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Alterations in DNA mismatch repair (MMR) proteins result in microsatellite instability (MSI), increased mutation accumulation at target genes and cancer development. About one-third of gastric cancers display high-level microsatellite instability (MSI-High) and low-level microsatellite instability (MSI-Low) is frequently detected. To determine whether variations in the levels of MMR proteins or mutations in the main DNA MMR genes are associated with MSI-Low and MSI-High in gastric cancer cell lines, the MSI status (MSI-High, MSI-Low or MS-Stable (MSS)) of 14 gastric cancer lines was determined using multiple clone analysis with a panel of five microsatellite markers. Protein levels of hMLH1, hMSH2, hMSH6, hPMS2 and hPMS1 were determined by Western blot. Sequence analysis of hMLH1 and hMSH2 was performed and the methylation status of the hMLH1 promoter was examined. The cell lines SNU1 and SNU638 showed MSI-High, decreased to essentially absent hMLH1 and hPMS2 and reduced hPMS1 and hMSH6 protein levels. The hMLH1 promoter region was hypermethylated in SNU638 cells. The MKN28, MKN87, KATOIII and SNU601 cell lines showed MSI-Low. The MMR protein levels of cells with MSI-Low status was similar to the levels detected in MSS cells. A marked decrease in the expression levels of MutL MMR proteins (hMLH1, hPMS2 and hPMS1) is associated with high levels of MSI mutations in gastric cancer cells. Gastric cancer cell lines with MSI-Low status do not show significant changes in the levels of the main DNA MMR proteins or mutations in the DNA mismatch repair genes hMSH2 and hMLH1. These well-characterized gastric cancer cell lines are a valuable resource to further our understanding of DNA MMR deficiency in cancer development, progression and prognosis.

Although regenerating gene (Reg) protein is reported to have a trophic effect on gastric epithelial cells, its involvement in human gastric diseases is not clear. We have recently shown that both gastrin and gastric mucosal inflammation enhance Reg gene expression in the fundic mucosa in rats. This study was designed to clarify whether Reg protein is involved in Helicobacter pylori-induced gastritis and whether Reg gene expression is linked to serum gastrin levels in this condition. Mongolian gerbils were inoculated with an H. pylori strain isolated from a gastric cancer patient. Four weeks later, some of the gerbils with H. pylori infection were eradicated by lansoprazole, amoxicillin, and clarithromycin. The time courses of changes in Reg gene expression, serum gastrin levels, gastric acidity, and histopathologic factors were examined. Four weeks after H. pylori infection, gastritis started spreading to the fundic mucosa, and gastric acidity started reducing. Serum gastrin levels and Reg mRNA expression in the fundus were significantly increased 6 weeks after infection. Reg mRNA expression in the fundus correlated significantly with both serum gastrin levels and the severity of fundic mucosal inflammation. After H. pylori eradication, serum gastrin levels and fundic mucosal inflammation were normalized, and the increase in Reg mRNA expression was abolished. The Reg gene is associated with hypergastrinemia and fundic mucosal inflammation and may be involved in H. pylori-induced gastritis.

We investigated the expression of cyclooxygenase-2 (COX-2) and COX-1 mRNAs during longterm Helicobacter pylori infection of the Mongolian gerbil (18 months) as well as the effect of eradication therapy and the cag pathogenicity island on COX mRNA expression. COX mRNA levels were determined by reverse transcription-polymerase chain reaction. Pyloric channel ulcers were noted in one of 10 gerbils (10%) at 3 months, 33% at 6 months, 50% at 9 months, 17% at 10 months, 40% at 12 months and 25% at 18 months after inoculation of parental strains. Nineteen of 21 gerbils had successful H. pylori eradication and showed significant reduction of inflammation and no ulcerations. There were no significant differences in COX-1 mRNA expression between the groups. COX-2 mRNA expression was significantly increased 1 and 3 months after inoculation and then decreased to basal levels. In control animals, COX-2 mRNA expression was significantly higher at 12 and 18 months compared to younger animals. cagE knockout mutants did not induce gastric inflammation and induced significantly lower COX-2 mRNA expression compared to parental strains. COX-2 mRNA was induced early in H. pylori infection and then declined. COX-2 mRNA expression was also induced with aging.

Nonsteroidal anti-inflammatory drugs decrease sporadic colorectal carcinoma and adenomas in patients with familial adenomatous polyposis and in rodent models of sporadic colon cancer and familial adenomatous polyposis. Similarly, selective cyclooxygenase 2 inhibitors decrease adenomas in humans and rodents. However, their effects on chronic colitis and colitis-associated neoplasia are unknown. Interleukin 10-/- mice (C57/B6) were fed regular chow (n = 20) or chow with celecoxib (1,500 ppm, n = 18) or rofecoxib (75 ppm, n = 20) for 12 weeks. Twenty-eight percent of the celecoxib group died versus 5% of the control and rofecoxib groups (p < 0.05 compared with control). Celecoxib and rofecoxib increased the incidence of colitis (26% vs. 92% and 68%, p < 0.01), colitis score (0.4 +/- 0.2 vs. 2.5 +/- 0.3 and 2 +/- 0.4, p < 0.01), aberrant crypt foci (0.5 +/- 0.3 vs. 3.7 +/- 2.6 and 2.8 +/- 0.7, p < 0.01), aberrant crypts per mouse (4.11 +/- 2.1 vs. 41.2 +/- 9.7 and 27.1 +/- 7.5, p < 0.01) and dysplasia (11% vs. 54% and 42%, p < 0.01). Similarly, indomethacin (9 ppm, n = 15) increased colitis score, aberrant crypt foci, and dysplasia after 27 days of treatment. Two selective cyclooxygenase 2 inhibitors exacerbate colitis and premalignant changes in the interleukin 10-/- mouse model of chronic colitis and colitis-associated colon carcinoma.

Chronic infections have been associated with cardiovascular disease. We used bacterial culture, polymerase chain reaction (PCR), and immunohistochemical staining with anti-vacA and anticagA antibodies to search for Helicobacter pylori and Chlamydiae pneumoniae in atherosclerotic plaques obtained at endarterectomy. Serum IgG antibodies to H. pylori and C. pneumoniae were also determined. Thirty-two patients were enrolled. Anti-H. pylori and anti-C. pneumoniae IgG were present in 72% and 81%, respectively. Culture and PCR for H. pylori of vessel walls and plaques were negative. Atherosclerotic plaque and normal vessel sections from H. pylori-negative and- positive patients showed reactivity with anti-vacA and anti-cagA antibodies. C. pneumoniae DNA was amplified in three atherosclerotic lesions. These findings suggest that the association between H. pylori infection and atherosclerosis does not result from continuing direct effects of H. pylori antigens in the vessel walls. Antigens within vessel atherosclerotic plaques cross-react with H. pylori virulence factors and could act as cofactors in determining instability for the atherosclerotic plaques.

Background: Expression of the angiogenic factor platelet-derived endothelial cell growth factor is induced in some gastric carcinomas. Whether angiogenesis is induced early in the development of gastric pre-neoplastic lesions and whether Helicobacter pylori infection affects platelet-derived endothelial cell growth factor expression is not known., Aim: To assess whether chronic gastritis, intestinal metaplasia, gastric dysplasia and gastric carcinomas express platelet-derived endothelial cell growth factor and whether Helicobacter pylori infection might affect the expression of platelet-derived endothelial cell growth factor in these lesions., Patients and Methods: Patients with gastric carcinomas, atrophic gastritis with associated intestinal metaplasia, dysplasia and controls without infection or carcinoma were studied., Results: Platelet-derived endothelial cell growth factor was detected by immunohistochemistry in 9 out 19 gastric carcinomas (45%). Only focal immunostaining was detected in intestinal metaplasia adjacent to dysplasia and in dysplastic cells. Of the tumours, 90% contained platelet-derived endothelial cell growth factor-positive interstitial cells. A significant correlation was found between active Helicobacter pylori infection and a larger number of platelet-derived endothelial cell growth factor-positive interstitial cells in areas of intestinal metaplasia (p<0.05)., Conclusion: Helicobacter pylori infection does not influence the expression of platelet-derived endothelial cell growth factor, once gastric cancer has developed. However, Helicobacter pylori infection may increase the extension of expression of platelet-derived endothelial cell growth factor by infiltrating interstitial cells in premalignant lesions, such as intestinal metaplasia, which may help create a favourable environment for tumour development. This may possibly be due to non-specific increase in recruitment of inflammatory cells caused by Helicobacter pylori infection. Further studies, with a larger number of samples, are now needed in order to confirm this finding.

Background & Aims: Helicobacter pylori infection is a major gastric cancer risk factor. H. pylori gastritis occurs more frequently in individuals with microsatellite instability-positive than those with microsatellite instability-negative gastric cancers, raising the possibility that H. pylori infection affects DNA mismatch repair (MMR). The aim of this study was to determine the effect of H. pylori on the expression of DNA MMR proteins and RNA in gastric epithelial cells., Methods: Gastric cancer cell lines were cocultured with H. pylori, bacterial extracts, and Campylobacter jejuni or Escherichia coli. MutS (hMSH2 and hMSH6) and MutL (hMLH1, hPMS2, and hPMS1) DNA MMR protein and RNA levels were determined., Results: All cell lines examined showed decreased levels of MutS and MutL DNA MMR proteins in a dose-dependent manner after coculture with H. pylori strains. The reduction in DNA MMR protein levels was caused by heat-sensitive H. pylori products. The levels of DNA MMR proteins were affected by C. jejuni but not by E. coli. RNA levels of hMSH2 and hMSH6 were also reduced after exposure to H. pylori., Conclusions: H. pylori infection of gastric epithelial cells leads to a decrease in DNA MMR proteins that is at least in part related to an H. pylori-induced decrease in messenger RNA levels of repair genes. These data suggest that H. pylori infection might lead to a deficiency of DNA MMR in gastric epithelial cells that may increase the risk of mutation accumulation in gastric mucosa cells and the risk of gastric cancer during chronic H. pylori infection.

Background: Helicobacter pylori-CagA positive strains have been shown to be associated with atherosclerosis. However, the pathogenesis is still undetermined. The aim of this study was to determine whether anti-CagA antibodies cross-react with antigens of normal and atherosclerotic arteries., Methods and Results: Eight umbilical cord sections, 14 atherosclerotic artery sections, and 10 gastrointestinal tract sections were examined by immunohistochemistry using polyclonal anti-CagA antibodies. Five atherosclerotic and 3 normal artery samples were also lysed in ice-cold lysis buffer containing protease inhibitors and were immunoprecipitated using the same antibodies. Anti-CagA antibodies reacted with cytoplasm and nuclei of smooth muscle cells in umbilical cord and atherosclerotic vessel sections, cytoplasm of fibroblasts-like cells in intimal atherosclerotic plaques, and the cell membranes of endothelial cells. Anti-CagA antibodies also specifically immunoprecipitated 2 high molecular weight antigens of 160 and 180 kDa from both normal and atherosclerotic artery lysates., Conclusions: Anti-CagA antibodies cross-react with antigens of both normal and atherosclerotic blood vessels. We speculate that the binding of anti-CagA antibodies to those antigens in injured arteries could influence the progression of atherosclerosis in CagA-positive H pylori-infected patients.

Chronic hepatitis may progress to cirrhosis and hepatocellular carcinoma (HCC). HCC represents one of the most common human cancers. Incidence rates for this tumor vary widely on a worldwide, suggesting that environmental factors such as infectious microorganisms, carcinogens, or nutrition play a role in its pathogenesis. Several Helicobacter spp. colonize the liver of animals and induce hepatitis. The aim of this study was to determine whether Helicobacter infection was associated with HCV-related liver diseases in humans. Liver tissue samples, including biopsy and surgically excised tissues, were collected from patients positive for hepatitis C viruses (HCV) RNA in the serum. Genomic DNA was extracted from sections of formalin-fixed paraffin-embedded tissues by using the QIAamp Tissue Kit and subjected to polymerase chain reaction (PCR) analysis using two sets of Helicobacter-specific 16S ribosomal RNA primers. To identify positive samples for H. pylori, a set of primers specific for a conserved region in the H. pylori vacA gene were also used. The patients' H. pylori status was determined by ELISA. Forty-one patients (mean age 54.9, range 19-78 years; 24 men) were studied. Thirty patients had chronic viral hepatitis (CH) without (N = 18) or with (N = 12) cirrhosis (CIR), and 11 patients had HCC. Anti-H. pylori IgG was detected in 54%. The expected 422- and 210-bp fragments of Helicobacter 16S rRNA were amplified from 27% of liver samples, including 17% of CH-CIR and 55% of HCC (P = 0.004). The vacA sequence was amplified in 10 of 41(24%) samples (27% of those with HCC). These data confirm the presence of H. pylori DNA sequences in human liver and suggest an association of Helicobacter spp. with HCV-related chronic liver diseases. Further studies are needed to ascertain whether Helicobacter spp. infection plays a role in the development of HCC.

Peptic ulcers and gastric malignancies are the two major complication of the course of Helicobacter pylori-associated chronic gastritis. Both gastric adenocarcinomas and MALT lymphomas occur in association with H. pylori infection, and studies support an etiological association. This article discusses the natural history of H. pylori-related gastric carcinogenesis and criteria to identify people susceptible to H. pylori infection-associated gastric cancer. It then reviews the molecular and genetic mechanisms underlying the malignant transformation of the gastric mucosa associated with H. pylori.

Background: H. pylori infection is a major risk factor in gastric cancer development. The availability of cDNA microarrays creates the unprecedented opportunity to examine simultaneously dynamic changes of multiple pathways affected by H. pylori infection., Aim: In this study we examined broad patterns of gene expression induced by H. pylori in the gastric cancer cell line 1739-CRL AGS cells in culture using the U95A microarray., Methods: H. pylori were cocultured with AGS cells for 4, 12, 24 and 48 h. Total RNA was extracted and after labelling was used for detection of genes represented in the human U95A microarray set. Data analyses were performed using GeneChip and CLUSFAVOR software., Results: Nearly 6000 genes present in the array were expressed by AGS cells. We report approximately 200 genes that showed the most marked changes. Our studies confirm the up-regulation of c-jun, jun-B, c-fos and cyclin D1 by H. pylori. We report for the first time the induction of the serine threonine kinase pim-1 and ATF3 by H. pylori infection of AGS cells., Conclusions: In this microarray analysis of gene expression induced by H. pylori in gastric epithelial cells, we identified a large number of unsuspected genes affected by H. pylori. Further, we show that unsupervised hierarchical cluster analysis can provide useful insight into the possible contribution of genes in specific pathways, based on their profile of expression.

The epidemiological link of Helicobacter pylori and gastric carcinoma was confirmed and the reported rate of gastric carcinoma development in the Japanese population with H. pylori gastritis was 2.9% during a 7.8-year period. Studies showed that gastric atrophy and intestinal metaplasia may partially regress after H. pylori eradication, but whether this will result in a decrease in the development of malignant lesions remains to be confirmed in large studies. Little progress was made towards the identification of criteria applicable to individualized populations that would benefit from H. pylori screening and surveillance of gastric malignancy. Studies of low-grade MALT lymphoma reported clinical and molecular features that may be useful to identify cases that may be responsive to H. pylori eradication therapy, namely evaluation of lymphoma stage by EUS and t(11;18). The role of H. pylori eradication therapy in high-grade MALT lymphoma needs to be evaluated in larger prospective studies. The application of methods of global analysis of gene expression (microarray studies) resulted in the explosion of information on the molecular pathways activated by H. pylori in gastric epithelial cells. New pathways that may play an important role in H. pylori carcinogenesis have been discovered in several studies.

Chronic hepatitis may progress to cirrhosis and hepatocellular carcinoma (HCC). Progressive accumulation of mutations and genomic instability in chronic viral hepatitis might flag an increased risk of HCC development. Genomic instability at dinucleotide microsatellite loci in chromosomes 2, 13, and 17 and at 2 mononucleotide repeat loci was examined in liver tissues from 41 patients, including 30 without HCC (18 patients with chronic hepatitis and 12 with cirrhosis) and 11 with HCC. Genomic instability was detected in 51% of the 41 cases. Allelic imbalance at informative dinucleotide loci occurred in 37% of the cases. In 14 cases (34%), allelic imbalance was detected in chronic hepatitis or cirrhosis without HCC. Allelic imbalance at the chromosome 13 locus was detected in 50% of the cases of chronic hepatitis C. Allelic imbalance at the TP53 chromosome locus and/or at the chromosome 13 locus was significantly more frequent than alterations at the chromosome 2 locus (P =.026). Low-level microsatellite instability was found in 20% of all cases examined and high-level microsatellite instability in 3 patients (7.5%), including 2 cases of chronic hepatitis and 1 case of cirrhosis. Our results show that allelic imbalance occurs frequently in hepatitis-related HCC as well as in chronic hepatitis in patients without HCC. Allelic imbalance at the D13S170 chromosome 13 locus (13q31.2) occurs frequently in chronic hepatitis, suggesting that genomic alterations affecting the long arm of chromosome 13 might be used to monitor the natural progression of chronic hepatitis-associated liver carcinogenesis., (Copyright 2001 by W.B. Saunders Company.)

Helicobacter pylori-associated gastritis may progress or be complicated by peptic ulcer and gastric malignancy, including gastric carcinoma and mucosa-associated lymphoid tissue lymphoma. Predicting who develops malignancy remains a clinical challenge. The molecular understanding of pathways that are associated with progression of the normal gastric epithelium to malignancy together with classic histologic parameters are promising ways of tackling this problem. Ideally, molecular tools used for screening should be available as noninvasive tests, such as examination of markers detectable in blood samples, but these are not currently available. In contrast, molecular markers that correlate with cancer risk can be examined in the epithelium after endoscopic biopsy and can be of importance in identifying individuals at risk, especially if combined with other parameters of gastric cancer risk.

Objectives: When and how Helicobacter pylori (H. pylori) originally entered the human population as well as how the infection is transmitted in different communities is unknown. We previously showed that Sardinian shepherds had almost a 100% prevalence of H. pylori and that the prevalence was higher than that of their same-household siblings., Aim: To examine whether H. pylori infection might be transmitted from sheep., Methods: Milk and gastric tissue were cultured and analyzed by PCR amplification using three sets of primers Helicobacter genus-specific 16S rRNA and two sets of primers specific for H. pylori vacA gene., Results: Helicobacter DNA was demonstrated in 60% (38/63) of milk samples and in 30% (6/20) of sheep tissue samples. H. pylori vacA gene was amplified in five of 38 milk samples, and in two of six sheep tissue samples respectively. H. pylori were cultured from sheep milk and tissue samples and confirmed as H. pylori on the basis of colony morphology, positive biochemical reactions, and negative Gram stain. Sequence analysis of 16S rRNA PCR products from these isolates demonstrated 99% identity with H. pylori., Conclusions: Together, the presence of H. pylori in sheep stomach in the absence of associated gastritis and recovery of H. pylori from sheep milk and gastric tissue suggest that sheep may be a natural host for H. pylori.

Deficiencies of MutL DNA mismatch repair-complex proteins (hMLH1, hPMS2, and hPMS1) typically result in microsatellite instability in human cancers. We examined the association patterns of MutL proteins in human epithelial cancer cell lines with (HCT-116, N87, SNU-1, and SNU-638) and without microsatellite instability (HeLa, AGS, KATO-III, and SNU-16). The analysis of hMLH1, hPMS2, and hPMS1 was performed using Northern blot, Western blot, and co-immunoprecipitation studies. Our data provide evidence that MutL proteins form two different complexes, MutL-alpha (hPMS2 and hMLH1) and MutL-beta (hPMS1 and hMLH1). Gastric and colorectal cancer cells lines with microsatellite instability lacked detectable hMLH1. Decreased levels of hMLH1 protein were associated with markedly reduced levels of hPMS2 and hPMS1 proteins, but the RNA levels of hPMS1 and hPMS2 were normal. In this study, we describe the association of hPMS1 with hMLH1 as a heterodimer, in human cells. Furthermore, normal levels of hMLH1 protein appear to be important in maintaining normal levels of hPMS1 and hPMS2 proteins.

Gastric carcinomas (GC) are heterogeneous tumors comprising variable amounts of cells of different lineage phenotype, including gastric mucous cells (surface--SMC or gland--GMC) and intestinal cells (IC). The evaluation of tumor behavior has classically depended on strictly morphological classifications of tumors. Microsatellite instability (MSI) is frequently detected in GC, but whether MSI affects all gastric cellular lineages or exclusively occurs in unique cellular lineages in GC is not known. The aims of this study were to test a combination of anti-mucin antibodies to classify gastric cancer into predominant cell lineage phenotype and to determine whether MSI in GC is associated with particular cellular tumor phenotypes. Fifty-five GC were immunophenotyped with antibodies specific for SMC, GMC, or IC. DNA was extracted from tumor and non-neoplastic gastric tissues and amplified with 5 microsatellite markers. A mixed cellular pattern was the most frequent phenotype of GC (61%) and was seen in both glandular (63%) and diffuse (58%)-type tumors. No significant difference in the rate of MSI was found in tumors with predominant gastric, intestinal or mixed phenotype. However, tumors with null or low-level expression of cellular lineage differentiation markers displayed MSI more frequently than tumors with high-level expression (40% v 20%). In conclusion, different gastric carcinoma cell lineage patterns can be easily identified with the 3 immunohistochemical markers used in this study. The 3 main cellular lineage components of gastric cancer can be similarly affected by microsatellite instability, consistent with the notion that MSI is an early event in gastric carcinogenesis.

The predominant H. pylori strain circulating among geographic locations differs with regard to the genomic structure. This study determined whether structural subtypes of the cagA 3' repeat region could be used to identify the population of origin of H. pylori isolates. We examined 600 cagA-positive H. pylori (Colombia, 100; USA, 100; France, 100; Canada, 20; Italy, 20; Korea, 100; Japan, 100; Hong Kong, 20; Taiwan, 20; Vietnam, 20). The cagA 3' region was amplified by PCR using primers specific to Japanese and Western 3' cagA gene sequences. PCR using Japanese cagA primers resulted in PCR products in 99-6 % of strains from East Asia but no non-Asian strains. Conversely, PCR using Western cagA primers resulted in amplicons in 100% of non-Asian strains, and only one from East Asia. cagA genotyping is useful for molecular epidemiological studies as strains can be completely separated by differences in the cagA 3' region.

The role and significance of microsatellite instability (MSI) in gastric carcinogenesis remain unknown. This study determined the chronology of MSI in gastric carcinogenesis by examining intestinal metaplasia (IM) from patients with and without gastric cancer. DNA was obtained from gastric specimens of 75 patients with gastric IM (30 cancer, 26 peptic ulcer, and 19 chronic gastritis patients) and was amplified with a set of eight microsatellite markers. Eight (26. 7%) tumors and seven (9.3%) IM samples (three from cancer-free patients) displayed high-level MSI (three or more loci altered). Low-level MSI (one or two loci altered) was detected in 50% of the tumors, in 40% of IM samples coexisting with cancer, and in 38% of IM tissues of cancer-free individuals. Among the 30 cancer patients, microsatellites were more frequently altered in IM coexisting with tumors that showed MSI (P = 0.003). In addition, patients with low-level MSI in the tumor tissues were more likely to have active Helicobacter pylori infection than those with stable tumors (P = 0.02). In conclusion, this study indicates that MSI occurs not only in gastric IM of patients with gastric carcinoma, but also in IM of cancer-free individuals. These data suggest that the progressive accumulation of MSI in areas of IM may contribute to gastric cancer development, representing an important molecular event in the multistep gastric carcinogenesis cascade.

Our purpose was to define the effect of pretreatment Helicobacter pylori resistance to metronidazole or to clarithromycin on the success of antimicrobial therapy. We used 75 key words to perform a literature search in MEDLINE as well as manual searches to identify clinical treatment trials that provided results in relation to H. pylori susceptibility to metronidazole and clarithromycin or both during the period 1984-1997 (abstracts were not included). Meta-analysis was done with both fixed- and random-effect models; results were shown using Galbraith's radial plots. We identified 49 papers with 65 arms for metronidazole (3594 patients, 2434 harboring H. pylori strains sensitive to metronidazole and 1160 harboring resistant strains). Metronidazole resistance reduced effectiveness by an average of 37.7% (95% CI = 29.6-45.7%). The variability in the risk difference for metronidazole was 122.0 to -90.6 and the chi-square value for heterogeneity was significant (P<0.001). Susceptibility tests for clarithromycin were performed in 12 studies (501 patients, 468 harboring H. pylori strains sensitive to clarithromycin and 33 harboring resistant strains). Clarithromycin resistance reduced effectiveness by an average of 55% (95% CI = 33-78%). We found no common factors that allowed patients to be divided into subgroups with additional factors significantly associated with resistance. In conclusion, metronidazole or clarithromycin pretreatment resistant H. pylori are the main factors responsible for treatment failure with regimens using these compounds. If H. pylori antibiotic resistance continues to increase, pretherapy antibiotic sensitivity testing might become necessary in many regions.

Objective: Previous studies have reported variable rates of microsatellite instability (MSI) in gastric cancer. We investigated the frequency of MSI in invasive gastric carcinoma of patients from three geographic regions., Methods: Genomic DNA from gastric cancer and nontumor tissue from 22 Korean, 20 Colombian, and 26 U.S. patients was amplified with five microsatellite markers., Results: MSI was more frequently seen in gastric cancer from Korea, affecting 50% of patients, in contrast with gastric cancers from the U.S. (7%) and Colombia (15%) (p = 0.003 and p = 0.03, respectively). MSI at one locus was significantly more frequent in gastric cancer from individuals >65 yr (p = 0.01). MSI was similarly associated with both diffuse and intestinal types of gastric cancer., Conclusions: MSI affects the two major histological types of gastric cancer, and was more frequent in gastric cancer from Korea than in the other countries, suggesting that the relative importance of different pathways of gastric carcinogenesis may vary in diverse regions of the world.

Microsatellite markers permit the analysis of microsatellite instability and loss of heterozygosity. Frequently, the allelotypes of microsatellites are interpreted in the presence of numerous bands in gels. The importance of different gel electrophoresis conditions in the interpretation of microsatellite patterns was tested. Microsatellite markers were used to amplify DNA from gastric cancer samples and adjacent gastric mucosa. Polymerase chain reaction (PCR) products were separated by electrophoresis through 7% polyacrylamide gels containing either 5.6 M urea and 32% formamide or 7 M urea. PCR reactions separated on urea/formamide gels resulted consistently in clear allele definition (one or two bands), whereas 7 M urea gels resulted in allele patterns that comprised multiple bands. Analysis of microsatellite abnormalities using nonformamide gels gave false negative results in just under a third of cases (four of 13). In conclusion, the interpretation of microsatellite alterations in cancer DNA is improved by using electrophoresis conditions that result in complete DNA denaturation, such as urea/formamide/acrylamide gel electrophoresis.

Background: The beta-lactam group of antibiotics kills bacteria by inhibiting the terminal stages of peptidoglycan metabolism. We have recently identified amoxicillin-resistant Helicobacter pylori, none of which expressed beta-lactamase. Penicillin-binding proteins (PBPs) represent a group of target enzymes for the beta-lactam antibiotic family, and alterations in PBPs have been described in other penicillin-resistant bacteria. The amoxicillin-resistant phenotype characteristically was lost after freezing but could be restored by consecutive transfers into gradient plates., Materials and Methods: To determine whether amoxicillin resistance in H. pylori was related to alterations in any of the H. pylori PBPs, five H. pylori strains resistant to amoxicillin and three amoxicillin-sensitive strains were tested. PBPs were extracted from bacteria grown to logarithmic phase, labeled in vivo with 3H-benzylpenicillin, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Four main PBPs were separated from all amoxicillin-sensitive H. pylori strains., Results: Only three of the four main PBPs were found in the amoxicillin-resistant H. pylori strains. The differentially detectable PBP (PBP D) had an apparent molecular weight of 30 to 32 kD., Conclusion: These results suggest that PBP D might play a role in the amoxicillin-resistant phenotype of H. pylori strains lacking beta-lactamase activity.

The sensitivities to penicillins and to a penicillin and beta-lactamase inhibitor combination agent were determined for Helicobacter pylori strains that were sensitive, moderately resistant, or highly resistant to amoxicillin. All strains were resistant to nafcillin and oxacillin. Moderately resistant strains showed an intermediate zone of inhibition to ticarcillin, mezlocillin, piperacillin, and amoxicillin-clavulanic acid. High-level resistance was associated with the smallest zone size for all penicillins tested.

The CagA protein of Helicobacter pylori is an immunogenic antigen of variable size and unknown function that has been associated with increased virulence as well as two mutually exclusive diseases, duodenal ulcer and gastric carcinoma. The 3' region of the cagA gene contains repeated sequences. To determine whether there are structural changes in the 3' region of cagA that predict outcome of H. pylori infection, we examined 155 cagA gene-positive H. pylori isolates from Japanese patients including 50 patients with simple gastritis, 40 with gastric ulcer, 35 with duodenal ulcer, and 30 with gastric cancer. The 3' region of the cagA gene was amplified by PCR followed by sequencing. CagA proteins were detected by immunoblotting using a polyclonal antibody against recombinant CagA. One hundred forty-five strains yielded PCR products of 642 to 651 bp; 10 strains had products of 756 to 813 bp. The sequence of the 3' region of the cagA gene in Japan differs markedly from the primary sequence of cagA genes from Western isolates. Sequence analysis of the PCR products showed four types of primary gene structure (designated types A, B, C, and D) depending on the type and number of repeats. Six of the seven type C strains were found in patients with gastric cancer (P < 0.01 in comparison to noncancer patients). Comparison of type A and type C strains from patients with gastric cancer showed that type C was associated with higher levels of CagA antibody and more severe degrees of atrophy. Differences in cagA genotype may be useful for molecular epidemiology and may provide a marker for differences in virulence among cagA-positive H. pylori strains.

Background: The efficacy of omeprazole and amoxycillin dual therapy to treat Helicobacter pylori infection has been inconsistent, suggesting the presence of host or bacterial factors influencing treatment success. The aim of this study was to assess the role of pre-treatment amoxycillin resistance in the efficacy of omeprazole and amoxycillin dual therapy., Methods: We studied 43 consecutive dyspeptic patients with H. pylori infection. Pre-treatment H. pylori infection was established by the combination of positive rapid urease test, culture and histology. Amoxycillin susceptibility testing was performed by an Epsilometer test (E-test) method and amoxycillin resistance was defined as minimum inhibitory concentration greater than 8 microg/mL. Patients received 20 mg omeprazole twice daily for 28 days and amoxycillin 1000 mg twice daily for 2 weeks. Adverse effects were documented using a questionnaire. H. pylori status was reassessed 6-8 weeks after the end of treatment by rapid urease testing and histological examination of gastric biopsies., Results: Forty-two dyspeptic patients completed the study, and one patient dropped out. H. pylori infection was cured in 2 3 of 42 patients (55%). The cure rate was higher in patients harbouring amoxycillin-sensitive organisms than in those with resistant strains: 66% (19/29) vs. 31% (4/13), respectively (P = 0.049). No significant differences in cure rates were evident in relation to age, sex, smoking habits or compliance., Conclusions: The effectiveness of amoxycillin-omeprazole dual therapy was greatly reduced in the presence of pre-treatment amoxycillin-resistant H. pylori. The success rate in patients with amoxycillin-sensitive H. pylori was only 66%, suggesting the presence of additional factors affecting the efficacy of this therapy.

The mouse gamma-glutamyl transpeptidase (GGT) gene encodes seven distinct mRNAs that are transcribed from seven separate promoters. Type II mRNA is the most abundant in kidney. We have developed a cell line with features of renal proximal tubular cells which expresses GGT mRNA types with a pattern similar to that of mouse kidney. Because a 346-bp sequence from the type II promoter directed the highest level of CAT activity in these cells, this region was used to drive the expression of a beta-galactosidase reporter gene in transgenic mice. Two transgenic mouse lines expressed beta-galactosidase limited to the renal proximal tubules. Site-directed deletions within this 346-bp promoter region demonstrated that cis-elements containing the consensus binding sites for AP2, a glucocorticoid response element (GRE)-like element, and the initiator region were required for transcriptional activity and were not additive. Purified AP2 bound and footprinted the AP2 consensus region, making it likely that transcription from the GGT type II promoter is regulated in part by AP2. These data suggest that transcription of the type II promoter requires multiple protein DNA interactions involving at least an AP2 element, and probably a GRE-like element and the initiator region.

gamma-Glutamyl transpeptidase is a key enzyme in glutathione (GSH) salvage, metabolism of endogenous mediators such as leukotrienes and prostaglandins, detoxification of xenobiotics including environmentally important compounds and carcinogens, and cellular processes dependent on the oxidation/reduction of glutathione. The enzyme is widely distributed, and these functions often occur in separate tissues and in response to different stimuli. Evidence indicates that gamma-glutamyl transpeptidase plays a direct role in some hepatic and renal responses to injury. In the mouse gamma-glutamyl transpeptidase is a single copy gene expressed from at least seven promoters, and many of the transcribed gamma-glutamyl transpeptidase RNAs are restricted in their expression. Studies that combine analyses of cellular processes with a knowledge of gene structure and expression hold promise for unravelling how these two different levels of function are integrated.

gamma-Glutamyl transpeptidase (gamma GT) is a crucial enzyme for the metabolism of xenobiotics and endogenous mediators of biological functions (leukotrienes, prostaglandins, and hepoxillins). Yet little is known about its potential role during development. It is a single copy gene expressed from at least seven promoters. Using histochemistry and immunohistochemistry we demonstrate that gamma GT first appears in the midgestational yolk sacs of mouse embryos. Established cell lines with phenotypic features of yolk sac endoderm (JC-44) or embryonic stem cells were also assayed for the expression of gamma GT. Significant levels were detected in JC-44 cells and higher levels were found in JC-44-derived embryoid bodies. Because this cell line appears to be a good in vitro counterpart of yolk sac differentiation, we characterized the gamma GT mRNA types expressed in JC-44 cells. By ribonuclease protection analysis, gamma GT RNA types IV and VI represent about 80% of the total gamma GT RNA in JC-44 embryoid bodies. Reverse transcription-mediated polymerase chain reaction detected low amounts of gamma GT RNA types I, III, and V. Expression of gamma GT in yolk sac follows a pattern seen in many tissues in which one or two gamma GT RNA types dominate the expression profile; however, the reason for this tissue specificity is unknown.

We have previously identified five promoters in the 5'-flanking region of the mouse gamma-glutamyl transpeptidase (gamma GT) gene. We now report the localization of a sixth promoter that supports the transcription of type III RNA, the major gamma GT RNA in fetal liver. We made a fetal liver cDNA library enriched for gamma GT RNA and obtained 12 gamma GT type III-specific clones. The longest clone is consistent with a transcription start site for type III RNA at a position 5' to the type IV promoter and about 5 kilobase(s) (kb) 5' to the first coding exon. We estimated by ribonuclease protection assay that about 80% of the gamma GT mRNA in fetal liver was type III. Primer extension and nuclease protection analyses mapped the 5' end of type III mRNA in fetal liver and kidney to a single cluster of potential major and minor transcription start sites. Deletion analysis using transient expression of chloramphenicol acetyltransferase constructs of the type III promoter region revealed the greatest activity with a 1-kb 5'-flanking fragment in mouse kidney proximal tubular cells and no detectable activity in NIH-3T3 fibroblasts. These studies demonstrate that the type III 5' region of the mouse gamma GT gene is organized into two distinct exons (IIIa and IIIb) and that type III RNA is expressed under the control of its own promoter.

To clarify differences between gamma-glutamyl transpeptidase (gamma GT) expression in the rat and the mouse small intestine, we cloned and sequenced 13 rat small intestine gamma GT cDNAs and compared them with those obtained from the mouse (Carter, et al. (1994) J. Biol. Chem. 269, 24581-24585). We found that all 13 were type VI, which in both species accounts for 80-90% of intestinal gamma GT. However, rat type VI gamma GT is substantially longer than mouse type VI RNA and includes at least 67 additional nucleotides at the 5' end that are non-homologous with the 5' end of the mouse type VI RNA; further, these show no homology to another mouse exon (VII) that splices to type VI RNA. In addition, a 168 nucleotide stretch is present in the middle of rat RNA that is absent in mouse type VI RNA.

Mouse gamma-glutamyl transpeptidase (gamma GT) is encoded by a single copy gene with at least five and probably six different promoters directing the transcription of six types of gamma GT RNAs. In mouse small intestine, only Type I, V, and VI gamma GT RNAs are detected, and ribonuclease protection assays reveal that Type VI represents more than 90% of gamma GT RNA. To investigate the structure of intestinal gamma GT RNA in greater detail, we cloned and sequenced mouse intestinal gamma GT cDNAs. Seven of eight informative clones were Type VI and consisted of Type VI unique exons, VIa and VIb (as described previously by us) (Rajagopalan, S., Wan, D.-F., Habib, G. M., Sepulveda, A. R., McLeod, M. R., Lebovitz, R. M., and Lieberman, M. W. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6179-6183) as well as common 3' sequences. Exon VIb contains two alternative splice acceptors, one previously identified by us and the other 17 bases 5' of this site. Another clone contained a previously unidentified gamma GT mRNA designated as Type VII. Type VII consists of a unique 5' exon which is 315 base pairs upstream of the exon VIa splice donor site and is spliced to exon VIb. Regulation of gamma GT expression in the small intestine is complex and involves at least three previously described promoters, alternative splicing, and a previously undescribed exonic sequence (Type VII RNA) 5' of promoter VI.

The mouse gamma-glutamyl transpeptidase (gamma GT) gene encodes six distinct mRNAs that differ in their 5'-untranslated regions but appear to code for the same protein. To elucidate the mechanisms that generate these different mRNAs we determined the transcription start sites of gamma GT kidney mRNAs and investigated the ability of the 5'-flanking regions of mRNAs I, II, IV, V, and VI to direct transcription of chloramphenicol acetyltransferase (CAT) reporter gene constructs in a mouse kidney cell line. Types I, II, and VI mRNAs show heterogeneous start sites, whereas types IV and V have more precise initiation sites. Only the type V 5'-flanking region contains a TATA-like element. The highest CAT activities were observed with 416 base pairs of type II and 240 base pairs of type IV flanking regions. We have also shown that types II and IV represent the predominant gamma GT mRNAs in kidney; therefore, these CAT activities correlate well with the relative amount of each gamma GT mRNA. This study shows that the mouse gamma GT gene is transcribed from at least five and possibly six different promoters. In addition, the gamma GT promoters show cell specificity because no significant CAT activity was detected when these constructs were introduced into NIH-3T3 fibroblasts.

The 5' region of the mouse gamma-glutamyltransferase (gamma GT; EC 2.3.2.2) gene has been cloned and analyzed. This analysis, combined with sequence information obtained from gamma GT cDNA clones, indicates that in mouse a single gamma GT gene codes for six different mRNAs that differ in their 5' sequences. Analysis of steady-state levels of gamma GT RNA reveals different expression patterns for these RNAs in different organs. The six different 5' sequences are widely separated within a 10-kb region and three of them show 75-86% identify with the three known rat gamma GT cDNAs. Although the heterogeneity of the 5' ends of gamma GT RNAs may be explained in part by alternative splicing, it is likely that multiple promoters are involved in their generation.

SV40 large T antigen (T-ag) is a potent oncogene able to transform many cell types. The human alpha-1-antitrypsin (AAT) gene encodes the major serine protease inhibitor in plasma. The 5' flanking sequence of the AAT gene contains cis-acting elements that specify the expression of reporter genes in transgenic mice. Although the AAT gene is expressed primarily in the liver, it also is expressed in epithelial cells of the stomach, kidney, intestine and pancreas. A fusion gene with the 5' flanking region of AAT driving the SV40 large T-ag gene was used to generate transgenic mice. Between 2 and 6 months of age, 6 of 7 founder mice developed hepatic neoplasms. Kidney hyperplasia (3/7) and carcinomas of the stomach (4/7) and pancreas (1/7) also were observed. Tumor cells in the different sites expressed T-ag that was detectable by immunohistochemical staining. A transgenic mouse line (1812) was established; liver tumors develop reproducibly in all offspring. Cellular changes progressing to liver tumor formation occur in discrete stages with predictable kinetics in animals of the 1812 line: (i) embryonal/fetal stage, no major histological changes; (ii) newborn to 2 weeks of age, hyperplastic livers with minor cellular changes; (iii) mice between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; (iv) mice 8 weeks of age and older, hepatocellular carcinomas and hepatoblastomas in a background of liver dysplasia.(ABSTRACT TRUNCATED AT 250 WORDS)

alpha-1-Antitrypsin (AAT) is the major antiprotease in human plasma; it is synthesized primarily in hepatocytes and to a lesser extent in several nonhepatic tissues. Under the control of regulatory elements of the human AAT gene, expression of SV40-large tumor antigen (T-ag) in transgenic mice occurred in the liver, stomach, pancreas, and kidney. Among seven founder transgenic animals, six developed liver carcinoma, four showed gastric neoplasia, and one developed pancreatic carcinoma. In three animals the kidneys showed glomerular or tubular epithelial hyperplasia but no malignancy. A stable transgenic line, 1812, was established. Members of this line reproducibly develop liver tumors by 10 weeks of age but do not exhibit any phenotypic changes in other tissues. Histological changes leading to liver tumor formation occurred with predictable kinetics and could be classified into four distinct stages: (a) embryonal/fetal stage, no recognizable histological changes; (b) newborn to 2 weeks of age, hyperplastic hepatocytes with reduced amounts of cytoplasm but no nuclear alterations; (c) between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; and (d) 8 weeks of age and thereafter, hepatocellular carcinomas in a background of liver dysplasia. Embryonic and newborn liver tissue showed uniform, high level expression of T-ag in the majority of hepatocytes by immunohistochemistry, whereas the dysplastic and tumoral stages were characterized by considerable variation in both the intensity of T-ag staining and the proportion of T-ag-positive cells. Immunoprecipitation analyses showed that T-ag was complexed with cellular protein p53 in all tumor samples. This study showed that SV40 T-ag expression in the liver resulted in cellular hyperplasia and dysplasia; additional event(s) apparently were required for progression to neoplasia. Those cooperating events occurred with predictable kinetics. This transgenic mouse system displays several similarities with human liver disease and provides a practical model for the study of separate steps in hepatocarcinogenesis.