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.

Review article: a conceptual approach to understanding the molecular mechanisms of cancer development in Barrett's oesophagus.

Oesophageal adenocarcinoma is one of the most deadly human malignancies. Gastro-oesophageal reflux disease (GERD) has been established as a strong risk factor for oesophageal adenocarcinoma, and more than 40% of adult Americans experience regular GERD symptoms. GERD can be complicated by oesophagitis, and by replacement of oesophageal squamous mucosa with metaplastic, intestinal-type epithelium (Barrett's oesophagus) that is predisposed to malignancy. Cancers in Barrett's oesophagus arise through a sequence of genetic alterations which endow unlimited proliferative capacity upon the cells by affecting components of the cell cycle clock apparatus-the pivotal molecular machinery in the cell nucleus that controls whether a cell will proliferate, differentiate, become quiescent or die. This report describes how the genetic abnormalities that have been recognized in Barrett's oesophagus might promote carcinogenesis through effects on the cell cycle clock machinery. The goal of this review is to provide the clinician with a useful conceptual basis for evaluating studies on the molecular mechanisms underlying the progression from metaplasia to carcinoma in Barrett's oesophagus.

Putative telomere-independent mechanisms of replicative aging reflect inadequate growth conditions.

Telomere shortening is the mechanism underlying replicative aging in fibroblasts. A variety of reports now claim that inactivation of the p16(INK4a)/pRB pathway is required in addition to telomere maintenance for the immortalization of cells such as skin keratinocytes and breast epithelial cells. We here show that the premature growth arrest of these cell types can be explained by an inadequate culture environment. Providing mesenchymal/epithelial interactions by cultivating the telomerase-expressing cells on feeder layers avoids the growth arrest associated with increased p16(INK4a). These results do not support a telomere-independent mechanism of replicative aging.

Detection of telomerase by in situ hybridization and by the polymerase chain reaction-based telomerase activity assay.

The onset of human cancer typically requires numerous genetic mutations, generally specific for the tissue type from which the cancer originates. Thus, it has been difficult to screen all tumor types for a single mutation. In recent years, telomerase activity has been associated with at least 85% of human malignancies as well as with some lesions considered preneoplastic by traditional cytology (1,2). Telomerase appears to be ubiquitously associated with a wide array of human cancers from a variety of tissue sources. Therefore, detection of telomerase activity relative to human cancer development is likely to be an important and novel method, in combination with cytology, for cancer diagnosis.

Absence of cancer-associated changes in human fibroblasts immortalized with telomerase.

The ectopic expression of telomerase in normal human cells results in an extended lifespan, indicating that telomere shortening regulates the timing of cellular senescence. As telomerase expression is a hallmark of cancer, we investigated the long-term effects of forced expression of human telomerase catalytic component (hTERT) in normal human fibroblasts. In vitro growth requirements, cell-cycle checkpoints and karyotypic stability in telomerase-expressing cells are similar to those of untransfected controls. In addition, co-expression of telomerase, the viral oncoproteins HPV16 E6/E7 (which inactivate p53 and pRB) and oncogenic HRAS does not result in growth in soft agar. Thus, although ectopic expression of telomerase in human fibroblasts is sufficient for immortalization, it does not result in changes typically associated with malignant transformation.

In situ hybridization for telomerase RNA in routine cytologic brushings for the diagnosis of pancreaticobiliary malignancies.

BACKGROUND: Brush cytology for the diagnosis of pancreaticobiliary malignancy has an overall sensitivity of 50%. Accurate and specific markers are therefore needed for the optimal evaluation of pancreaticobiliary strictures. Telomerase activity is present in 85% to 90% of all human cancers. We sought to determine the utility of in situ hybridization for telomerase RNA in endoscopic brushings for the diagnosis of pancreaticobiliary malignancy. METHODS: Endoscopic brushings from 18 patients with pancreatic or biliary strictures were evaluated by routine cytology and in situ hybridization for telomerase RNA. RESULTS: Eight of 18 strictures were malignant. Cytology was positive in 5 patients, whereas telomerase RNA was positive in 6. All malignancies were diagnosed by either cytology or telomerase RNA; however, both studies were positive in only 3. There were no false-positive results by either technique. CONCLUSION: The detection of telomerase RNA in endoscopic brushings may be an important adjunct to cytology for cancer diagnosis in pancreaticobiliary strictures.

In situ hybridization for the detection of telomerase RNA in the progression from Barrett's esophagus to esophageal adenocarcinoma.

BACKGROUND: The rate of incidence of esophageal adenocarcinoma has surpassed that of all other malignancies in the U.S. Esophageal adenocarcinoma arises from Barrett's esophagus through the multistep accumulation of genetic mutations in metaplastic cells, accompanied by histologic changes from metaplasia to low grade and high grade dysplasia and finally to carcinoma. Although telomerase activity has been demonstrated in 85-90% of human cancers, to the authors' knowledge there are no reports regarding esophageal adenocarcinoma. The objective of this study was to determine whether telomerase RNA is increased in esophageal adenocarcinoma and at what point in the histologic progression such an increase occurs. METHODS: Formalin-fixed, paraffin-embedded esophageal biopsies from patients with Barrett's esophagus and surgical resection specimens of esophageal adenocarcinoma containing varying stages of neoplastic progression were obtained. Adjacent sections from each specimen were evaluated by routine histology and in situ hybridization for the RNA component of human telomerase. RESULTS: The authors found that 100% of esophageal adenocarcinomas and high grade dysplasias were strongly positive for telomerase RNA. Basal crypt cells of Barrett's metaplasia demonstrated weak to moderate telomerase RNA in 70% of cases, whereas 90% of low grade dysplasias had moderate levels of telomerase RNA. CONCLUSIONS: The results of this study showed that 1) Barrett's epithelium may contain a population of immortalized cells, 2) a marked increase in the level of telomerase RNA accompanies the transition from low grade to high grade dysplasia, and 3) high levels of telomerase RNA accompany the development of esophageal adenocarcinoma in the vast majority of cases.