点击显示 收起
1Second Department of Internal Medicine, Fukui Medical University, Fukui; 2Division of Molecular Oncology, Institute for Genetic Medicine and Graduate School of Science, Hokkaido University, Sapporo, Japan
Received 19 August 2002; revised 27 September 2002; electronically published 19 December 2002.
Recent experiments have indicated that CagA of Helicobacter pylori is injected into epithelial cells via the type IV secretion system and undergoes tyrosine phosphorylation in cells and that translocated CagA binds the SRC homology 2 domaincontaining tyrosine phosphatase (SHP-2). We investigated these phenomena in in vivo human gastric mucosa. Tyrosine-phosphorylated CagA and CagA-coimmunoprecipitated SHP-2 were detected in gastric mucosa from H. pyloripositive patients with atrophic gastritis and in noncancerous tissues from H. pyloripositive patients with early gastric cancer. In contrast, CagA was not detected in gastric mucosa with either intestinal metaplasia or cancer. Our results provide the first evidence that CagA is translocated into the gastric epithelial cells, receives tyrosine phosphorylation, and binds SHP-2 in in vivo human gastric mucosa. Deregulation of SHP-2 by CagA may play a role in the acquisition of a cellular-transformed phenotype at a relatively early stage of multistep gastric carcinogenesis.
This study was performed according to the principles of the Declaration of Helsinki, and consent was obtained from each individual after a full description of the nature and protocol of the study.
Financial support: Ministry of Education, Culture, Sports, Science and Technology, Japan (grant-in-aid 13670502 for Scientific Research on Priority Areas [C]); Japan Society for the Promotion of Science (grant-in-aid 14031210 for Scientific Research [C]).
Reprints or correspondence: Dr. Takeshi Azuma, Second Dept. of Internal Medicine, Fukui Medical University Matsuoka-cho, Yoshida-gun, Fukui 910-1193, Japan ().
Helicobacter pylori has been implicated in gastric carcinogenesis, on the basis of various epidemiological studies [1, 2]. A working group of the World Health Organization International Agency for Research on Cancer concluded in 1994 that H. pylori is a group 1 carcinogen in humans [3]. CagA is the product of the cagA gene, which is carried in virulent type I strains of H. pylori [4]. One-half to two-thirds of Western isolates are type I strain. cagA-positive H. pylori infection is associated with gastric mucosal atrophy and gastric cancer [5]. In contrast, nearly all eastern-Asian strains carry the cagA gene, regardless of clinical outcome. Recent in vitro studies have provided a molecular basis for the pathological effects that CagA has on gastric epithelial cells. After attachment of cagA-positive H. pylori to gastric epithelial cells, CagA is directly injected from the bacteria into the cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the host cells [68]. A recent study demonstrated that Src is the kinase of CagA phosphorylation [9]. The cagA-positive H. pylorihost cell interaction also triggers morphological changes similar to those induced by a growth factor: the hummingbird phenotype, characterized by elongation and spreading of cells [6]. Translocated CagA may be involved in dysregulation of host cell functions, thereby contributing to pathogenesis. Furthermore, CagA forms a physical complex with the SRC homology 2 domaincontaining tyrosine phosphatase (SHP-2) in a phosphorylation-dependent manner and stimulates phosphatase activity [10]. SHP-2 is actively involved in regulation of the spreading, migration, and adhesion of cells. Deregulation of SHP-2 by CagA may induce abnormal proliferation and movement of gastric epithelial cells. However, these findings were observed in in vitro systems such as in vitro H. pylori infection and in vitro transfection studies, which have used a human gastric cancer cell line, AGS. We therefore investigated CagA translocation, phosphorylation, and binding activity to SHP-2 in in vivo human gastric mucosa.
Subjects. Ten patients (6 men and 4 women [mean age, 52.5 years]) with atrophic gastritis and 5 patients (4 men and 1 woman [mean age, 54.8 years]; all had intestinal-type cancer) with early gastric cancer, all of whom were diagnosed by endoscopy, participated in the study. Five H. pylorinegative normal control subjects (3 men and 2 woman; mean age, 51.8 years) were also chosen from subjects who visited multiphasic health testing services at the Second Department of Internal Medicine, Fukui Medical University. The services included upper gastrointestinal endoscopy to screen for gastric cancer. A total of 8 biopsy specimens were obtained from each patient and normal control subjects: 4 from the greater curvature of the gastric antral mucosa and 4 from the greater curvature of the gastric fundic mucosa. Two from the antral or fundic mucosa were subjected to immunoblot analysis. One from the antral or fundic mucosa was subjected to histological analysis. One from the antral or fundic mucosa was subjected to culture for H. pylori. Endoscopic mucosal resection was performed on 5 patients with early gastric cancer. Tissue samples, 1 from a part of cancer tissue and 1 from noncancerous tissue surrounding cancer tissue, were also obtained from resected gastric mucosa.
Antibodies. The primary antibodies for immunoprecipitation and immunoblotting were an anti-CagA polyclonal antibody (Austral Biologicals), an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology), and an antiSHP-2 antibody (C-18; Santa Cruz Biotech).
Immunoprecipitation and immunoblotting. Biopsy and resected-tissue specimens were washed 3 times with 0.01 M PBS (pH 7.5) containing 2 mM Na3VO4 and were homogenized in an ice-cold lysis buffer (50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 5 mM EDTA, and 1% Triton X-100) that contained 2 mM Na3VO4, 2 mM phenylmethylsulfonyl sulfoxide, 10 g of leupeptin/mL, 10 g of trypsin inhibitor/mL, and 10 g of aprotinine/mL. The homogenized samples were centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was subsequently immunoprecipitated, with either the anti-CagA polyclonal antibody or normal control IgG, for 30 min at 4°C, after which protein GSepharose beads (Amersham Pharmacia Biotech) were added for 90 min at 4°C. The immunoprecipitates were washed 4 times with the lysis buffer and then were boiled with an electrophoresis SDS sample buffer (2% SDS, 10% glycerol, 6% 2-mercaptoethanol, 0.003% bromophenol blue, and 50 mM Tris/HCl [pH 6.8]) for 5 min. Equal amounts of samples from immunoprecipitates were separated by SDS-PAGE (7.5% polyacrylamide) and were blotted onto Immobilon P (Millipore). The membranes were blocked with either 1% bovine serum albumin or 5% skim milk in T-TBS (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, and 0.5% Tween 20) and were incubated with a primary antibody in T-TBS overnight at 4°C. After being washed with T-TBS, the membranes were incubated with horseradish peroxidaseconjugated goat anti-rabbit or anti-mouse IgG polyclonal antibodies in T-PBS for 1 h and were visualized with an enhanced chemiluminescence-detection system, according to directions supplied by the manufacturer (Amersham Pharmacia Biotech).
H. pylori culture. Gastric biopsy specimens from each patient were inoculated onto a trypticase soy agar (TSA)II/5% sheep blood plate and cultured under microaerobic conditions (5% O2, 15% CO2, and 80% N2) at 37°C for 5 days. A single colony was picked from each primary culture plate, inoculated onto a fresh TSA-II plate, and cultured under the conditions described above. H. pylori was identified as a gram-negative spiral bacilli with urease activity. A few colonies were picked from each plate and were transferred into 20 mL of Brucella broth liquid culture medium that contained 10% fetal calf serum and were cultured for 3 days under the same conditions as described above. DNA from each H. pylori isolate was extracted from the pellet of the liquid culture sample by the protease/phenol-chloroform method, suspended in 300 L of a TE buffer (10 mM Tris HCl and 1 mM EDTA), and stored at 4°C until PCR amplification.
PCR analysis of the cagA gene. We examined the cagA gene by PCR among isolates from the patients. The 3 region of cagA was amplified by PCR using the following primers: forward primer 5-GAATTGTCTGATAAACTTGAAA and reverse primer 5-GCGTATGTGGCTGTTAGTAGCG. PCR conditions were as follows: heating at 94°C for 5 min, followed by 25 cycles consisting of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The tubes were kept at 72°C for 7 min before storage at 4°C. PCR products were examined by 2% agarose gel electrophoresis to detect the cagA gene.
Results. Recent in vitro studies have provided a molecular basis for the pathological effects that CagA has on gastric epithelial cells. CagA protein of H. pylori is injected into epithelial cells via the bacterial type IV secretion system and undergoes tyrosine phosphorylation in the cells, and that translocated CagA forms a physical complex with SHP-2. In the present study, we detected the tyrosine-phosphorylated CagA protein and CagA-coimmunoprecipitated endogenous SHP-2 in both gastric fundic and gastric antral mucosa from all H. pyloripositive patients with atrophic gastritis but not from H. pylorinegative control subjects (). H. pylori strains from these patients were cagA-positive strains. The cagA gene was detected by PCR in all H. pylori strains examined in the study. Of interest, the CagA protein, tyrosine-phosphorylated CagA, and CagA-coimmunoprecipitated endogenous SHP-2 were not detected in the gastric mucosa with intestinal metaplasia or cancer, although they were detected in noncancerous mucosal tissue from all H. pyloripositive patients with early gastric cancer who were examined in the study ().
fig.ommitted
Figure 1. Immunoblot analysis of human gastric mucosa by anti-CagA, anti-phosphotyrosine (Antip-Tyr), and antiSHP-2 antibody. Tyrosine-phosphorylated CagA and CagA-coimmunoprecipitated endogenous SHP-2 were detected in both gastric fundic (lane 1) and gastric antral mucosa (lane 2) from H. pyloripositive atrophic gastritis patients but not in tissue from H. pylorinegative control subjects (fundic mucosa [lane 3] and antral mucosa [lane 4]).
fig.ommitted
Figure 2. Top (full-color panels), Histological findings in tissue samples adjacent to samples used for the immunoblot analysis shown in figure 1 (scale bars, 20 M), including (A) a biopsy specimen from fundic mucosa, showing moderate inflammatory infiltration; (B) a biopsy specimen from antral mucosa, severe inflammatory infiltration; (C) a part of resected gastric mucosa surrounding cancer tissue, showing accompanying intestinal metaplasia; and (D) a part of cancer tissue, showing tubular adenocarcinoma without submucosal invasion. Tyrosine-phosphorylated CagA and CagA-coimmunoprecipitated endogenous SHP-2 were not detected in the gastric mucosa of those with either intestinal metaplasia (C) or cancer (D), although they were detected in noncancerous mucosal tissue in the same patient (A [fundic mucosa] and B [antral mucosa]). Bottom (3 gels), Immunoblot analysis of human gastric mucosa by anti-CagA, anti-phosphotyrosine (Antip-Tyr), and antiSHP-2 antibody in a case of intestinal-type early gastric cancer.
Discussion. To our knowledge, the present study provides the first compelling evidence that CagA is actively translocated from bacteria to gastric epithelial cells, receives tyrosine phosphorylation, and binds SHP-2 in in vivo human gastric mucosa. Correa's model of gastric carcinogenesis suggests that the process may be a continuous progression whereby lesions of increasing severity develop over 2 or 3 decades: acute gastritis progresses to chronic gastritis, then to chronic atrophic gastritis with the development of intestinal metaplasia, and finally to frank gastric carcinoma [11]. It has been estimated that 10% of patients with chronic atrophic gastritis develop gastric cancer within 15 years. Intestinal metaplasia with goblet cells has been associated with gastric cancer in up to 90% of cases and is considered to be a good histological marker of premalignancy. Chronic gastritis induced by H. pylori infection usually progresses to atrophic gastritis. The risk of gastric cancer increases with the degree and the extent of atrophic gastritis. Severe glandular atrophy develops, and intestinal metaplasia occurs, accompanied by the disappearance of H. pylori colonization. This is the reason of our finding that CagA protein, tyrosine-phosphorylated CagA, and CagA-coimmunoprecipitated endogenous SHP-2 were not detected in the gastric mucosa with intestinal metaplasia or cancer. It is therefore suspected that H. pylori infection plays a causative role at a relatively early phase of gastric carcinogenesis.
CagA translocated from H. pylori into gastric epithelial cells can perturb mammalian signal transduction machinery and modify cellular function by physically interacting with a host cell protein, SHP-2, during the infection [10]. SHP-2, like its Drosophila homolog Corkscrew, is known to play an important positive role in the mitogenic signal transduction that connects receptor tyrosine kinases and ras [12]. SHP-2 is also actively involved in regulation of the spreading, migration, and adhesion of cells. Translocated CagA may play important roles in the pathogenicity of H. pylori infection. It is possible that deregulation of SHP-2 by translocated CagA may play a role in the acquisition of a cellular-transformed phenotype at a relatively early stage of multistep carcinogenesis in gastric cancer.
An animal model using Mongolian gerbils has successfully demonstrated that long-term infection with H. pylori induces gastric adenocarcinoma [13]. H. pylori infection also has been found to enhance N-methyl-N-nitrosoureainduced adenocarcinoma of the glandular stomach in Mongolian gerbils [14]. These Mongolian-gerbil models demonstrate the initiator and promoter effect of H. pylori infection on gastric carcinogenesis. Although the pathogenesis of the tumor is unknown, one possible explanation is increased epithelial turnover in the inflamed mucosa. Enhanced cell replication increases the frequency of mutation, by errors in gene replication, by conversion of endogenous or exogenous DNA adducts to mutation, or by decreasing the time allowed for DNA-repair processes. In the Mongolian-gerbil model, prominent regenerative hyperplasia is a characteristic feature and is consistently observed. This is suggestive of persistently and highly enhanced cellular proliferation in the gastric mucosa in this model. It has also been reported that H. pylori infection induces an increase in regenerative proliferation activity in human gastric mucosa [15]. It is possible that deregulation of SHP-2 signaling pathways by translocated CagA may induce abnormal proliferation and movement of gastric epithelial cells, initiating or promoting gastric carcinogenesis. Further molecular analysis is necessary to clarify the pathological mechanism of gastric adenocarcinoma induced by H. pylori infection.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Correa P. A human model of gastric carcinogenesis. Cancer Res 1988; 48:355460.
12.
13.
14.
15.