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The breast cancer susceptibility gene (BRCA2) is localized mainly in the nucleus where it plays an important role in DNA damage repair. Some BRCA2 protein is also present in the centrosome. Here, we demonstrate that BRCA2 interacts with plectin, a cytoskeletal cross-linker protein, and that this interaction controls the position of the centrosome. Phosphorylation of plectin by cyclin-dependent kinase 1/cyclin B (CDK1/CycB) kinase has been reported to abolish its cross-linking function during mitosis. Here, we induced phosphorylation of plectin in prepared fractions of HeLa cells by adding activated CDK1/CycB kinase. Consequently, there was significant dissociation of the centrosome from the nuclear membrane. Plectin has six homologous ankyrin-like repeat domains (termed PLEC M1-M6). Using a pull-down assay, we found that GST-PLEC M1 and a GST-C-terminal region fusion protein (which comprised PLEC M6, along with an adjacent vimentin site) interacted with BRCA2. Since each PLEC module exhibits high homology to the others, the possibility of all six domains participating in this interaction was indicated. Moreover, when PLEC M1 was overexpressed in HeLa cells, it competed with endogenous plectin and inhibited the BRCA2-plectin interaction. This inhibitory effect resulted in dissociation of the centrosomes from the nucleus and increased the rate of micronuclei formation which may lead to carcinogenesis. In addition, when either BRCA2 or plectin was suppressed by the appropriate siRNA, a similar change in centrosomal positioning was observed. We suggest that the BRCA2-plectin interaction plays an important role in the regulation of centrosome localization and also that displacement of the centrosome may result in genomic instability and cancer development.

Gypican-3 (GPC3) has been recognized as an oncofetal protein in hepatic neoplasms and yolk sac tumors. To characterize a distinct subgroup of gastric carcinoma (GC) expressing GPC3 (GPC3-GC), primary and metastatic GC tissues were evaluated by immunohistochemistry with special focus on their related entities: hepatoid, clear-cell, and alpha-fetoprotein-producing GC. GPC3-GC was defined as focal GPC3-GC when 10-49% of neoplastic cells were positive, and as diffuse GPC3-GC when more than 50% of cells were positive. Among 926 GC cases, 101 (11%) were GPC3-GC, of which 45 were diffuse and 56 were focal GPC3-GC. Specific histological patterns, such as the hepatoid and clear-cell patterns, were frequently observed in diffuse GPC3-GC (38 and 49%, respectively) and in focal GPC3-GC (4 and 25%, respectively), whereas these patterns were extremely rare in GPC3-negative GC. Immunoreactive alpha-fetoprotein was only identified in GPC3-GC (38% of diffuse and 14% of focal GPC3-GC). Both diffuse and focal GPC3-GC showed nodal metastasis more frequently (67 and 55%, respectively) than GPC3-negative GC (34%), and the diffuse GPC3-GC had significantly more T2-4 and M1 stage cases. GPC3 immunostaining was present in 57 out of 61 nodal metastases (93%) and in all four liver metastases examined. Importantly, diffuse GPC3 expression was observed in the liver metastasis, even if the primary tumor was focal GPC3-GC. GPC3-GC is a distinctive group of GC, which unifies hepatoid, clear-cell, and alpha-fetoprotein-producing GC. GPC3 is expected to be a target of forthcoming immunotherapy for a patient bearing this specific type of GC.

Mongolian gerbils infected with Helicobacter pylori (H. pylori ) develop heterotopic proliferative glands (HPGs) in the glandular stomach submucosa. To investigate the effects of H. pylori eradication on cell turnover in HPGs, three antibiotics, lansoprazole, amoxicillin and clarithromycin, were administered at 50 or 75 weeks after inoculation of H. pylori, and the stomachs were excised for histological examination at 1, 2, 4, 8 or 25 weeks thereafter. The HPGs were classified into gastric type (G-type) and others (GI + I-type), which included both pure intestinal (I-type) and gastric-and-intestinal mixed type (GI-type). Apoptosis and cell proliferation were evaluated by means of TUNEL assay and BrdU labeling, respectively. At 8 weeks post-eradication, apoptotic indices were significantly increased in the eradication group (G-type: 2.5%; GI + I-type: 7.2%) compared to the non-eradication group (G-type: 0.6%; GI + I-type: 2.1%: P < 0.01), while BrdU labeling indices were significantly decreased (G-type: 1.9%; GI + I-type: 6.8% as compared with 4.3% and 13.2%, respectively, P < 0.01 for both). At 25 weeks, the apoptotic indices were similarly higher [G-type: 0.4 (eradication group) vs. 0.2% (non-eradication group); GI + I-type: 5.8 vs. 1.1%, both P < 0.01], and the BrdU labeling indices (G-type: 0.8 vs. 2.2%, P < 0.01; GI + I-type: 5.1 vs. 11%, P < 0.05) continued to be lower in HPGs. Furthermore, there were highly significant reductions in the areas of HPGs at 8 and 25 weeks post-eradication. These findings demonstrated that eradication results in apoptosis and reduction of proliferation of HPGs in H. pylori-infected gerbils, these lesions thus being apparently reversible through regulation of cell kinetics.

The goal of this study was to elucidate whether beta-catenin gene mutations might contribute to glandular stomach carcinogenesis in Helicobacter pylori (H.pylori)-infected Mongolian gerbils. Firstly, exon 3 of gerbil beta-catenin cDNA, a mutation hot spot, was cloned and sequenced and found to have 89.3% homology with the human form and 95.5% with the rat and mouse forms. Peptide sequence in this region was shown to be 100% conserved in these mammals. Then, 45 stomach adenocarcinomas induced with N-methyl-N-nitrosourea (MNU) plus H. pylori infection and 7 induced with MNU alone were examined for beta-catenin expression by immunohistochemistry and for DNA mutations using a combination of microdissection and PCR-single strand conformation polymorphism analysis. One gastric cancer in the MNU + H. pylori group (2.2%) displayed nuclear (N) beta-catenin localization, 3 (6.7%) showed cytoplasmic (C) distribution in local regions, and 41 (91.1%) demonstrated cell membrane (M) localization. Tumors induced by MNU alone showed only membranous beta-catenin localization (7/7). Analysis of exon 3 of the beta-catenin gene dem-onstrated all tumors with membrane or cytoplasmic staining as well as surrounding normal mucosa (S) to feature wild-type beta-catenin. In contrast, the lesion with nuclear staining had a missense mutation at codon 34 [GAC (Gly) --> GAA (Glu)] in exon 3 (1/1 = 100%, N vs. M, P < 0.05; and N vs. S, P < 0.05). In conclusion, these results suggest that beta-catenin may not be a frequent target for mutation in stomach carcinogenesis in MNU + H. pylori-treated gerbils.

Both tolerance to nutrient starvation and angiogenesis are essential for cancer progression because of the insufficient supply of nutrients to tumor tissue. Since chronic nutrient starvation seldom occurs in normal tissue, cancer's tolerance to nutrient starvation should provide a novel target for cancer therapy. In this study, we propose an anti-austerity strategy to exploit the ability of agents to eliminate cancer cells' tolerance to nutrient starvation. We established a simple screening method for agents that inhibit cancer cell viability preferentially during nutrient starvation, using PANC-1 cell line cultured in nutrient-rich and nutrient-deprived media. After screening over 2000 culture media of actinomycetes, we identified a new compound, kigamicin D (C(48)H(59)NO(19)), which shows preferential cytotoxicity to cancer cells under nutrient-deprived conditions, but hardly any cytotoxicity under nutrient-rich conditions. Both subcutaneous and oral administration of kigamicin D strongly suppressed the tumor growth of several tested pancreatic cancer cell lines in nude mice. Moreover, kigamicin D was observed to block the activation of Akt induced by nutrient starvation. Therefore, our results suggest that kigamicin D be a candidate for implementing our novel concept, anti-austerity, which may serve as a new strategy for cancer therapy.

In 2005, a Japanese epidemiological study showed that increase in plasma glucose levels is a risk factor for gastric cancer. However, no animal model has hitherto shown any association between diabetes mellitus and neoplasia in the stomach. Diabetic (db/db) mice have obese and diabetic phenotypes, including hyperglycemia, because of disruption of the leptin receptor. In the present study, effects of hyperglycemia and/or hyperinsulinemia on the development of proliferative lesions were therefore examined in db/db mice given N-methyl-N-nitrosourea (MNU). A total of 120 mice were assigned to four groups: Group A, 40 db/db mice with MNU; Group B, 40 + /db mice with MNU; Group C, 30 misty (wild-type) mice with MNU; Group D, 10 db/db mice without MNU. MNU was given at 60 ppm in drinking water for 20 weeks. Subgroups of animals were sacrificed at weeks 21 and 30 and blood samples were collected to measure glucose, insulin, leptin, and adiponectin concentrations. The removed stomachs were fixed in formalin, and embedded in paraffin for histological examination and immunohistochemistry. At week 30 in Groups A, B, C and D, hyperplasia was observed in 100, 79, 57, and 0%, and dysplasia in 91, 43, 71, and 0%, respectively. Adenocarcinomas and pepsinogen-altered pyloric glands (PAPG), putative preneoplastic lesions, were observed only in Group A, at an incidence of 45%. The serum levels of insulin and leptin were also elevated in Group A. Gastric carcinogenesis by MNU was enhanced in db/db mice, possibly in association with hyperinsulinemia and hyperleptinemia.

Intestinal metaplasia has been investigated extensively as a possible premalignant condition for stomach cancer but its pathogenesis is still not fully understood. In the present study, we examined the relationship between endocrine and mucous cell marker expression periodically after Helicobacter pylori infection in the Mongolian gerbil model. The numbers of chromogranin A (CgA)-positive, gastrin-positive and gastric inhibitory polypeptide (GIP)-positive cells in H. pylori-infected groups was increased significantly compared with the non-infected case. However, CgA-positive and gastrin-positive cells then decreased from 50 through 100 experimental weeks after H. pylori infection, whereas GIP-positive cells increased. Coexistence of gastrin-positive and GIP-positive cells was detected in the same gastric and intestinal mixed phenotypic glandular-type glands. In conclusion, the endocrine cell phenotype is in line with that of the mucous counterpart in the glands of H. pylori-infected Mongolian gerbil stomach, supporting the concept that development of intestinal metaplasia is due to the abnormal differentiation of a stem cell.

The Helicobacter pylori-infected Mongolian gerbil (MG) has been established as an appropriate animal model for studies of stomach cancer development. However, there have hitherto been no data on the phenotypic classification of glandular stomach cancers in H. pylori-infected and non-infected MG. We therefore examined the phenotypes of 50 and six advanced glandular stomach cancers in H. pylori-infected and non-infected MG, respectively, as well as adjacent non-neoplastic mucosa, using several gastrointestinal epithelial phenotypic markers. The lesions were divided phenotypically into 21 gastric, 24 gastric-and-intestinal mixed, four intestinal and one null types, with 90.0% of the lesions harboring gastric elements and 56.0% demonstrating intestinal phenotypic expression in H. pylori-infected MG. All six lesions were classified as gastric type in non-infected MG. There was no clear correlation with the presence of intestinal metaplasia in surrounding mucosa. In conclusion, our data suggest that most advanced adenocarcinomas retain a gastric cellular phenotype in the glandular MG stomach. Thus, it might be proposed that intestinal metaplasia is a paracancerous phenomenon rather than a premalignant condition. H. pylori infection may trigger intestinalization of both stomach cancers and non-neoplastic mucosa.

Mongolian gerbils are an ideal animal model to explore the role of H. pylori on cancer development. However, there have been no established adenocarcinoma cell lines from this model animal. In the present study, we have established cancer cell lines from a primary gastric cancer tissue of a Mongolian gerbil. The derived cells could be stably attached with H. pylori, revealed under a scanning electron microscope, and easily transplanted to the nude mice. Rapid phosphorylation of IκB, Erk1/2, and AKT of these cells was observed by Interleukin-1 beta stimulation, and luciferase reporter gene assay on transcriptional activation of Nuclear Factor kappa B after challenging with either H. pylori NCTC11637 or its isogenic cagE-knockout mutant, H. pylori revealed the cagE-dependent NF-κB transcriptional activation. The newly established cancer cell lines from the in vivo gastric carcinogenesis model animal, the Mongolian gerbil, can be used to develop effective therapeutic strategies against gastric cancer, especially in exploring the effect of H. pylori, and thus might greatly contribute to gastric cancer prevention and treatment in humans. (Cancer Sci 2005; 96: 170–175)

Hypomethylation of the global genome, considered to be composed mainly of repetitive sequences, is consistently observed in cancers, and aberrant hypo- and hypermethylation of CpG islands (CGIs) in promoter regions are also observed. Since methylation alterations in unique promoter sequences and in other genomic regions have distinct consequences, we analyzed the relationship between the global hypomethylation and the hypomethylation of unique promoter CGIs using human gastric cancers. Seven of ten gastric cancer cell lines showed marked decreases in 5-methylcytosine content, which correlated with hypomethylation of the LINE1 repetitive sequence. Six of the seven cell lines showed hypomethylation in five or all of the six normally methylated CGIs in promoter regions of six genes, and this was associated with induction of aberrant expression. The remaining three cell lines without global hypomethylation showed promoter hypomethylation in one or none of the six CGIs. Frequent promoter hypomethylation, however, did not correlate with frequent promoter hypermethylation. In primary gastric cancers too, global hypomethylation was associated with hypomethylation of LINE1 repetitive sequence and promoter hypomethylation. Of 93 gastric cancers, 33 cancers with frequent promoter hypomethylation and 27 cancers with frequent promoter hypermethylation constituted different groups. These findings represent experimental evidence that frequent hypomethylation of normally methylated promoter CGIs is associated with global hypomethylation, and that these hypomethylations occur independently of frequent promoter CGI hypermethylation.