Demonstration and characterization of mutations induced by Helicobacter pylori organisms in gastric epithelial cells.
BACKGROUND
Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability-type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability-positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection.


METHODS
Mutations were detected using a green fluorescent protein reporter vector (pEGFP-CA13). Gastric cancer AGS cells transfected with pEGFP-CA13 were cocultured with H. pylori or Escherichia coli. The numbers of green fluorescent protein (GFP)-positive cells were determined, and GFP, hMSH2, and hMLH1 protein levels were measured by Western blot. The effect of H. pylori on CpG methylation status of hMLH1 was determined by methylation-specific polymerase chain reaction.


RESULTS
GFP levels and GFP-positive cell numbers in AGS cells cocultured with H. pylori significantly increased, as the levels of hMLH1 and hMSH2 dropped. H. pylori cocultures induced low-level CpG methylation of the hMLH1 promoter. Sequence analysis of cells cocultured with H. pylori showed an increased number of frameshift mutations and point mutations as compared to cells not cocultured with H. pylori (p = .03 and p = .001, respectively).


CONCLUSIONS
This is the first report showing that H. pylori bacteria may lead to accumulation of genomic mutations, independently of underlying inflammation. This is associated with reduced DNA mismatch repair, and is at least in part associated with CpG methylation of the hMLH1 promoter. These data support the notion that H. pylori-induced mutations and epigenetic alterations in gastric epithelial cells during chronic gastritis may contribute to an increased risk of gastric cancer associated with H. pylori infection.
Yao
Yuan
Y
Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Tao
Hong
H
Park
Dong Il
DI
Sepulveda
Jorge L
JL
Sepulveda
Antonia R
AR
D048868:Adaptor Proteins, Signal Transducing; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis; D002460:Cell Line; D018920:Coculture Techniques; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D004847:Epithelial Cells / Q000382:microbiology*; D004926:Escherichia coli; D005753:Gastric Mucosa / Q000382:microbiology*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D016480:Helicobacter pylori / Q000472:pathogenicity*; D006801:Humans; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein / Q000032:analysis; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000032:analysis; D011401:Promoter Regions, Genetic; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein