Tohru Niwa

Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Altered patterns of DNA methylation associated with Helicobacter pylori (HP) infection of gastric epithelial cells are thought to contribute to gastric cancer risk. However, it is unclear whether this increased risk reflects an infection-associated inflammatory response or the infection itself. In this study, we sought to clarify mechanisms in a gerbil model of gastric cancer where we showed that HP infection is causally involved in induction of aberrant DNA methylation. By genome-wide screening, CpG islands that were aberrantly methylated in gerbil gastric cancer cell lines were isolated, and 10 islands were shown to be specifically methylated only in gastric mucosae infected with HP. By temporal analysis, methylation levels in gastric epithelial cells started to increase at 5 to 10 weeks after infection and reached high levels by 50 weeks. When HP was eradicated, methylation levels markedly decreased 10 and 20 weeks later, but they remained higher than those in gerbils that were not infected by HP. Expression levels of several inflammation-related genes (CXCL2, IL-1beta, NOS2, and TNF-alpha) paralleled the temporal changes of methylation levels. Significantly suppressing inflammation with the immunosuppressive drug cyclosporin A did not affect colonization by HP but blocked the induction of altered DNA methylation. Our findings argue that DNA methylation alterations that occur in gastric mucosae after HP infection are composed of transient components and permanent components, and that it is the infection-associated inflammatory response, rather than HP itself, which is responsible for inducing the altered DNA methylation.

The presence of RNA polymerase II, active or stalled, predicts epigenetic fate of promoter CpG islands

Instructive mechanisms are present for induction of DNA methylation, as shown by methylation of specific CpG islands (CGIs) by specific inducers and in specific cancers. However, instructive factors involved are poorly understood, except for involvement of low transcription and trimethylation of histone H3 lysine 27 (H3K27me3). Here, we used methylated DNA immunoprecipitation (MeDIP) combined with a CGI oligonucleotide microarray analysis, and identified 5510 and 521 genes with promoter CGIs resistant and susceptible, respectively, to DNA methylation in prostate cancer cell lines. Expression analysis revealed that the susceptible genes had low transcription in a normal prostatic epithelial cell line. Chromatin immunoprecipitation with microarray hybridization (CHiP-chip) analysis of RNA polymerase II (Pol II) and histone modifications showed that, even among the genes with low transcription, the presence of Pol II was associated with marked resistance to DNA methylation (OR = 0.22; 95% CI = 0.12-0.38), and H3K27me3 was associated with increased susceptibility (OR = 11.20; 95% CI = 7.14-17.55). The same was true in normal human mammary epithelial cells for 5430 and 733 genes resistant and susceptible, respectively, to DNA methylation in breast cancer cell lines. These results showed that the presence of Pol II, active or stalled, and H3K27me3 can predict the epigenetic fate of promoter CGIs independently of transcription levels.

The presence of a methylation fingerprint of Helicobacter pylori infection in human gastric mucosae

Aberrant DNA methylation is deeply involved in human cancers, but its inducers and targets are still mostly unclear. Helicobacter pylori infection was recently shown to induce aberrant methylation in gastric mucosae, and produce a predisposed field for cancerization. Here, we analyzed the presence of target genes in methylation induction by H. pylori and the mechanism for the gene specificity. Noncancerous gastric mucosae were collected from 4 groups of individuals (with and without a gastric cancer, and with and without current H. pylori infection; N = 11 for each group), and methylation of promoter CpG islands of 48 genes that can be methylated in gastric cancer cell lines was analyzed by methylation‐specific PCR. In total, 26 genes were consistently methylated in individuals with current or past infection by H. pylori, whereas 7 genes were not methylated at all. In addition, 14 genes were randomly or intermediately methylated in individuals with gastric cancers and the remaining 1 gene was methylated in all the cases. The methylation‐susceptible genes had significantly lower mRNA expression levels than the methylation‐resistant genes. H. pylori infection did not induce mRNA and protein expression of DNA methyltransferases; DNMT1, DNMT3A or DNMT3B. Gene specificity was present in the induction of aberrant DNA methylation by H. pylori infection, and low mRNA expression, which could precede methylation, was one of the mechanisms for the gene specificity. These findings open up the possibility that a methylation fingerprint can be used as a novel marker for past exposure to a specific carcinogenic factor.

Suppression of C/EBPα expression in periportal hepatoblasts may stimulate biliary cell differentiation through increased Hnf6 and Hnf1b expression

The expression of C/EBPα, which may govern transcription of mature hepatocyte marker genes, was suppressed in periportal hepatoblasts in mouse liver development, leading to biliary cell differentiation. This study was undertaken to analyze how inactivation of the Cebpa gene affects biliary cell differentiation and gene expression of the regulatory genes for that differentiation, including Hnf1b and Hnf6. In the knockout mouse liver at midgestation stages, pseudoglandular structures were abundantly induced in the parenchyma with elevated expression of Hnf6 and Hnf1b mRNAs. The wild-type liver parenchyma expressed mRNAs of these transcription factors at low levels, though periportal biliary progenitors had strong expression of them. These results suggest that expression of Hnf6 and Hnf1b is downstream of C/EBPα action in fetal liver development, and that the suppression of C/EBPα expression in periportal hepatoblasts may lead to expression of Hnf6 and Hnf1b mRNAs. Immunohistochemical studies with biliary cell markers in knockout livers demonstrated that differentiated biliary epithelial cells were confined to around the portal veins. The suppression of C/EBPα expression may result in upregulation of Hnf6 and Hnf1b gene expression, but be insufficient for biliary cell differentiation. When liver fragments of Cebpa-knockout fetuses, in which hepatoblasts were contained as an endodermal component, were transplanted in the testis of Scid (Prkdc) male mice, almost all hepatoblasts gave rise to biliary epithelial cells. Wild-type hepatoblasts constructed mature hepatic tissue accompanied by biliary cell differentiation. These results also demonstrate that the suppression of C/EBPα expression may stimulate biliary cell differentiation.

Insufficient role of cell proliferation in aberrant DNA methylation induction, and involvement of specific types of inflammation

Chronic inflammation is deeply involved in induction of aberrant DNA methylation, but it is unclear whether any type of persistent inflammation can induce methylation and how induction of cell proliferation is involved. In this study, Mongolian gerbils were treated with five kinds of inflammation inducers [Helicobacter pylori with cytotoxin-associated gene A (CagA), H.pylori without CagA, Helicobacter felis, 50% ethanol (EtOH) and saturated sodium chloride (NaCl) solution]. Two control groups were treated with a mutagenic carcinogen that induces little inflammation (20 p.p.m. of N-methyl-N-nitrosourea) and without any treatment. After 20 weeks, chronic inflammation with lymphocyte and macrophage infiltration was prominent in the three Helicobacter groups, whereas neutrophil infiltration was mainly observed in the EtOH and NaCl groups. Methylation levels of eight CpG islands significantly increased only in the three Helicobacter groups. By Ki-67 staining, cell proliferation was most strongly induced in the NaCl group, demonstrating that induction of cell proliferation is not sufficient for methylation induction. Among the inflammation-related genes, Il1b, Nos2 and Tnf showed increased expression specifically in the three Helicobacter groups. In human gastric mucosae infected by H.pylori, NOS2 and TNF were also increased. These data showed that inflammation due to infection of the three Helicobacter strains has a strong potential to induce methylation, regardless of their CagA statuses, and increased cell proliferation was not sufficient for methylation induction. It was suggested that specific types of inflammation characterized by expression of specific inflammation-related genes, along with increased cell proliferation, are necessary for methylation induction.

Alu and Satα hypomethylation in Helicobacter pylori-infected gastric mucosae.

Global hypomethylation and regional hypermethylation are supposed to be hallmarks of cancer cells. During gastric carcinogenesis, in which Helicobacter pylori infection is causally involved, aberrant hypermethylation is already present in H. pylori‐infected gastric mucosae. In contrast, little is known about global hypomethylation, which can be caused by hypomethylation of individual repetitive elements and other sequences. We, therefore, investigated hypomethylation of individual repetitive elements and the global 5‐methylcytosine content in four groups of gastric mucosal samples that represented the time course of H. pylori infection and gastric carcinogenesis [gastric mucosae of H. pylori‐negative healthy volunteers (G1, n = 34), H. pylori‐positive healthy volunteers (G2, n = 42), H. pylori‐positive gastric cancer patients (G3, n = 34) and H. pylori‐negative gastric cancer patients (G4, n = 20)] and 52 primary gastric cancers. Major variants of Alu, LINE1 and Satα were identified, and their methylation levels were quantified by bisulfite pyrosequencing. Compared with G1, the Alu methylation level was decreased in G2, G3, G4 and cancers (89.2–97.1% of that in G1, p < 0.05). The Satα methylation level was decreased in G2 (91.6%, p < 0.05) and G3 (94.3%, p = 0.08) but not in G4 and cancers. The LINE1 methylation level was decreased only in cancers. The 5‐methylcytosine content was at similar levels in G2, G3 and G4 and highly variable in cancers. These results showed that Alu and Satα hypomethylation is induced in gastric mucosae by H. pylori infection during gastric carcinogenesis, possibly in different target cells, and that global hypomethylation is not always present in human gastric cancers.

Prevention of Helicobacter pylori–Induced Gastric Cancers in Gerbils by a DNA Demethylating Agent

Suppression of aberrant DNA methylation is a novel approach to cancer prevention, but, so far, the efficacy of the strategy has not been evaluated in cancers associated with chronic inflammation. Gastric cancers induced by Helicobacter pylori infection are known to involve aberrant DNA methylation and associated with severe chronic inflammation in their early stages. Here, we aimed to clarify whether suppression of aberrant DNA methylation can prevent H. pylori–induced gastric cancers using a Mongolian gerbil model. Administration of a DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC), to gerbils (0.125 mg/kg for 50–55 weeks) decreased the incidence of gastric cancers induced by H. pylori infection and N-methyl-N-nitrosourea (MNU) treatment from 55.2% to 23.3% (P < 0.05). In gastric epithelial cells, DNA methylation levels of six CpG islands (HE6, HG2, SB1, SB5, SF12, and SH6) decreased to 46% to 68% (P < 0.05) of gerbils without 5-aza-dC treatment. Also, the global DNA methylation level decreased from 83.0% ± 4.5% to 80.3% ± 4.4% (mean ± SD) by 5-aza-dC treatment (P < 0.05). By 5-aza-dC treatment, Il1b and Nos2 were downregulated (42% and 58% of gerbils without, respectively) but Tnf was upregulated (187%), suggesting that 5-aza-dC treatment induced dysregulation of inflammatory responses. No obvious adverse effect of 5-aza-dC treatment was observed, besides testicular atrophy. These results showed that 5-aza-dC treatment can prevent H. pylori–induced gastric cancers and suggested that removal of induced DNA methylation and/or suppression of DNA methylation induction can become a target for prevention of chronic inflammation–associated cancers. Cancer Prev Res; 6(4); 263–70. ©2013 AACR.

Preferential expression of connexin37 and connexin40 in the endothelium of the portal veins during mouse liver development

Hepatic blood vessels consist of the hepatic artery and three types of venous channels (the portal veins, the sinusoids, and the hepatic veins). This study was undertaken to analyze, by immunohistochemistry, connexin expression throughout the vascular development of the fetal mouse liver with special attention being given to portal vein development. In the adult liver, connexin37 and connexin40 were expressed in the endothelium of the portal vein and hepatic artery, but not in those of the hepatic vein and sinusoids. Connexin43 was expressed in mesothelial cells and smooth muscle cells of the portal veins. The preferential expression of connexin37 and connexin40 in portal veins was seen throughout liver development, including its primordium formation stage (10.5-day or 11.5-day stage), although connexin37 expression was transiently seen in free nonparenchymal cells in fetal stages. The differentiation of each blood vessel in the hepatic vascular system may occur in early developmental stages, soon after hepatic primordium formation.

Whole-genome analyses of loss of heterozygosity and methylation analysis of four tumor-suppressor genes in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach carcinomas.

N‐Methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG)‐induced rat stomach carcinomas are considered to be a good model for differentiated‐type human stomach carcinomas. However, as for their molecular basis, only infrequent mutations of Catnb (β‐catenin) and Trp53 (p53) have been observed. Here, we carried out a whole‐genome analysis of loss of heterozygosity (LOH) using 21 stomach carcinomas induced by MNNG in F1 hybrids of ACI and BUF rats, and also analyzed promoter methylation of four tumor‐suppressor genes. LOH analysis was performed using 130 polymorphic markers covering rat chromosomes 1–20 with an average interval of 20 Mbp. Despite adapting conditions so that LOH could be detected with up to a 50% contamination of stromal cells, no LOH was detected at any loci. CpG islands in putative promoter regions of four tumor‐suppressor genes, Cdh1 (E‐cadherin), Cdkn2a (p16), Mlh1, and Rassf1a, were analyzed by methylation‐specific polymerase chain reaction (PCR). However, no methylation was detected. In contrast, the promoter region of Pgc (pepsinogen C), which lacks a CpG island, was methylated in all 21‐cancer samples. These results indicated that LOH spanning a chromosomal region larger than 30–40 Mbp or silencing of Cdh1, Cdkn2a, Mlh1, and Rassf1a, was not involved in MNNG‐induced rat stomach carcinomas. The search for other genes involved in these carcinomas needs to be continued. (Cancer Sci 2005; 96: 409  – 413)

Altered mucosal DNA methylation in parallel with highly active Helicobacter pylori-related gastritis

BackgroundChronic inflammation triggered by Helicobacter pylori causes altered DNA methylation in stomach mucosae, which is deeply involved in gastric carcinogenesis. This study aimed to elucidate the correlation between altered mucosal DNA methylation levels and activity of H. pylori-related gastritis, because inflammatory activity shows particular correlations with the development of diffuse-type cancer.MethodsMethylation levels in stomach mucosae of 78 healthy volunteers were determined by real-time methylation-specific PCR or bisulfite pyrosequencing. Examined loci were the promoter CpG islands of six genes (FLNc, HAND1, THBD, p41ARC, HRASLS, and LOX) and the CpG sites of non-coding repetitive elements (Alu and Satα) that are reportedly altered by H. pylori infection. Activity of H. pylori-related gastritis was evaluated using two serum markers: H. pylori antibody titer and pepsinogen II.ResultsMethylation levels of the six CpG islands were consistently increased, and those of the two repetitive elements were consistently decreased in a stepwise manner with the activity of gastric inflammation as represented by serum marker levels. Each serum marker level was well correlated with the overall DNA methylation status of stomach mucosa, and these two serologic markers were additive in the detection of the mucosa with severely altered DNA methylation.ConclusionsAlteration in mucosal DNA methylation level was closely correlated with activity of H. pylori-related gastritis as evaluated by serum markers. The observed correlation between altered DNA methylation levels and activity of H. pylori-related gastritis appears to be one of the relevant molecular mechanisms underlying the development of diffuse-type cancer.