Dear Sir, Yuasa et al. published that specific lifestyle factors, described to be potentially preventive of gastric cancer on epidemiological basis, may directly influence the carcinogenic process by affecting demethylation or maintenance of unmethylated status of selected genes. Specifically, it was shown that CDX2 was methylated in 23.6% of primary gastric carcinomas, whereas no methylation was seen in adjacent noncancerous gastric tissue of some of the cancer patients studied. They also demonstrated a significant inverse association between intake of green tea (more than 7 cups/day) and the methylation status of CDX2 in gastric cancer patients. However, in the same article, the authors did not show that the methylation status was correlated with CDX2 expression. In our study, we investigated if methylation of the CDX2 promoter could be a mechanism involved in the regulation of CDX2 expression in gastric carcinomas using gastric carcinoma cell lines as models and also in intestinal metaplasia (IM) of the stomach. CDX2 is a transcription factor that plays a major role in intestinal differentiation both in the intestine and in aberrant locations, such as in IM of the stomach. We show that there is no association between CDX2 promoter methylation and CDX2 expression, suggesting that methylation does not constitute a primary mechanism regulating CDX2 expression both in gastric carcinoma cell lines and in gastric preneoplastic lesions. Thus, our findings do not support the conclusions taken by Yuasa et al. We used the bioinformatic tool CpGPlot to search for potential CpG islands in the proximal region of the CDX2 promoter and identified two regions that fulfil the criteria (Fig. 1a). Then we studied the basal CDX2 mRNA expression and the methylation status of the CDX2 promoter in a panel of four human gastric carcinoma cell lines (AGS, GP202, IPA220 and MKN45). CDX2 mRNA expression analysis in the gastric carcinoma cell lines showed that AGS and IPA220 strongly express CDX2, while GP202 and MKN45 present equally low levels of CDX2 expression (Fig. 1b). The methylation of the CDX2 promoter in these cell lines was determined using the bisulfite-genome sequencing method (Fig. 1c). CpG island 1 was methylated in all cell lines, whereas CpG island 2 was methylated in GP202 and AGS cell lines and unmethylated in IPA220 and MKN45 cell lines. These results did not correlate with CDX2 mRNA expression. To further confirm if methylation could be in any way involved in CDX2 regulation, we treated these cell lines with the demethylating agent 5-aza-2’deoxycytidine (Fig. 1d). Once more, the results obtained suggest that methylation is not directly regulating CDX2 expression since cell lines with similar basal methylation status, AGS and GP202, react differently to the treatment with a demethylating agent. Although there is an increase of expression in GP202 after treatment, CDX2 remains unchanged in AGS. Strikingly, MKN45 which shows unmethylated CDX2 promoter also reacts to the treatment with an increased expression. Furthermore, we examined CDX2 expression and the methylation status of 42 CpG sites, contained in CpG island 2 of the CDX2 promoter, in two specimens of normal gastric mucosa, adjacent IM foci and in two normal colonic mucosa samples. As expected no CDX2 expression was observed in normal gastric mucosa in contrast to IM and colonic tissue (data not shown). The methylation pattern was inconsistent and no correlation was found with CDX2 expression in all samples (Fig. 2). In summary, and although previous studies show that methylation might be involved in CDX2 regulation, our in vitro and in vivo results demonstrate that the pattern of methylation at the CDX2 promoter is random and unrelated with its expression. The increase in CDX2 expression observed in some of the cell lines studied is more likely due to the effect of the demethylating agent over epigenetically modified regulators, such as silenced transcription factors or modified histones, and not directly over CDX2. Yours sincerely, Bruno PEREIRA, Carla OLIVEIRA, Leonor DAVID, Raquel ALMEIDA*
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May 23, 2011
Myoung Kim 
Open Access
