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

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.

Characterisation of telomerase immortalised normal human oesophageal squamous cells.

BACKGROUND AND AIMS: Oesophageal cell lines derived from malignancies have numerous genetic abnormalities and therefore are of limited value for studying the early events in carcinogenesis. Reported attempts to establish normal human oesophageal cell lines either have failed to achieve immortalisation or have achieved it by disrupting important cell functions. We have used telomerase technology to establish normal human oesophageal cell lines. METHODS: Endoscopic biopsy specimens of normal oesophageal squamous epithelium were trypsinised, dispersed into single cell suspensions, and cocultivated with ATCC Swiss 3T3 cells. Oesophageal cells were infected with the catalytic subunit of human telomerase (hTERT) using a defective retroviral vector. The integrity of cell cycle checkpoints was tested by measuring p53 response to UV irradiation, and p16 response to infection with H-RasGV12. Expression of a differentiation marker was tested by measuring involucrin response to calcium exposure. RESULTS: Cultures of uninfected oesophageal cells had weak telomerase activity at baseline but exhibited loss of telomerase activity and progressive telomere shortening before undergoing senescence between population doublings (PD) 40-45. In contrast, hTERT infected cells exhibited sustained telomerase activity and stabilisation of telomere length. These cells have reached PD 100 with no diminution in growth rate, while cell cycle checkpoint integrity and involucrin response to calcium exposure have remained intact. CONCLUSIONS: By introducing telomerase into normal human oesophageal squamous cells cocultivated with feeder layers, we have established a cell line that retains normal cell cycle checkpoints and normal differentiation markers. This cell line may be useful for studying the early events in oesophageal carcinogenesis.