W1575 Effect of Eosinophil Granule Proteins On P27 Expression in Esophageal Epithelial Cells

Abstract

INTRODUCTION: p27, a tumor suppressor that regulates the G1 to S phase transition of the cell cycle, is reduced or mislocalized in Barrett's associated adenocarcinoma (BAA). Mislocalization of p27 from the nucleus to cytoplasm disrupts its normal function, and has been associated with poor prognosis in BAA. We have shown that bile acids and hydrochloric acid (HCl) cause mislocalization of p27 from the nucleus to the cytoplasm in normal squamous esophageal epithelial cells (HET-1A) in culture, without affecting total cell p27 levels. Both bile and acid reflux have been associated with an increased risk for development of Barrett's esophagus (BE), but not every patient with BE has a history of GERD. Many patients with symptoms mimicking GERD are later found to have eosinophilic esophagitis (EoE). Eosinophil granule proteins implicated in EoE [major basic protein (MBP) and eosinophil peroxidase (EPO)], have been shown to damage human pneumocytes and guinea pig tracheal epithelium. We sought to determine whether MBP or EPO affects p27 expression in HET-1A cells similar to bile acids and HCl. METHODS: HET-1A cells were incubated with MBP at 5-100μg/ml or EPO at 0.8-80μg/ml for 24 hours. Cells were harvested for cytotoxicity studies and immunoblot analysis to determine p27 expression. Indirect immunofluorescence (IF) was performed to determine the subcellular localization of p27. Flow cytometric analysis was used to study theMBP and EPO effects on cell proliferation. RESULTS: Exposure of HET-1A cells to MBP or EPO at various concentrations did not affect total cell p27 levels. When HET-1A cells were treated with either MBP or EPO, increased p27 was detected in the cytoplasm, along with reduced but not absent p27 nuclear staining. Flow cytometric profiles were similar for untreated cells and cells treated with MBP or EPO. CONCLUSIONS: 1) Exposure of HET-1A cells toMBP or EPO results in increased cytoplasmic p27. 2) The presence of p27 in the cytoplasm may disrupt its normal cell cycle inhibitory function and be an important early marker of dysplasia.