Personne : Têtu, Bernard
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Université Laval. Département de biologie moléculaire, de biochimie médicale et de pathologie
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- PublicationRestreintFactors affecting interindividual variability of hepatic UGT2B17 protein expression examined using a novel specific monoclonal antibody(American Society for Pharmacology and Experimental Therapeutics, etc., 2019-02-28) Lévesque, Éric; Rouleau, Michèle; Labriet, Adrien; Emond, Jean-Philippe; Hovington, Hélène; Périgny, Martine; Desjardins, Sylvie; Têtu, Bernard; Lacombe, Louis; Brisson, Hervé.; Guillemette, Chantal; Caron, Patrick; Fallon, John K.; Villeneuve, Lyne; Klein, Kathrin; Simonyan, David; Smith, Philip; Zanger, Ulrich M.The accurate quantification of the metabolic enzyme UGT2B17 has been hampered by the high sequence identity with other UGT2B enzymes (as high as 94%) and by the lack of a specific antibody. Knowing the significance of the UGT2B17 pathway in drug and hormone metabolism and cancer, we developed a specific monoclonal antibody (EL-2B17mAb), initially validated by the lack of detection in liver microsomes of an individual carrying no UGT2B17 gene copy and in supersomes expressing UGT2B enzymes. Immunohistochemical detection in livers reveals a strong labeling of bile ducts and variable labeling of hepatocytes. Expression levels assessed by immunoblotting were highly correlated to mass spectrometry-based quantification (r = 0.93) and three major expression patterns (absent, low or high) were evidenced. Livers with very low expression were carriers of the functional rs59678213 G variant, which is located in the binding site for the transcription factor Forkhead Box A1 (FOXA1) of the UGT2B17 promoter. The highest expression was observed for individuals carrying at least one rs59678213 A allele. A multiple regression analysis indicated that the number of gene copies explained only 8% of UGT2B17 protein expression, 49% when adding rs59678213 and reached 54% when including sex. The novel EL-2B17mAb antibody allowed specific UGT2B17 quantification and exposed different patterns of hepatic expression. It further suggests that FOXA1 is a key driver of UGT2B17 expression in the liver. The availability of this molecular tool will help characterize UGT2B17 level in various disease states and establish more precisely the UGT2B17 enzyme contribution to drug and hormone metabolism
- PublicationRestreintProduction of bioengineered cancer tissue constructs in vitro : epithelium–mesenchyme heterotypic interactions(Tissue Culture Association, 2001-07-01) Tremblay, Nathalie; Germain, Lucie; Wang, Chang Shu; Auger, François A.; Têtu, Bernard; Goulet, FrancineA few models have been established to study cancer cells in vitro. However, the cellular interactions have rarely been studied specifically using bioengineered cancer constructs combining human carcinoma cells and tumor-associated fibroblasts. We developed an in vitro model of tridimensional bioengineered cancer tissue constructs (bCTC) by seeding mammary epithelial cancer cells or normal keratinocytes over a mesenchymal layer containing tumor-derived fibroblastic cells or normal skin fibroblasts. After the introduction of epithelial cells, each construct was cultured for another 10 d. Histologic analyses showed that carcinoma cell lines could invade the subjacent mesenchymal layer and that the capacity to migrate was related to the invasive potential of cancer cells and the type of fibroblasts used, while noninvasive populations did not. Of the tested epithelial cells, MDA-MB-231 and, to a lesser degree, HDQ-P1 cell lines were invasive, and the invasion was deeper into the mesenchymal component containing tumor-derived fibroblasts. However, with normal skin fibroblasts, the mesenchymal layer was degraded twice faster than with tumor-derived fibroblastic cells. MDA-MB-231 cells and normal keratinocytes induced the highest level of gelatinase B, and the level was lowest with the MCF-7 cell line. The activated form of gelatinase B was, however, induced to the highest levels in the keratinocyte-seeded bCTC containing tumor-derived but not normal fibroblasts. MDA-MB-231 was the only epithelial cancer cell line whose activity of gelatinase A was reduced when cocultured with tumor-derived fibroblasts but not under normal fibroblast stimulation. Finally, a 50/48-kDa gelatinase band has been observed in bCTCs with noninvasive epithelial cells only. Our study demonstrates the selective secretion of gelatinases according to the phenotype of the cells seeded in the various bCTCs.
- PublicationRestreintSelective culture of epithelial cells from primary breast carcinomas using irradiated 3T3 cells as feeder layer(Elsevier, 2001-03-01) Tremblay, Nathalie; Germain, Lucie; Wang, Chang Shu; Auger, François A.; Têtu, Bernard; Goulet, FrancineThe main drawback of the selective culture of human mammary epithelial cells from primary breast cancer is the overgrowth of tumor-associated stromal fibroblasts. This drawback may be overcome by using, in primary culture, lethally irradiated 3T3 cells which act as a feeder layer to maintain tumor-derived epithelial cell proliferation. These 3T3 cells, exposed to 60 Gy at confluence, form a specific cellular substrate which constitutes an obstacle to fibroblast attachment. Enzyme-disaggregated breast cells from six primary breast carcinomas were cocultured over lethally irradiated but living 3T3 cells. The method led to the purification of tumor-derived epithelial cells from all six cancer samples, and long-term culture was obtained in one. The epithelial nature of these purified tumor-derived epithelial cells was demonstrated by their general morphology and by the expression of cytokeratins and Epithelial Membrane Antigen. These results confirm the stimulatory effect of a this stromal feeder layer on breast epithelial cell growth and show that this stromal feeder layer can also control the fibroblast overgrowth. Our results provide an alternative approach in the selective culture of epithelial cells from primary breast carcinoma.
- PublicationRestreintEstablishment and characterization of a new cell line derived from a human primary breast carcinoma(Elsevier, 2000-07-01) Germain, Lucie; Wang, Chang Shu; Lavoie, Josée; Drouin, Régen; Auger, François; Têtu, Bernard; Champetier, Serge.; Goulet, FrancineA new cell line, designated HDQ-P1, was successfully established from a primary ductal infiltrating mammary carcinoma by using a 3T3 feeder layer lethally irradiated to 60 Gy. The HDQ-P1 cells have been grown in culture for over 115 passages and have a doubling time of 60 hours. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, telomerase expression, tumor antigen expression, xenograft implantation into nude mice, colony formation in soft agar, TP53 sequencing, and gene copy number of C-MYC, C-ERBB-2, and C-H-RAS oncogenes. The epithelial nature of this cell line was confirmed by ultrastructural analysis, expression of cytokeratins, and epithelial membrane antigen. The HDQ-P1 cells possess an extensively rearranged and polyploid karyotype, with an average of 20 recurrent marker chromosomes. Scatchard analysis demonstrated that both primary tumor and HDQ-P1 cells were estrogen- and progesterone-receptor negative. The HDQ-P1 cells had the same expression of human telomerase reverse transcriptase as other established breast cancer cell lines such as MDA-MB-231, SK-BR-3, and MCF-7. Direct DNA sequencing showed a point mutation which yielded to a stop codon at the amino acid 213 in exon 6 of the TP53 gene. A five-fold amplification of C-MYC was observed in HDQ-P1 cells. No amplification of C-ERBB-2 and C-H-RAS genes were observed. This cell line presents unique characteristics and may prove to be a good experimental model for investigating breast cancer biology.