40- to 100-kD Protein(s) of Helicobacter pylori Stimulate DNA Synthesis in Epithelial Cell Lines without Affecting Apoptosis

Abstract

Background: Previous in vitro studies have demonstrated that water extracts and sonicates of Helicobacter pylori increase DNA synthesis in a small intestinal epithelial cell line. The aim of this study was to identify mitogenic factor(s) in a water extract of a H. pylori strain and to examine their effects on DNA synthesis and apoptosis in vitro. Methods: IEC-6 and FHs 74 cells were incubated for 24 h with different dilutions of a water extract of H. pylori (cytotoxic strain 88–23) or with 6 protein fractions obtained by gel filtration. Cells were labeled with tritiated thymidine and processed for autoradiography. DNA synthesis was evaluated by the labeling index (LI%). The proportion of IEC-6 cells undergoing apoptosis and/or necrosis was evaluated by flow cytometry using fluorescein isothiocyanate (FITC)-labeled annexin-V and propidium iodide. In vitro caspase activity was also determined as an alternative method for detection of apoptosis. Results: The water extract of H. pylori 88-23 markedly increased DNA synthesis in both epithelial cell lines (p < 0.01). A marked stimulation of DNA synthesis was also observed in IEC-6 cells incubated with fraction II- containing proteins of a molecular weight ranging between 40 and 100 kD (p < 0.01). A lesser stimulation of DNA synthesis was observed in cells incubated with higher concentrations of the other protein fractions (p < 0.01). Neither the water extract of H. pylori 88-23 nor the protein fraction II (40–100 kD) induced apoptosis in IEC-6 cells. Conclusion: A water extract of H. pylori 88-23 and a protein fraction containing proteins with molecular weights of 40–100 kD stimulate DNA synthesis in a rat and human small intestinal cell line. Apoptosis was unaffected by the water extract and by protein fraction II, which indicate that the H. pylori-derived mitogen(s) have the capacity to directly enhance epithelial cell proliferation in vitro.