Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model

Abstract

As there has been no previous information on the consequences of telomerase expression in genetically altered, mortal cells derived from pre-malignant tissue, we sought to determine the effect of hTERT (human catalytic subunit of telomerase reverse transcriptase) transduction of pre-malignant cell strains from Barrett's esophagus that do not contain telomerase activity and possess a finite lifespan. Primary cultures of Barrett's esophageal epithelium transduced with a retrovirus containing hTERT were characterized by growth factor requirements, cytogenetics and flow cytometry. Expression of telomerase lengthened telomeres and greatly extended the lifespan of hTERT transduced (hTERT+) Barrett's esophagus cells. Growth factor dependency of the hTERT+ cultures remained largely similar to the parental cultures, although there was a modest increase in the ability to grow in agar. Chromosomal instability, measured by both karyotypic and FISH (fluorescence in situ hybridization) analyses, was reduced but not abrogated by hTERT transduction, suggesting that telomerase expression can enhance genomic stability. However, the persistence of residual instability gave rise to new clonal and non-clonal genetic variants, and in one hTERT+ culture a new DNA aneuploid population was observed, the only time such a ploidy shift has been seen in Barrett's cell strains in vitro. These in vitro observations are analogous to the clinical progression to aneuploidy that often precedes cancer in Barrett's esophagus, and suggest that reactivation of telomerase may be permissive for continued genetic evolution to cancer. Long-lived Barrett's esophagus epithelial cultures should provide a useful in vitro model for studies of neoplastic evolution and chemopreventive therapies.

Glycine-extended gastrin inhibits apoptosis in Barrett's oesophageal and oesophageal adenocarcinoma cells through JAK2/STAT3 activation

“…The characteristics of this cell line have been described previously (Palanca-Wessels et al 2003). G-Gly-17 was from NeoMPS (Strasbourg, France) and amidated gastrin-17 was from Bachem (St Helens, UK).…”

“…QhERT cells were maintained in MCDB 153 medium supplemented with 5% FBS, 20 ng/ml epidermal growth factor (Bachem), 140 mg/ml bovine pituitary extract, 5 mg/ml insulin, 5 mg/ml transferrin, 0 . 4 mg/ml hydrocortisone, 100 pM cholera toxin, 20 mg/ml adenine, 4 mM glutamine, 0.25 mg/ml amphoteracin B, 100 u/ml penicillin and 100 u/ml streptomycin (Palanca-Wessels et al 2003).…”

Chromosomal Instability in Barrett's Esophagus Is Related to Telomere Shortening

“…Anaphase bridge quantification was done as described previously (26) using two primary, mortal epithelial cell cultures from Barrett's esophagus, designated CP-A and CP-C, and the same two cell cultures after transduction with the reverse transcriptase component of human telomerase; these cells and their culture are as described previously (36). The cells were 4 ¶,6-diamidino-2-phenylindole stained and cells with G 2 /4N DNA content were sorted onto glass slides.…”

“…To determine if this mechanism is plausible in Barrett's esophagus, we evaluated the presence of anaphase bridges in two primary cultures of Barrett's esophagus epithelium, because metaphase cells are infrequent in Barrett's biopsies and the size of biopsies is limited. These cell cultures have been shown previously to have shortened telomeres and high levels of CIN (36). We also examined the same two cell cultures after their transduction with telomerase, which resulted in lengthened telomeres (36).…”

A novel role for the retinoic acid-catabolizing enzyme CYP26A1 in Barrett's associated adenocarcinoma

“…The hTERT-immortalized non-dysplastic Barrett's cell line BAR (gifts from Rhonda F Souza, University of Texas Southwestern Medical Center, TX, USA) was grown on a 3T3 sorry feeder layer in KBM-2 (Cambrex, UK) medium and supplemented as previously described (Hormi-Carver et al, 2007). In addition, an hTERTimmortalized non-dysplastic cell line, QhTERT (CP-A), and three dysplastic cell lines, ChTERT (CP-B), GohTERT (CP-D) and GihTERT (CP-D) (gifts from P Rabinovich, University of Washington, WA, USA), were maintained in MCDB 153 medium (Sigma, St Louis, MO, USA) and supplemented as described previously (Palanca-Wessels et al, 2003).…”