Immortalization of human esophageal epithelial cells by human telomerase reverse transcriptase (hTERT)

Abstract

Carcinogenesis is a multistep process involving accumulation of numerous cellular abnormalities over a lengthy time-frame. One of the major steps is to achieve unlimited replicative potential (immortalization). The proliferative life-span of normal somatic cells is limited by an intrinsic mechanism associated with progressive shortening of telomeres during cell propagation, which induces the cells to undergo replicative senescence. We have established an immortal human esophageal epithelial cell line, NE2-hTERT, by ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase (a holoenzyme responsible for the maintenance of telomere length). During the immortalization process, the percentage of senescence associated β-galactosidase positive cells was monitored and was found to decrease with increasing passages as the cells escaped senescence and became immortalized. So far, the cell line has undergone over 100 population doublings. The NE2-hTERT cell line retained epithelial morphology and expressed cytokeratins typical for esophageal epithelium. It also had a near-diploid clonal karyotype. Analysis of expression profiles of cell cycle regulatory proteins in this cell line at different passages showed loss of p16, down-regulation of p53, as well as elevated p21 and p27 expressions during immortalization. Treatment with the demethylating agent, 5-aza-deoxycytidine, restored the p16 expression, which indicated that the spontaneous loss of p16 during immortalization was due to promoter methylation. Upregulation of both p53 and p21 in response to irradiation and mitomycin C treatment indicated that the cell line had intact DNA damage response. The altered p21 and p27 expressionsmight reflect a feedback mechanism to maintain certain ‘normality’ in cell-cycle regulation in these cells. Immortalized cell lines, especially ones with minimal genetic alterations, are invaluable models for studying the molecular basis of carcinogenesis. We observed that late passage NE2-hTERT cells showed increased tumorigenic potential in being able to form tightly-packed colonies in soft agar. This increased anchorage-independency suggests that the cell line may be just one step away from neoplastic transformation and is therefore particularly useful for studying genes associated with tumorigenic transformation of esophageal epithelial cells. [This study was supported by grants from the Research Grants Council of the Hong Kong Special Administrative Region, China, Project No. HKU 7385/03M and HKU 7556/06M, and the HKU URC Seed Funding Programme for Basic Research, Project No. 200411159061]