Malignant Transformation of Non-Neoplastic Barrett's Epithelial Cells through Well-Defined Genetic Manipulations

Xi Zhang,Shelby D Melton,Rhonda F Souza,Hui Ying Zhang,Chunhua Yu,David H Wang,Stuart J Spechler,Robert M Genta,Kathleen Wilson,Xiaofang Huo,Hana Algül

doi:10.1371/journal.pone.0013093

Abstract

BackgroundHuman Barrett's cancer cell lines have numerous, poorly-characterized genetic abnormalities and, consequently, those lines have limited utility as models for studying the early molecular events in carcinogenesis. Cell lines with well-defined genetic lesions that recapitulate various stages of neoplastic progression in Barrett's esophagus would be most useful for such studies.Methodology/Principal FindingsTo develop such model cell lines, we started with telomerase-immortalized, non-neoplastic Barrett's epithelial (BAR-T) cells, which are spontaneously deficient in p16, and proceeded to knock down p53 using RNAi, to activate Ras by introducing oncogenic H-RasG12V, or both. BAR-T cells infected with either p53 RNAi or oncogenic H-RasG12V alone maintained cell-to-cell contact inhibition and did not exhibit anchorage-independent growth in soft agar. In contrast, the combination of p53 RNAi knockdown with expression of oncogenic H-RasG12V transformed the p16-deficient BAR-T cells, as evidenced by their loss of contact inhibition, by their formation of colonies in soft agar, and by their generation of tumors in immunodeficient mice.Conclusions/SignificanceThrough these experiments, we have generated a number of transformed and non-transformed cell lines with well-characterized genetic abnormalities recapitulating various stages of carcinogenesis in Barrett's esophagus. These lines should be useful models for the study of carcinogenesis in Barrett's esophagus, and for testing the efficacy of chemopreventive and chemotherapeutic agents.

Highlights

The incidence of esophageal adenocarcinoma, a lethal tumor that develops from the metaplastic epithelium of Barrett’s esophagus, has increased profoundly in the United States over the past several decades [1,2]
Human Barrett’s cancer cells have sustained numerous, poorly characterized genetic injuries and, cell lines derived from those malignancies are of limited value for studying early molecular events in carcinogenesis
Well-defined genetic alterations that target the p53 and Ras pathways, we have induced the malignant transformation of our telomerase-immortalized human Barrett’s epithelial cells, and we have developed a number of non-transformed cell lines with welldefined, growth-promoting genetic changes that might recapitulate various stages of neoplastic progression in Barrett’s esophagus

Summary

Introduction

The incidence of esophageal adenocarcinoma, a lethal tumor that develops from the metaplastic epithelium of Barrett’s esophagus, has increased profoundly in the United States over the past several decades [1,2]. A durable, in vitro model that starts with non-malignant, human, Barrett’s metaplastic cells to recapitulate the various stages of neoplastic progression might be most useful for studying the early molecular mechanisms underlying malignancy in Barrett’s esophagus. We have developed cultures of human Barrett’s epithelial cells from esophageal biopsy specimens of non-neoplastic Barrett’s metaplasia, and we have immortalized those cells by forcing them to express telomerase. Well-defined genetic alterations that target the p53 and Ras pathways, we have induced the malignant transformation of our telomerase-immortalized human Barrett’s epithelial cells, and we have developed a number of non-transformed cell lines with welldefined, growth-promoting genetic changes that might recapitulate various stages of neoplastic progression in Barrett’s esophagus

Results

Discussion

Findings

Materials and Methods