Genomic loss and epigenetic silencing of very-low-density lipoprotein receptor involved in gastric carcinogenesis.

Homozygous loss in the genomic sequence, a mechanism for inactivating tumor-suppressor genes (TSGs) in cancer, has been used as a tag for the identification of novel TSGs, and array-based comparative genomic hybridization (array-CGH) has a great potential for high-throughput identification of this change. We identified a homozygous loss of the very-low-density lipoprotein receptor (VLDLR) gene (9p24.2) from genome-wide screening for copy-number alterations in 32 gastric cancer (GC) cell lines using array-CGH. Although previous reports demonstrated mRNA or protein expression of VLDLR in various cancers including GC, the association between genomic losses or epigenetic silencing of this gene and carcinogenesis has never been reported before. Homozygous deletion of VLDLR was also seen in primary GCs, albeit infrequently, and about half of GC cell lines showed lost or reduced VLDLR expression. The VLDLR expression was restored in gene-silenced GC cells after treatment with 5-aza 2'-deoxycytidine. According to methylation analyses, hypermethylation of the VLDLR promoter region, which all of GC lines without its expression showed, occurred in some primary GCs. Restoration of VLDLR type I expression in GC cells reduced colony formation. These results suggest that not only the expression of VLDLR but also genetic or epigenetic silencing of this gene may contribute to tumor formation and be involved in gastric carcinogenesis.

Histogenesis of hyperplastic polyps of the stomach in terms of cellular proliferation.

We investigated the histogenesis of hyperplastic polyps of the stomach, in terms of cellular proliferation, by studying endoscopically removed and gastrectomized human gastric polyps either labeled with bromodeoxyuridine (BrdU) by in vitro flash labeling techniques or labeled in an isolated organ circulation system, in both of which, perfluorochemical artificial blood was employed. Immunohistochemistry with antibodies against BrdU and proliferating cell nuclear antigen (PCNA) was simultaneously employed. The generative cell zone of pedunculated and semipedunculated polyps was markedly expanded compared with that of the background mucosa, and this change also appeared in sessile polyps, although to a lesser degree. Enhanced proliferative activity was observed in both epithelial and stromal cells in areas of erosion. Our results demonstrate that the initial change in the histogenesis of hyperplastic polyps is an expansion of the generative cell zone, followed by interstitial edema and stromal cell proliferation, and that erosion can facilitate these changes. No correlation was found between the size of the polyps and the labeling indices. This finding explains, in part, the diversity of chronological changes in the size and shape of hyperplastic polyps.

[Analysis of proliferative activity in gastric biopsy specimens by combined BrdU and DNA polymerase alpha immunohistochemistry].

To evaluate correctly the proliferative activity of tumor cells, it is necessary to clarify not only S-phase fraction but also the growth fraction of tumor tissues. We used combined BrdU and DNA polymerase alpha (pol-alpha) immunohistochemistry to gastric biopsy specimens, and analyzed the proliferation of the neoplastic lesions of various degrees of malignancy. The results were as follows: The distribution of pol-alpha positive cells were almost the same as that of BrdU positive cells, but the percentage of pol-alpha positive cells was higher than that of BrdU positive cells irrespective of the mucosal specimens. In the adenomas, both BrdU and pol-alpha positive cells distributed generally superficially in the mucosal layer. In the well differentiated adenocarcinomas, both BrdU and pol-alpha positive cells distributed diffusely in the deeper layer of the mucosa. The ratio of the number of BrdU positive cells to that of pol-alpha positive cells, which means the S-phase fraction in the growth fraction, was higher in the tumor and that higher in the well differentiated adenocarcinomas than that of the adenomas. In conclusion, the combined BrdU and pol-alpha immunohistochemistry in gastric biopsy specimens are useful to evaluate the degree of malignancy.

[Cell proliferation and maturation of ectopic submucosal glands of the stomach].

Stomachs with multiple submucosal glands keep our attention because of their high risk for having gastric carcinomas, but little is known about the relationship between submucosal glands and occurring cancers. So we investigated the structure of submucosal glands including location of proliferating cells. We labeled proliferating cells of three stomachs with cancerous lesions by circulating artificial blood through the isolated organs with bromodeoxyuridine (BrdU). Serial sections of submucosal glands were stained with hematoxylin eosin, periodic acid Schiff, Alcian blue, high iron diamine-Alcian blue, concanavalin A paradox, galactose oxidase Schiff as well as anti-BrdU immunohistochemically. Results showed that most of submucosal glands were composed of functionally matured cells and these cells made regular structure just like those of mucosal layer. Labeling index of BrdU of these submucosal glands were low (0.2%-0.7%). Two atypical glands were seen, and labeling index were 2.6% and 22.2% respectively. In conclusion, submucosal glands of the stomachs were thought to be made by moving proliferating cells from mucosal layer to submucosal layer. So we saw these submucosal glands paracancerous lesions rather than precancerous ones. But we couldn't deny the possibility to occur cancerous lesions from atypical glands in submucosal glands.

Phosphodiesterase isozymes involved in regulating acid secretion in the isolated mouse stomach.

The effect of subtype-selective phosphodiesterase (PDE) inhibitors on acid secretion was examined in mouse stomachs to investigate which PDE isozymes are involved in the local regulation of this secretion. Male DDY mice were used after 18 h fasting. An isolated stomach was incubated in an organ bath containing buffered solution gassed with 95% O(2)/5% CO(2), while the lumen was perfused with unbuffered solution gassed with 100% O(2). Acid secretion was measured at pH 5.4 using a pH-stat method. Histamine or pituitary adenylate cyclase activating polypeptide (PACAP) was added to the serosal solution. PDE inhibitors were added to the serosal solution 30 min before histamine or PACAP. The secretion of acid in the isolated stomach was increased by histamine or PACAP, and these responses were totally inhibited by famotidine. IBMX alone increased basal acid secretion and significantly enhanced the acid responses to histamine and PACAP. Among the PDE inhibitors tested, only rolipram (PDE4 inhibitor) significantly increased basal acid secretion and potentiated the acid responses to histamine and PACAP. The latter peptide increased histamine release into the medium, and this response was also enhanced by rolipram. Furthermore, rolipram significantly increased cAMP production induced in the isolated stomach by histamine and PACAP. These results suggest that PDE4 is involved in the local regulation of gastric acid secretion via the degradation of cAMP and that the PDE4 inhibitor rolipram increases the secretion of acid by potentiating acid production in parietal cells and enhancing histamine release from enterochromaffin-like cells.

Defective expression of polarity protein PAR-3 gene (PARD3) in esophageal squamous cell carcinoma.

The partition-defective 3 (PAR-3) protein is implicated in the formation of tight junctions at epithelial cell-cell contacts. We investigated DNA copy number aberrations in human esophageal squamous cell carcinoma (ESCC) cell lines using a high-density oligonucleotide microarray and found a homozygous deletion of PARD3 (the gene encoding PAR-3). Exogenous expression of PARD3 in ESCC cells lacking this gene enhanced the recruitment of zonula occludens 1 (ZO-1), a marker of tight junctions, to sites of cell-cell contact. Conversely, knockdown of PARD3 in ESCC cells expressing this gene caused a disruption of ZO-1 localization at cell-cell borders. A copy number loss of PARD3 was observed in 15% of primary ESCC cells. Expression of PARD3 was significantly reduced in primary ESCC tumors compared with their nontumorous counterparts, and this reduced expression was associated with positive lymph node metastasis and poor differentiation. Our results suggest that deletion and reduced expression of PARD3 may be a novel mechanism that drives the progression of ESCC.

Regression of idiopathic thrombocytopenic purpura after endoscopic mucosal resection of gastric mucosa associated lymphoid tissue lymphoma.

Recent reports have suggested an association between Helicobacter pylori infection and both gastric mucosa associated lymphoid tissue (MALT) lymphoma and thrombocytopenic purpura. Although treatments eradicating H pylori lead to regression of these diseases in some cases, the exact mechanisms are still controversial. This case report describes a patient with thrombocytopenic purpura accompanied by an early stage gastric MALT lymphoma. Endoscopic mucosal resection of the lesion in this patient led to dramatic regression of thrombocytopenic purpura, and t(11;18)(q21;q21), which means resistance more likely to H pylori eradication therapy, was confirmed by fluorescence in situ hybridisation. There is no evidence of recurrence and his platelet count is within normal limits after 24 months of follow up. This is the first case report describing regression of thrombocytopenic purpura after mucosal resection of a gastric MALT lymphoma. We suggest that while some cases of thrombocytopenic purpura may be induced by H pylori, others may be due to an autoreactive antibody produced by MALT lymphoma B cells.

Endoscopic mucosal resection using a partial transparent hood for lesions located tangentially to the endoscope.

BACKGROUND: Numerous methods have been developed to resect early-stage gastric and esophageal cancers, but it is difficult to resect lesions viewed tangentially with the endoscope. METHODS: We have designed and developed an original method of endoscopic mucosal resection using a partial transparent hood to treat difficult cases in which the lesions are located tangentially to the endoscope. The hood was attached on the right side of the endoscope and, after insertion into the stomach or the esophagus, was lightly pressed on the orad side of the lesion. Then the lesion was resected using grasping forceps and electrosurgical current snare. RESULTS: The average diameter of specimens was 26 +/- 8 mm in gastric lesions and 20 +/- 3 mm in esophageal lesions, both 6 mm larger than those obtained by previous methods. CONCLUSION: This device and technique were extremely useful for mucosal resection of lesions located tangentially to the endoscope.

Comparison of PCR-restriction fragment length polymorphism analysis and PCR-direct sequencing methods for differentiating Helicobacter pylori ureB gene variants.

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the Helicobacter pylori genes is widely used to differentiate strains. However, with this typing method only a single base change at a specific restriction site can be detected. In addition, it is unclear whether the nucleotide base change recognized by RFLP is related to a substitution of encoded amino acid. To examine the validity of the PCR-RFLP method, 933-bp PCR products were obtained from 41 different clinical H. pylori isolates and were digested with Sau3A restriction endonuclease. Furthermore, the nucleotides of the same region in the ureB gene were directly sequenced and compared. PCR-RFLP confirmed that there was genetic diversity within the ureB gene with three distinct types, one being well conserved and the other two being variations. However, the direct sequencing method revealed that there was no difference at the nucleotide level among these RFLP types. Base substitutions recognized by Sau3A occurred in the third-base position and did not change the encoded amino acid. In addition, many nucleotide mutations, which could not be recognized by Sau3A, were frequently found. These results suggest that the PCR-RFLP method provides for an easy typing scheme of isolates, but does not reveal the true extent of genetic diversity. It is proposed that careful observation is required for the interpretation of results when clinical isolates are differentiated.