Transformation of benign Barrett's epithelium by repeated acid and bile exposure over 65 weeks: A novel in vitro model

Abstract

The mechanism by which gastroesophageal reflux promotes metaplasia→dysplasia→ carcinoma is unknown. The aim of the study is to determine if repeated exposure to acid and bile confers a tumorigenic phenotype in a telomerase (hTERT)-immortalized benign Barrett’s cell line, BAR-T. BAR-T cells were exposed to acid (pH 4) (A) and bile salt (200µM glycochenodeoxycholic acid) (B) daily for 5 min up to 65+ wks. The control cells were grown in parallel without any A or B treatment. Cell morphology, proliferation, transformation, and molecular changes in the gene expression for COX-2, TC22, p53 and p53 target genes were analyzed at 8–12 wks intervals. At 46 wks BAR-T cells exposed to (A+B) showed distinct phenotypic changes: forming clusters and acini, and at 65 wks displayed foci in monolayer, and formed distinct colonies in soft agar. Untreated cells did not show any such changes. In A+B treated BAR-T cells, COX-2 mRNA increased 10-20-fold, TC22 mRNA increased by 2-3-fold at 22 – 65 wks, p53, MDM2, PERP and p21mRNA increased 2.5-, 6.4-, 4- and 2.6- fold respectively when compared to untreated cells at 34 weeks. However, at 58 wks onward, there was a sharp decline of p53 and its target genes to the baseline level. At 65 weeks A+B treated BAR-T cells formed tumor in nude mice whereas untreated cells did not. We demonstrate a novel in-vitro model of transformation of a benign Barrett’s cell line following repeated exposure to A+B over the course of 65 wks.

Characterization of squamous esophageal cells resistant to bile acids at acidic pH: implication for Barrett's esophagus pathogenesis

“…These markers are also increased in BE metaplasia (19,38). Furthermore, these results are in agreement with recent studies, indicating that bile acids at low pH may induce an increase in the expression of CDX2, CK8/18, and villin in other model systems (5,6,13,24,25,66). At this point, it is not clear why we did not observe an increase in CDX2 mRNA, while villin mRNA was significantly increased.…”

“…HET1A is a normal human esophageal epithelial cell line immortalized by transfection of the SV40 T antigen early region gene. These cells have epithelial morphology, stain positively for cytokeratin 13, and have remained nontumorigenic in athymic, nude mice for more than 12 mo (59). The cells were cultured in BRFF-EPM2 medium (Athena Environmental Sciences, Baltimore, MD) supplemented with 50 g/ml gentamicin and 0.25 g/ml fungizone.…”

Toll-like Receptor 2 Signalling and the Lysosomal Machinery in Barrett’s Esophagus

“…1). This acid and bile salts mixture was chosen based on previous studies and were optimized in cell culture experiments (data available upon request) [24][25][26]. Subsequently, samples were washed and culture medium with or without the TLR2 agonist was added.…”

Barrett’s to Oesophageal Cancer Sequence: A Model of Inflammatory-Driven Upper Gastrointestinal Cancer

“…In a BO cell line, knocking down p53 and overexpressing the oncogene Ras caused malignant transformation [66]. Bile and acid treatment, which can induce ROS production in the oesophagus [67], increased p53 expression in BO cells initially, but this expression subsequently decreased with malignant transformation [68], suggesting that reflux may play a role in p53 alterations.…”