Characterization of telomerase-immortalized, non-neoplastic, human Barrett’s cell line (BAR-T)

Abstract

Barrett's esophagus, a metaplasia predisposed to malignant transformation, has been studied in vitro using esophageal adenocarcinoma cell lines. However, findings in such transformed cells may not be applicable to the non-neoplastic cells of benign Barrett's esophagus. Therefore, we have developed and characterized a Barrett's cell line derived from a patient without malignancy or dysplasia. Human Barrett's epithelial cells were immortalized with the insertion of hTERT (human telomerase reverse transcriptase) using a Cre-lox recombination system. We then examined properties of this continuous cell line, such as in vitro tumorigenicity, growth patterns, histological differentiation characteristics, karyotype, and checkpoint arrest mechanisms (e.g., p16, p21, and p53). Non-neoplastic Barrett's epithelial cells infected with hTERT (BAR-T cells) have been sustained in culture beyond 200 population doublings. BAR-T cells maintain a diploid chromosome number and exhibit non-neoplastic properties, such as contact inhibition and anchorage-dependent growth. BAR-T cells express differentiation Barrett's epithelial markers, such as villin and cytokeratins 4, 8 and 18, and stain positive for Alcian blue, indicating the presence of mucin-producing cells. Expression of checkpoint arrest proteins p21 and p53 are intact, while p16 expression is lost. Thus, we have created a human Barrett's cell line that is not malignantly transformed, and yet can be maintained indefinitely in culture. BAR-T cells are diploid, have histological differentiation markers characteristic of benign Barrett's epithelium, and also maintain appropriate expression of p21 and p53. This cell line should be a useful model for the study of the early events in carcinogenesis in Barrett's esophagus.

Repeated exposure to acid and bile selectively induces colonic phenotype expression in a heterogeneous Barrett's epithelial cell line

“…In summary, using a non-neoplastic Barrett's cell line (BAR-T), which is composed of both squamous (CK4) and metaplastic/columnar cells (villin-and alcian blue-positive), 52 we demonstrate selective increase of CK8/18 type columnar cells, colonic phenotype (mAbDas-1-positive) and Cox-2 expression following chronic (up to 6 weeks) repeated exposure to acid and bile salt. Bile and acid together (A þ B pH 4) were found to be more potent than either alone.…”

“…The BAR-T cell line is reported to be a heterogeneous mix of cells of different phenotypes 52 and/or cells in different stages of differentiation. We observed an increase in CK8/18 expression in the BAR-T cell line significantly in response to bile at pH 4, as compared to acid alone or bile salt at neutral pH.…”

“…51 This focal multi-layered epithelium within BE comprises cells that concurrently express both squamous and columnar cytokeratin markers, much like the esophageal mucosal gland duct epithelium 34 and the non-neoplastic telomerase-immortalized Barrett's cell line, (BAR-T). 52 We utilized the BAR-T cell line as a model to identify the specific cell phenotype in a heterogeneous 52 cell population that could demonstrate molecular and/or cell phenotypic changes following chronic exposure to acid and bile. We utilized specific cell-phenotype markers: CK8/18 for columnar, CK4 for squamous, mAbDas-1 for colonic phenotype and p75NTR suggested as marker for esophageal progenitors 53,54 and quantified the cells displaying each phenotype.…”

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett’s metaplasia

“…Human esophageal epithelial cells were isolated from endoscopic biopsies of squamous and Barrett's epithelium (defined as the presence of specialized intestinal metaplasia) and telomerase immortalized as previously described (46). Squamous cell lines from patients with GERD with (NES-B3T, NES-B10T) and without Barrett's esophagus (NES-G2T, NES-G4T) and metaplastic Barrett's esophagus cell lines (BAR-T, BAR-10T) were used in experiments.…”

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

“…Souza and grown in a special supplemented keratinocyte basal medium-2 (Lonza, Breda, The Netherlands) according to Jaiswal et al [21,22]. The human embryonic kidney cell line HEK293 does not express TLR2 and was accordingly used as a negative control and cultured as described previously [22].…”