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[Cancer Research 63, 6350-6356, October 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Coexpression of Helicobacter Pylori’s Proteins CagA and HspB Induces Cell Proliferation in AGS Gastric Epithelial Cells, Independently from the Bacterial Infection1

Antonio De Luca2, Alfonso Baldi, Patrizia Russo, Anna Todisco, Lucia Altucci, Nicola Giardullo, Luigi Pasquale, Salvatore Iaquinto, Vittorio D’Onofrio, Maria Caterina Parodi, Marco G. Paggi and Gaetano Iaquinto2

Department of Medicine and Public Health, Section of Clinical Anatomy [A. D. L.], Department of Biochemistry and Biophysics "F. Cedrangolo," Section of Anatomical Pathology [A. B.], and Department of General Pathology and Oncology, "Centro sperimentale S. Andrea delle Dame," [L. A.], Second University of Naples, 80138 Naples; Laboratory C, Department for the Development of Therapeutic Programs, Regina Elena Cancer Institute, 00158 Rome [A. B., P. R., M. G. P.]; Pathology and Clinical Laboratory [A. T.] and Division of Gastroenterology [N. G., L. P., V. D., S. I., G. I.], San G. Moscati Hospital, 83100 Avellino; and Digestive Endoscopy and Gastroenterology Division, San Martino Hospital, 16100 Genoa [M. C. P.], Italy


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenocarcinoma of the stomach is the second most common cause of cancer mortality in the world. The purpose of this study was to evaluate the potential role in carcinogenesis of two secreted Helicobacter pylori’s proteins, CagA and HspB, both shown to increase the risk of gastric carcinoma in patients infected with H. pylori-positive strain. The effects of these two proteins on cell kinetics and the ability to selectively affect the expression of cell cycle-related proteins by transfection of a human gastric epithelial cell line (AGS) were analyzed. Using a genomic library of H. pylori, we isolated and cloned CagA and HspB. The effects of the overexpression of these proteins on cell growth were analyzed in AGS cells by immunoblots, proliferation assay, and flow cytometry. Coexpression of CagA and HspB in AGS cells in the first 48 h caused an increase of the level of E2F transcription factor, cyclin D3, and phosphorylated retinoblastoma protein, all involved in the G1-S checkpoint of the cell cycle. Consistently, an increase of cell proliferation, corresponding to an augment of the fraction of the cells in the S-G2-M phase of the cell cycle, was also demonstrated. Moreover, an increase of c-jun protein levels, but not of c-fos, was also found after coexpression of CagA and HspB. All these data suggest that CagA and HspB, independently from the bacterial infection, have a direct effect on the cell growth of the gastric cells acting on the G1-S checkpoint of the cell cycle.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gastric cancer is currently the second most prevalent cancer in the world (1, 2, 3) . Epidemiological studies have demonstrated an up to 6-fold increased risk of developing adenocarcinoma in patients infected with Helicobacter pylori, suggesting a link between persistent gastric H. pylori infection in human patients and the development of gastric carcinoma (4, 5, 6, 7, 8) . Because of this strong epidemiological association, H. pylori has been classified as a definite carcinogen by the International Agency for Research on Cancer (9) . The first observation suggesting that primary bacterial infection can lead to carcinogenesis has come from two independent groups, using Mongolian gerbils. In this animal model, long-term infection with H. pylori induced gastric cancer (10 , 11) . Moreover, topographic location and histology of these tumors were very similar to those observed in humans. Despite these advances, the molecular mechanisms by which bacterial infection leads to malignant transformation is not known at present. It has been suggested that H. pylori is not directly mutagenic but rather acts more indirectly favoring the formation of mutagenic substances (12 , 13) . Alternatively, it has been proposed that H. pylori infection increases the mutation rate in gastric mucosa by altering cell proliferation control (14) .

H. pylori has a colonization range restricted to gastric mucosa and produces a number of protein products, including urease, cytotoxin, flagella, CagA protein, Hsps, and adherence factors that contribute to its ability to colonize, to avoid host defenses, and to inflict damage to the host. Some strains of H. pylori have been shown to be more pathogenic than others. For example, strains that possess CagA are associated with increased severity of gastritis and with additional risk for developing atrophic gastritis and cancer (15) . Moreover, it has been demonstrated that after H. pylori infection, CagA is translocated into the epithelial cells, and it undergoes tyrosine phosphorylation in the host cells (16, 17, 18) . The phosphorylated form of CagA might function as a phosphatase that regulates host cell growth changes by H. pylori (19) .

CagA is not the only protein product of H. pylori that has been demonstrated to be associated with an increase ability of the bacterium to cause damage to the human organism. A few years ago, a protein of Mr 58,000 was cloned and characterized in some strains of H. pylori and was named upon its homology with the family of the heat shock proteins, HspB (20 , 21) . This protein has been shown to increase the risk of gastric carcinoma in patients with this H. pylori-positive strain (22) . In fact, separate groups obtained similar data showing that patients with adenocarcinoma of the stomach were serum positive to HspB (22 , 23) . Interestingly, in these cancer patients, the prevalence of serum IgG antibody to CagA was not significantly higher than in other gastric pathologies related to H. pylori. These data point out that H. pylori strain HspB+ could be associated with an increased risk of developing gastric cancer. However, the function of this protein in the pathogenesis of gastric cancer has not yet been investigated.

The ability of a cell to control its own replication is very important for the maintenance of the structure and functions of the organ it belongs to and, in final analysis, of the organism it is a part of. In fact, cellular replication must occur in faithful respect of a program, which establishes exact space-time repetition. Many pathologies are at the moment connected to an altered control of cellular replication. In the last 10 years, we have started to understand the critical phases of cellular replication that regulate cell cycle progression and the mechanisms through which the checkpoints operate. It is not a surprise that genes encoding the key proteins’ synthesis for the progression of cell cycle appear modified, deleted, or expressed in an abnormal way in cancer cells (24) .

This is the case of positive regulators such as cyclins and cdks3 or of negative regulators such as the inhibitors of kinases and oncosuppressor genes. Mammalian cdks are numerous, and each one of them acts in different parts of the cell cycle. These kinases are activated by larger proteins called cyclins, according to their cyclical expression and degradation. The catalytic subunits of these complexes are named cdks. The external environment of the cell manipulates cellular proliferation and differentiation by stimulating and/or inhibiting certain signal transduction pathways. However, each component of the cell cycle machinery, as it relates to cell division, has the potential to elicit or to contribute to a neoplastic phenotype (25) .

In the light of these findings, we performed several experiments to better elucidate the mechanisms and relationships between H. pylori proteins and their effect on cell cycle control of gastric epithelia. We examined in detail the effect of two H. pylori proteins (CagA and HspB) on cell kinetics and their ability to selectively affect the expression of cell cycle-related proteins using a human gastric epithelial cell line (AGS). Our data taken together provide additional information to further enhance our understanding of the molecular mechanism by which H. pylori proteins alter the growth status of the cell.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids.
Plasmid expression constructs for CagA (GeneBank accession no. AB090089) and HspB (accession no. X73840) were prepared by the insertion of PCR fragments into pcDNA3T7Tag, purchased by Invitrogen (Carlsbad, CA), using genomic DNA from H. pylori strain CCUG 17874 as a template. All oligonucleotides used as primers for PCR were synthesized by MWG Biotech (Ebersberg, Germany). Primers for HspB were 5'-gatcggatccatggcaaaagaaatcaaattttca-3' (sense) and 5'-gatcgaattcttacatcatgccgcccatgcctc-3' (antisense). Primers for CagA were 5'-gatcgaattcatgactaacgaaacc-3' (sense) and 5'-gatcggatccatagggggttgtatg-3' (antisense). PCR was performed according to the instructions of the GeneAmp kit used (Perkin-Perkin-Elmer Corp., Slelton, CT). Briefly, 30 cycles, including 1-min denaturation at 94°C, 1-min annealing at 48°C, and 2-min extension at 72°C, were followed by a 10-min extension at 72°C. DNA sequences of PCR-generated fragments and of plasmid constructs were confirmed by sequencing.

Cell Culture and Transfection.
AGS human gastric epithelial cells (American Type Culture Collection, Manassas, VA) were maintained in Ham’s F-12 medium supplemented with 10% FBS and 50 µg/ml penicillin-streptomycin (Life Technologies, Inc.). AGS cells were transfected with pcDNA3T7Tag, pcDNA3T7Tag-CagA, or pcDNA3T7Tag-HspB by use of Lipofectamine (Invitrogen) according to the manufacturer’s protocol. The total amount of plasmids was adjusted by using the empty vector plasmid in each assay. Briefly, 1 x 105 cells were plated in Ham’s F12 containing 10% FBS in 6-well plates 24 h before transfection. Transfections were performed with serum-free Ham’s containing 1 µg of plasmid constructs and 3 µl of Lipofectamine. After 5 h, fresh Ham’s F12 containing 10% FBS was added until 2 ml of final volume. Cells were collected at 24 and 48 h and were used to prepare protein extracts or to perform cell proliferation assay and cell cycle analysis. A transfection efficiency of at least 40% was reached for all of the experiments.

Immunoblotting.
Plates of 70–80% confluent AGS cells were transfected with 1 µg of plasmid constructs/plates by the Lipofectamine protocol. After 24 and 48 h, the cells were collected and lysed in 100 µl lysis buffer [50 mM Tris-Cl (pH 7.4), 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.1% Triton X-100, 0.1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin] for 30 min in ice. Lysates were centrifuged at 14,000 x g for 10 min at 4°C. Thirty µg of protein were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Millipore) in 3-(cyclohexylamino)propanesulfonic acid buffer [10 mM 3-(cyclohexylamino)propanesulfonic acid and 20% methanol (pH 11.0)]. The membrane was blocked with 5% milk in TBS-T buffer [2 mM Tris, 13.7 mM NaCl, and 0.1% Tween 20 (pH 7.6)] and then washed in TBS-T. Primary antibody for cyclins A, B1, B2, D1, D2, D3, E, cdks 1, 2, 4, 5, c-jun, c-fos, E2F1, and for the cdks inhibitors proteins p16, p21 and p27 were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The monoclonal antibody against TAG was purchased from Novagen (Darmstadt, Germany), and the monoclonal antiphosphotyrosine antibody (clone 4G150) was purchased from Upstate (Lake Placid, NY). Protein levels were tested by Bradford assay and normalized by Coomassie blue staining.

The primary antibodies, except the monoclonal antiphosphotyrosine, were incubated with the membrane in 3% milk for 2 h and then washed in TBS-T. The membrane was then incubated with anti-immunoglobulin coupled with horseradish peroxidase (Amersham Biosciences, Inc., Piscataway, NJ) and washed in TBS-T.

The transfected cells used to investigate the phosphotyrosine were collected at 24 and 48 h and lysed in 100 µl of lysis buffer HNTG (50 mM HEPES, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 mM EGTA, 1 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4) for 5 min in ice. Lysates were centrifuged at 14000 rpm for 10 min at 4°C. Thirty µg of protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane (Amersham Biosciences, Inc.) in Tris-glycine buffer (Bio-Rad). The membrane was blocked with 2% milk in NET 1x buffer [1.5 M NaCl, 500 mM Tris (pH 7.5), 50 mM EDTA (pH 7.5), and 0.5% Triton] and then washed in NET 1x. The primary antibody against phosphotyrosine was incubated with the membrane in 0.2% gelatin for 2 h and then washed in NET 1x. The membrane was then incubated with anti-immunoglobulin coupled with horseradish peroxidase (Amersham Biosciences, Inc.) and washed in NET 1x.

The presence of secondary antibody bound to the membrane was detected using the enhanced chemiluminescence system (DuPont NEN, Wilmington, DE).

Northern Blot.
Northern blot experiments to evaluate E2F1 expression at transcriptional level in AGS cells collected 48 h after transfection were performed three times in according to the protocol described previously (26) .

Cell Proliferation Assays.
AGS cells (1 x 105) were transfected with 0.5 µg of pcDNA3T7tag-CagA plus 0.5 µg of pcDNA3T7tag-HspB or with 1 µg of the empty vector alone. They were seeded (7 x 103) in complete medium on five replicate 96-well plates 24 and 48 h later. Cell number was determined by use of the cell proliferation II nonradioactive assay (Roche, Basel, Switzerland), which relies on the ability of a living cell to convert tetrazolium salts [XTT and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into a formazan product. The assay was performed in according to the manufacturer’s protocol. Briefly, after 3 h from cell seeding, 50 µl of tetrazolium salt (XTT-labeling mixture) was added and the cells were incubated for 4 h at 37°C. The formazan dye was measured after 4 h in an optimal visible spectrophotometric range of 450–500 nm (492 nm) with a Multiscan MS primary EIA. All of the experiments were performed three times.

Cell Cycle Analysis.
Unsynchronized cells in the mid-log phase were seeded at a density of 1 x 105 cells/6-well plate. After 24 h, cells were transfected by the Lipofectamine protocol. At 24 and 48 h, cells were collected by mild trypsination and gentle centrifugation and washed twice in PBS. A total of 2.5 x105 cells was collected and resuspended in 500 µl of a hypotonic buffer (0.1% Triton X-100, 0.1% sodium citrate, and 50 µg/ml propidium iodide). Cells were incubated in the dark at 4°C for at least 12 h. Samples were acquired on a FACSCalibur flow cytometer using the Cell Quest software (Becton Dickinson) and analyzed with standard procedures using the Cell Quest software and the ModFit LT Software (Becton Dickinson; Ref. 27 ). All of the experiments were performed three times.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CagA and HspB Can Be Efficiently Expressed in AGS Cells.
To determine whether CagA and HspB proteins were efficiently expressed in AGS cells, we transfected pcDNA3T7Tag, pcDNA3T7Tag-CagA, or pcDNA3T7Tag-HspB constructs. Cell cultured were collected at 24 and 48 h, and cell lysates were immunoblotted to detect the expression of both H. pylori proteins by using an anti-TAG monoclonal antibody. As depicted in Fig. 1ACitation , both CagA and HspB were expressed in AGS cells, after transfection. The NCTC 11638 CagA protein, derived from H. pylori strain CCUG17874, possesses several potential tyrosine phosphorylation sites. Of these, residues -893 and -912 are made by the EPIYA sequences that have been already demonstrated to be in vivo tyrosine phosphorylation sites of CagA in AGS cells (28) . Immunoblotting with an antiphosphotyrosine antibody on AGS revealed that the transfected CagA protein underwent tyrosine phosphorylation (Fig. 1B)Citation .



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  		Fig. 1. AGS cells were transiently transfected with an empty vector, with CagA, or with HspB. Western blotting was performed using an anti-TAG antibody (A) or an antiphosphotyrosine antibody (B) on cell extracts at 24 and 48 h after transfection.

 
Interestingly, no effect on the morphology of the transfected cells, compared with the wt-AGS cells, was ever detected.

Effects of CagA and HspB Overexpression on Cell Cycle-related Proteins in AGS Cells.
We examined the effects of CagA and HspB on the level of expression of proteins involved in the control of the cell cycle machinery. AGS cells were transfected with CagA alone, HspB alone, both proteins together and the empty vector alone as a control. Cells cultured in the same conditions were collected at 24 and 48 h. Cell lysates were immunoblotted to detect several members of the cell cycle machinery: cyclins D family members, cdks 4, 5, 6 and cdk inhibitors p16, p21, p27. These experiments were performed three times with the same results.

Overexpression of each protein alone led to a slight increase of cyclin D3 protein. Instead, over-expressing of CagA and HspB together led to an increase more than double of this cyclin compared with cells transfected to the empty vector (Fig. 2A)Citation . Statistical analysis (t-student test) confirmed that cyclin D3 differences in expression induced by CagA or HspB alone and by the two proteins together respect to the control were all statistically significant (P < 0.02). Intriguingly, among the cyclin D family members (cyclins D1, D2, and D3), only cyclin D3 was increased after over-expressing the two Helicobacter proteins (Fig. 2B)Citation .



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  		Fig. 2. AGS cells were transiently transfected with the empty vector, with CagA, with HspB and with CagA and HspB together and tested for the expression of cyclin D3 (A, left side), of the three members of the cyclin D family (B), of the cdk’s members cdks 4, 5, 6 (C), and for the cdk’s inhibitors p16-p21-p27 (D) on cell extracts at 24 and 48 h after transfection. On the right side of A, the densitometric analysis of cyclin D3 bands is shown.

 
On the basis of these observations we decided to perform additional experiments to evaluate the expression of proteins present in complex with cyclin D3. No changes of expression were observed either in the cdk partners of the cyclin D family: cdks 4, 5, and 6 (Fig. 2C)Citation or in the cdk inhibitors: p16, p21, and p27 (Fig. 2D)Citation .

However, the phosphorylated products of the tumor suppressor Rb, a substrate of the cdk-cyclin D complexes, were increased in AGS cells overexpressing the two proteins, as shown in Fig. 3ACitation . Because it has been shown that infection with H. pylori induces the activation of the AP-1 family of transcription factors in gastric epithelial cells (29) , we decided to look at the expression level of c-fos and c-jun in the AGS cells transfected with CagA and HspB. Indeed, we found that AGS cells overexpressing CagA, HspB, or both proteins displayed a statistically significant by (P < 0.02 with Student t test) higher level of expression of c-jun respect to control (Fig. 3B)Citation . Interestingly, the level of c-fos did not change significantly (data not shown).



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  		Fig. 3. AGS cells were transiently transfected with the empty vector, with CagA, with HspB and with CagA and HspB together. Western blotting was performed using an anti-Rb antibody (A) or anti-c-Jun antibody on cell extracts at 24 and 48 h after transfection (B, left side). On the right side of B, the densitometric analysis of c-jun bands is shown.

 
These data are compatible with an increase in cell proliferation, probably caused by a deregulation of the G1-S checkpoint of the cell cycle. Consistently, we found higher levels of the transcription factor E2F1, the function of which is to activate the transcription of genes required for the G1-S passage in the cell cycle in the transfected cells at 24 and 48 h. This increase in expression was observed both at transcriptional and translational level (Fig. 4)Citation . Interestingly, the increase in the transcriptional level of E2F was also observed when CagA or HspB alone were transfected, being CagA more effective. Statistical analysis (Student t test) confirmed that E2F1 differences in expression caused by overexpression of CagA and HspB were all statistically significant (P < 0.02).



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  		Fig. 4. AGS cells were transiently transfected with the empty vector, with CagA, with HspB and with CagA and HspB together. Differential expression of E2F1 was shown both at RNA level (Northern blot in A, left side; Lane 1, pcDNA3; Lane 2, CagA; Lane 3, HspB; Lane 4, CagA+HspB) and at protein level (Western blot in B, left side). On the right side of A and B, the densitometric analysis of E2F1 bands is shown.

 
Similar results on cell cycle-related proteins have been obtained using the NCI-N87: human epithelial from gastric carcinoma cell line and HeLa cell line, derived from a carcinoma of the uterine cervix (A. De Luca and G. Iaquinto, unpublished data).

H. Pylori Proteins CagA and HspB Induce Cell Proliferation Deregulating the Cell Division Cycle in AGS Cells.
We examined the direct effects of CagA and HspB on cell cycle-related events in the AGS gastric cancer cell line by a cell proliferation assay. This test is based on the spectrophotometric quantification of cell growth and viability because of the detection of the ability of a living cell to convert a tetrazolium salts [XTT and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] into a formazan product. We observed that the gastric cells cotransfected with CagA and HspB grew faster than the cells transfected with the empty vector alone or with only one of the two proteins, reaching a peak ~48 h (Fig. 5A)Citation . All of the experiments were performed three times with the same results. Statistical analysis by Student t student test confirmed that increase in the rate of cell proliferation caused by cotransfection of CagA and HspB was statistically significant respect to the other points (P < 0.02).



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  		Fig. 5. Cell proliferation assay on AGS cells. Coexpression of CagA and HspB after 48 h was able to induce an increase in cell proliferation compared with the empty vector (pcDNA3) and to the cells transfected with HspB or CagA alone. Data are representative of three independent experiments. SDs are shown (A). Fluorescence-activated cell sorting analysis: only coexpression of CagA and HspB was able to increase the S-G2-M fraction in AGS cells after 48 h (B). The percentage given is the average value obtained over three independent experiments. SDs are shown.

 
We used flow cytometry to analyze in greater detail the mechanisms by which CagA and HspB affect AGS cell proliferation. Indeed, analysis of unsynchronized AGS cells 48 h after cotransfection with CagA and HspB revealed an increase of the fraction of the cells in the S-G2-M phase of the cell cycle, compared with the controls. Interestingly, overexpression of CagA or HspB alone was not able to reach the same effect (Fig. 5B)Citation . All of the experiments were performed three times with the same results. Student t test showed that, indeed, the increase of the fraction of cells in the S-G2-M phase of the cell cycle caused by cotransfection of CagA and HspB was statistically significant (P < 0.02).

Taken together, our results suggest that H. pylori proteins CagA and HspB, independently of the mechanism of infection, influences AGS cell growth at least, in part, by altering the cell cycle control.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
At the end of 1983, several groups almost simultaneously reported the presence of spiral bacteria in patients with chronic gastritis and peptic ulceration. Now it is recognized and accepted that H. pylori infection is the most frequent cause of gastritis, peptic ulcer disease, and gastric adenocarcinoma around the world.

The molecular mechanisms by which H. pylori causes disease in humans remain unclear. The development of new molecular approaches based on the in vitro/in vivo studies of all of the major proteins of the H. pylori and the use of animal models is clarifying H. pylori pathogenesis and its role in the development of gastric cancer.

It has been shown that H. pylori strains expressing the cytotoxin-associated protein (CagA) induce a proto-oncogene activation that may represent an important step in the mechanism of H. pylori-induced neoplasia (29) . Moreover, it has been recently demonstrated that CagA protein secreted in the gastric cells is able to form a physical complex with SHP-2 tyrosine phosphatase, inducing a growth factor-like response in gastric epithelial cells (28) .

On the other hand, strains of H. pylori carrying HspB have been shown to increase the risk of gastric carcinoma (22 , 23) . Furthermore, several studies have shown that HspB is secreted by H. pylori and that it is detectable on the mucosal surface and within epithelial cells (30, 31, 32, 33) . Drawing from this background, we decided to look at the effects of the overexpression of these proteins in AGS cells, independently from any pathogenetic mechanism induced by the infection. We first demonstrated that both CagA and HspB were efficiently expressed in AGS cells under the described experimental conditions. Moreover, because it has been shown that after translocation in the gastric epithelial cells, CagA protein undergoes tyrosine phosphorylation (17 , 18) , we indeed demonstrated that the CagA protein transfected was tyrosine phosphorylated in our model.

Indeed, we have found that this efficient coexpression of CagA and HspB was able to influence AGS cell growth by inducing cell cycle proliferation through an increase in the S-G2-M phase of the cell cycle, as revealed by fluorescence-activated cell sorting analysis. The control of the G1-S-phase of the cell cycle is mainly governed by the functional unit composed by D-type cyclins, cdk4/6 kinases, p16, and Rb protein (25) . Indeed, the induction of cell proliferation in this experimental system was associated with a specific increase only in cyclin D3 protein and Rb gene product, in its phosphorylated forms. Consistently, an increase in the E2F1 transcription factor level, whose function is to activate the transcription of genes required for the G1-S passage in the cell cycle, was demonstrated. Interestingly, we found that both proteins were able to increase the transcriptional levels of E2F, being CagA more effective, and that cotransfection of CagA and HspB was able to reach a stronger increase. This does not exclude the possibility that also other molecular mechanisms such as the ubiquitin-proteasome-dependent degradation pathway are responsible for the high protein levels of E2F1 caused by transfection of these two proteins. Indeed, it has been recently demonstrated that E2FI protein levels are regulated by the ubiquitin-proteasome-dependent degradation pathway (34) . Because the three D-type cyclins are not functionally redundant but play distinct roles in the cell cycle (33) , the specific involvement of cyclin D3 in our system deserves additional investigation. Overexpression of cyclin D3 has been specifically linked to the progression of several tumors, including pancreatic adenocarcinoma, breast cancer, non-Hodgkin lymphoma, and thyroid carcinoma (35, 36, 37, 38, 39) . In particular, it has been proposed that elevated levels of cyclin D3 can titrate p21 and/or p27 away from cyclin D-cdk complexes, this resulting at the end in increased kinase activity (36 , 37) . The elevated levels of phosphorylated Rb protein found in our system is in agreement with this functional hypothesis.

D-Cyclins appear to be activated by growth factors at the transcriptional level in several cell types (25 , 27) . Moreover, H. pylori infection induces expression of the AP-1 family of transcription factors through the extracellular signal-regulated/mitogen-activated protein kinase cascade (28) . Recently, it has been demonstrated by Northern blot analysis that cyclin D1 transcription in AGS cells is enhanced by coculture with H. pylori and inhibited by mitogen-activated protein/extracellular signal-regulated kinase inhibitors, indicating that the mitogen-activated protein kinase pathway may be involved in intracellular signal transduction during H. pylori infection (40) . Therefore, we looked at the expression of c-fos and c-jun in our model. We found a significant increase in c-jun expression in AGS cells overexpressing both CagA and HspB, whereas c-fos level was unaffected. These data suggest that these H. pylori proteins induce expression of the proto-oncogene c-jun and this causes G1-S transition, specifically increasing cyclin D3 expression. Consistently, AP-1 sites have been found in the cyclin D3 promoter (41) .

Interestingly, expression of CagA or HspB proteins alone was not able to induce these effects in AGS cells. This observation may suggest that cooperation among different H. pylori’s proteins is necessary to induce cell cycle alterations in the infected cells.

In conclusion, we report here that the epithelial hyperproliferation observed in chronic H. pylori infection can be specifically stimulated in AGS cells by some of the bacterial protein products. Assuming that similar changes occur in nontransformed gastric epithelial cells, our findings suggest that the development of cancer is not attributable to a nonspecific accumulation of random mutations mainly caused by the synthesis of reactive oxygen species after the infection (42) or to an induction of hypergastrinemia caused by the host response to H. pylori infection (43) but may be triggered by hyperproliferative effects specifically because of some proteins produced by the pathogenic strains of H. pylori. Our experimental strategy based on the transfection of these proteins, indeed, eliminated all of the insults to the cells eventually caused by the infection of the culture with H. pylori. To the best of our knowledge, this is the first study showing a specific hyperproliferative effect on gastric epithelial cells caused by the action of two bacterial protein products, independently from any effect attributable to the mechanism of infection.

On the basis of recent published studies, the direct pathogenetic effect of CagA on gastric epithelial cells seems to involve the deregulation of SHP-2 pathway. However, the molecular mechanisms responsible of the pathogenic effect of HspB on gastric epithelial cells remains still unclear (44) . Finally, some limitations of this study should be acknowledged. First of all, this experimental system ignores possible effects of CagA or HspB on the fibroblasts of the gastric lamina propria that might also be targets of their action. Secondly, it is possible that with this transfection model, higher intracellular levels of CagA or HspB are obtained than are actually seen with H. pylori infection. Finally, AGS cells are derived from a tumor and this might partially affect the results because these cells are already transformed. Nevertheless, the mayor contribution of this work, which is the demonstration of a direct effect of two secreted H. pylori proteins on the cell cycle regulation of a gastric carcinoma cell line, is not weakened by these limitations. This experimental approach will allow to better understand the role of CagA and HspB. Moreover, similar effects of CagA and HspB on the expression of cell cycle-related proteins have been obtained using the HeLa cell line (derived from human cervix epithelial cell line). This suggests that CagA and HspB act on universal pathways involved in cell cycle regulation and that their effect is not specific only for gastric carcinoma cell lines. Nevertheless, the effects of CagA and HspB on apoptosis should be evaluated.

In conclusion, this study additionally contributes to the elucidation of the molecular mechanisms involved in the effects of H. pylori on cell cycle control and provides insights into the roles played by the organism’s proteins in gastric carcinogenesis. Nevertheless, it indicates possible molecular targets of diagnosis and therapy for this kind of neoplasm.


   ACKNOWLEDGMENTS
 
We thank Michael Latronico for editing the manuscript and Tullio Battista (Regina Elena Institute, Rome, Italy) for his excellent technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from Cheli Foundation, Malesci spa., San G. Moscati Hospital, Avellino; Second University of Naples, Futura-Onlus, and International Society for the Study of Comparative Oncology, Silver Spring, MD, USA. Back

2 To whom requests for reprints should be addressed: Dept. of Medicine and Public Health, Via L. Armanni 5, 80138 Naples, e-mail: antonio.deluca{at}unina2.it or Division of Gastroenterology, San G. Moscati Hospital, Viati, Italia, 83100 Avellino; e-mail: iaquintog{at}yahoo.it Back

3 The abbreviations used are: cdk, cyclin-dependent kinase; FBS, fetal bovine serum; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt; AP-1, activator protein 1; Rb, retinoblastoma; TBS-T, Tris Buffered Saline +0.1% Tween 20. Back

Received 3/12/03. Revised 7/ 7/03. Accepted 7/21/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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