40- to 100-kD Protein(s) of <i>Helicobacter pylori</i> 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.

Effect of Helicobacter pylori on DNA synthesis of human epithelial cells

“…There have been several reports that water extracts of H. pylori stimulate DNA synthesis in rat small intestinal IEC-6 cells. [19][20][21] However, Kurosawa et al 22 reported that water extracts of H. pylori inhibited cell proliferation of human umbilical vein epithelial cells with decreased labeling index of BrdU. The cause for the difference in the effect of the water extract on DNA synthesis is unclear, but it is possible that the difference in H. pylori strains, cell lines, and methods of quantifying DNA synthesis might account for the discrepancy in the results between previous reports and the present study.…”