40- to 100-kD protein(s) of Helicobacter pylori stimulate DNA synthesis in epithelial cell lines without affecting apoptosis.
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.


CONCLUSIONS
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.
Neopikhanov
V
V
Division of Internal Medicine, Section of Gastroenterology and Hepatology, Karolinska Institutet, Danderyd Hospital, Stockholm, Sweden.
Samali
A
A
Wreiber
K
K
Engstrand
L
L
Uribe
A
A
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D001426:Bacterial Proteins / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D004247:DNA / Q000096:biosynthesis*; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D016480:Helicobacter pylori / Q000737:chemistry*; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats
D017304:Annexin A5; D001426:Bacterial Proteins; D004247:DNA; D020169:Caspases