Biomedical subjects

Christian Laboisse

Publications and source records attributed to Christian Laboisse.

10 recordsLinked to original sources

PubMed2006

Fasting induces hyperlipidemia in mice overexpressing proprotein convertase subtilisin kexin type 9: lack of modulation of very-low-density lipoprotein hepatic output by the low-density lipoprotein receptor.

Several proprotein convertase subtilisin kexin type 9 (PCSK9) mutations lead to familial hypercholesterolemia by virtue of its role as a negative modulator of the low-density lipoprotein receptor (LDLr). Here, we uncover that upon dietary challenge, the down-regulation of the LDLr is also a key mechanism by which PCSK9 modulates the hepatic production of apolipoprotein-B-containing lipoproteins. Thus, adenoviral-mediated overexpression of PCSK9 in 24-h fasted mice results in massive hyperlipidemia, due to a striking increase in very-low-density lipoprotein (VLDL) triglycerides and apolipoprotein B100 hepatic output. Similar studies in LDLr (-/-) mice demonstrate that PCSK9-mediated alteration of VLDL output in the fasted state requires the LDLr. This increased production of VLDL was associated with a concomitant reduction of intrahepatic lipid stores as well as a lack of down-regulation of peroxisome proliferator-activated receptor-alpha activity and target genes expression. Finally, we show that PCSK9 hepatic expression is inhibited by the hypotriglyceridemic peroxisome proliferator-activated receptor-alpha agonist fenofibrate. In summary, the negative modulation of LDLr expression by PCSK9, which decreases plasma LDL clearance, also promotes an overproduction of nascent VLDL in vivo upon fasting.

Adenoviridae↗

PubMed2005

Expression of human TFF3 in relation to growth of HT-29 cell subpopulations: involvement of PI3-K but not STAT6.

The trefoil factor family (TFF) peptides 1 and 2 (TFF1 and 2) are expressed in mucus cells of the stomach, whereas TFF3 is localized in goblet cells of the intestine. In the present study, we aimed to determine whether phosphatidylinositol 3-kinase (PI3-K) or signal transducer and activator of transcription protein 6 (STAT6) is involved in the expression of goblet cell specific markers. TFF3 expression was analyzed by RT-PCR, Northern blot, and radioimmunoassay (RIA) in relation to cell growth in subclones of HT-29 cells including the CL.16E and methotrexate (MTX) cell lines, which both exhibit a phenotype of mucus-secreting intestinal cells. A 30-fold increase in TFF3 mRNA levels and a 10-fold increase in TFF3-cell content were observed between the early proliferative and the late confluency states. The levels of MUC2 and MUC3 mRNA were also increased in the course of the differentiation process. A three to fourfold increase in PI3-K and Akt activities was observed in early post-confluent cells as compared with pre-confluent cells. Exposure of pre- and post-confluent cells to LY294002, a specific PI3-K inhibitor, for 1-4 days profoundly reduced TFF3 and MUC2 expression. A marked reduction in mucin granules content was also observed in LY-treated cells. Inhibition of the mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 did not modify the course of differentiation of the goblet cell lines. Moreover, stable transfection of HT-29 CL.16E cells with a dominant negative form of STAT6 had no effect on TFF3 induction. Together, these data indicate that PI3-K promotes the expression of TFF3 and MUC2 and that the PI3-K/Akt pathway may play a pivotal role in intestinal goblet cell differentiation.

Cell Differentiation↗

PubMed2004

Quantitative rather than qualitative differences in gene expression predominate in intestinal cell maturation along distinct cell lineages.

Several cell types are present in the intestinal epithelium that likely arise from a common precursor, the stem cell, and each mature cell type expresses a unique set of genes that characterizes its functional phenotype. Although the process of differentiation is intimately linked to the cessation of proliferation, the mechanisms that dictate intestinal cell fate determination are not well characterized. To investigate the reprogramming of gene expression during the cell lineage allocation/differentiation process, we took advantage of a unique system of two clonal derivatives of HT29 cells, Cl16E and Cl19A cells, which spontaneously differentiate as mucus producing goblet and chloride-secreting cells, respectively, as a function of time. By profiling gene expression, we found that these two cell lines show remarkably similar kinetics of change in gene expression and common clusters of coordinately regulated genes. This demonstrates that lineage-specific differentiation of intestinal epithelial cells is characterized overall by the sequential recruitment of functionally similar gene sets independent of the final phenotype of the mature cells.

Animals↗

PubMed2004

Orexins acting at native OX(1) receptor in colon cancer and neuroblastoma cells or at recombinant OX(1) receptor suppress cell growth by inducing apoptosis.

Screening of 26 gut peptides for their ability to inhibit growth of human colon cancer HT29-D4 cells grown in 10% fetal calf serum identified orexin-A and orexin-B as anti-growth factors. Upon addition of either orexin (1 microM), suppression of cell growth was total after 24 h and >70% after 48 or 72 h, with an EC(50) of 5 nm peptide. Orexins did not alter proliferation but promoted apoptosis as demonstrated by morphological changes in cell shape, DNA fragmentation, chromatin condensation, cytochrome c release into cytosol, and activation of caspase-3 and caspase-7. The serpentine G protein-coupled orexin receptor OX(1)R but not OX(2)R was expressed in HT29-D4 cells and mediated orexin-induced Ca(2+) transients in HT29-D4 cells. The expression of OX(1)R and the pro-apoptotic effects of orexins were also indicated in other colon cancer cell lines including Caco-2, SW480, and LoVo but, most interestingly, not in normal colonic epithelial cells. The role of OX(1)R in mediating apoptosis was further demonstrated by transfecting Chinese hamster ovary cells with OX(1)R cDNA, which conferred the ability of orexins to promote apoptosis. A neuroblastoma cell line SK-N-MC, which expresses OX(1)R, also underwent growth suppression and apoptosis upon treatment with orexins. Promotion of apoptosis appears to be an intrinsic property of OX(1)R regardless of the cell type where it is expressed. In conclusion, orexins, acting at native or recombinant OX(1)R, are pro-apoptotic peptides. These findings add a new dimension to the biological activities of these neuropeptides, which may have important implications in health and disease, in particular colon cancer.

Animals↗

PubMed2004

Real-time allele-specific amplification for sensitive detection of the BRAF mutation V600E.

BRAF is a cytoplasmic serine/threonine kinase in the MAPK pathway that transduces signals from RAS family members to MEK1/2. Mutations in the BRAF gene have been described in the majority of cutaneous melanomas, papillary thyroid carcinoma and to a lesser extent in other cancers. The predominant mutation reported is a single transversion in exon 15 (T1799A). We designed a real-time allele-specific PCR to detect this mutation. This assay allowed us to detect this alteration in samples containing 2% of cells harboring this mutation, which is equivalent to 1% mutated DNA, assuming heterozygosity for the allele. Using this assay, we then tested 44 human primary colorectal tumors. We found the V600E mutation in four samples (9.1%). Analysis of DNA extracted from paraffin-embedded sections gave similar results, indicating that archived tissues can also be analyzed using this assay. The advantages of this method include: (1) rapidity; (2) sensitivity; (3) ease; (4) large throughput; (5) low cost and (6) the small quantities of DNA needed.

Alleles↗

PubMed2003

Repression of MUC2 gene expression by butyrate, a physiological regulator of intestinal cell maturation.

Sodium butyrate (NaB) inhibits proliferation, stimulates apoptosis, and promotes differentiation of human colon cancer cells along the absorptive phenotype. In vitro, butyrate induces a switch from cells with a secretory to an absorptive phenotype. Here, we report that NaB specifically represses the expression of the MUC2 gene, a differentiation marker of the secretory goblet cell lineage, in forskolin- and 12-O-tetradecanoylphorbol 13-acetate-induced HT29 cells, and Cl.16E cells, a clonal derivative of HT29 cells that spontaneously differentiates into goblet cells. Thus, NaB repression is independent of the nature of the stimulus that triggers MUC2 expression. Further, repression was independent of new protein synthesis. Our results suggest that inhibition of MUC2 is linked to the ability of butyrate to repress histone deacetylase activity, since trichostatin A, another inhibitor of histone deacetylases, also inhibited MUC2 expression in induced HT29 cells. Finally, we demonstrate that the NaB effect is specific for this marker of the secretory cell lineage, since carcinoembryonic antigen, which is expressed in both the secretory and absorptive cells, is induced by NaB. Thus, the NaB repression of a definitive function of the secretory cell lineage is a further mechanism, in addition to the effects on proliferation and apoptotic pathways, through which butyrate can regulate intestinal homeostasis.

Butyrates↗

PubMed2003

Application of gene expression profiling to colon cell maturation, transformation and chemoprevention.

Methods for high-throughput analysis of profiles of gene expression that assay thousands of genes simultaneously are powerful approaches for understanding and classifying cell and tissue phenotype. This includes analysis of normal pathways of cell maturation and their perturbation in transformation, the sensitivity and mechanism of response of normal and tumor cells to physiological and pharmacological agents, and modulation of tumor risk and progression by nutritional factors. However, the complex data generated by such approaches raise difficulties in analysis. We will describe some of the methods we have used in analyzing databases generated in a number of projects in our laboratories. These include: the role of k-ras mutations in colon cell transformation; the role of p21(WAF1/cip1) in intestinal tumor formation and response to sulindac; the development of the absorptive and goblet cell lineages; sensitivity of colonic cells to chemotherapeutic agents; mechanisms that regulate c-myc expression utilizing novel methods of transcriptional imaging; and interaction of nutritional and genetic factors in modulation of intestinal tumor formation.

Animals↗

PubMed2003

Glutamine preserves protein synthesis and paracellular permeability in Caco-2 cells submitted to "luminal fasting".

This study used polarized cell line Caco-2 as a model of human enterocytes to determine: 1) whether deprivation of nutrients on the apical (luminal) side of the epithelium (fasting) alters protein synthesis in enterocytes; 2) if so, whether glutamine can attenuate the effects of fasting; and 3) whether the effects of glutamine depend on its route (i.e., apical vs. basolateral) of supply. Caco-2 cells were submitted to nutrient deprivation on the apical side to mimic the effects of fasting, whereas the basolateral side of the epithelium remained exposed to regular medium. Cells were then incubated with [2H3]leucine with or without glutamine, and the fractional synthesis rate (FSR) of total cell protein was determined from [2H3]leucine enrichments in protein-bound and intracellular free leucine measured by gas chromatography/mass spectrometry. A 24-h apical nutrient deprivation (luminal fasting) was associated with a decline in intracellular glutamine, glutamate, and glutathione concentrations (-38, -40, and -40%, respectively), protein FSR (-20%), and a rise in passage of dextran, an index of transepithelial permeability. In fasted cells, basolateral or luminal glutamine supplementation did not alter the glutathione pool, but it restored protein FSR and improved permeability. The effects of glutamine were abolished by 6-diazo-oxo-l-norleucine, an inhibitor of glutaminase, and was mimicked by glutamate. We conclude that in Caco-2 cells, protein synthesis depends on nutrient supply on the apical side, and glutamine regardless of the route of supply corrects some of the deleterious effects of fasting in a model of human enterocytes through its deamidation into glutamate.

Caco-2 Cells↗

PubMed2003

[Value of the uCyt+ test for the detection and followup of bladder tumors].

INTRODUCTION: Superficial bladder tumours are common tumours that frequently relapse, generally with deterioration of the grade. Cystoscopic surveillance is unpleasant for the patient and is associated with considerable morbidity, while urine cytology, although very easy to poor, has a low sensitivity. According to the literature, the uCyt+ test is very effective for the detection of urothelial tumours. This immunofluorescent technique detects desquamated tumour cells previously labelled by specific antibodies. MATERIAL AND METHOD: This prospective study was conducted in 92 patients undergoing screening or follow-up cystoscopy. Voided urine was collected prior to cystoscopy to perform standard cytology and a uCyt+ test. These examinations were interpreted independently under blinded conditions. RESULTS: The sensitivity and specificity of uCyt+ were 68.7% and 81.7%, versus 40.6% and 96.7% for cytology. The sensitivity of uCyt+ was higher for low-grade tumours (87.5%), in contrast with cytology (37.5%). The results of uCyt+ were comparable to those of cytology for high-grade tumours (80%). CONCLUSION: The uCyt+ test presented better performances than standard cytology. It would allow less frequent surveillance cystoscopy examinations, especially in patients with low-grade lesions.

Cystoscopy↗

PubMed2003

In situ immunophenotype of the inflammatory infiltrate in eosinophilic fasciitis.

OBJECTIVE: Eosinophilic fasciitis (EF) is histologically characterized by a fibrous and inflammatory thickening of subcutaneous septal-fascial-perimysial collagenous scaffold. This study aims to define the immunophenotype of inflammatory cells of fascia and muscle underlying the in situ immune response in EF. METHODS: In 11 cases of EF, we determined the phenotype of inflammatory cells, expression of MHC class I and class II antigens, and C5b9 membranolytic attack complex (MAC) deposits by immunohistochemistry analysis of fascia tissue. Muscle biopsies from 9 patients with active dermatomyositis and 5 with active polymyositis were used as controls. Results. In all patients but one, the inflammatory infiltrate was mainly composed of macrophages associated with CD8+ T lymphocytes (CD4/CD8 ratio < 1) and few eosinophils. Cytotoxic properties were found in 14% of CD8+ T lymphocytes, as shown by granzyme B expression. MHC Class I antigens were overexpressed (5/7) by muscle fibers, with a paratrabecular reinforcement in 4 cases. MHC class II antigens were not expressed by muscle fibers except in one case. C5b9 MAC deposits were not detected. CONCLUSION: Our in situ characterization of inflammatory infiltrate demonstrates the predominancy of macrophages and CD8+ T lymphocytes. Some of these CD8+ lymphocytes contain granzyme B, thus suggesting a cytotoxic cellular immune response in EF, which could be triggered by infectious or environmental agents.

CD4-CD8 Ratio↗