| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Carcinogenesis |
Division of Infectious Diseases, Departments of Medicine and Microbiology and Immunology [T. L. C.], and Division of Gastroenterology, Departments of Medicine and Cancer Biology [U. S. K., D. A. I., R. M. P.], Vanderbilt University School of Medicine, Nashville, Tennessee 3722-2605, and Department of Veterans Affairs Medical Center, Nashville, Tennessee 37212 [T. L. C., R. M. P.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
|---|
6–27) that lacks a unique strongly hydrophobic region near the VacA NH2 terminus. Moreover, when an equimolar mixture of purified VacA 6–27 and purified wild-type VacA were added simultaneously to AGS cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the apoptosis-inducing activity of wild-type VacA. These results indicate that VacA induces gastric epithelial cell apoptosis and suggest that differences in levels of gastric mucosal epithelial apoptosis among H. pylori-infected persons may result from strain-dependent variations in VacA structure. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
|---|
One strain-specific H. pylori locus that has been associated with an increased risk for carcinogenesis is vacA, which encodes a secreted bacterial toxin (VacA; Refs. 9, 10, 11, 12, 13, 14 ). When added to mammalian cells in vitro, VacA induces multiple structural and functional alterations in cells, the most prominent of which is the formation of large intracellular vacuoles (15) . Vacuole formation in response to purified VacA is dependent on the presence of weak bases such as ammonia, as well as on internalization of the toxin by cells (16, 17, 18, 19, 20, 21) .
A vacA gene is present in virtually all of the H. pylori strains examined (10 , 22) ; however, strains vary considerably in the production of vacuolating cytotoxin activity. This variation is primarily attributable to variations in vacA gene structure. The regions of greatest diversity are localized near the 5' end of vacA (allele families s1a, s1b, s1c, or s2) and in the mid-region of vacA (allele families m1 or m2; 22, 23, 24 ). Most type s1 VacA toxins possess detectable vacuolating cytotoxic activity in vitro, whereas type s2 VacA proteins possess little if any cytotoxic activity (22) . This is attributable to the presence of a 12-amino-acid hydrophilic segment at the NH2 terminus of type s2 toxins, which abolishes cytotoxic activity (25 , 26) .
H. pylori strains that possess a type s1/m1 vacA allele are associated with an increased risk of gastric cancer (27, 28, 29, 30) and enhanced gastric epithelial cell injury (31 , 32) compared with vacA s2/m2 strains. This relationship between s1/m1 alleles and gastric cancer is consistent with investigations that have examined the distribution of vacA genotypes throughout the world. In regions in which the background rate of distal gastric cancer is high, such as Colombia and Japan, most H. pylori strains contain type s1/m1 alleles (24) .
Host responses to the presence of H. pylori are likely to be important in affecting the threshold for carcinogenesis. In several studies, H. pylori has been associated with increased levels of apoptosis in human gastric mucosa (33, 34, 35, 36) . The capacity of H. pylori to induce apoptosis of gastric epithelial cells also has been demonstrated in murine (37, 38, 39) and Mongolian gerbil (40 , 41) models of infection.
Cell culture model systems have been used to investigate mechanisms through which H. pylori induces apoptosis. These studies have demonstrated that multiple H. pylori factors, including urease (42)
, products of the cag pathogenicity island (43)
, and lipopolysaccharide (44)
contribute to apoptosis. H. pylori urease has been shown to bind to class II MHC molecules and induce apoptosis in KatoIII and N87 gastric epithelial cells (42)
. H. pylori can also stimulate apoptosis in vitro by inducing expression of the cell-surface receptor Fas and Fas ligand (36
, 45, 46, 47)
, or by activating the transcription factor NF-
B (48)
.
In addition to these pathways, several lines of evidence suggest that H. pylori VacA may induce gastric epithelial cell apoptosis. In a previous study, we demonstrated that a wild-type VacA-producing strain induced apoptosis of gastric epithelial cells to a greater extent than did an isogenic mutant strain, based on flow cytometry analysis (43) . Another study has shown that broth culture supernatant from a toxin-producing H. pylori strain induced apoptosis of AGS cells (49) . However, at present, it is unclear whether VacA alone is sufficient to induce apoptosis or whether additional H. pylori factors are required. Thus, the goal of this study was to investigate further the capacity of VacA to induce gastric epithelial cell apoptosis.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
|---|
6–27) containing a 22-amino-acid internal deletion near the NH2 terminus (52)
. An isogenic ureB mutant was generated by insertional mutagenesis as previously described (43)
, using pMK180, which contains aphA (conferring kanamycin resistance) cloned into ureB (53)
. Isogenic ureB mutants were selected on Brucella agar with kanamycin (25 µg/ml) and confirmed to lack urease activity.
H. pylori Culture
For coculture experiments with gastric epithelial cells, H. pylori were grown in Brucella broth with 5% FBS for 24–48 h, harvested by centrifugation (2000 x g), and resuspended in antibiotic-free RPMI 1640 with 10% FBS to a concentration of 1 x 108 colony forming units/ml. For all of the experiments, H. pylori were added to cells at a bacteria:cell concentration of 100:1, based on previous reports that H. pylori reproducibly induce apoptosis in AGS cells at this ratio (43
, 45)
. Bacteria were identified as H. pylori by urease and oxidase activity as well as by Gram’s stain morphology.
Preparation of H. pylori Broth Culture Supernatants and VacA Purification
H. pylori strains were grown in sulfite-free broth (50)
containing either 5% FBS or 0.5% activated charcoal. Cultures were incubated on a rotary shaker for 48 h at 37°C in ambient air containing 5% CO2, and the cultures then were centrifuged. Broth culture supernatants from cultures containing FBS were concentrated 30-fold by ultrafiltration (Millipore) and passed through a 0.2-µm filter. VacA was purified from broth culture supernatants as described previously (50)
and then was dialyzed in PBS. Protein concentrations were determined using a Micro-BCA assay (Pierce). Acid activation of VacA was accomplished by dropwise addition of 250 mM HCl to the purified toxin until a pH of 3.0 was reached (54)
.
Cell Culture
AGS human gastric epithelial cells (ATCC CRL 1739) were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FBS and 20 µg/ml gentamicin in an atmosphere of 5% CO2 at 37°C. Coculture experiments, in which AGS cells were incubated with viable H. pylori, were performed in antibiotic-free media containing 10% FBS, using 12- or 96-well polypropylene tissue culture plates (Nunc, Denmark). AGS cells were not serum starved, and remained subconfluent during each assay. Purified, acid-activated VacA preparations were added to AGS cells in medium with or without 5 mM ammonium chloride. The addition of ammonium chloride did not change the pH of the tissue culture medium.
Assessment of Apoptosis
DNA Fragmentation ELISA.
DNA fragmentation was quantified using a commercially available ELISA (Roche, Indianapolis, IN) that detects nucleosomal fragments in cytoplasmic fractions of cells undergoing apoptosis but not in cells undergoing necrosis (41)
. For these experiments, 5 x 103 AGS cells/well in 96-well plates were incubated in triplicate with H. pylori (5 x 105 colony forming units/well), filtered supernatants, purified VacA, or medium alone for 24–48 h. Cells were then lysed according to the manufacturer’s instructions and centrifuged, and supernatants were analyzed by ELISA exactly as described previously (41)
. Absorbance was measured at 405 nm.
DNA-specific Fluorochrome Staining.
AGS cells were cultured with or without purified VacA for 48 h, fixed with ice-cold methanol for 10 min, incubated with 1 µg/ml propidium iodide for 5 min, and visualized by fluorescence microscopy. For each sample, at least 100 cells were evaluated by an independent observer (U.S.K.) unaware of experimental conditions. Results are expressed as the mean number of cells with highly condensed nuclei and fragmented chromatin/total number of cells counted.
|
Statistics |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
|---|
0.05 were considered significant. |
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
|---|
), compared with AGS cells incubated in medium alone. In contrast, coculture with the vacA null mutant did not significantly increase apoptosis compared with controls (Fig. 1)
. The addition of exogenous ammonium chloride did not alter apoptosis induced by the live wild-type H. pylori strain (Fig. 1)
. Because H. pylori urease has been reported to induce apoptosis in other experimental systems (42)
, we also tested an isogenic urease-negative mutant of 60190 and found that apoptosis after coculture with the urease-negative mutant did not differ substantially from apoptosis after coculture with the wild-type strain (data not shown).
|
. These findings indicate that culture supernatant from a toxigenic H. pylori strain, but not from its isogenic vacA null derivative, can induce DNA fragmentation, a phenotype consistent with apoptosis.
|
. In contrast, in the absence of ammonium chloride, VacA failed to increase DNA fragmentation (Fig. 3)
. As expected, treatment of purified VacA with protease (attached to agarose beads) abolished the capacity of the preparation to induce apoptosis, which indicates that the apoptotic activity was attributable to a protein and not attributable to any potential nonprotein contaminants, such as lipopolysaccharide.
|
, features consistent with apoptosis. Under the experimental conditions of this study, VacA induced detectable nuclear morphological abnormalities in 28 ± 6% of cells (Fig. 4B)
. Thus, results of both a DNA fragmentation assay and propidium iodide staining indicate that purified VacA can induce apoptosis in AGS cells, but these events require acid activation of the purified toxin and the presence of supplemental ammonium chloride.
|
.
|
. In contrast, the s2/m1 VacA protein induced relatively little cell vacuolation, and apoptosis was not altered by the acidified s2/m1 chimera (Fig. 6)
.
|
6–27) that lacks a critical hydrophobic region near the VacA NH2 terminus (52)
. VacA 6–27 is secreted by H. pylori and does not exhibit any defects in binding or uptake by cells; however, it is unable to induce vacuolation (52)
. Because addition of a type s2 sequence to the VacA NH2 terminus abolished the ability to stimulate apoptosis (Fig. 6)
, we next investigated whether VacA 6–27 could induce apoptosis. AGS cells were incubated with acidified wild-type VacA, VacA 6–27, or buffer alone for 48 h in the presence of ammonium chloride; and apoptosis was quantified by ELISA. As expected, based on previous experiments with HeLa cells, wild-type VacA induced extensive vacuolation of AGS cells, whereas VacA 6–27 did not (data not shown), confirming that this hydrophobic region was required for cell-vacuolating activity (52)
. VacA 6–27 also lacked the capacity to induce apoptosis (Fig. 6)
.
VacA 6–27 Exerts a Dominant Negative Effect on Apoptosis Induced by Wild-Type VacA.
We have shown previously that when an equimolar mixture of wild-type VacA and VacA 6–27 are added to HeLa cells, the mutant toxin exhibits a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA (52)
. Having established that VacA 6–27 failed to stimulate apoptosis, we next tested the possibility that VacA 6–27 could inhibit apoptosis induced by wild-type VacA. As expected, acid-activated wild-type toxin significantly increased apoptosis compared with buffer-treated controls (Table 1)
. When premixed with wild-type VacA at ratios ranging from 1:1 to 1:16, acid-activated VacA 6–27 inhibited DNA fragmentation induced by wild-type VacA in a dose-dependent manner; complete inhibition of apoptotic activity was detected when the ratio of mutant:wild-type VacA was 1:2 (data not shown). These coincubation experiments indicate that VacA 6–27 can effectively block the apoptotic activity of wild-type VacA and suggest that the molecular mechanisms underpinning VacA-induced vacuolation also regulate VacA-dependent apoptosis.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
|---|
B and induction of IL-8 (60, 61, 62)
, similar to the effects of H. pylori observed within colonized gastric mucosa (60
, 63)
. Therefore, this in vitro system provides useful insights into the interaction of H. pylori with gastric epithelial cells in the human stomach.
We and others have previously reported that VacA contributes to the induction of apoptosis in AGS cells by demonstrating a loss of phenotype using isogenic vacA null mutant strains (43
, 49)
. In the present study, we demonstrate that purified VacA can induce apoptosis in the absence of other H. pylori factors. Importantly, acid-activation of VacA was required in order for the purified toxin to exert its apoptotic effects. In previous studies, we have shown that acidification of VacA results in disassembly of the toxin’s oligomeric structure and enhances its internalization into mammalian cells (21
, 50) . Thus, it seems likely that the internalization of VacA may be required for the toxin to induce apoptosis. Galmiche et al. (64)
provided additional insights into these events by showing that VacA fragments that are expressed in transiently transfected nongastric (HeLa) cells, insert into mitochondrial membranes, induce cytochrome c release, and activate the caspase-3-dependent cell-death-signaling cascade. These findings are consistent with recent observations that activated caspase-3 is increased within gastric mucosa of H. pylori-infected humans and monkeys compared with uninfected tissue (65)
. Interestingly, Staphylococcus aureus 
-toxin, a soluble pore-forming toxin, has been reported to induce mitochondrial release of cytochrome c into the cytoplasm of lymphocytes, which is followed by the sequential activation of caspases 8, 9, and 3, ultimately leading to apoptosis (66)
. Although there may be similarities between the mechanisms by which VacA and S. aureus -toxin induce apoptosis, the specific intracellular signaling cascades that are activated by full-length VacA in gastric epithelial cells remain to be determined.
In the present studies, supplementation of tissue culture medium with ammonium chloride was required in order for purified VacA to induce apoptosis. Similarly, supplemental ammonium chloride is required for purified VacA to induce cell vacuolation. Recent studies have reported that ammonium alone is able to induce apoptosis in gastric epithelial cells (67 , 68) ; and in one study, ammonium-induced apoptosis was accompanied by cytochrome c release and the activation of caspases 9 and 3 (68) . H. pylori produces ammonia via multiple pathways, one of the most important of which is urease-mediated hydrolysis of urea (69) . In addition to generating ammonia, urease has been shown to induce apoptosis in KATOIII and N87 gastric epithelial cells in vitro by binding to MHC class II antigens (42) , which are expressed on gastric epithelial cell surfaces and up-regulated in the presence of H. pylori (70) . However, inactivation of ureB in the present study had no effect on the ability of H. pylori to induce apoptosis in AGS cells. Therefore, the capacity of urease to induce apoptosis, either directly by binding to MHC class II antigens or indirectly by generating a requisite cofactor (ammonia) for VacA-dependent apoptosis, may be cell-line dependent.
Two forms of VacA that fail to induce cell vacuolation (VacA 6–27 and s2/m1 VacA; Refs. 26
and 52
) also failed to induce apoptosis. In comparison with wild-type VacA, these two inactive forms each contain a mutation or modification near the NH2 terminus of the toxin (26
, 52)
. This suggests that functions attributable to the NH2 terminus of VacA are required for both cell-vacuolating and apoptosis-inducing activity. VacA 6–27 and s2/m1 VacA each form anion-selective membrane channels significantly less efficiently than does wild-type VacA (26
, 52)
. Thus, it seems likely that membrane channel formation may be required for both VacA-induced cell vacuolation and VacA-induced apoptosis. In previous studies, we have shown that VacA 6–27 acts as a dominant negative mutant protein (26
, 52)
. We now show that, in addition to inhibiting the vacuolating activity of wild-type VacA, this dominant negative mutant inhibits the apoptotic activity of wild-type VacA. The mechanism of inhibition likely involves the formation of hetero-oligomeric complexes, comprised of both wild-type VacA and mutant VacA (26)
, which are defective in both vacuolating activity and apoptotic activity.
Although VacA and various other H. pylori factors can independently induce apoptosis in isolated in vitro systems, apoptosis in vivo is likely to be influenced by numerous host mediators present within inflamed mucosa. IFN-
, a cytokine that is increased within colonized mucosa, is synergistic with H. pylori in inducing Fas-Fas ligand-regulated apoptosis of gastric epithelial cells in vitro (45
, 71)
. H. pylori infection of IFN--deficient mice leads to decreased levels of gastric inflammation compared with levels in infected wild-type mice (72)
, and Helicobacter felis infection of Fas-deficient mice is also associated with reduced levels of inflammation compared with levels in infected wild-type mice; mucosal apoptosis scores are decreased in parallel (38)
. In contrast, H. felis infection of mice lacking secretory phospholipase A2 leads to increased levels of epithelial cell apoptosis compared with levels in infected wild-type mice (37)
. We have recently shown that H. pylori can induce apoptosis in gastric epithelial cells in vitro by activating the transcription factor NF-B (48)
, and activated NF-B is present within gastric epithelial cells of infected but not of uninfected persons (60)
. IL-1ß, which is increased within inflamed mucosa (73)
, can stimulate multiple intracellular signaling pathways involved in apoptosis, including Fas and NF-B (45
, 60
, 71)
. Thus, differing levels of IL-1ß expression within H. pylori-colonized gastric mucosa, associated with host-specific IL-1ß polymorphisms (74)
, may contribute to various levels of apoptosis in vivo.
The development of intestinal-type gastric adenocarcinoma involves progression through a well-defined series of histological steps, initiated by the transition from normal mucosa to chronic superficial gastritis, followed by the appearance of atrophic gastritis and intestinal metaplasia, and, finally, dysplasia and adenocarcinoma (1) . The risk for developing gastric cancer is >90-fold higher in patients with severe multifocal atrophic gastritis than in patients with normal mucosa (75) . Apoptosis in response to VacA and other H. pylori factors may play an important role in the process by which gastric cancer develops in H. pylori-infected humans. The exact mechanisms by which H. pylori-associated apoptosis may predispose to gastric cancer are not yet entirely clear, but enhanced rates of cell loss could potentially accelerate the development of gastric atrophy or intestinal metaplasia (76) . In addition, apoptosis may contribute to compensatory hyperproliferation, which may also increase the risk for gastric cancer.
In conclusion, the present studies provide strong evidence that VacA stimulates apoptosis in AGS cells. Addition of a type s2 sequence to the VacA NH2 terminus abolishes the capacity of the toxin to stimulate apoptosis. These results suggest that heterogeneity in levels of gastric mucosal cell apoptosis among H. pylori-infected persons may result from differences in the structure and activity of VacA proteins produced by different H. pylori strains.
|
FOOTNOTES |
|---|
1 Supported in part by NIH Grants DK 58587,CA77955, DK 53623, AI 39657, and by the Medical Research Service of the Department of Veterans Affairs. 
2 To whom requests for reprints should be addressed, at Division of Gastroenterology, Vanderbilt University School of Medicine, 1161 21st Avenue South, C-2104 Medical Center North, Nashville, TN 37232-2279. Phone: (615) 322-5200; Fax: (615) 343-6229; E-mail: richard.peek{at}vanderbilt.edu
3 The abbreviations used are: IL, interleukin; NF, nuclear factor; FBS, fetal bovine serum.
Received 8/26/02. Accepted 1/ 6/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSStatisticsRESULTSDISCUSSIONREFERENCES |
|---|
B-mediated apoptosis induced by Helicobacter pylori in gastric epithelial cells. J. Biol. Chem., 276: 31059-31066, 2001.B in gastric epithelial cells. Gastroenterology, 113: 1099-1109, 1997.[Medline]
B in gastric epithelial cells. J. Immunol., 160: 2401-2407, 1998.B through a signaling pathway involving IB kinases. NF-B-inducing kinase, TRAF2, and TRAF6 in gastric cancer cells. Gastroenterology, 119: 97-108, 2000.[Medline]
-Toxin is a mediator of Staphylococcus aureus-induced cell death and activates caspases via the intrinsic death pathway independently of death receptor signaling. J. Cell Biol., 155: 637-648, 2001., gene-deficient mice. J. Immunol., 165: 1022-1029, 2000., interleukin-1ß, and interleukin-8 production in patients with Helicobacter pylori infection. Scand. J. Gastroenterol., 29: 425-429, 1994.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. T. Franco, D. B. Friedman, T. A. Nagy, J. Romero-Gallo, U. Krishna, A. Kendall, D. A. Israel, N. Tegtmeyer, M. K. Washington, and R. M. Peek Jr. Delineation of a Carcinogenic Helicobacter pylori Proteome Mol. Cell. Proteomics, August 1, 2009; 8(8): 1947 - 1958. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-R. KI, I.-H. HONG, J.-K. PARK, K.-S. HONG, O.-K. HWANG, J.-Y. HAN, A.-R. JI, S.-I. PARK, S.-K. LEE, S.-E. YOO, et al. Potent Neutralization of Vacuolating Cytotoxin (VacA) of Helicobacter pylori by Immunoglobulins Against the Soluble Recombinant VacA Anticancer Res, June 1, 2009; 29(6): 2393 - 2402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Haesebrouck, F. Pasmans, B. Flahou, K. Chiers, M. Baele, T. Meyns, A. Decostere, and R. Ducatelle Gastric Helicobacters in Domestic Animals and Nonhuman Primates and Their Significance for Human Health Clin. Microbiol. Rev., April 1, 2009; 22(2): 202 - 223. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. P. Tobin, G. T. Henehan, R. P. Murphy, J. C. Atherton, A. F. Guinan, S. W. Kerrigan, D. Cox, P. A. Cahill, and P. M. Cummins Helicobacter pylori-induced inhibition of vascular endothelial cell functions: a role for VacA-dependent nitric oxide reduction Am J Physiol Heart Circ Physiol, October 1, 2008; 295(4): H1403 - H1413. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Peek Jr Helicobacter pylori infection and disease: from humans to animal models Dis. Model. Mech., July 1, 2008; 1(1): 50 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-R. Ki, H.-R. Lee, M.-J. Goo, I.-H. Hong, S.-H. Do, D.-H. Jeong, H.-J. Yang, D.-W. Yuan, J.-K. Park, and K.-S. Jeong Differential regulation of ERK1/2 and p38 MAP kinases in VacA-induced apoptosis of gastric epithelial cells Am J Physiol Gastrointest Liver Physiol, March 1, 2008; 294(3): G635 - G647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. T. Franco, E. Johnston, U. Krishna, Y. Yamaoka, D. A. Israel, T. A. Nagy, L. E. Wroblewski, M. B. Piazuelo, P. Correa, and R. M. Peek Jr. Regulation of Gastric Carcinogenesis by Helicobacter pylori Virulence Factors Cancer Res., January 15, 2008; 68(2): 379 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Ayala, J. Torres-Mena, and L. Lopez-Carrillo Association between Helicobacter pylori VacA antigens and gastric cancer depends on the detection method used: immunoblot versus neutralization of the vacuolating activity of VacA J. Med. Microbiol., January 1, 2008; 57(1): 9 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Hisatsune, E. Yamasaki, M. Nakayama, D. Shirasaka, H. Kurazono, Y. Katagata, H. Inoue, J. Han, J. Sap, K. Yahiro, et al. Helicobacter pylori VacA Enhances Prostaglandin E2 Production through Induction of Cyclooxygenase 2 Expression via a p38 Mitogen-Activated Protein Kinase/Activating Transcription Factor 2 Cascade in AZ-521 Cells Infect. Immun., September 1, 2007; 75(9): 4472 - 4481. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Hofman, S. Lassalle, E. Selva, K. Kalem, A. Steff, X. Hebuterne, D. Sicard, P. Auberger, and P. Hofman Involvement of mast cells in gastritis caused by Helicobacter pylori: a potential role in epithelial cell apoptosis J. Clin. Pathol., June 1, 2007; 60(6): 600 - 607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Caruso, D. Fina, I. Peluso, M. C. Fantini, C. Tosti, G. D. V. Blanco, O. A. Paoluzi, F. Caprioli, F. Andrei, C. Stolfi, et al. IL-21 Is Highly Produced in Helicobacter pylori-Infected Gastric Mucosa and Promotes Gelatinases Synthesis J. Immunol., May 1, 2007; 178(9): 5957 - 5965. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Izzotti, S. De Flora, C. Cartiglia, B. M. Are, M. Longobardi, A. Camoirano, I. Mura, M. P. Dore, A. M. Scanu, P. C. Rocca, et al. Interplay between Helicobacter pylori and host gene polymorphisms in inducing oxidative DNA damage in the gastric mucosa Carcinogenesis, April 1, 2007; 28(4): 892 - 898. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Matarrese, L. Falzano, A. Fabbri, L. Gambardella, C. Frank, B. Geny, M. R. Popoff, W. Malorni, and C. Fiorentini Clostridium difficile Toxin B Causes Apoptosis in Epithelial Cells by Thrilling Mitochondria: INVOLVEMENT OF ATP-SENSITIVE MITOCHONDRIAL POTASSIUM CHANNELS J. Biol. Chem., March 23, 2007; 282(12): 9029 - 9041. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. M. S. Algood, V. J. Torres, D. Unutmaz, and T. L. Cover Resistance of Primary Murine CD4+ T Cells to Helicobacter pylori Vacuolating Cytotoxin Infect. Immun., January 1, 2007; 75(1): 334 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Terebiznik, C. L. Vazquez, K. Torbicki, D. Banks, T. Wang, W. Hong, S. R. Blanke, M. I. Colombo, and N. L. Jones Helicobacter pylori VacA Toxin Promotes Bacterial Intracellular Survival in Gastric Epithelial Cells Infect. Immun., December 1, 2006; 74(12): 6599 - 6614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nakayama, J. Hisatsune, E. Yamasaki, Y. Nishi, A. Wada, H. Kurazono, J. Sap, K. Yahiro, J. Moss, and T. Hirayama Clustering of Helicobacter pylori VacA in Lipid Rafts, Mediated by Its Receptor, Receptor-Like Protein Tyrosine Phosphatase {beta}, Is Required for Intoxication in AZ-521 Cells Infect. Immun., December 1, 2006; 74(12): 6571 - 6580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Kusters, A. H. M. van Vliet, and E. J. Kuipers Pathogenesis of Helicobacter pylori Infection Clin. Microbiol. Rev., July 1, 2006; 19(3): 449 - 490. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Falzano, P. Filippini, S. Travaglione, A. G. Miraglia, A. Fabbri, and C. Fiorentini Escherichia coli Cytotoxic Necrotizing Factor 1 Blocks Cell Cycle G2/M Transition in Uroepithelial Cells Infect. Immun., July 1, 2006; 74(7): 3765 - 3772. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. O'Brien, D. A. Israel, U. Krishna, J. Romero-Gallo, J. Nedrud, M. E. Medof, F. Lin, R. Redline, D. M. Lublin, B. J. Nowicki, et al. The Role of Decay-accelerating Factor as a Receptor for Helicobacter pylori and a Mediator of Gastric Inflammation J. Biol. Chem., May 12, 2006; 281(19): 13317 - 13323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Yamasaki, A. Wada, A. Kumatori, I. Nakagawa, J. Funao, M. Nakayama, J. Hisatsune, M. Kimura, J. Moss, and T. Hirayama Helicobacter pylori Vacuolating Cytotoxin Induces Activation of the Proapoptotic Proteins Bax and Bak, Leading to Cytochrome c Release and Cell Death, Independent of Vacuolation J. Biol. Chem., April 21, 2006; 281(16): 11250 - 11259. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. J. Torres, M. S. McClain, and T. L. Cover Mapping of a Domain Required for Protein-Protein Interactions and Inhibitory Activity of a Helicobacter pylori Dominant-Negative VacA Mutant Protein Infect. Immun., April 1, 2006; 74(4): 2093 - 2101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Genisset, C. L. Galeotti, P. Lupetti, D. Mercati, D. A. G. Skibinski, S. Barone, R. Battistutta, M. de Bernard, and J. L. Telford A Helicobacter pylori Vacuolating Toxin Mutant That Fails To Oligomerize Has a Dominant Negative Phenotype Infect. Immun., March 1, 2006; 74(3): 1786 - 1794. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pinto-Santini and N. R. Salama The Biology of Helicobacter pylori Infection, a Major Risk Factor for Gastric Adenocarcinoma Cancer Epidemiol. Biomarkers Prev., August 1, 2005; 14(8): 1853 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.W. Kaplan, R. Lux, T. Huynh, A. Jewett, W. Shi, and S. K. Haake Fusobacterium nucleatum Apoptosis-inducing Outer Membrane Protein Journal of Dental Research, August 1, 2005; 84(8): 700 - 704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. J. Torres, S. E. Ivie, M. S. McClain, and T. L. Cover Functional Properties of the p33 and p55 Domains of the Helicobacter pylori Vacuolating Cytotoxin J. Biol. Chem., June 3, 2005; 280(22): 21107 - 21114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Yahiro, A. Wada, E. Yamasaki, M. Nakayama, Y. Nishi, J. Hisatsune, N. Morinaga, J. Sap, M. Noda, J. Moss, et al. Essential Domain of Receptor Tyrosine Phosphatase {beta} (RPTP{beta}) for Interaction with Helicobacter pylori Vacuolating Cytotoxin J. Biol. Chem., December 3, 2004; 279(49): 51013 - 51021. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Wada, E. Yamasaki, and T. Hirayama Helicobacter pylori Vacuolating Cytotoxin, VacA, Is Responsible for Gastric Ulceration J. Biochem., December 1, 2004; 136(6): 741 - 746. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Galgani, I. Busiello, S. Censini, S. Zappacosta, L. Racioppi, and R. Zarrilli Helicobacter pylori Induces Apoptosis of Human Monocytes but Not Monocyte-Derived Dendritic Cells: Role of the cag Pathogenicity Island Infect. Immun., August 1, 2004; 72(8): 4480 - 4485. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Yilmaz, T. Jungas, P. Verbeke, and D. M. Ojcius Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway Contributes to Survival of Primary Epithelial Cells Infected with the Periodontal Pathogen Porphyromonas gingivalis Infect. Immun., July 1, 2004; 72(7): 3743 - 3751. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Sundrud, V. J. Torres, D. Unutmaz, and T. L. Cover Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion PNAS, May 18, 2004; 101(20): 7727 - 7732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Li, A. Wandinger-Ness, J. R. Goldenring, and T. L. Cover Clustering and Redistribution of Late Endocytic Compartments in Response to Helicobacter pylori Vacuolating Toxin Mol. Biol. Cell, April 1, 2004; 15(4): 1946 - 1959. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nakayama, M. Kimura, A. Wada, K. Yahiro, K.-i. Ogushi, T. Niidome, A. Fujikawa, D. Shirasaka, N. Aoyama, H. Kurazono, et al. Helicobacter pylori VacA Activates the p38/Activating Transcription Factor 2-mediated Signal Pathway in AZ-521 Cells J. Biol. Chem., February 20, 2004; 279(8): 7024 - 7028. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. J. Torres, M. S. McClain, and T. L. Cover Interactions between p-33 and p-55 Domains of the Helicobacter pylori Vacuolating Cytotoxin (VacA) J. Biol. Chem., January 16, 2004; 279(3): 2324 - 2331. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C. Willhite, T. L. Cover, and S. R. Blanke Cellular Vacuolation and Mitochondrial Cytochrome c Release Are Independent Outcomes of Helicobacter pylori Vacuolating Cytotoxin Activity That Are Each Dependent on Membrane Channel Formation J. Biol. Chem., November 28, 2003; 278(48): 48204 - 48209. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ismail, M. B. Hampton, and J. I. Keenan Helicobacter pylori Outer Membrane Vesicles Modulate Proliferation and Interleukin-8 Production by Gastric Epithelial Cells Infect. Immun., October 1, 2003; 71(10): 5670 - 5675. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) or equal volumes of acidified buffer (
) in the presence of ammonium chloride (5 mM). Apoptosis was quantified by DNA fragmentation ELISA and results are expressed as the levels of nucleosomal release relative to AGS cells in medium without additives. Data represent results from two independent experiments performed in triplicate and are expressed as mean ± SD.