**5. Effects of** *COX-2* **methylation on its mRNA expression in MGs**  *in vitro*

methylation and *H. pylori* infection in gastric ulcer healing, *COX* mRNA levels in samples prepared from acetic acid-induced gastric ulcers in MGs were examined. Then, the effects of *COX-2* methylation on *COX-2* mRNA expression were also investigated *in vitro* using an

Gastric ulcers were produced by injecting 25% of acetic acid (0.03 mL) into the submucosal layer of the gastric wall of the antral-oxyntic border in MGs 48 weeks after inoculation with an *H. pylori* suspension in Brucella broth. The MGs were killed at 5, 10, and 20 days after ulcer induction, and the stomachs were dissected and removed. The maximum and minimum diameters of the ulcers were measured, and the area of each ulcer, which was approximately elliptical, was calculated and compared between MGs with and without *H. pylori* infection. The ulcer area was largest on day 5, and then gradually decreased. In accordance with previous reports [29], the gastric ulcer area was larger in *H. pylori*-infected MGs than in uninfected MGs (**Figure 3B**–**D**). While *COX-2* mRNA expression at the ulcer edge was increased 5 days after acetic acid injection in MGs without *H. pylori* infection, as was reported in rats and mice [31], no increases in *COX-2* mRNA levels were observed in *H. pylori*-infected MGs. In contrast, gastric ulceration was not associated with a change in *COX-1* mRNA levels in MGs with or without *H. pylori* infection (**Figure 4A,B**). Thus, *H. pylori* infection caused delayed ulcer heal-

**Figure 4.** *COX* mRNA levels during healing of an acetic acid-induced gastric ulcer in MGs with or without *H. pylori* infection. *COX* mRNA levels in acetic-acid-induced gastric ulcers in MGs were measured by real-time PCR. First-strand cDNA was prepared by reverse transcription of 5 μg of total RNA using superscript III reverse transcriptase (Applied Biosystems, Carlsbad, CA, USA). Real-time quantitative reverse transcription PCR was carried out using TaqMan Gene Expression Assays with a 7500 real-time PCR system (Applied Biosystems) according to the manufacturer's instructions. Primers for *COX-1*, *COX-2*, and *β-actin* were designed based on their cDNA sequences (GenBank accession nos. AB 044783, AB044784, and AB040445, respectively) and according to previous reports [33]. SDS2.1 software (Applied Biosystems) was used to perform the comparative Δ-Ct analysis. Glyceraldehyde-3-phosphate dehydrogenase was used as an endogenous control. Values are the mean ± SE. β-Actin left: gctacagcttcaccaccaca, right: ccatctcttgctcgaagtcc, 93 bp. COX-1 left: gtggctatttcctgcagctc, right: agtgggtgccagtggtagag, 112 bp. COX-2 left: tgggcgtgaaaggaaataag, right:

*H. pylori-*infected MG stomach-derived cell line.

212 Chromatin and Epigenetics

ing and impaired COX-2 induction in MG stomachs.

ggggatcagggatgaacttt, 87 bp.

As discussed above, *COX-2* mRNA expression is regulated by an epigenetic mechanism in KATO-III human gastric carcinoma cells in which *COX-2* is densely methylated [22]. To investigate the role of methylation in *COX-2* mRNA expression in *H. pylori*-infected gastric mucosa of MGs, we treated MGC2 cells with 5-aza-dC, a methyltransferase inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor. MGC2 is an adenocarcinoma cell line established from the gastric cancer tissue of a *H. pylori*-infected MG [32]. *COX-2* mRNA expression levels in these cells were restored after the addition of 5-aza-dC. In contrast, treatment with TSA did not induce *COX-2* mRNA expression (**Figure 5**). These results indicated that *COX-2* mRNA expression in MGs is regulated via both transcriptional and epigenetic mechanisms. Histone acetylation was not involved in silencing *COX-2* expression in these cells.

**Figure 5.** Effects of 5-aza-dC, a methyltransferase inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor, on *COX-2* mRNA expression in MG gastric adenocarcinoma MGC2 cells. The MGC2 cells [32] were a generous gift from Dr. Tatematsu and were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) plus serum expander MITO (0.1%, Collaborative Biomedical Products Bedford, MA, USA) on a type I collagen-coated dish (Asahi Techno Glass, Japan). All the cultures were incubated at 37°C with 95% air and 5% CO2 . *COX-2* mRNA expression was measured by real-time PCR in the cell lines grown in the presence of vehicle with or without 5-Aza-dC (1 μmol/L) or TSA for 5 days. Values shown are the mean ± SE.
