Helicobacter pylori infection is associated with increased gastric epithelial cell turnover and is a risk factor for noncardia gastric cancer. H. pylori reduces the expression of p27 protein, a cyclin-dependent kinase inhibitor of the G1 to S-phase cell cycle transition and gastric tumor suppressor gene. Although cell cycle dysregulation associated with decreased p27 may contribute to gastric carcinogenesis, how H. pylori reduces p27 in gastric epithelial cells remains unknown. In the present study, we investigated the mechanisms of the p27 decrease, using AGS and MKN28 gastric epithelial cells cocultured with H. pylori strains under conditions of defined cell cycle distribution. The expression of p27 protein was reduced by H. pylori in a dose- and time-dependent manner. Northern blot and pulse-chase analyses revealed that this reduction was not regulated at a transcriptional level but by accelerated p27 degradation via a proteasome-dependent pathway. Despite up-regulation of the proteasome-dependent degradation of p27 protein, neither threonine 187-phosphorylated p27 nor skp2 (the ubiquitin ligase for p27) were increased. Furthermore, H. pylori impaired p27 ubiquitination and did not increase global proteasomal function. These results indicate that H. pylori increases the degradation of p27 through a proteasomal pathway distinct from the physiological pathway that degrades p27 during cell cycle progression. Putative virulence genes of H. pylori (cagA, cagE, or vacA) played no role in reducing p27 expression. Increased degradation of p27 by H. pylori through a proteasome-dependent, ubiquitin-independent pathway may contribute to the increased risk of gastric cancer associated with chronic H. pylori infection.

Epidemiological studies have demonstrated that gastric infection with the Gram-negative bacterium Helicobacter pylori is associated with an increased risk of developing noncardia gastric cancer (1, 2, 3). Furthermore, eradication of H. pylori may prevent gastric cancer in high-risk populations with chronic gastritis (4) and can reduce the rate of progression from intermediate preneoplastic gastric lesions of intestinal metaplasia and atrophy (5). More direct evidence implicating H. pylori as a gastric carcinogen has come from the demonstration of gastric cancer in the Mongolian gerbil following experimental infection by H. pylori(6, 7) and from C57/BL6 mice infected with the related gastric bacterium H. felis(8).

How H. pylori promotes gastric carcinogenesis is not known. However, studies in humans and in animal models have demonstrated that H. pylori increases the percentage of gastric mucosal epithelial cells displaying markers of proliferation and apoptosis (9, 10). Because increased cell turnover is a common precursor of neoplastic transformation (11), investigating the link between H. pylori and altered gastric epithelial cell cycling may therefore provide important information regarding the mechanisms of gastric carcinogenesis associated with chronic H. pylori infection.

Progression of cells through the cell cycle is controlled by interactions between cell cycle control proteins (cyclins) and their catalytically active CDKs.3 The activity of each cyclin-CDK complex is in turn regulated by several different mechanisms, the most important being negative regulation by CDK inhibitors (12). Mutation or aberrant expression of specific cell cycle-regulatory proteins is common in tumors, suggesting that these proteins are critical targets during carcinogenesis (13). p27kip1 (p27) is a CDK inhibitor, whose major targets are the cyclin E-CDK2 and cyclin D-CDK4/6 complexes, that governs cell cycle transition from late G1 to S phase (14). The amount of p27 is mainly regulated by posttranslational ubiquitin-proteasome-mediated proteolysis. The cell cycle-dependent degradation of p27 is dependent on phosphorylation at Thr187 in late G1 phase by CDK2, under positive regulation by cyclin E. Thr187 phosphorylation is a necessary prerequisite for the sequential addition of ubiquitin molecules by a ubiquitin ligase complex containing the F-box protein skp2 (for review, see Ref. 15). Polyubiquitination of p27 then targets p27 for degradation in the proteasome, thus removing the p27 cell cycle “brake,” allowing cells to transition from G1 to S phase. Loss of p27 function therefore accelerates cell cycle progression and predisposes cells to malignant transformation, as is well illustrated by the observation of increased tumor incidence in hemizygous and homozygous p27-deleted mutant mice after carcinogen exposure (16). For this reason, p27 has been termed a haploinsufficient tumor suppressor gene (16), and decreased expression of p27, probably due to increased proteasomal degradation of p27 after ubiquitination (17, 18), has been demonstrated to be associated with a poor prognosis in several types of cancer, including gastric cancer (14, 19, 20).

Recent studies by our group and others have shown that the level of p27 protein in gastric epithelial cells is decreased by H. pylori, both in chronic gastritis (21) and in intestinal metaplasia (22). Furthermore, p27 expression is reduced in gastric epithelial cells exposed to H. pylori acutely (23) and chronically (21), suggesting that the bacterium itself rather than the associated inflammatory response present in the gastric mucosa can rapidly and permanently down-regulate epithelial cell p27. Because decreased p27 may be an important step linking H. pylori to hyperproliferation and gastric carcinogenesis, we investigated the cellular and molecular mechanisms responsible for this effect of H. pylori.

Cell Lines and Culture Conditions.

AGS human gastric epithelial cells (CRL-1739; American Type Culture Collection, Manassas, VA) and MKN28 human gastric epithelial cells (JCRB0253; Japan Health Sciences Foundation) were maintained in an atmosphere of 5% CO2 at 37°C in Ham’s F-12 medium (AGS) or RPMI 1640 (MKN28) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) without antibiotics in 75-cm2 tissue culture flasks (BD Biosciences, San Jose, CA). For synchronization of the cell cycle at G0-G1 phase, cells were serum deprived for 48 h.

H. pylori Strains and Culture Conditions.

Coculture experiments were performed, as described previously (23), using wild-type H. pylori strain 60190 (ATCC49503), a cagA-positive and vacA-positive strain isolated from a patient with nonulcer dyspepsia, its isogenic cagA-negative mutant, its isogenic vacA-negative mutant, or its isogenic cagE (=picB)-negative mutant [kindly provided by M. Blaser (New York University); Refs. 24, 25, 26]. Five minimally passaged clinical strains [GC102 and A855 (provided by Mark Kidd; Yale University, New Haven, CT) and J68, B107, and B166 (provided by Richard Peek; Vanderbilt University, Nashville, TN] were also used (27, 28). GC102 was isolated from a 58-year-old man with a diffuse-type antral adenocarcinoma, A855 was isolated from a 37-year-old woman with chronic atrophic pangastritis, J68 was isolated from a 33-year-old woman who had a duodenal ulcer, B107 was isolated from a 60-year-old woman with gastric and duodenal erythema, and B166 was isolated from a 61-year-old woman with duodenal erythema. Bacteria were maintained on trypticase soy agar containing 5% sheep blood (BD Biosciences) incubated at 37°C in 5% CO2 for a minimum of two and a maximum of four passages from frozen stocks. Inocula for coculture were diluted from suspensions that had been prepared from 48-h subcultures and adjusted by comparison of absorbance to McFarland standards. H. pylori bacteria were added to AGS cells at a ratio of 1:200 in all experiments, unless otherwise stated in the figure legends. To verify the viability of H. pylori after incubation in culture media, the media were serially diluted 10-fold, 100 μl of each dilution were plated on agar plates that allow optimal growth of H. pylori, and colony counts were determined 48 h after plating.

Cell Cycle Analysis.

Adherent and floating epithelial cells were collected together and fixed in 70% ethanol. Cell pellets were suspended in 400 μl of 0.2 mg/ml propidium iodide containing 0.6% NP40 (ICN Pharmaceuticals, Costa Mesa, CA) plus the same volume of 2 mg/ml RNase (Sigma Chemical Co., St. Louis, MO) and then incubated in the dark at room temperature for 30 min. Data acquisition and analysis were performed on a FACSort instrument equipped with CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA). Cell cycle analysis was performed with ModFIT software (Becton Dickinson Immunocytometry Systems). All experiments were performed at least three times and gave similar results.

Protein Extraction and Western Blotting.

Cells were collected with a rubber policeman, washed twice in ice-cold PBS, and resuspended in lysis buffer containing 50 mm HEPES, 150 mm NaCl, 2.5 mm EGTA, 1.0 mm EDTA, 1.0 mm DTT, 0.1% Tween 20, 10% glycerol, 10 mm β-glycerophosphate, 1.0 mm sodium fluoride, and 0.1 mm sodium orthovanadate (adjusted to pH 7.5), plus the protease inhibitors leupeptin (10 mg/ml), aprotinin (10 mg/ml), and 1.0 mm phenylmethylsulfonyl fluoride. The suspended cells were sonicated on ice (twice for 15 s) with a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) and then centrifuged at 13,000 × g for 10 min at 4°C to yield soluble cell lysates. Protein concentrations were determined using the BCA Protein Assay (Pierce Chemical Co., Rockford, IL). For Western blotting, 30 μg of a total cell lysate were subjected to SDS-PAGE. The proteins were then transferred to Immobilin-P membranes (Millipore, Bedford, MA). The primary antibodies used were mouse monoclonal antibodies to p27kip1 (clone 57; BD Biosciences), cyclin A (Upstate Biotechnology, Lake Placid, NY), cyclin D1/2 (Upstate Biotechnology), ubiquitin (clone P4D1; Santa Cruz Biotechnology Inc., Santa Cruz, CA), and β-actin (Sigma Chemical Co.) and rabbit polyclonal antibodies to Thr187-phosphorylated p27 (PT-187; Zymed Laboratories Inc., San Francisco, CA), p45skp2 (clone SKP2–2B12; Zymed Laboratories Inc.), cyclin E [kindly provided by J. Singer (Brown University)], CDK2 (Upstate Biotechnology), and p21cip1 (sc-397, Santa Cruz Biotechnology Inc.). Immune detection was performed using the enhanced chemiluminescence Western blotting detection system (Perkin-Elmer Life Sciences Inc., Boston, MA). β-Actin immunoblotting was performed to verify that equal amounts of protein had been loaded in each lane. Quantitative densitometric analysis was performed using NIH Image software.

Northern Blotting.

Total RNA was prepared with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Ten μg of total RNA were electrophoresed on a 1% agarose denaturing gel, transferred to Hybond-N membrane (Amersham Biosciences Corp., Piscataway, NJ) by capillary electrophoresis, and fixed with UV light. The membrane was hybridized with a 32P-labeled human p27 cDNA probe (29). After washing, the membrane was exposed to film with intensifying screens at −80°C.

Pulse-chase Analysis of p27 Degradation.

Cells were serum starved for 48 h and metabolically radiolabeled with [35S]methionine and [35S]cysteine (200 μm; Perkin-Elmer Life Sciences Inc.) for 2 h. The culture medium was then replaced by serum-free, methionine-containing medium. After 2, 4, and 6 h of coculture with H pylori, cells were harvested, and lysates were immunoprecipitated with anti-p27 antibody. Immunoprecipitates were subjected to SDS-PAGE, and signals were surveyed by autoradiography.

In Vitro p27 Ubiquitination Assay.

[35S]Methionine-labeled p27 was prepared with the coupled in vitro transcription/translation system (Promega, Madison, WI) using plasmid pcDNA3/p27 (30). Cells were serum starved for 48 h and then cocultured with or without H. pylori for 6 h. Extracts were prepared as described by Nguyen et al.(31), with minor modifications. Briefly, plates were washed twice with ice-cold PBS and once with cold hypotonic buffer [20 mm HEPES (pH 7.5), 1.5 mm MgCl2, 5 mm KCl, and 1 mm DTT], resuspended in hypotonic buffer, left to stand on ice for 30 min, and then centrifuged at 13,000 × g for 30 min at 4°C, and supernatants were collected. Ten μl of radiolabeled p27 were then incubated with 200 μg of cell extracts supplemented with 1 mg/ml methylated ubiquitin (Boston Biochem, Cambridge, MA), 2 μm ubiquitin aldehyde (Boston Biochem), 200 μm MG-132 (Calbiochem, San Diego, CA), and an ATP regeneration system (25 mm phosphocreatine, 10 μg/ml creatine kinase, and 1 mm ATP) at 30°C in a total volume of 50 μl for the times indicated in the figure legends. The reactions were stopped by the addition of SDS sample buffer, and the products were resolved by SDS-PAGE. Ubiquitinated p27 forms were identified by fluorography.

In Vitro Proteasome Activity Assay.

Cells were collected and resuspended in a lysis buffer containing 50 mm Tris (pH 8.0), 5 mm EDTA, 150 mm NaCl, 0.5% NP40, 0.5 mm phenylmethylsulfonyl fluoride, and 0.5 mm DTT. They were then sonicated twice for 15 s and centrifuged at 13,000 × g for 10 min, and the supernatants were collected as whole cell extracts. Twenty-μg aliquots of cell extracts were then incubated in reaction buffer [25 mm HEPES, 0.5 mm EDTA (pH 7.6)] in quadruplicate for 30 min at 37°C with the following fluorogenic substrates: (a) chymotrypsin-like activity, Suc-Leu-Leu-Val-Tyr-AMC; (b) peptidylglutamyl peptide hydrolyzing activity, Z-Leu-Leu-Glu-AMC; and (c) trypsin-like activity, Z-Ala-Arg-Arg-AMC, 2HCl (all from Calbiochem). The amount of product (free AMC) was measured by Fluorocounter (Packard Bio Science Co., Meriden, CT) with an excitation filter of 360 nm and an emission filter of 460 nm.

In Vitro Assay for CDK2-associated Activity.

The in vitro CDK2-associated kinase assay was performed as described previously (32). Cells were collected, sonicated twice for 15 s in lysis buffer, and centrifuged, and the supernatant fraction was collected. Immunoprecipitation with 1 μg of anti-CDK2 polyclonal antibody was performed using protein A-Sepharose beads (Sigma Chemical Co.), followed by washing of the beads four times with lysis buffer and twice with reaction buffer [50 mm HEPES, 10 mm MgCl2, 1 mm DTT, 2.5 mm EGTA, 10 mm β-glycerophosphate, 1 mm NaF, and 0.1 mm sodium orthovanadate (pH 7.5)]. The final pellets were resuspended in 45 μl of reaction buffer containing 2 mg of histone H1 (Calbiochem) and 5 mCi of [γ-32P]ATP and incubated for 30 min at 30°C. The reaction mixture was then subjected to SDS-PAGE, and the intensity of phosphorylation of the histone H1 substrate was determined by autoradiography.

Cell Cycle-dependent Expression of p27 Protein in AGS Cells.

The expression of p27 protein was low in AGS cells during the exponential phase of growth in medium containing 10% serum (Fig. 1,A). Under these conditions, expression of skp2, the ubiquitin ligase for p27, was abundant. In comparison, after 48 h of serum deprivation, AGS cells accumulated in the G0-G1 phase of the cell cycle (Fig. 1 B) in association with high expression of p27 and low expression of phosphorylated p27 and skp2, consistent with reduced degradation of p27 protein in the G0-G1 phase of the cell cycle. Cyclin E, the major positive regulator of the G1 to S-phase transition was also at a low level in these cells. After treatment of AGS cells with the proteasome inhibitor MG-132, both p27 and phosphorylated p27 expression were markedly increased, consistent with a decrease in proteasomally mediated degradation of p27.

H. pylori Reduces p27 Protein Expression Dose-dependently, Irrespective of Cell Cycle Effects.

The level of p27 protein changes considerably during normal cell cycle progression. To examine the effects of H. pylori on p27, it was therefore necessary to separate changes in p27 levels due to altered cell cycle progression induced by H. pylori from changes in p27 due to effects of H. pylori that are not related to H. pylori’s effects on the cell cycle. To do this, we deprived AGS cells of serum for 48 h to inhibit the G1 to S-phase cell cycle transition and then added H. pylori in the absence of serum. Under these conditions, the addition of H. pylori did not alter cell cycle phase distribution significantly (Fig. 2,A), yet there was a dose-dependent decrease of p27 protein (Fig. 2,B, Lanes 1–5). Thus, the reduction of p27 induced by H. pylori was not due to the physiological down-regulation of p27 during cell cycle progression. The addition of H. pylori to serum-fed, exponentially growing AGS cells also resulted in a dose-dependent decrease in p27 expression (Fig. 2 B, Lanes 6–9), but the effect was less marked in these proliferating cells.

Reduction of p27 Protein Is due to Its Increased Degradation.

The addition of H. pylori did not alter the abundance of steady-state p27 mRNA as determined by Northern blotting (Fig. 3,A). Because the regulation of p27 protein abundance under physiological (cell cycle-related) conditions is known to be predominantly posttranslational, we therefore examined the effect of H. pylori on the degradation of p27 protein. Metabolically radiolabeled p27 was more rapidly degraded in the presence of H. pylori (Fig. 3 B), indicating that the down-regulation of p27 by H. pylori is due to increased p27 protein degradation.

Effect of H. pylori on Expression of p27 Protein Degradation Intermediates and Its Ubiquitin Ligase, skp2.

The degradation of p27 during the cell cycle is known to be largely through the ubiquitin- proteasome pathway. The addition of the proteasome inhibitor MG-132 at a concentration of 20 μm that did not influence H. pylori viability (as assessed by colony counts 48 h after plating the media; data not shown) abolished the decrease of p27 induced by H. pylori (Fig. 4,A). This result indicates that the decrease in p27 induced by H. pylori depends on proteasomal activity. However, the reduction in levels of p27 protein caused by H. pylori was accompanied by decreased expression of phosphorylated p27 and skp2 (Fig. 4 B), thus the decrease in p27 induced by H. pylori was proteasome dependent, but not through the physiological phosphorylation-ubiquitination pathway associated with increased skp2 expression.

Although Shirane et al.(33) reported that under some circumstances p27 may be degraded into a Mr 22,000 proteolytic product through a ubiquitin proteasome-independent pathway, the Mr 22,000 proteolytic product of p27 was not observed in any of these experiments (data not shown).

H. pylori Paradoxically Decreases CDK2-associated Kinase Activity.

Phosphorylation of p27 at Thr187 in the physiological ubiquitin-proteasome pathway is mediated by cyclin E-associated CDK2 activity, and a reciprocal relationship exists between cyclin E-associated CDK2 activity and the expression of p27. However, the reduction of p27 and phosphorylated p27 that we observed after the addition of H. pylori was associated with a decrease in CDK2 kinase activity (Fig. 5,A). This was not due to an inhibition of the binding of CDK2 by p27 because the expression of p27 was reduced to a similar extent by H. pylori in both the CDK2 immunoprecipitate and the supernatant (Fig. 5,B). To determine the mechanism of the decrease of CDK2 activity associated with decreased p27, we examined the expression of other regulatory partners of CDK2 activity and of other proteins involved in the transition from the G1 to S phase of the cell cycle (Fig. 5 C). Both cyclin E and cyclin A were also decreased by H. pylori. In contrast to these reductions in cyclin E, cyclin A, and p27, H. pylori did not alter cyclin D1/2 or the CDK inhibitor p21.

H. pylori Inhibits Ubiquitination of p27.

During the cell cycle-dependent regulation of p27, the degradation of p27 via the ubiquitin-proteasome pathway is dependent on p27 ubiquitination mediated by skp2. Because a skp2-independent ubiquitination pathway has also been reported (34), the decrease of skp2 expression by H. pylori that we observed does not preclude H. pylori from increasing the ubiquitination of p27. However, the in vitro p27 ubiquitination assay demonstrated that H. pylori decreased the ability of AGS cells to ubiquitinate p27 (evident from comparing Lanes 5 and 6 in Fig. 6,A with Lanes 2 and 3, respectively). As a control in this assay, p27 ubiquitination activity was greater in lysates from exponentially growing AGS cells (Lane 7) compared with serum-starved cells (Lane 3), consistent with increased ubiquitin-dependent p27 degradation during S phase. Additionally, the expression of several ubiquitinated proteins that may normally be detected by anti-ubiquitin antiserum when proteasomal function is inhibited were decreased by H. pylori (Fig. 6 B). Taken together, these results indicate that H. pylori does not increase but instead decreases the ubiquitination of p27 and possibly other proteins.

Effects of H. pylori on Proteasomal Activity.

Our results show that the proteasome-dependent degradation of p27 by H. pylori is associated by neither up-regulation of phosphorylation nor increased ubiquitination of p27. However, it is conceivable that our findings could be explained by H. pylori markedly accelerating p27 degradation at the level of increased proteasomal function, despite low concentrations of the intermediary forms of p27 (Thr187-phosphorylated p27 and ubiquitinated p27). We therefore measured the in vitro proteasomal activity of lysates from AGS cells that were cultured with H. pylori to test the hypothesis that H. pylori increases proteasomal activity nonspecifically. However, after the addition of H. pylori, the three major proteasome activities examined were decreased (Fig. 7), as determined by proteolytic cleavage of representative proteasomal substrates. These results indicate that the increased degradation of p27 by H. pylori is not due to an increase in global proteasomal function, but rather due to a ubiquitin-independent, proteasome-dependent pathway.

Effects of H. pylori on MKN28 Cells.

We have previously reported that H. pylori inhibits the G1 to S-phase cell cycle progression through a p53-independent mechanism (23). Because AGS cells possess wild-type p53 (35), to determine whether any of the changes we observed in AGS cells were dependent on functional p53, we also examined the effect of H. pylori on p27 and related molecules in MKN28 gastric epithelial cells that have a missense mutation in p53 (codon 251, isoleucine to leucine; Ref. 36). As seen in Fig. 8, MKN28 cells showed reductions in p27, cyclin E, and cyclin A that were similar to those observed in AGS cells (Fig. 5 C), indicating that these changes are independent of p53 function.

Effect of H. pylori-related Factors on p27 Expression.

The reduction of p27 induced by H. pylori was dependent on direct contact between live H. pylori and AGS cells because it could be abolished by separating AGS cells from H. pylori by a Transwell membrane (Nalge Nunc International Corp., Naperville, IL; data not shown). Putative H. pylori virulence-associated genes include the cagA and cagE genes within the cag pathogenicity island and the vacA gene (37). Incubation of AGS cells with isogenic H. pylori strains with loss of function deletions in each of these three genes resulted in a decrease in p27 expression similar to that observed with the wild-type strain (Fig. 9,A). All of the five clinical strains tested also decreased the level of p27 protein, but strains lacking the entire cag island (strains J68, B107, and B166) showed a small decrease in their ability to decrease p27 (Fig. 9 B), indicating that genes within the cag pathogenicity island may play a minor role in decreasing the level of p27 protein. Taken together, these data suggest that the ability of H. pylori to reduce p27 is not related to cagA, cage, or allelotypes of vacA or iceA but may be partially dependent on other cag island genes.

Chronic infection with H. pylori results in chronic gastritis, which progresses in some susceptible individuals to gastric cancer. Experiments reported here suggest that a pathway that may be relevant to malignant transformation induced by H. pylori involves the down-regulation of p27, a cell cycle inhibitor, tumor suppressor, and apoptosis regulator. Previous work from our group (21) and others (22) has demonstrated that chronic H. pylori infection is associated with a reduction in the expression of p27 protein in gastric epithelial cells in biopsy specimens, as detected by immunohistochemistry. We now demonstrate that the reduction in levels of p27 protein expression can be reproduced by the coculture of H. pylori with gastric epithelial cells and that it is dose and time dependent. Our data also reveal that the decrease in p27 protein is mediated through increased p27 protein breakdown rather than decreased transcription, based on the findings that steady-state p27 mRNA levels were unchanged, whereas pulse-chase analysis demonstrated more rapid protein degradation after the addition of H. pylori to gastric epithelial cells.

It is noteworthy that the reduction in p27 that we observed with H. pylori occurred independent of H. pylori’s effects on the cell cycle. Previous work has established that although chronic H. pylori infection in vivo is associated with a hyperproliferative state (10), short-term coculture of H. pylori with gastric epithelial cells in vitro results in an inhibition of cell cycle progression at the G1-S phase of the cycle (10, 38) and at G2-M (28). The level of p27 protein is highly cell cycle phase dependent. Therefore we took particular attention in designing experiments that demonstrated that H. pylori was capable of down-regulating p27 expression in cells that were not actively proliferating (because p27 expression falls during S phase), thus dissociating the effect of H. pylori on p27 expression from the effects of H. pylori on cell cycle-dependent changes in p27 expression.

What are the molecular mechanisms responsible for the increased p27 degradation by H. pylori? Increased activation of Thr187-dependent ubiquitin-proteasome pathway associated with increased skp2 expression has been reported in several human cancers, including colon and gastric cancer (17, 19), and low p27 expression is generally associated with a poor prognosis in cancer (14). These observations are consistent with increased cell proliferation in cancer cells and the demonstration of p27 as a tumor suppressor (16) in an animal model. We demonstrate here that H. pylori decreases p27 protein in epithelial cells by up-regulating proteasome-dependent degradation. Our results also suggest that this degradation is associated with neither increased phosphorylation of p27 at Thr187, increased skp2 expression, nor increased p27 ubiquitination. Indeed, the expressions of skp2, Thr187-phosphorylated p27, and ubiquitinated p27 were all decreased by the addition of H. pylori to AGS cells. Thus, H. pylori increases p27 protein degradation by a mechanism that it is quite different from that responsible for the degradation of p27 during normal cell cycling.

Several alternative pathways that regulate the function and/or expression of p27 protein at a posttranslational level have been described recently, including some that are proteasome independent (33, 39), some that are skp2 independent (34), and some that are Thr187 phosphorylation independent (34, 40). Furthermore, in addition to the onset of p27 degradation in late G1 phase, two groups have reported a separate and distinct p27 degradative pathway active in the G0-G1 phase of the cell cycle that is independent of phosphorylation at Thr187(34, 40), although there is disagreement regarding the requirement for skp2 in this pathway. Our data also demonstrate that the reduction of p27 by H. pylori is less evident in exponentially growing AGS cells than in serum-starved cells, consistent with H. pylori decreasing p27 predominantly or entirely in cells in G0-G1 phase, possibly through this Thr187-independent pathway. It is not yet clear whether this more recently discovered pathway is dependent on the phosphorylation of p27 at sites other than Thr187; several other phosphorylated forms of p27 have been described, including p27 phosphorylation downstream of mitogen-activated protein kinase signaling pathways (39) and phosphorylation of serine 10 (41, 42), threonine 157 (43, 44, 45), and threonine 198 (by Akt; Ref. 46), which have each been reported to be associated with nuclear export of p27 and reduced p27 protein stability. Whether the apparent abundance of “alternative” p27 degradative pathways, as well as one involving the neddylation of skp2 that obviates the need for p27 phosphorylation before ubiquitination (47), are cell type dependent, cell cycle phase dependent, and stimulus dependent remains to be determined.

The above-mentioned mechanisms of increased p27 degradation all converge on ubiquitination as a final common step before proteasome-mediated degradation. However, our data support a proteasome- dependent but ubiquitin-independent degradation pathway. Such a pathway has not previously been described for p27, although it has been described for several other short-lived proteins including p21cip1(48, 49), ornithine decarboxylase (50), and c-Jun (51). We speculate that H. pylori may stimulate such a novel proteasome-dependent, ubiquitin-independent pathway of p27 degradation.

Although our results indicate that H. pylori regulates the expression of p27 through increased degradation, this may not be the only mechanism responsible for decreasing p27 protein expression during chronic infection by H. pylori. In a long-term coculture model during which H. pylori selects for apoptosis-resistant gastric epithelial cells, we have documented that the expression of p27 mRNA is reduced in these apoptosis-resistant cells by about 30% by Northern blot (21) and have recently confirmed this result by cDNA microarray analysis.4 Thus, p27 protein may also be transcriptionally regulated by H. pylori during chronic infection.

H. pylori is highly prevalent in human populations and is genetically diverse (37). The ability of H. pylori to promote gastric carcinogenesis in only a subset of infected persons is therefore thought to be related to both specific bacterial virulence factors and genetically determined host responses to infection, particularly polymorphisms of cytokines and cytokine receptors (52, 53). Bacterial virulence factors with a potential to influence gastric epithelial cell cycling and epithelial cell signal transduction include H. pylori’s VacA exotoxin and genes within its cag pathogenicity island (10, 28). The cag gene products include several that form a type IV bacterial secretory apparatus capable of translocating H. pylori products, such as CagA, directly into gastric epithelial cells (54, 55), resulting in the induction of signal transduction pathways of potential relevance to malignant transformation (56). Our results indicate that the reduction of p27 is not dependent on H. pylori’s cagA, cagE, or vacA genes, although the requirement for adherence and the results using five clinical strains suggest that other genes within the cag pathogenicity island may be responsible for the effects of H. pylori on p27. We note with interest the recent report by Sommi et al.(57) that H. pylori broth culture filtrate may increase the expression of p27, as determined by immunofluorescence. This effect was associated with the inhibition of cell cycle progression in two of the four gastric cell lines examined. However, because in vivo p27 expression is decreased in H. pylori-infected patients (21, 22), the pathophysiological significance of these findings related to a putative soluble factor secreted by H. pylori are currently uncertain. Furthermore, in our experiments, the expression of p27 protein was unchanged when the attachment of H. pylori to gastric cells was inhibited by a Transwell membrane, thus indicating that secreted H. pylori products are unlikely to be responsible for altering p27. Of interest, this factor studied from broth culture filtrates also did not inhibit cell cycle progression in AGS cells (57), although it is well established that live H. pylori bacteria do have this effect (23, 28, 38). Therefore, although these discrepant results may be due to methodological differences, such as the use of highly concentrated broth culture filtrate or the measurement of p27 by flow cytometric analysis rather than immunoblotting, it is conceivable that H. pylori does have more than one effect on p27 protein—one that requires attachment, and one that does not.

Regardless of the precise mechanisms by which H. pylori alters the expression of the cell cycle inhibitor p27, the functional consequences of decreased p27 are likely to be relevant to gastric carcinogenesis. In an animal model, homozygous or heterozygous loss of p27 causes tissue hyperplasia associated with increased cell proliferation and increased susceptibility to cancer after exposure to exogenous carcinogens (16, 58). Additionally, loss of p27 may reduce cell death through apoptosis (59), thus potentially causing inappropriate and excessive tissue growth.

In summary, our results indicate that H. pylori activates pathways leading to the increased degradation of p27 in gastric epithelial cells through a proteasome-dependent, ubiquitin-independent mechanism that is distinct from the physiological pathway of p27 degradation known to be up-regulated in some cancers. Analysis of several H. pylori cancer-associated genes suggests that neither cagA, cagE, nor vacA is involved in this process but that other cag island genes may play a modest role in the reduction of p27. Further work may define the specific H. pylori factors responsible. Regardless of mechanisms, the down-regulation of p27 by H. pylori may lead to increased gastric epithelial cell proliferation, decreased apoptosis, and increased risk for gastric cancer.

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

Supported by grants from the Concern Foundation and the R. Robert and Sally D. Funderburg Research Scholar Award from the American Gastroenterology Association’s Foundation for Digestive Health and Nutrition and from the U.S. National Institutes of Health (COBRE grant 1P20RR17695-01) (to S. F. M.).

3

The abbreviations used are: CDK, cyclin-dependent kinase; AMC, 7-amino-4-methylcoumarin.

4

H. Eguchi, S. Carpentier, S-S. Kim, S. F. Moss. Regulates the apoptotic response of gastric epithelial cells to Helicobacter pylori, manuscript in preparation.

Fig. 1.

Correlation of p27-related protein expression with specific cell cycle distributions in AGS cells. AGS cells were cultured (a) in normal medium [Serum (+)], (b) for 48 h in serum-free medium [Serum (−)], and (c) in the presence of the proteasome inhibitor MG-132 (10 μm). The bottom graphs in A show the densitometric analysis of protein expression under these conditions. A, protein analysis by Western blot, with β-actin as control. B, flow cytometric analysis of cell cycle phase distribution, with percentages of cells in each phase as shown.

Fig. 1.

Correlation of p27-related protein expression with specific cell cycle distributions in AGS cells. AGS cells were cultured (a) in normal medium [Serum (+)], (b) for 48 h in serum-free medium [Serum (−)], and (c) in the presence of the proteasome inhibitor MG-132 (10 μm). The bottom graphs in A show the densitometric analysis of protein expression under these conditions. A, protein analysis by Western blot, with β-actin as control. B, flow cytometric analysis of cell cycle phase distribution, with percentages of cells in each phase as shown.

Close modal
Fig. 2.

A, cell cycle phase distribution of AGS cells in the presence or absence of H. pylori. AGS cells were synchronized by 48 h of serum starvation (0 h) and harvested for flow cytometry 0, 6, 12, and 24 h after the addition of H. pylori (+) or medium alone (−). The first column illustrates the results for AGS cells growing exponentially in 10% serum. [Serum (+)]. B, alteration of p27 protein expression by H. pylori. AGS cells were starved of serum for 48 h to induce synchronization (Lanes 1–5) or grown continually in medium containing serum (Lanes 6–9), and H. pylori was added at the indicated ratios. The expression of p27 protein was measured by immunoblot after 12 h of coculture. The bottom graph shows the densitometric analysis of the expression of p27. The decrease of p27 was dependent on the concentration of H. pylori and was more marked in the serum-starved cells.

Fig. 2.

A, cell cycle phase distribution of AGS cells in the presence or absence of H. pylori. AGS cells were synchronized by 48 h of serum starvation (0 h) and harvested for flow cytometry 0, 6, 12, and 24 h after the addition of H. pylori (+) or medium alone (−). The first column illustrates the results for AGS cells growing exponentially in 10% serum. [Serum (+)]. B, alteration of p27 protein expression by H. pylori. AGS cells were starved of serum for 48 h to induce synchronization (Lanes 1–5) or grown continually in medium containing serum (Lanes 6–9), and H. pylori was added at the indicated ratios. The expression of p27 protein was measured by immunoblot after 12 h of coculture. The bottom graph shows the densitometric analysis of the expression of p27. The decrease of p27 was dependent on the concentration of H. pylori and was more marked in the serum-starved cells.

Close modal
Fig. 3.

A, Northern blot analysis of p27 mRNA in AGS cells after 0, 3, 6, and 12 h of coculture with H. pylori, demonstrating that the abundance of p27 mRNA was not altered by H. pylori. B, pulse-chase analysis of the turnover rate of p27. The bottom graph shows the densitometric analysis of the radiolabeled p27, relative to time 0 (100%). The addition of H. pylori increases the rate of degradation of 35S-labeled p27.

Fig. 3.

A, Northern blot analysis of p27 mRNA in AGS cells after 0, 3, 6, and 12 h of coculture with H. pylori, demonstrating that the abundance of p27 mRNA was not altered by H. pylori. B, pulse-chase analysis of the turnover rate of p27. The bottom graph shows the densitometric analysis of the radiolabeled p27, relative to time 0 (100%). The addition of H. pylori increases the rate of degradation of 35S-labeled p27.

Close modal
Fig. 4.

A, p27 degradation by H. pylori is proteasome dependent. AGS cells and H. pylori were cocultured for 6 h in the absence (−) or presence (+) of the proteasome inhibitor MG-132 (20 μm). Western blot demonstrates that the decrease in p27 expression induced by H. pylori is abolished by MG-132. The bottom graph shows the densitometric analysis of p27 expression in this blot, normalized for β-actin. B, effects of H. pylori on total p27, Thr187-phosphorylated p27, and skp2 ubiquitin ligase. AGS cells were serum deprived for 48 h, and then H. pylori (+) or serum-free medium only (−) was added, and cells were harvested at the times indicated. The bottom graph shows the densitometric analysis of specific protein expression by immunoblot after normalization for β-actin.

Fig. 4.

A, p27 degradation by H. pylori is proteasome dependent. AGS cells and H. pylori were cocultured for 6 h in the absence (−) or presence (+) of the proteasome inhibitor MG-132 (20 μm). Western blot demonstrates that the decrease in p27 expression induced by H. pylori is abolished by MG-132. The bottom graph shows the densitometric analysis of p27 expression in this blot, normalized for β-actin. B, effects of H. pylori on total p27, Thr187-phosphorylated p27, and skp2 ubiquitin ligase. AGS cells were serum deprived for 48 h, and then H. pylori (+) or serum-free medium only (−) was added, and cells were harvested at the times indicated. The bottom graph shows the densitometric analysis of specific protein expression by immunoblot after normalization for β-actin.

Close modal
Fig. 5.

Effects of H. pylori on regulators of the G1-S transition. AGS cells were serum deprived for 48 h, and then H. pylori or serum-free medium only was added, and cells were harvested at the indicated time points. A, decrease in CDK2-associated kinase activity by H. pylori [HP (+)]. B, the binding of p27 to CDK2 was not decreased by H. pylori. Cell lysates were immunoprecipitated using anti-CDK2 antibody, and immunoprecipitates (IP) and 10% aliquots of supernatant (SUP) were subjected to electrophoresis. p27 was evaluated by Western blot. C, immunoblot demonstrating expression of G1-S-phase-regulatory proteins in AGS cells. H. pylori was added at the indicated AGS:bacterial cell ratios. The bottom graphs show the densitometric analysis of protein expression after the addition of H. pylori.

Fig. 5.

Effects of H. pylori on regulators of the G1-S transition. AGS cells were serum deprived for 48 h, and then H. pylori or serum-free medium only was added, and cells were harvested at the indicated time points. A, decrease in CDK2-associated kinase activity by H. pylori [HP (+)]. B, the binding of p27 to CDK2 was not decreased by H. pylori. Cell lysates were immunoprecipitated using anti-CDK2 antibody, and immunoprecipitates (IP) and 10% aliquots of supernatant (SUP) were subjected to electrophoresis. p27 was evaluated by Western blot. C, immunoblot demonstrating expression of G1-S-phase-regulatory proteins in AGS cells. H. pylori was added at the indicated AGS:bacterial cell ratios. The bottom graphs show the densitometric analysis of protein expression after the addition of H. pylori.

Close modal
Fig. 6.

H. pylori decreases protein ubiquitination. A, in vitro assay of p27 ubiquitination by lysates of AGS cells grown in the absence or presence of H. pylori. The incubation time of in vitro-translated p27 with the cell lysates is indicated in minutes at the top. Mono- and polyubiquitinated p27 species are decreased in lysates from serum-starved AGS cells cocultured with H. pylori and increased in lysates from exponentially growing cells. B, Western blot analysis of total cell lysates using anti-ubiquitin antibody demonstrating that H. pylori decreases ubiquitinated protein expression, detectable when the proteasome activity is inhibited with 50 μmN-acetyl-Leu-Leu-norleucinal (ALLN; Sigma Chemical Co.).

Fig. 6.

H. pylori decreases protein ubiquitination. A, in vitro assay of p27 ubiquitination by lysates of AGS cells grown in the absence or presence of H. pylori. The incubation time of in vitro-translated p27 with the cell lysates is indicated in minutes at the top. Mono- and polyubiquitinated p27 species are decreased in lysates from serum-starved AGS cells cocultured with H. pylori and increased in lysates from exponentially growing cells. B, Western blot analysis of total cell lysates using anti-ubiquitin antibody demonstrating that H. pylori decreases ubiquitinated protein expression, detectable when the proteasome activity is inhibited with 50 μmN-acetyl-Leu-Leu-norleucinal (ALLN; Sigma Chemical Co.).

Close modal
Fig. 7.

Proteasomal activities measured in vitro. A, chymotrypsin-like activity; B, peptidylglutamyl peptide hydrolyzing-like activity; C, trypsin-like activity. AGS cells were cocultured with H. pylori for the indicated times. Gray columns show proteasome activity results in the absence of H. pylori. All data have been normalized to time 0 (100%). ∗, P < 0.01 by two-way ANOVA with Bonferroni-adjusted t tests compared with the absence of H. pylori.

Fig. 7.

Proteasomal activities measured in vitro. A, chymotrypsin-like activity; B, peptidylglutamyl peptide hydrolyzing-like activity; C, trypsin-like activity. AGS cells were cocultured with H. pylori for the indicated times. Gray columns show proteasome activity results in the absence of H. pylori. All data have been normalized to time 0 (100%). ∗, P < 0.01 by two-way ANOVA with Bonferroni-adjusted t tests compared with the absence of H. pylori.

Close modal
Fig. 8.

Effect of H. pylori on p27, cyclin E, and cyclin A in MKN28 gastric epithelial cells. H. pylori was added to MKN28 cells that had been serum deprived for 48 h. Cells were harvested for immunoblotting at 12, 24, and 36 h. The bottom graphs show the densitometric analysis of protein expression.

Fig. 8.

Effect of H. pylori on p27, cyclin E, and cyclin A in MKN28 gastric epithelial cells. H. pylori was added to MKN28 cells that had been serum deprived for 48 h. Cells were harvested for immunoblotting at 12, 24, and 36 h. The bottom graphs show the densitometric analysis of protein expression.

Close modal
Fig. 9.

A, AGS cells and wild-type (WT) H. pylori or its isogenic mutants (cagA negative, cagE negative, or vacA negative) were cocultured for 12 h. Expression of p27 protein was decreased to the same manner by wild-type H. pylori or its isogenic mutants. The right panel shows the densitometric analysis of p27 expression. B, strain 60190 and five clinical strains of H. pylori were cocultured for 12 h. cagA, vacA, and iceA status of the strains examined is listed in the table. The bottom graph shows the densitometric analysis of protein expression. −, no H. pylori (control).

Fig. 9.

A, AGS cells and wild-type (WT) H. pylori or its isogenic mutants (cagA negative, cagE negative, or vacA negative) were cocultured for 12 h. Expression of p27 protein was decreased to the same manner by wild-type H. pylori or its isogenic mutants. The right panel shows the densitometric analysis of p27 expression. B, strain 60190 and five clinical strains of H. pylori were cocultured for 12 h. cagA, vacA, and iceA status of the strains examined is listed in the table. The bottom graph shows the densitometric analysis of protein expression. −, no H. pylori (control).

Close modal

We are grateful to Suzanne de la Monte for technical advice and Jeffrey D. Singer for helpful discussions and manuscript review.

1
Huang J. Q., Sridhar S., Chen Y., Hunt R. H. Meta-analysis of the relationship between Helicobacter pylori seropositivity and gastric cancer.
Gastroenterology
,
114
:
1169
-1179,  
1998
.
2
Helicobacter and Cancer Collaborative Group. Gastric cancer and Helicobacter pylori: a combined analysis of 12 case control studies nested within prospective cohorts.
Gut
,
49
:
347
-353,  
2001
.
3
Ekstrom A. M., Held M., Hansson L. E., Engstrand L., Nyren O. Helicobacter pylori in gastric cancer established by CagA immunoblot as a marker of past infection.
Gastroenterology
,
121
:
784
-791,  
2001
.
4
Uemura N., Okamoto S., Yamamoto S., Matsumura N., Yamaguchi S., Yamakido M., Taniyama K., Sasaki N., Schlemper R. J. Helicobacter pylori infection and the development of gastric cancer.
N. Engl. J. Med.
,
345
:
784
-789,  
2001
.
5
Correa P., Fontham E. T., Bravo J. C., Bravo L. E., Ruiz B., Zarama G., Realpe J. L., Malcom G. T., Li D., Johnson W. D., Mera R. Chemoprevention of gastric dysplasia: randomized trial of antioxidant supplements and anti-Helicobacter pylori therapy.
J. Natl. Cancer Inst. (Bethesda)
,
92
:
1881
-1888,  
2000
.
6
Honda S., Fujioka T., Tokieda M., Satoh R., Nishizono A., Nasu M. Development of Helicobacter pylori-induced gastric carcinoma in Mongolian gerbils.
Cancer Res.
,
58
:
4255
-4259,  
1998
.
7
Watanabe T., Tada M., Nagai H., Sasaki S., Nakao M. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils.
Gastroenterology
,
115
:
642
-648,  
1998
.
8
Fox J. G., Sheppard B. J., Dangler C. A., Whary M. T., Ihrig M., Wang T. C. Germ-line p53-targeted disruption inhibits Helicobacter-induced premalignant lesions and invasive gastric carcinoma through down-regulation of Th1 proinflammatory responses.
Cancer Res.
,
62
:
696
-702,  
2002
.
9
Mannick E. E., Bravo L. E., Zarama G., Realpe J. L., Zhang X. J., Ruiz B., Fontham E. T., Mera R., Miller M. J., Correa P. Inducible nitric oxide synthase, nitrotyrosine, and apoptosis in Helicobacter pylori gastritis: effect of antibiotics and antioxidants.
Cancer Res.
,
56
:
3238
-3243,  
1996
.
10
Shirin H., Weinstein I. B., Moss S. F. Effects of H. pylori infection of gastric epithelial cells on cell cycle control.
Front. Biosci.
,
6
:
E104
-E118,  
2001
.
11
Preston-Martin S., Pike M. C., Ross R. K., Jones P. A., Henderson B. E. Increased cell division as a cause of human cancer.
Cancer Res.
,
50
:
7415
-7421,  
1990
.
12
Sherr C. J. Cancer cell cycles.
Science (Wash. DC)
,
274
:
1672
-1677,  
1996
.
13
Sandhu C., Slingerland J. Deregulation of the cell cycle in cancer.
Cancer Detect. Prev.
,
24
:
107
-118,  
2000
.
14
Philipp-Staheli J., Payne S. R., Kemp C. J. p27Kip1: regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer.
Exp. Cell Res.
,
264
:
148
-168,  
2001
.
15
Slingerland J., Pagano M. Regulation of the cdk inhibitor p27 and its deregulation in cancer.
J. Cell. Physiol.
,
183
:
10
-17,  
2000
.
16
Fero M. L., Randel E., Gurley K. E., Roberts J. M., Kemp C. J. The murine gene p27Kip1 is haplo-insufficient for tumour suppression.
Nature (Lond.)
,
396
:
177
-180,  
1998
.
17
Loda M., Cukor B., Tam S. W., Lavin P., Fiorentino M., Draetta G. F., Jessup J. M., Pagano M. Increased proteasome-dependent degradation of the cyclin-dependent kinase inhibitor p27 in aggressive colorectal carcinomas.
Nat. Med.
,
3
:
231
-234,  
1997
.
18
Masuda T. A., Inoue H., Sonoda H., Mine S., Yoshikawa Y., Nakayama K., Mori M. Clinical and biological significance of S-phase kinase-associated protein 2 (Skp2) gene expression in gastric carcinoma: modulation of malignant phenotype by Skp2 overexpression, possibly via p27 proteolysis.
Cancer Res.
,
62
:
3819
-3825,  
2002
.
19
Mori M., Mimori K., Shiraishi T., Tanaka S., Ueo H., Sugimachi K., Akiyoshi T. p27 expression and gastric carcinoma.
Nat. Med.
,
3
:
593
1997
.
20
Ohtani M., Isozaki H., Fujii K., Nomura E., Niki M., Mabuchi H., Nishiguchi K., Toyoda M., Ishibashi T., Tanigawa N. Impact of the expression of cyclin-dependent kinase inhibitor p27Kip1 and apoptosis in tumor cells on the overall survival of patients with non-early stage gastric carcinoma.
Cancer (Phila.)
,
85
:
1711
-1718,  
1999
.
21
Shirin H., Sordillo E. M., Kolevska T. K., Hibshoosh H., Kawabata Y., Oh S. H., Kuebler J. F., Delohery T., Weghorst C. M., Weinstein I. B., Moss S. F. Chronic Helicobacter pylori infection induces an apoptosis-resistant phenotype associated with decreased expression of p27kip1.
Infect. Immun.
,
68
:
5321
-5328,  
2000
.
22
Yu J., Leung W. K., Ng E. K., To K. F., Ebert M. P., Go M. Y., Chan W. Y., Chan F. K., Chung S. C., Malfertheiner P., Sung J. J. Effect of Helicobacter pylori eradication on expression of cyclin D2 and p27 in gastric intestinal metaplasia.
Aliment. Pharmacol. Ther.
,
15
:
1505
-1511,  
2001
.
23
Shirin H., Sordillo E. M., Oh S. H., Yamamoto H., Delohery T., Weinstein I. B., Moss S. F. Helicobacter pylori inhibits the G1 to S transition in AGS gastric epithelial cells.
Cancer Res.
,
59
:
2277
-2281,  
1999
.
24
Tummuru M. K., Cover T. L., Blaser M. J. Mutation of the cytotoxin-associated cagA gene does not affect the vacuolating cytotoxin activity of Helicobacter pylori..
Infect. Immun.
,
62
:
2609
-2613,  
1994
.
25
Cover T. L., Tummuru M. K., Cao P., Thompson S. A., Blaser M. J. Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains.
J. Biol. Chem.
,
269
:
10566
-10573,  
1994
.
26
Tummuru M. K., Sharma S. A., Blaser M. J. Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells.
Mol. Microbiol.
,
18
:
867
-876,  
1995
.
27
Kidd M., Lastovica A. J., Atherton J. C., Louw J. A. Heterogeneity in the Helicobacter pylori vacA and cagA genes: association with gastroduodenal disease in South Africa?.
Gut
,
45
:
499
-502,  
1999
.
28
Peek R. M., Jr., Blaser M. J., Mays D. J., Forsyth M. H., Cover T. L., Song S. Y., Krishna U., Pietenpol J. A. Helicobacter pylori strain-specific genotypes and modulation of the gastric epithelial cell cycle.
Cancer Res.
,
59
:
6124
-6131,  
1999
.
29
Polyak K., Lee M. H., Erdjument-Bromage H., Koff A., Roberts J. M., Tempst P., Massague J. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals.
Cell
,
78
:
59
-66,  
1994
.
30
Sgambato A., Zhang Y. J., Ciaparrone M., Soh J. W., Cittadini A., Santella R. M., Weinstein I. B. Overexpression of p27Kip1 inhibits the growth of both normal and transformed human mammary epithelial cells.
Cancer Res.
,
58
:
3448
-3454,  
1998
.
31
Nguyen H., Gitig D. M., Koff A. Cell-free degradation of p27kip1, a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome.
Mol. Cell. Biol.
,
19
:
1190
-1201,  
1999
.
32
Eguchi H., Nagano H., Yamamoto H., Miyamoto A., Kondo M., Dono K., Nakamori S., Umeshita K., Sakon M., Monden M. Augmentation of antitumor activity of 5-fluorouracil by interferon α is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells.
Clin. Cancer Res.
,
6
:
2881
-2890,  
2000
.
33
Shirane M., Harumiya Y., Ishida N., Hirai A., Miyamoto C., Hatakeyama S., Nakayama K., Kitagawa M. Down-regulation of p27Kip1 by two mechanisms, ubiquitin-mediated degradation and proteolytic processing.
J. Biol. Chem.
,
274
:
13886
-13893,  
1999
.
34
Hara T., Kamura T., Nakayama K., Oshikawa K., Hatakeyama S. Degradation of p27Kip1 at the G0-G1 transition mediated by a Skp2-independent ubiquitination pathway.
J. Biol. Chem.
,
276
:
48937
-48943,  
2001
.
35
Chen G., Sordillo E. M., Ramey W. G., Reidy J., Holt P. R., Krajewski S., Reed J. C., Blaser M. J., Moss S. F. Apoptosis in gastric epithelial cells is induced by Helicobacter pylori and accompanied by increased expression of BAK.
Biochem. Biophys. Res. Commun.
,
239
:
626
-632,  
1997
.
36
Matozaki T., Sakamoto C., Matsuda K., Suzuki T., Konda Y., Nakano O., Wada K., Uchida T., Nishisaki H., Nagao M., Kasuga M. Missense mutations and a deletion of the p53 gene in human gastric cancer.
Biochem. Biophys. Res. Commun.
,
182
:
215
-223,  
1992
.
37
Suerbaum S., Michetti P. Helicobacter pylori infection.
N. Engl. J. Med.
,
347
:
1175
-1186,  
2002
.
38
Ahmed A., Smoot D., Littleton G., Tackey R., Walters C. S., Kashanchi F., Allen C. R., Ashktorab H. Helicobacter pylori inhibits gastric cell cycle progression.
Microbes Infect.
,
2
:
1159
-1169,  
2000
.
39
Kawada M., Yamagoe S., Murakami Y., Suzuki K., Mizuno S., Uehara Y. Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway.
Oncogene
,
15
:
629
-637,  
1997
.
40
Malek N. P., Sundberg H., McGrew S., Nakayama K., Kyriakides T. R., Roberts J. M., Kyriakidis T. R. A mouse knock-in model exposes sequential proteolytic pathways that regulate p27Kip1 in G1 and S phase.
Nature (Lond.)
,
413
:
323
-327,  
2001
.
41
Ishida N., Kitagawa M., Hatakeyama S., Nakayama K. Phosphorylation at serine 10, a major phosphorylation site of p27Kip1, increases its protein stability.
J. Biol. Chem.
,
275
:
25146
-25154,  
2000
.
42
Ishida N., Hara T., Kamura T., Yoshida M., Nakayama K., Nakayama K. I. Phosphorylation of p27Kip1 on serine 10 is required for its binding to CRM1 and nuclear export.
J. Biol. Chem.
,
277
:
14355
-14358,  
2002
.
43
Viglietto G., Motti M. L., Bruni P., Melillo R. M., D’Alessio A., Califano D., Vinci F., Chiappetta G., Tsichlis P., Bellacosa A., Fusco A., Santoro M. Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by PKB/Akt-mediated phosphorylation in breast cancer.
Nat. Med.
,
8
:
1136
-1144,  
2002
.
44
Shin I., Yakes F. M., Rojo F., Shin N. Y., Bakin A. V., Baselga J., Arteaga C. L. PKB/Akt mediates cell-cycle progression by phosphorylation of p27Kip1 at threonine 157 and modulation of its cellular localization.
Nat. Med.
,
8
:
1145
-1152,  
2002
.
45
Liang J., Zubovitz J., Petrocelli T., Kotchetkov R., Connor M. K., Han K., Lee J. H., Ciarallo S., Catzavelos C., Beniston R., Franssen E., Slingerland J. M. PKB/Akt phosphorylates p27, impairs nuclear import of p27 and opposes p27-mediated G1 arrest.
Nat. Med.
,
8
:
1153
-1160,  
2002
.
46
Fujita N., Sato S., Katayama K., Tsuruo T. Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization.
J. Biol. Chem.
,
277
:
28706
-28713,  
2002
.
47
Podust V. N., Brownell J. E., Gladysheva T. B., Luo R. S., Wang C., Coggins M. B., Pierce J. W., Lightcap E. S., Chau V. A Nedd8 conjugation pathway is essential for proteolytic targeting of p27Kip1 by ubiquitination.
Proc. Natl. Acad. Sci. USA
,
97
:
4579
-4584,  
2000
.
48
Sheaff R. J., Singer J. D., Swanger J., Smitherman M., Roberts J. M., Clurman B. E. Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination.
Mol. Cell
,
5
:
403
-410,  
2000
.
49
Bao W., Thullberg M., Zhang H., Onischenko A., Stromblad S. Cell attachment to the extracellular matrix induces proteasomal degradation of p21CIP1 via Cdc42/Rac1 signaling.
Mol. Cell. Biol.
,
22
:
4587
-4597,  
2002
.
50
Murakami Y., Matsufuji S., Kameji T., Hayashi S., Igarashi K., Tamura T., Tanaka K., Ichihara A. Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination.
Nature (Lond.)
,
360
:
597
-599,  
1992
.
51
Jariel-Encontre I., Pariat M., Martin F., Carillo S., Salvat C., Piechaczyk M. Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26 S proteasome.
J. Biol. Chem.
,
270
:
11623
-11627,  
1995
.
52
El-Omar E. M., Carrington M., Chow W. H., McColl K. E., Bream J. H., Young H. A., Herrera J., Lissowska J., Yuan C. C., Rothman N., Lanyon G., Martin M., Fraumeni J. F., Jr., Rabkin C. S. Interleukin-1 polymorphisms associated with increased risk of gastric cancer.
Nature (Lond.)
,
404
:
398
-402,  
2000
.
53
Figueiredo C., Machado J. C., Pharoah P., Seruca R., Sousa S., Carvalho R., Capelinha A. F., Quint W., Caldas C., van Doorn L. J., Carneiro F., Sobrinho-Simoes M. Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma.
J. Natl. Cancer Inst. (Bethesda)
,
94
:
1680
-1687,  
2002
.
54
Censini S., Lange C., Xiang Z., Crabtree J. E., Ghiara P., Borodovsky M., Rappuoli R., Covacci A. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors.
Proc. Natl. Acad. Sci. USA
,
93
:
14648
-14653,  
1996
.
55
Censini S., Stein M., Covacci A. Cellular responses induced after contact with Helicobacter pylori..
Curr. Opin. Microbiol.
,
4
:
41
-46,  
2001
.
56
Higashi H., Tsutsumi R., Muto S., Sugiyama T., Azuma T., Asaka M., Hatakeyama M. SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein.
Science (Wash. DC)
,
295
:
683
-686,  
2002
.
57
Sommi P., Savio M., Stivala L. A., Scotti C., Mignosi P., Prosperi E., Vannini V., Solcia E. Helicobacter pylori releases a factor(s) inhibiting cell cycle progression of human gastric cell lines by affecting cyclin E/cdk2 kinase activity and Rb protein phosphorylation through enhanced p27KIP1 protein expression.
Exp. Cell Res.
,
281
:
128
-139,  
2002
.
58
Kiyokawa H., Kineman R. D., Manova-Todorova K. O., Soares V. C., Hoffman E. S., Ono M., Khanam D., Hayday A. C., Frohman L. A., Koff A. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27Kip1.
Cell
,
85
:
721
-732,  
1996
.
59
Katayose Y., Kim M., Rakkar A. N., Li Z., Cowan K. H., Seth P. Promoting apoptosis: a novel activity associated with the cyclin-dependent kinase inhibitor p27.
Cancer Res.
,
57
:
5441
-5445,  
1997
.