Malignant transformation of non-neoplastic Barrett's epithelial cells through well-defined genetic manipulations

Abstract

Background: Human 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 Findings: To develop such model cell lines, we started with telomerase-immortalized, nonneoplastic 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/Significance: Through 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.