List the title and complete references (author(s), journal or book, year, page number) of all publications directly resulting from studies supported by the project (i.e., with citation of this grant in the acknowledgement section). List the publications for the project in accordance to the following categories: (1) manuscripts published and accepted for publications; (2) manuscripts submitted; and (3) conference proceedings. Provide one copy of each publication not previously reported to the National Science Council in the Appendix.
Distinct Gene Expression Profiles in Gastric Epithelial Cells Induced by Different Clinical Isolates of Helicobacter pylori---- Implication of Bacteria and Host Interaction in Gastric
Carcinogenesis( manuscript submitted )(See appendix)
Distinct Gene Expression Profiles in Microsatellite Instable and
Stable Gastric Cancers Analyzed by Microarray (manuscript in
preparation)
6b. Patents
List all inventions disclosed, patents filed, and patents granted. Please note
the inventors, assignee, title of patent, country or area where patent applied
for, filing or issued number and date.
Appendix
Distinct Gene Expression Profiles in Gastric Epithelial Cells Induced by Different Clinical Isolates of Helicobacter pylori---- Implication of Bacteria and Host Interaction in Gastric
Carcinogenesis
Yu-Ting Changa, Ming-Shiang Wua,Ya-Jen Changb, Ching-Chow Chenb, Yi-Shing Linc, Tracy Hsiehc, Pan-Chyr Yanga, and Jaw-Town Lina. Departments of aInternal Medicine and bPharmacology,National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei;
cGenasia Biotechnology, Ltd., Taipei, Taiwan
Corresponding author: Jaw-Town Lin, M.D., Ph. D.
Address: Department of Internal Medicine, National Taiwan University Hospital, No.7, Chung-Shan South Road, Taipei, Taiwan
Telephone: 886-2-23123456 ext.5695 Fax: 886-2-23947899
e-Mail address: [email protected]
Running title: Gene Expression in Gastric Epithelial Cells Induced by Helicobacter pylori
Key words: Helicobacter pylori (H. pylori), gastric cancer, microarray, C/EBPβ
Abstract
Helicobacter pylori (H. pylori) infection is a major risk factor of peptic ulcer, gastric cancer, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The interplay between H. pylori and host determines the various outcomes after infection and is an important issue for further elucidation of pathogenesis of H. pylori-related diseases. The availability of cDNA microarray creates the unprecedented opportunity to examine simultaneously dynamic changes of multiple molecular pathways affected by different H. pylori strains infection. To elucidate the cross-talk of H. pylori and gastric epithelial cells, three different clinical isolated H. pylori strains, GC, DU and MA strain, isolated from patients of gastric cancer(GC), duodenal ulcer(DU), and gastric MALT lymphoma (MA), were cocultured with AGS cells for 6 hours. Total RNA was extracted and used for detection of genes represented in the Human 1 cDNA Microarray (Agilent).Validation of microarray data were done by Western blot analysis of selected genes. Of the 12,814 clones on the Human 1 cDNA microarray, there were 522 genes expressed differently in the three groups. Of the 522 genes, there were 4 genes, 4 genes and 13 genes, either up- or down-regulated more than twofold change, in AGS cells induced specifically by GC, MA and DU strain respectively. The GC and DU strains induced more genes involving in carcinogenesis, such as pim-1, jun B, and VEGF. In the study, we provide evidence of bacterial factors may determine the outcomes of H.
pylori infection. Different bacterial virulent factors, other than cagA and vacA,
are critical in the development of various gastroduodenal diseases. The expression profiles of cDNA microarray provide clues for diagnosis, treatment and prevention of H. pylori-related gastroduodenal diseases.
Introduction
Helicobacter pylori (H. pylori) is a prevalent gram-negative bacterium. The infection of H. pylori has been recognized as a major risk factor in the development of chronic active gastritis, peptic ulcer disease, and gastric cancer[1]. The prevalence of H. pylori-related diseases varies in different geographic regions and patient populations[2]. However, the majority of H.
pylori colonized individuals remain asymptomatic; approximately 20% of
infected patients develop clinically significant diseases such as peptic ulcer, gastric adenocarcinoma, or gastric mucosa-associated lymphoid tissue (MALT) lymphoma[1, 3]. This variability in clinical outcomes may result from differences in inflammatory responses governed by host genetics, and specific interactions between host and microbial determinants[3].Bacterial virulence, host genetic, and environmental factors are among the critical determinants that predispose to the clinical manifestation of H. pylori infection.
H. pylori populations are extremely diverse[4, 5] because H. pylori has a
very plastic genome, reflecting its high rate of recombination and point mutation.
Different H. pylori strains have different effects on cellular turnover and level of apoptosis of gastric mucosal epithelial cells [6-10]. Previous studies have shown that the presence of cag pathogenicity island (PAI) and specific genotype of vacA or iceA gene are more frequently associated with H.
pylori-related diseases [11, 12]. It implied that differences in virulence factors
that characterize different strains of H. pylori could influence the clinical outcome of H. pylori infection. However, the virulent markers for H. pylori strains that affect Western populations have little or no predicative power in East Asian populations[13, 14]. The molecular pathways of gastric carcinogenesis and H.
pylori-related diseases are multiple, producing complex pattern of molecular
changes in gastric epithelial cells. It is of fundamental importance to understand the main molecular pathways affected by H. pylori in epithelial cells in order to identify the critical steps in induction of H. pylori-related diseases, such as duodenal ulcer, gastric caner, and gastric MALT lymphoma. The availability of cDNA microarray reference creates the unprecedented opportunity to examine simultaneously dynamic changes of multiple pathways affected by different H.
pylori strains infection. In this study, we apply the microarray technology to
examine the broad patterns of gene expression of gastric epithelial cell lines which co-culture with three different clinical isolates of H. pylori. Understanding the molecular pathway affected by different H. pylori in the gastric epithelium may provide novel approaches to screening the high risk group and identification of molecular targets for further prevention of peptic ulcer disease, gastric cancer, and gastric MALT lymphoma.
Materials and Methods
Bacteria and growth conditions
The H. pylori strains employed in this study included gastric cancer (GC) strains, duodenal ulcer (DU) strains, and gastric MALT lymphoma (MA) strains originally isolated from patients with gastric adenocarcinoma, duodenal ulcer, and gastric MALT lymphoma, respectively. H. pylori strains were grown on Columbia agar plates containing 5% (vol/vol) sheep blood and antibiotics supplement (GIBCO BRL, Rockville, Md.) at 370C in a microaerophilic chamber (Don Whitley, West Yorkshire, England ) containing 5% O2, 85% N2, and 10%
CO2 for 48 hours. Bacterial cells were collected and washed with PBS buffer (pH 7.4) and pelleted. Cell pellets were then resuspended in PBS buffer (pH7.4) and used for infection experiment immediately. Bacterial numbers were measured by spectrophotometry at 600 nm.
Cell culture
The gastric cancer cell line 1739-CRL (AGS cell) was used to perform in vitro co-culture experiment with H. pylori. The AGS cells were maintained in RPMI 1640 (Sigma Co.) supplemented with 10% fetal bovine serum (FBS) (GIBCO BRL, Rockville, MD., USA), 100U/ml of penicillin, and 100µg/ml of streptomycin. Co-culture with bacteria was prepared by seeding 1x106 AGS cells in 10cm plates in RPMI 1640 without antibiotics. The cell lines were treated with three different clinically isolated H. pylori strains for 6 hours with MOI of 300 or a similar volume of PBS as control. After washing the cell lines with PBS and removing the bacteria, total RNA were extracted from AGS cells by TRIzol reagent (Gibco BRL) and quantified by OD 260nm and qualified by Bioanalyzer (Agilent Technology, USA).
Probe Labeling and microarray hybridization and scanning
Total 15µg of purified RNA are converted to cDNA using a 3DNATM Array 50 Expression Array Detection Kit (Genisphere, U. S. A.). Correspondingly synthesized cDNA products are combined and concentrated by ethanol precipitation and suspended in hybridization buffer. Hybridization of labeled cDNA is hybridized to Human 1 cDNA microarray (Agilent Technologies, USA) at 650C for 17 hours. After hybridization, slides were washed by 0.5X SSC/0.01% SDS at room temperature for 5 minutes, and 0.06X SSC at room temperature for 2 minutes. Washed microarrays are then hybridized with Cy3 and Cy5 dendrimers in formamide-based buffer at 530C for 3 hours. After hybridization with dendrimers, slides were washed by 2X SSC/0.01% SDS at 420C for 15 minutes, 2X SSC at room temperature for 10 minutes, and 0.2X SSC at room temperature for 10 minutes. After washing and drying by centrifugation, microarrays are scanned with a Virtek fluorescence reader (Virtek, CA) at 535 nm for Cy3 and 625 nm for Cy5. Scanned images are analyzed by Array-Pro image acquisition software (Media Cybernetics, USA).
An image analysis algorithm is used to quantify signal and background intensity for each target element. The targets which signal to background intensity ratio more than 2.5 were selected for further analysis. Data normalization is performed by Lowess method using R package (written by Terry Speeds Microarray Data Analysis Group, University of Berkerley, USA). The microarray data were analyzed by Spotfire software (Someville, MA., USA).
Preparation of Cell Extracts and Western Blot Analysis
Rabbit polyclonal antibodies specific for CCAAT enhancer-binding protein (C/EBP β) and goat polyclonal antibody specific for actin were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA., USA). After incubation with various Helicobacter pylori strains for 3, 6, 12, 18, and 24 hours, AGS cells were rapidly washed with PBS to remove bacteria, and then lysed with ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EGTA, 1 mM NaF, 150 mM NaCl, 1 mM PMSF, 5 µg/ml of leupeptin, 20 µg/ml of aprotinin, 1 mM Na3VO4, 10mM
β-glycerophosphate, 5mM Na-pyrophosphate, 1% Triton X-100). The cell lysate (100 µg total protein) was subjected to SDS-PAGE using 10 % running gels. The proteins were transferred to nitrocellulose paper. The membranes were blocked for 1 h at 25oC with 0.1% milk in Tris-buffered saline/Tween 20 (TTBS) ) and then incubated for 1 h at 25oC with rabbit antibodies specific for C/EBPβ or goat antibodies specific for actin. Then, the membrane was incubated for 30 min at 25oC with horseradish peroxidase-labeled secondary antibody against rabbit or goat. After each incubation, the membrane was washed extensively with TTBS.
The immunoreactive band was detected using ECL detection reagents (Amersham) and visualized using Hyperfilm-ECL. Quantitative data were obtained using a computing densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA., USA).
Genotyping and sequencing of vacA
DNAs from three clinically isolated H. pylori were extracted for vacA analysis as previously described[15, 16].
Statistical Analysis
The average of the two replicate experiments was determined and used for fold change determination by comparing the gene expression fold change relative to the non-infected control. Pearson correlation method was used to analyze the similarity of gene expression between each two groups.
Discriminate genes and differences between the three groups were analyzed using two-tailed ANOVA test. After ANOVA test, those genes shown to undergo more than twofold up- or down-regulation induced by H. pylori were selected for further analysis. P<0.05 was considered statistically significant.
Results
Comparative analysis of gene expression of AGS cells induced by different clinical isolated H. pylori infection
The intensity of 99% targets of the 12,814 clones on the six Human 1 cDNA microarray were more than or equal to 2.5 times of background. We found the gene expression profile was more similar between the GC strain and DU strain (r=0.552) than that between GC strain and MA strain (r=0.393), and that between DU strain and MA strain (r=0.355). ANOVA analysis showed that there were 522 genes expressed differently in the three groups. There were 4 genes up- or down-regulated expression in AGS cells induced specifically by GC strain (Table 1), 4 genes up- or down-regulated expression in AGS cells induced specifically by DU strain (Table 2), and 13 genes up- or down-regulated expression in AGS cells induced specifically by MA strain (Table 3). Those genes shown to undergo more than twofold up- or down-regulation induced by H. pylori were further analyzed. We found that there were 32 genes, including pim-1 oncogene, vascular endothelial growth factor (VEGF), jun B, CCAAT/enhancer binding protein β (C/EBPβ), and cyclin G2, etc. up- or down-regulated expression in AGS cells induced both by GC and DU strains but not by MA strain (Table 4).
Validation of microarray data by Western blot analysis
For verifying the results of microarray, the protein expression of C/EBPβ in AGS cells induced by three H. pylori strains at 3, 6, 12, 18, and 24 hours were quantified by Western blot analysis. We found that the GC and DU strain induced C/EBPβ expression in AGS cells at 6, 12, 18 and 24hours (Figure 1).
The MA strain did not induced C/EBPβ expression in AGS cells at different time
point in our experiments. The result was consistent with our microarray data.
Genotypes of Helicobacter pylori
All of the three strains of Helicobacter pylori express cagA. The genotype of vacA of the three strains is s1m2.
Discussion
Although H. pylori is associated with chronic gastritis, duodenal ulcer, gastric cancer, and gastric MALT lymphoma, only a small portion of infected population developed theses diseases while the majority of infected people remained asymptomatic. Therefore is crucial to investigate the distinct response of gastric epithelial cells to different H. pylori strains. Examination of the effects of H. pylori on gene expression in the gastric epithelial cells or mucosa has been reported in literature [17-19]. However, only a single strain or isogenic mutant strains of H. pylori were used in the previous studies. With the involvement of the bacterial factors, host susceptibility, and interaction of bacteria, environment and host immune response in vitro experiments using gastric cancer cell lines in co-cultured with a single strain of H. pylori can not fully elucidate the pathogenesis of H. pylori-related gastric diseases.
Microarray has been used to identify differences in gene content between H.
pylori strains that induce distinct pathological outcomes in an animal model [20].
In the present study, we report the different clinically isolated H. pylori strains would induce distinct gene expression patterns in gastric epithelial cells.
Understanding the interaction between H. pylori and gastric epithelial cells is an essential prerequisite in elucidating the underlying mechanisms of different H.
pylori-related diseases. The conventional method is to analyze the defined
signal pathway in co-culture model. This approach provides a reliable but limited clue. Applying the technology of microarray to study host-microbe interaction can generate a holistic view of signal alterations in a single experiment. This is the first study to show that the mRNA expression of gastric epithelial cells after infection by different strains of H. pylori is not general but
rather strain-specific. Of the three clinical isolates, the DU strain from the patient with duodenal ulcer and the GC strain from the patient of gastric cancer induced more oncogenes, protooncognes, or genes involved in carcinogenesis, such as fos, jun B, pim-1 and VEGF, etc. The expressions of these genes in AGS cells induced by H. pylori were similar to the result of previous study [17].However, the MA strain from the patient of gastric MALT lymphoma did not induce those genes. The result implied that the strain-specific factors play an important role in gastric carcinogenesis and the role of H. pylori strain in the pathogenesis of gastric MALT lymphoma is quite different to that of duodenal ulcer and gastric cancer. In addition to the difference of H. pylori strains, the genes up- or down-regulated in AGS cells induced by H. pylori are related to the MOI of bacteria, duration of coculture, and the type of microarray we used. This is why there are some differences in the genes induced by H. pylori between our report and previous studies[17-19].
C/EBPβ is one of the family of transcription factor, CAAT box enhancer binding proteins (C/EBPs). C/EBPβ is strongly upregulated at the transcription level by inflammatory stimuli, participates in both inflammatory and metabolic regulations, and controls cell cycle progression[21]. The increase in C/EBPβ results in accelerated entrance of liver cells to S phase for replication [22]. Our experiment showed that the GC and DU strains induced higher C/EBPβ transcription and translation in AGS cells than MA strains did. The result implied that the H. pylori may induce the signal pathway related to C/EBPβ in gastric carcinogenesis. Previous study had shown that the COX-2 expression was regulated by the expression of the C/EBPβ [23]. The production of COX-2 has been linked to different gastroduodenal diseases including ulcer and cancer. In
addition, increased activation of C/EBPβ has been reported to promote tumor invasiveness and render a malignant phenotype of renal cell carcinoma [24]
and C/EBPβ may be a target for tumor inhibition[25]. The role of C/EBPβ in the pathogenesis of H. pylori-related diseases has not been studied yet.
Collectively, these results suggest C/EBPβ may play a distinct role in the pathogenesis of H. pylori-related gastric carcinogenesis and ulcer formation and indicate microarray is a rapid and reliable way to identify targets involved in pathogenesis of H. pylori-related diseases. Further study for the role of C/EBPβ in gastric carcinogenesis is needed.
It is of fundamental importance to understand the main molecular pathways affected by H. pylori in epithelial cells in order to identify the critical steps of gastric carcinogenesis. In this study, we have identified distinct genes involved in different clinicaly isolated H. pylori infection. The results provide evidence of bacterial factors and molecular cross-talk between bacteria and the epithelial cells may determine the outcomes of H. pylori infection. Further studies in this line including the identification of the bacterial virulent factors, and elucidation of the different pathways involved, are critical in understanding gastric carcinogenesis and provide novel clues for diagnosis, treatment, and prevention of gastric cancer.
Acknowledgement
The study was supported in part by the grant from National Science Council (NSC91-3112-B002-007).
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