Effect of Helicobacter pylori VacA on gene expression of gastric cancer cells.

AIM: To determine the effect of Helicobacter pylori VacA on gene expression of gastric cancer cells. METHODS: Gene expression profile of a gastric cancer cell line, SGC7901, after challenged by VacA+ and VacA- H pylori broth culture supernatants (BCS), was detected by the cDNA microarray technique. Cytoskeleton changes of SGC7901 and HeLa cells were observed through high-resolution laser scanning confocal microscopy. RESULTS: A total of 16,000 cDNA clones were detected. The percentage of genes with heterogeneous expression in SGC7901 cells challenged by VacA+ BCS reached 5%, compared with that challenged by VacA- BCS. There were 865 genes/EST with 2-fold differential expression levels and 198 genes/EST with 3-fold differential expression levels. Most of these genes were involved in vital cell events including signal transduction, regulation of gene expression, cytoskeleton, apoptosis, stress response and inflammation, cell cycle and tumor development. Cells co-cultured with VacA+ BCS showed collapsed and disrupted microtubular cytoarchitecture. CONCLUSION: VacA+ BCS can disrupt cytoskeletal architecture, likely through affecting the expression of cytoskeleton-associated genes, directly induce the expression of tumor promoter-related genes and inhibit the expression of tumor suppressor genes, thus favoring the development of tumors. VacA+ BCS can also alter the expression of inflammation and stress response genes. This suggests that VacA may play an important role in the pathogenicity of H pylori.

Analysis of gene expression profile in gastric cancer cells stimulated with Helicobacter pylori isogenic strains.

To understand the biological processes within host cells induced by VacA, isogenic strains of Helicobacter pylori (NCTC 11638 or 11638-DeltavacA) were used to stimulate gastric cancer cells SGC7901, and differentially expressed genes in host cells were identified using cDNA microarray technology. More than 300 genes were found to alter their mRNA expression at different time points, among which 68 were related to the cytoskeleton, 87 were associated with cell cycle, cell death and proliferation, IL8 expression was also found to be up-regulated. Cells co-cultured with broth-culture supernatant (BCS) of NCTC 11638 showed more alteration in microtubule cytoskeleton morphology, as observed by laser scanning confocal microscopy, and a lower apoptosis rate, detected by flow cytometry, compared with those co-cultured with BCS of 11638-DeltavacA. The supernatants of cells co-cultured with NCTC 11638 showed significantly higher IL8 expression than those co-cultured with 11638-DeltavacA. It is concluded that VacA disrupts cytoskeletal architecture by influencing the expression of cytoskeleton-associated genes. VacA breaks the balance between cell proliferation and cell death by inducing the maladjustment of genes related to cell cycle. VacA is also able to induce the inflammatory response.

mRNA expression profiling reveals a role of Helicobacter pylori vacuolating toxin in escaping host defense.

AIM: To study the immune response of host to Helicobacter pylori VacA. METHODS: The monocyte/macrophage-like U937 cells were infected with Helicobacter pylori vacA-positive strain NCTC 11638 or isogenic vacA-negative mutant. Differentially expressed genes were identified at 2, 6, 10, and 24 h post-infection by cDNA microarray. Differential expressions of some genes were confirmed by Northern blot. RESULTS: More than 100 genes altered their mRNA expression at different time points respectively, many of which were identified to be related to immune evasion. CONCLUSION: VacA is a crucial element for H pylori to escape from host immune defense by means of differentially regulating the expression of some related genes. These genes, previously known or unknown to be involved in the mechanism of immune evasion, deserve further investigation to unearth much more information complicated in the immune response.

Deletion of Helicobacter pylori vacuolating cytotoxin gene by introduction of directed mutagenesis.

AIM: To construct a vacA-knockout Helicobacter pylori mutant strain, whose only difference from the wild strain is its disrupted vacA gene. METHODS AND RESULTS: A clone containing kanamycin resistance gene used for homologous recombination was constructed in a directional cloning procedure into pBluescript II SK, and then transformed into vacA+ H pylori by electroporation. Colonies growing on the selective media containing kanamycin were harvested for chromosomal DNA extraction, and the allelic exchange was determined by polymerase chain reactions and sequencing. Loss of vacuolating activity of the vacA-knockout strain was confirmed by examining the gastric cells co-cultured with cell-free supernatants from H pylori wild strain or the mutant. CONCLUSION: We constructed a vacA-knockout strain of H pylori through direct mutagenesis, which creates an important precondition for the future research on virulence comparison with gene expression analysis.

Coxsackievirus B3-induced apoptosis and caspase-3.

Cell death can be classified into two categories: apoptosis and necrosis. Apoptotic pathway can be either caspase-dependent or caspase-independent. Caspase-independent cytopathic effect (CPE) has been described. In order to evaluate the pattern of HeLa cell death induced by Coxsackievirus B3 (CVB3) and whether apoptosis involves caspase activation, we co-cultivated HeLa cells with CVB3 and detected the cytopathic changes, the alteration of mRNA and protein expression of caspase-3 gene plus caspase-3 activity, as well as analyzing DNA fragmentation before and after caspase-3 activity inhibition. According to the results, we propose that CVB3 may induce apoptosis and necrosis in HeLa cells, the latter appearing much earlier. Caspase-3 is activated at the levels of both transcription and translation, and procaspase-3 is proteolytically cleaved, thus leading to the continuous increasing of both caspase-3 precursor protein and its subunit. However, besides CPE, apoptosis induced by CVB3 is not a direct consequence of the activation of caspase-3, or caspase-3 is not the only effector molecule in apoptotic cell death, for caspase-3 inhibitor can not decrease DNA fragmentation. Some other biochemical mechanisms may participate in the process, whose role weakens the effect of inhibiting caspase-3 activity.

Cyclosporin A protects Balb/c mice from liver damage induced by superan tigen SEB and D-GalN.

AIM:To investigate the pathogenic effect of SEB and D-GalN on liver and the protection of cyclosporin A, the relationship between hepatic apoptosis and necrosis and the possible mechanism of acute hepatic necrosis.METHODS:After staphylococcal enterotoxin B (SEB) mixed with D-galactosamine (D-GalN) were injected intraperitoneally into Balb/c mice and those previously treated with cyclosporin A, blood samples were collected and livers were isolated at 2, 6, 12, 24h. Patterns of hepatocellular death were studied morphologically and biochemically, circulating cytokines (TNF-alpha, IFN-gamma) and mice mortality within 24h was assessed.RESULTS: The SEB could induce the typical apoptotic changes of hepatocytes, the D-GalN could induce hepatocytes apoptosis and degeneration at the same time, and the mice having received the SEB+D-GalN injections developed apoptosis at 2 and 6h, but after 12h hepatocytes were characterized by severe injury, whereas all the examinations in the cyclosporin A treated mice were normal.CONCLUSION:Hepatic cell apoptosis might be related to necrosis, and massive hepatocyte apoptosis is likely the initiating step of acute hepatic necrosis in mice. The effects induced by SEB and D-GalN on hepatocytes might be mediated by T cells, and could be prevented by cyclosporin A.