Carcinogenesis Advance Access originally published online on October 8, 2008
Carcinogenesis 2009 30(1):122-130; doi:10.1093/carcin/bgn227
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Induction of intestinalization in human esophageal keratinocytes is a multistep process
1 Department of Medicine, Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA 19104, USA
2 AstraZeneca LP, Wilmington, DE 19850-5437, USA
* To whom correspondence should be addressed. Division of Gastroenterology, University of Pennsylvania, 650 Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104, USA. Tel: +1 215 898 0161; Fax: +1 215 573 2024; Email: lynchj{at}mail.med.upenn.edu
Barrett's esophagus (BE) is the replacement of normal squamous esophageal mucosa with an intestinalized columnar epithelium. The molecular mechanisms underlying its development are not understood. Cdx2 is an intestine-specific transcription factor that is ectopically expressed in BE, but its role in this process is unclear. Herein, we describe a novel cell culture model for BE. Retroviral-mediated Cdx2 expression in immortalized human esophageal keratinocytes [EPC-human telomerase reverse transcriptase (hTERT)] could transiently be established but not maintained and was associated with a reduction in cell proliferation. Coexpression of cyclin D1, but not a dominant-negative p53, rescued proliferation in the Cdx2-expressing cells. Cdx2 expression in the EPC-hTERT.D1 cells decreased cell proliferation but did not induce intestinalization. We investigated for other treatments to enhance intestinalization and found that acidic culture conditions uniformly killed EPC-hTERT.D1.Cdx2 cells. However, treatment with 5-aza-2-deoxycytidine (5-AzaC) to demethylate epigenetically silenced genes did appear to be tolerated. Multiple Cdx2 target genes, markers of intestinal differentiation and markers of BE, were induced by this 5-AzaC treatment. More interestingly, the expression level of several of these genes was enhanced only in the EPC-hTERT.D1-Cdx2 cells treated with 5-AzaC. Two of these, SLC26a3/DRA (downregulated in adenoma) and Na+/H+ exchanger 2 (NHE2), were not previously known to be elevated in BE; however, we confirmed their elevation in BE tissue samples. 5-AzaC treatment also induced cell senescence, even at low doses. We conclude that ectopic proliferation signals, alterations in epigenetic gene regulation and the inhibition of tumor suppressor mechanisms are required for Cdx2-mediated intestinalization of human esophageal keratinocytes in BE.
Abbreviations: 5-AzaC, 5-aza-2-deoxycytidine; BE, Barrett’s esophagus; cDNA, complementary DNA; DRA, downregulated in adenoma; EAC, esophageal adenocarcinoma; GFP, green fluorescent protein; hTERT, human telomerase reverse transcriptase; mRNA, messenger RNA; NHE2, Na+/H+ exchanger 2; PCR, polymerase chain reaction; pRb, retinoblastoma protein; RT, reverse transcription
Received May 9, 2008; revised August 28, 2008; accepted September 24, 2008.