as determined by two statistical models (a global error assessment model and a classic ANOVA, both with a P < 0.01). Concordance with semiquantitative real-time PCR was high. Analyzing the promoters of the differentially expressed ABC transporters did not identify common transcriptional motifs between family members or with other genes; however, the expression profile for Abcb9 was highly correlated with fibulin-1, and both genes share a common complex promoter model involving the NFkappaB, zinc binding protein factor (ZBPF), GC-box factors SP1/GC (SP1F), and early growth response factor (EGRF) transcription binding motifs. The cellular location of another of the differentially expressed ABC transporters, Abcc3, was examined by immunohistochemistry. Staining revealed that the protein is consistently expressed in the basolateral compartment of enterocytes along the anterior-posterior axis of the intestine. Furthermore, the intensity of the staining pattern is concordant with the expression profile. This agrees with previous findings in which the mRNA, protein, and transport function of Abcc3 were increased in the rat distal intestine. These data reveal regional differences in gene expression profiles along the intestinal tract and demonstrate that a complete understanding of intestinal ABC transporter function can only be achieved by examining the physiologically distinct regions of the gut.

ATP-Binding Cassette Transporters↗
PubMed2004

Intestinal membrane transport of drugs and nutrients: genomics of membrane transporters using expression microarrays.

Carrier-mediated transport across membranes plays an important role in drug and nutrient absorption. However, relevant transporters remain largely unknown for most substrates. Their identification requires global analysis of expressed mRNAs in intestinal tissues. Microarray technologies capable of measuring mRNA profiles have proven useful in detecting the expression of genes encoding transporters and ion channels in intestines and Caco-2 cells. This colon carcinoma cell line with characteristics of absorptive enterocytes serves as a common model for drug absorption studies. Gene expression patterns of membrane transporters and channels define the cell's overall transport capacity. Moreover, transporter mRNA profiles provide a basis for assessing drug-drug and drug-food interactions in intestinal absorption. To determine relevant transporters for any given substrate, chemogenomic methods have emerged correlating mRNA expression in multiple tissues to drug transport or response. The resultant drug-transporter databases permit the search for transporter-drug relationships at a genomic scale.

Animals↗
PubMed2004

How the gut links innate and adaptive immunity.

Mucosal surfaces represent the main sites in which environmental microorganisms and antigens interact with the host. Sentinel cells, including epithelial cells, lumenal macrophages, and intraepithelial dendritic cells, continuously sense the environment and coordinate defenses for the protection of mucosal tissues. The mucosal epithelial cells are crucial actors in coordinating defenses. They sense the outside world and respond to environmental signals by releasing chemokines and cytokines that recruit inflammatory and immune cells to control potential infectious agents and to attract cells able to trigger immune responses. Among immune cells, dendritic cells (DC) play a key role in controlling adaptive immune responses, due to their capacity to internalize foreign materials and to present antigens to naive T and B lymphocytes, locally or in draining organized lymphoid tissues. Immune cells recruited in epithelial tissues can, in turn, act upon the epithelial cells and change their phenotype in a process referred to as epithelial metaplasia.

Adaptation, Physiological↗
PubMed2003

Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines.

PURPOSE: The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. METHOD: Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. RESULTS: Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. CONCLUSIONS: Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.

Adult↗
PubMed2003

Intra- and interindividual variabilities of valacyclovir oral bioavailability and effect of coadministration of an hPEPT1 inhibitor.

Variability in valacyclovir bioavailability and the potential for cephalexin-valacyclovir interaction were evaluated. The intraindividual acyclovir area under the concentration-time curve (AUC) varied minimally, whereas interindividual differences were substantial. Coadministration of the human peptide transporter 1 (hPEPT1) substrates valacyclovir and cephalexin minimally reduced the acyclovir AUC. These results suggest a stable valacyclovir absorption phenotype, significant interindividual variability, and minimal interaction between these hPEPT1 substrates.

Acyclovir↗
PubMed2003

Gene expression databases and data mining.

The DNA microarray technology has arguably caught the attention of the worldwide life science community and is now systematically supporting major discoveries in many fields of study. The majority of the initial technical challenges of conducting experiments are being resolved, only to be replaced with new informatics hurdles, including statistical analysis, data visualization, interpretation, and storage. Two systems of databases, one containing expression data and one containing annotation data are quickly becoming essential knowledge repositories of the research community. This present paper surveys several databases, which are considered "pillars" of research and important nodes in the network. This paper focuses on a generalized workflow scheme typical for microarray experiments using two examples related to cancer research. The workflow is used to reference appropriate databases and tools for each step in the process of array experimentation. Additionally, benefits and drawbacks of current array databases are addressed, and suggestions are made for their improvement.

Breast Neoplasms↗
PubMed2002

In vitro assessment of intestinal IGF-I stability.

Insulin-like growth factor (IGF-I) is a 7648-Da polypeptide consisting of 70 amino acids. Clinically, IGF-I might be used in type II diabetes, which requires a life-long treatment. Therefore, delivery routes other than parenteral injections are highly desirable. For convenience, the peroral route is the most attractive. Therefore, in an attempt to answer the feasibility of oral delivery of IGF-I we examined the metabolism of this polypeptide in the gut in the presence of crude porcine pancreatic enzymes (CPPE) and flushings of the small and large intestine from pig, rat, and dog. Moreover, incubation studies with purified pancreatic enzymes that are present in the intestine were performed to determine the most active enzymes responsible for the intestinal cleavage of IGF-I. IGF-I was mainly degraded by chymotrypsin (t(1/2) = 2.7 min) and trypsin (t(1/2) = 34.6 min), whereas in the presence of aminopeptidase M and carboxypeptidase A IGF-I was stable up to 90 min. IGF-I was degraded in flushings from the jejunum, ileum, and colon. However, there were no significant differences in the stability of IGF-I between the examined intestinal segments. The addition of serine protease inhibitors such as a combination of aprotinin, soybean trypsin inhibitor, and Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), as well as casein profoundly improved the stability. Because we were able to improve the stability of IGF-I in vitro in all species at the same degree we speculate that a similar extension of half-life might also be possible in the human intestinal system.

Animals↗