Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.     

Urokinase plasminogen activator receptor is upregulated by Helicobacter pylori in human gastric cancer AGS cells via ERK, JNK, and AP-1

The gastric pathogen Helicobacter pylori (H. pylori) is suggested to be associated with gastric cancer progression. In this study, we investigated the effect of H. pylori on urokinase plasminogen activator receptor (uPAR) expression which has been known to correlate closely with gastric cancer invasion. H. pylori induced the uPAR expression in a time- and concentration-dependent manner. Specific inhibitors and inactive mutants of MEK-1 and JNK were found to suppress the H. pylori-induced uPAR expression and the uPAR promoter activity. Electrophoretic mobility shift assay and transient transfection study using an AP-1 decoy oligonucleotide confirmed that the activation of AP-1 is involved in the H. pylori-induced uPAR upregulation. The AGS cells treated with H. pylori showed a remarkably enhanced invasiveness, and this effect was partially abrogated by uPAR-neutralizing antibodies. These results suggest that H. pylori induces uPAR expression via Erk-1/2, JNK, and AP-1 signaling pathways and, in turn, stimulates the cell invasiveness in human gastric cancer AGS cells.     

The Helicobacter pylori's protein VacA has direct effects on the regulation of cell cycle and apoptosis in gastric epithelial cells

In this study, we have evaluated the effects on cell cycle regulation of VacA alone and in combination with other two Helicobacter pylori proteins, cytotoxin-associated protein (CagA) and HspB, using the human gastric epithelial cells (AGS). Our results indicate that VacA alone was able to inhibit the G1 to S progression of the cell cycle. The VacA capacity of inhibiting cell progression from G1 to S phase was also observed when cells were co-transfected with CagA or HspB. Moreover, VacA over-expression caused apoptosis in AGS cells through activation of caspase 8 and even more of caspase 9, thus indicating an involvement of both the receptor-mediated and the mitochondrial pathways of apoptosis. Indeed, the two pathways probably can co-operate to execute cell death with a prevalence of the mitochondrial pathways. 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 human gastric epithelial cells.     

Helicobacter pylori infection influences expression of genes related to angiogenesis and invasion in human gastric carcinoma cells

Infection with Helicobacter pylori (H. pylori) is considered a risk factor for gastric carcinoma. The purpose of this study was to clarify whether H. pylori infection plays a role in progression of gastric carcinoma. We examined the expression of genes encoding angiogenic factors and proteases by human gastric carcinoma cell lines (MKN-1 and TMK-1) co-cultured with or without H. pylori by cDNA microarray analysis. Co-culture with H. pylori increased expression of mRNAs encoding interleukin (IL)-8, vascular endothelial growth factor (VEGF), angiogenin, urokinase-type plasminogen activator (uPA), and metalloproteinase (MMP)-9 by gastric carcinoma cells. Up-regulation of these genes at the mRNA and protein levels was confirmed by Northern blot analysis, semi-quantitative RT-PCR analysis, and ELISA. In vitro angiogenic and collagenase activities of conditioned medium from the gastric carcinoma cells were also stimulated by co-culture with H. pylori. These results indicate that H. pylori infection may regulate angiogenesis and invasion of human gastric carcinoma.     

The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells.     

Differential regulation of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) by phosphorylation directed by the cyclin encoded by Murine Herpesvirus 68

Members of the gamma2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21(Cip) and p27(Kip). Furthermore, M cyclin directed cdk2 to phosphorylate p27(Kip1) on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27(Kip1) and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27(Kip1)-imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21(Cip1) and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.     

Helicobacter pylori and mitogen-activated protein kinases mediate activator protein-1 (AP-1) subcomponent protein expression and DNA-binding activity in gastric epithelial cells

Emerging evidence has suggested a critical role for activator protein-1 (AP)-1 in regulating various cellular functions. The goal of this study was to investigate the effects of Helicobacter pylori and mitogen-activated protein kinases (MAPK) on AP-1 subcomponents expression and AP-1 DNA-binding activity in gastric epithelial cells. We found that H. pylori infection resulted in a time- and dose-dependent increase in the expression of the proteins c-Jun, JunB, JunD, Fra-1, and c-Fos, which make up the major AP-1 DNA-binding proteins in AGS and MKN45 cells, while the expression levels of Fra-2 and FosB remained unchanged. Helicobacter pylori infection and MAPK inhibition altered AP-1 subcomponent protein expression and AP-1 DNA-binding activity, but did not change the overall subcomponent composition. Different clinical isolates of H. pylori showed various abilities to induce AP-1 DNA binding. Mutation of cagA, cagPAI, or vacA, and the nonphosphorylateable CagA mutant (cagA(EPISA)) resulted in less H. pylori-induced AP-1 DNA-binding activity, while mutation of the H. pylori flagella had no effect. extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) each selectively regulated AP-1 subcomponent expression and DNA-binding activity. These results provide more insight into how H. pylori and MAPK modulate AP-1 subcomponents in gastric epithelial cells to alter the expression of downstream target genes and affect cellular functions.     

Mechanisms of cell cycle arrest in response to TGF-beta in progestin-dependent and -independent growth of mammary tumors

TGF-beta1 modulation of cell cycle components was assessed in an experimental model in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary tumors in Balb/c mice. TGF-beta1 inhibited both MPA-induced proliferation of progestin-dependent C4HD epithelial cells and proliferation of the progestin-independent variant cell type C4HI, arresting cells in G(1) phase of the cell cycle. Progestin-independent 60 epithelial cells evidenced reduced response to TGF-beta1 antiproliferative effects. TGF-beta1 inhibition of cyclins D1 and A expression and up-regulation of p21(CIP1) levels were the common findings in all three cell types. In addition, a significant content reduction of cyclin D1/cdk4 and cyclin A/cdk2 complexes was found after TGF-beta1 inhibition of MPA-dependent and -independent proliferation. TGF-beta1 inhibited cyclin D2 expression and up-regulated p27(KIP1) levels only when acting as inhibitor of MPA-induced proliferation of C4HD cells. Regulation of these two cell cycle components resulted in decreased cyclin D2/cdk2 complex and in increased p27(KIP1) association with cdk2 in C4HD cells treated with TGF-beta1. These two molecular mechanisms, unobserved in progestin-independent growth of C4HI or 60 cells, were associated with a significantly higher degree of inhibition of cdk2 kinase activity in C4HD cells compared to that found in TGF-beta-treated C4HI or 60 cells. Reduced sensitivity of 60 cells to the growth-inhibitory effects of TGF-beta1 correlated with significantly lower levels of p15(INK4B), p21(CIP1), and p27(KIP1) expressed in these cells, compared to the levels present in C4HD or C4HI cells, and correlated as well with lack of expression of p16(INK4). Thus, common targets were found to exist in TGF-beta1 inhibitory action on breast cancer cells, but regulation of specific targets was found when TGF-beta1-inhibited proliferation driven by the progesterone receptor.     

Cooperative regulation of the cell division cycle by the protein kinases RAF and AKT

The RAS-activated RAF-->MEK-->extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)-->PDK1-->AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G(1) cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27(Kip1) expression. Repression of p27(Kip1) was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27(Kip1). Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27(Kip1) in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21(Cip1) from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAF-->MEK-->ERK and PI3'K-->PDK-->AKT signaling pathways can cooperate to promote G(0)-->G(1)-->S-phase cell cycle progression in both normal and cancer cells.     

p27(kip1) upregulated by hnRNPC1/2 antagonizes CagA (a virulence factor of Helicobacter pylori)-mediated pathogenesis

Background and Aims: Infection by Helicobacter pylori is one of the major contributing factors of chronic active gastritis and peptic ulcer and is closely associated with the occurrence and progression of gastric cancer. CagA protein is a major virulence factor of H. pylori that interacts with SHP‐2, a true oncogene, to interfere with cellular signaling pathways; CagA also plays a crucial role in promoting the carcinogenesis of gastric epithelial cells. However, currently, the molecular mechanisms of gastric epithelial cells that antagonize CagA pathogenesis remain inconclusive. Methods: We showed that AGS gastric cancer cells transfected with CagA exhibited the inhibition of proliferation and increased activity of caspase 3/7 using two‐dimensional gel electrophoresis and secondary mass spectrometry (MS/MS). Results: It was found that the AGS gastric cancer cells stably expressing CagA displayed significantly increased the expression of 16 proteins, including hnRNPC1/2. Further analysis revealed that hnRNPC1/2 significantly boosted the expression of the p27^kip1 protein. Conclusion: Our data suggested that hnRNPC1/2 upregulates p27^kip1 expression and the subsequent suppression of cell proliferation and induction of apoptosis, thereby providing an important mechanism whereby gastric epithelial cells antagonize CagA‐mediated pathogenesis.     

Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. This is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of human prostate carcinoma cells by EGCG.     

p57KIP2 control of actin cytoskeleton dynamics is responsible for its mitochondrial pro-apoptotic effect

p57 (Kip2, cyclin-dependent kinase inhibitor 1C), often found downregulated in cancer, is reported to hold tumor suppressor properties. Originally described as a cyclin-dependent kinase (cdk) inhibitor, p57^KIP2 has since been shown to influence other cellular processes, beyond cell cycle regulation, including cell death and cell migration. Inhibition of cell migration by p57^KIP2 is attributed to the stabilization of the actin cytoskeleton through the activation of LIM domain kinase-1 (LIMK-1). Furthermore, p57^KIP2 is able to enhance mitochondrial-mediated apoptosis. Here, we report that the cell death promoting effect of p57^KIP2 is linked to its effect on the actin cytoskeleton. Indeed, whereas Jasplakinolide, an actin cytoskeleton-stabilizing agent, mimicked p57^KIP2''s pro-apoptotic effect, destabilizing the actin cytoskeleton with cytochalsin D reversed p57^KIP2''s pro-apoptotic function. Conversely, LIMK-1, the enzyme mediating p57^KIP2''s effect on the actin cytoskeleton, was required for p57^KIP2''s death promoting effect. Finally, p57^KIP2-mediated stabilization of the actin cytoskeleton was associated with the displacement of hexokinase-1, an inhibitor of the mitochondrial voltage-dependent anion channel, from the mitochondria, providing a possible mechanism for the promotion of the mitochondrial apoptotic cell death pathway. Altogether, our findings link together two tumor suppressor properties of p57^KIP2, by showing that the promotion of cell death by p57^KIP2 requires its actin cytoskeleton stabilization function.     

Accumulation of 8-nitroguanine in human gastric epithelium induced by Helicobacter pylori infection

Helicobacter pylori infection causes chronic inflammation, which can lead to gastric carcinoma. A double immunofluorescence labeling study demonstrated that the level of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) apparent in gastric gland epithelium was significantly higher in gastritis patients with H. pylori infection than in those without infection. A significant accumulation of proliferating cell nuclear antigen, a prognostic factor for gastric cancer, was observed in gastric gland epithelial cells in patients with H. pylori infection as compared to those without infection, and its accumulation was closely correlated with the formation of 8-nitroguanine and 8-oxodG. These results suggest that nitrosative and oxidative DNA damage in gastric epithelial cells and their proliferation by H. pylori infection may lead to gastric carcinoma. 8-Nitroguanine could be not only a promising biomarker for inflammation but also a useful indicator of the risk of gastric cancer development in response to chronic H. pylori infection.