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Characterization of Alterations of DNA Repair MGMT Gene Expression in Gastric Epithelial Cells Induced by H. pylori Organisms
Author(s) -
Yao Yuan,
DiPaola James E,
Sepulveda Antonia R.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a764
Subject(s) - methylation , microbiology and biotechnology , dna methylation , biology , cpg site , bisulfite sequencing , dna methyltransferase , helicobacter pylori , dna repair , methyltransferase , gene , messenger rna , western blot , gene expression , genetics
Background and Aims: Increased mutagenesis occurs in gastric mucosa during chronic gastritis associated with H. pylori infection (Hp). The DNA repair protein O6‐Methylguanine DNA methyltransferase (MGMT) can prevent G to A mutations. The aim of this study was to determine whether Hp affects expression of MGMT in gastric epithelial cells and to determine the underlying mechanisms. Methods: Two cycles of co‐culture of Hp (strain 43504) and AGS gastric epithelial cells were performed with increasing Hp (MOI 0, 100 and 1000) for 36 hours. MGMT mRNA and protein levels were analyzed by real‐time RT‐PCR and western blot, respectively. Quantitative SYBR GREEN methylation specific PCR, and bisulfite sequence analysis of the MGMT promoter were performed. Results: Hp caused a significant reduction of MGMT protein levels in AGS cells with increasing bacterial numbers, to 30% at MOI 100, and to 12% at MOI 1000, compared to controls. MGMT mRNA levels were reduced by Hp, to 80% at MOI 100, and to 60% at MOI 1000, compared to controls. Hp induced CpG methylation in AGS cells, however, in only a small proportion of MGMT promoter regions (<2%). Conclusions: H. pylori organisms can lead to markedly reduced levels of MGMT protein, making the cells more likely to accumulate mutations. Although Hp organisms induce a drop in MGMT mRNA levels and CpG promoter methylation, the greater reduction in protein levels, suggests that post‐transcriptional mechanisms are likely to underlie the effects of Hp on MGMT function. Supported in part by funds from R01 DK062185 , PI: Antonia Sepulveda.