DNA Hypermethylation Downregulates Telomerase Reverse Transcriptase (TERT) during H. pylori-Induced Chronic Inflammation

Figure 2

H. pylori inhibits hTERT gene expression and telomerase activity in gastric epithelial cells. (a) hTERT gene expression (upper panel) was measured by real-time qPCR, and protein levels were analysed by western blot (lower panel) in human gastric epithelial cell lines AGS infected with H. pylori B38 at MOI 20, 50, and 100 for 24 h. (b) hTERT gene expression measured in AGS cells treated with H. pylori B38 bacterial extracts (20 μg·ml⁻¹) for 24 h. (c) MKN45 and KatoIII gastric epithelial cell lines infected with H. pylori B38 for 24 h and 48 h at MOI 100. (d) Telomerase activity analysed by TRAPeze® assay in AGS cell extracts prepared from cells infected with H. pylori B38 for 12 h and 24 h (MOI 100). C+, positive control using commercial telomerase-positive cell extracts; C−, negative control. For each analysed condition, heat-inactivated cell extracts obtained after incubation at 85°C for 10 min were also analysed. The displayed gel is representative of 2 independent experiments performed in duplicate. (e) hTERT gene expression is also inhibited in AGS cells infected by the H. pylori strain 7.13 (MOI 50 and 100) in a CagA- and CagE-independent manner. , one-way ANOVA analysis followed by Dunn’s multiple comparison (infected versus uninfected ; ). (f) Oxidative stress generated by exposure of cells to H₂O₂ 10 mM for 24 h inhibits the hTERT gene expression. (g) Representative western blot of AGS cells infected 24 h with H. pylori 7.13 as in (d) and treated with lycopene 5 μM. Lycopene abolished the H. pylori-mediated inhibition of TERT. Results are expressed as mean ± SD of three independent experiments (infected versus uninfected ; ; ). , one-way ANOVA Kruskal–Wallis followed by Dunn’s multiple comparison (infected versus uninfected ; ) (a and e).

(a)

(b)

(c)

(d)

(e)

(f)

(g)