Main

Epidemiological, clinico-pathological, and animal studies performed over the last 20 years have provided increasing evidence for the importance of prior infection by the gastric bacterium Helicobacter pylori in the genesis of gastric cancer.1, 2 It has been estimated that 59% of gastric cancers worldwide are directly attributable to H. pylori.3 Both host-related genetic susceptibility and bacterial virulence factors contribute to the tendency of H. pylori to induce cancer in only a minority of all individuals at risk.4, 5 For the most common histological subtype of gastric cancer (the ‘intestinal’ subtype6), a pre-neoplastic sequence manifests from chronic superficial gastritis through atrophic gastritis, intestinal metaplasia, and dysplasia to cancer, as defined originally by Correa et al.7 Progression through these histological cancer precursors is accompanied by the accumulation of mutations, including that of p53, typical of a multi-step process of carcinogenesis.8 The ‘diffuse’ subtype of gastric cancer6 may also be preceded by years of chronic gastritis,8 but typically without progression through recognizable atrophic change or metaplasia. Reduction or loss of expression of E-cadherin and catenins by both mutation and CpG hypermethylation are frequently noted molecular changes in diffuse gastric carcinogenesis.8 Although the precise molecular and cellular events responsible for the promotion of gastric cancer by H. pylori remain poorly defined, the ability of H. pylori to increase gastric epithelial cell turnover9 is a potentially important step, based on the widespread association of neoplastic transformation with hyperproliferative states.10

p27kip1 is a cyclin-dependent kinase inhibitor that binds to cyclinE/cdk2, thereby blocking the G1/S transition necessary for cell cycle progression.11 The level of p27 is upregulated under stress conditions, leading to cell cycle inhibition and apoptosis.11 Additional roles for p27 have also been proposed recently, including tumor suppression12 and the regulation of cell migration13 and mitosis.14 p27 protein levels are mainly regulated through degradation by ubiquitin-dependent proteolysis.15 Protein ubiquitination requires a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase, or E3.16 The E3 ligase activity for p27 is provided by the SCF complex, composed of Skp1, cullin-1, rbx-1, and the p27-specific F-box protein Skp2.16 Skp2 specifically interacts with the extreme COOH-terminus of p27 only when Thr-187 is phosphorylated by cyclin E/CDK2.17, 18 This association of Skp2 with p27 results in the recruitment of p27 to the SCF core complex, thereby promoting its ubiquitination and degradation.19 Mice lacking Skp2 accumulate p27, suggesting a pivotal role for Skp2 in the degradation of p27 in vivo.20 However, other reports suggest the existence of an Skp2-independent mechanism of p27 degradation.21

Low p27 expression has been reported in many cancers, including those of the colon, breast, prostate, lung, and brain, as well as in gastric carcinoma of both intestinal and diffuse subtypes.11, 22, 23, 24 Decreased p27 in cancer is associated with a poorer prognosis.23, 25 p27 protein is also decreased in H. pylori infection, both in areas of gastritis and in intestinal metaplasia.26, 27 In vitro, the addition of H. pylori to cultured gastric epithelial cells increases proteasome-dependent p27 protein degradation without altering p27 gene transcription.28 However, it is unknown whether similar mechanisms occur in vivo or whether decreased p27 is restored to normal following H. pylori eradication in chronic gastritis.

Skp2 is upregulated and is also related to a poor prognosis in gastric carcinoma,29 but it remains uncertain whether increased Skp2 mediates accelerated degradation of p27 protein or whether Skp2 influences p27 through other mechanisms. Skp2 regulates the expression of many intracellular proteins by ubiquitination, including p27, c-Myc, p57kip2, Smad4, and E2F-1, thus leading to their increased proteasomal degradation.20, 30, 31, 32, 33, 34 The effect of H. pylori on Skp2 has not been previously evaluated.

The c-Myc oncoprotein is a basic helix–loop–helix zipper transcriptional factor that regulates several genes intimately involved in cell proliferation, differentiation, apoptosis, and oncogenesis.35 c-Myc decreases not only transcription but also the nuclear transport of p27 from the cytoplasm, thus regulating the G1/S transition for cell cycle progression through p27.36, 37 The SCF component Skp2 accelerates c-Myc ubiquitination and destruction, as well as stimulates c-Myc transcription in cells progressing to S phase.30, 31 Thus, several mechanisms may regulate p27 expression through c-Myc and Skp2 in the context of H. pylori infection, and the ability of Skp2 to both activate c-Myc and destroy p27 may be relevant to the development of H. pylori-associated gastric cancer. Thus far, no evidence exists that the p27–Skp2–c-Myc axis is related on a molecular basis or epidemiologically to the established p53 and E-cadherin pathways of gastric carcinogenesis.8

Owing to the importance of translating these observations regarding p27, Skp2, and c-Myc derived from in vitro cellular models to understand the mechanisms of H. pylori-associated gastric carcinogenesis, we investigated whether in vivo low p27 levels are reversible after the eradication of H. pylori, whether p27 protein in H. pylori infection is related to low p27 mRNA expression, and whether low p27 protein expression in H. pylori infection is associated with altered expression of Skp2 and c-Myc.

Materials and methods

Patients and Tissue Collection

A total of 22 patients with chronic gastritis who had successfully been treated for H. pylori in Uijongbu St. Mary's Hospital, Korea, were included in this study. Their mean age was 53 years (range 33–71); 12 were male. Two endoscopic antral biopsies were taken before and 2 months after eradication of H. pylori. Tissues were fixed in neutral-buffered formalin and embedded in paraffin blocks for RNA extraction and immunohistochemical staining. H. pylori infection was diagnosed in biopsies by a rapid urease test, H&E staining, and immunohistochemistry. H. pylori was eradicated using a proton pump inhibitor (omeprazole, lansoprazole, or rabeprazole) with amoxicillin and clarithromycin for 7–14 days.38 Eradication was confirmed by a negative urea breath test, and by the assessment of visible H. pylori bacteria on H&E staining and immunohistochemistry.38

Laser Capture Microdissection (LCM) and RNA Extraction

Formalin-fixed, paraffin-embedded tissues were sectioned at 8 μm thickness for RNA extraction. The sections were deparaffinized with xylene, and rehydrated with diluted ethanols in RNase-free water. Slides were then stained with HistoGene staining solution (Arcturus, Mountain View, CA, USA), dehydrated for 30 s in 75, 95%, and for 60 s in anhydrous 100% ethanol serially, and placed in two changes of xylene (5 min each). After 5 min drying at room temperature, 5–8 thousand epithelial cells were microdissected using the AutoPix LCM instrument (Arcturus) and harvested onto a capsure Macro LCM cap (Arcturus). Glands exhibiting intestinal metaplasia were specifically excluded.

RNA was then extracted from the harvested cells using the Optimum FFPE RNA Isolation kit (Ambion, Austin, TX, USA) as recommended in the manufacturer's instructions with modifications. Briefly, cells were digested with 110 μl (1.81 μg/μl) proteinase K at 42°C for 2 h. In all, 100 μl of RNA extraction buffer was then added, and 105 μl of 100% ethanol was admixed with vortexing. This mixture was transferred to the Filter Cartridge Assembly, and centrifuged at 13 200 rpm for 30 s. The cartridge was washed serially with Washing Solutions 1 and 2/3, and 14 μl of RNA was eluted at 75°C. In all, 1 μl of RNA was used to check the concentration using the BioPhotometer (Eppendorf, Germany), and the remaining RNA was stored at −80°C.

mRNA Quantification Using Real-Time Reverse Transcription Polymerase Chain Reaction (PCR)

Total RNA extracted from microdissected cells was treated with RNase-free DNase I (Roche Diagnostics Corporation, Indianapolis, IN, USA) to remove contaminating genomic DNA. Reverse transcription of RNA was performed in a final volume of 20 μl containing 4 μl of 5 × reverse transcription buffer (250 mM Tris-HCl, 40 mM MgCl2, 150 mM KCl, 5 mM dithiothreitol, pH 8.5), 2.5 mM each deoxynucleotide triphosphate, 40 U of RNase inhibitor, 3.2 μg random hexamers, 20 U of avian myeloblastosis virus reverse transcriptase (all from Roche), and 250 ng/10 μl of total RNA. Samples were incubated at 25°C for 10 min and 42°C for 1 h, the reverse transcriptase was then inactivated by heating at 99°C for 5 min, and the samples were kept on ice until use.

Real-time quantitative PCR was performed using the iCycler iQ Multi-Color Real Time PCR Detection System (Bio-Rad, Hercules, CA, USA) in a 25 μl reaction mix containing 5 μl of diluted reverse transcribed samples (5.0 ng of equivalent total RNA), 12.5 μl of QuantiTec™ SYBR Green PCR Master Mix (Qiagen, Valencia, CA, USA), and serially diluted plasmid constructs of pcDNA3-p27,39 pcDNA3-skp2 (provided by Michele Pagano, New York University, NY, USA), pLXSH-c-myc,40 or pcDNA2.1-18SrRNA41 in the standard curve. cDNA reverse transcribed from RNA from the AGS gastric carcinoma cell line was used to construct the standard curve for interleukin-8 (IL-8). The sequence and specific volume of each primers used are listed in Table 1. Thermocycling was carried out for 45 cycles, with denaturation at 95°C for 15 s, annealing for 30 s at the temperature listed in Table 1, and extension at 72°C for 30 s. Standard serial 10 × dilutions were prepared from the plasmid constructs or the AGS cell line for IL-8 to relate the threshold cycle to the log input amount of template. The relative amounts of gene transcripts for p27, skp2, c-myc, and IL-8 were determined using the relative standard curve method, and were normalized for 18SrRNA.41

Table 1 PCR primers used in this study
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Immunohistochemistry for p27, Skp2, c-Myc, and H. pylori and Evaluation of Inflammation

Formalin-fixed, paraffin-embedded antral biopsy specimens were sectioned at 5 μm thickness, and mounted on Fisherbrand Superfrost plus slides (Fisher Scientific, Pittsburgh, PA, USA). After antigen retrieval (for c-Myc in 1 mM EDTA (pH 8.0) and for Skp-2 in 10 mM citrate buffer (pH 6.0)), sections were incubated overnight at 4°C with monoclonal mouse antibodies to c-Myc (1:100 dilution, Abcam Inc., Cambridge, MA, USA), Skp-2 (1:30 dilution, Zymed, San Francisco, CA, USA), or p27kip1 (1:100 dilution, Zymed) or rabbit polyclonal anti-H. pylori antibody (1:500 dilution, DakoCytomation, Carpinteria, CA, USA). Immunoreactivity was detected using the DAB Map Kit (PIERCE, Woburn, MA, USA), based on the avidin–biotin complex immunoperoxidase technique.

Positively stained cells were evaluated using image analysis (Image-Pro Plus, version 4.5.1, Media Cybernetics, Silver Spring, MD, USA) to reduce observer variation. After thresholding, positively stained nuclei were counted in a total of 200 epithelial cells in each compartment of the gastric gland (surface, neck, and basal portion), and expressed as a percentage for each compartment and for the entire gland. Acute and chronic inflammation and the H. pylori colonization density of chronic gastritis were scored by Resnick using the updated Sydney system.42

Statistics

Differences in mRNA and/or protein expression of p27, Skp2, c-Myc, and IL-8 before and after H. pylori eradication were compared by Wilcoxon's signed rank test. Correlations between expression of these genes and gastric inflammation in the pre-eradication values were analyzed using Spearman's correlation coefficient. A P-value less than 0.05 was regarded as statistically significant.

Results

Histology

In all, 19 of the 22 patients had atrophic gastritis (11 moderate, eight mild) with intestinal metaplasia in 12 cases. Only three patients had neither atrophy nor intestinal metaplasia. After H. pylori eradication, scores of chronic inflammation were significantly lower and scores of acute inflammation and H. pylori colonization were zero in all cases (Table 2).

Table 2 Scores of inflammation and H. pylori colonization in gastric mucosa (mean±s.e.m.)
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Immunohistochemistry of p27, Skp2, and c-Myc

p27, Skp2, and c-Myc immunohistochemistry resulted in dark brown nuclear staining patterns in the epithelial and interstitial inflammatory cells of the gastric antral mucosa. Expression of p27 protein in gastric epithelial cells was higher after the eradication of H. pylori (Figure 1a and b). In contrast, the percentages of Skp2 (Figure 1c and d) and c-Myc (Figure 1e and f) positive epithelial cell nuclei were significantly lower after H. pylori eradication. Summarizing the data for the entire group of 22 patients, the percentage of gastric antral epithelial cells expressing p27 protein was significantly higher after the eradication of H. pylori (mean±s.e.m. 37±2.4% pre-eradication vs 55±2.8% post-eradication; P<0.001) (Figure 2a). In contrast, Skp2 protein expression in these same cells was lower after H. pylori eradication (35±3.8 vs 23±2.6%, P=0.009) (Figure 2b), and so was the expression of c-Myc (47±3.6 vs 30±3.8%, P<0.001) (Figure 2c). No differences were observed in expression of p27, Skp2, or c-Myc either before or after the eradication of H. pylori between cases with atrophic gastritis and the three cases with non-atrophic gastritis. The changes in p27 and c-Myc protein following H. pylori eradication were most evident in epithelial cells of the glandular surface (for p27: 44±4.1% pre-eradication vs 67±4.1% post-eradication; P<0.001, for c-Myc: 44±4.9 vs 26±4.9%; P=0.02, respectively) and in the neck (p27: 27±3.0 vs 43±3.0%; P=0.002, c-Myc: 56±4.5 vs 32±4.1%; P<0.001, respectively). Skp2 was significantly lower only in the neck of the gastric glands (36±4.3% pre-eradication vs 31±2.1% post-eradication; P=0.03).

Figure 1
figure 1

Immunohistochemistry for p27, Skp2, and c-Myc. p27, Skp2, and c-Myc proteins were detected in the nuclei of epithelial and interstitial inflammatory cells of the gastric mucosa. Expression of p27 protein was higher in gastric antral epithelial cells after the eradication of H. pylori. In contrast, the expression of both Skp2 and c-Myc proteins was lower after the eradication of H. pylori. Original magnification: × 400.

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Figure 2
figure 2

Changes in expression of p27, Skp2, and c-Myc proteins in gastric antral epithelial cells of 22 patients following the eradication of H. pylori. The percentage of epithelial cells expressing p27 protein was significantly higher after eradication of H. pylori (a). Gastric epithelial cells expressing Skp2 (b) or c-Myc (c) were significantly lower after H. pylori eradication. The percentage of epithelial cells expressing each specific protein was determined using image analysis software on representative areas of well-oriented immunostained sections. The thick black bars indicate the mean values, **P<0.05.

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LCM and mRNA Quantification

LCM of epithelial cells was successful from a single 8-μm section in all specimens, and yielded on average 441 ng RNA from 5000–8000 epithelial cells (Figure 3). IL-8 mRNA was significantly lower after eradication of H. pylori (the mean±s.e.m. IL-8 mRNA/18SrRNA ratio was 111±25.1 before vs 16±5.3 after eradication, P<0.001) (Figure 4); this fall in IL-8 is consistent with previous reports.43, 44 The IL-8 mRNA pre-eradication value correlated significantly with histological scores of acute inflammation (R=0.55, P=0.004). In contrast, mRNA levels of p27, skp2, and c-myc were not altered significantly by H. pylori eradication. The mRNA expression of p27, skp2, and c-myc (normalized by 18SrRNA) was not significantly different between before and after eradication of H. pylori (p27 mRNA 19±2.6 vs 23±5.1, P=0.26, skp2 mRNA 33±6.2 vs 27±5.1, P=0.34, and c-myc mRNA 44±6.2 vs 49±14.1, P=0.72, respectively) (Figure 5). There was no correlation between mRNA expression and protein expression in Helicobacter-associated gastritis for each gene evaluated, nor was there a difference between cases with atrophic gastritis and cases without atrophy.

Figure 3
figure 3

Example of LCM. (a) The target gastric epithelium was marked digitally, shown here as a red color. (b) After laser microdissection, gastric epithelial cells were captured on a Capsure Macro LCM cap. (c) Residual tissue after microdissection has removed the gastric epithelial cells of interest.

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Figure 4
figure 4

IL-8 mRNA was significantly lower in gastric epithelial cells after the eradication of H. pylori. IL-8 mRNA was measured by real-time PCR of laser-captured microdissected RNA and normalized for 18SrRNA. Thick black bars indicates the mean values, **P<0.001.

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Figure 5
figure 5

Changes in mRNA transcript number for (a) p27, (B) Skp2, and (c) c-Myc in gastric epithelial cells of 22 patients after the eradication of H. pylori. RNA was extracted from laser-captured microdissected epithelial cells, measured by real-time PCR, and normalized for 18SrRNA. Thick black bars indicates the mean values. None was significantly changed following H. pylori eradication.

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Discussion

The main findings of our study are that the decrease in epithelial expression of p27 associated with H. pylori is reversible following H. pylori eradication, and that this decrease in p27 associated with chronic H. pylori-associated gastritis is mainly at the level of p27 protein, without significant change in p27 mRNA. These findings are consistent with the major effect of H. pylori on p27 observed in vitro, namely that it decreases p27 expression through increasing p27 protein degradation.28 In co-culture, H. pylori alone produces this effect, suggesting that the decrease observed in vivo is due to the bacterium itself rather than the associated inflammatory response present in the gastric mucosa. However, the effect of H. pylori on p27 expression may be modulated by inflammatory cells and cytokines present in the gastric mucosa colonized by H. pylori. Although p27 was reported to be increased in the proliferative zone of gastric glands,45 we found that this region expressed very little p27 compared to both the epithelial surface and the glandular base.

The expression of Skp2, the ubiquitin ligase responsible for p27 degradation during cell cycle progression, is increased in many cancers, including gastric cancer.29 We also found a reciprocal relationship between low p27 and elevated Skp2 expression in our study. While p27 increased following the eradication of H. pylori, Skp2 decreased. As with p27, the change in expression level was confined to Skp2 protein and not mRNA, which again favors a predominantly post-translational level of regulation for Skp2, consistent with prior in vitro studies.46, 47, 48 Whether the inverse relationship between Skp2 and p27 is a causal one remains to be determined, since the reduction of p27 protein by H. pylori in vitro was not associated with increased Skp2.28 However, in co-culture H. pylori inhibits cell cycle progression,49 whereas in vivo H. pylori-associated gastritis is a hyperproliferative condition;9 thus, increased Skp2 in H. pylori-infected patients may just reflect increased cell proliferation.

H. pylori infection was associated with higher expression of both Skp2 and c-Myc. c-Myc has previously been reported to be increased in H. pylori-associated gastritis, is associated with increased cell proliferation, and, as in our current study, was lower after the eradication of H. pylori.50, 51 Increased c-Myc may be responsible for decreasing p27 at multiple levels, including transcriptional regulation and cytoplasmic sequestration of p27, allowing for cell cycle progression from G1 to S phases.36, 37 We did not observe cytoplasmic p27 expression in gastric epithelial cells even before eradication when c-Myc was increased, but this may be due to a relative lack of sensitivity of immunostaining. The relationship between c-Myc, Skp2, and p27 is complex. Skp2 has ubiquitin ligase activity for c-Myc, leading to its ubiquitination and destruction in the proteasome, but Skp2 also can stimulate c-Myc transcription.30, 31 As cells progress to S phase, increasing levels of Skp2 therefore lead not only to the targeting of p27 for proteasomal degradation but also the transient activation of c-Myc. c-Myc then synergizes with the decrease in p27 protein level to enforce the commitment of cells to enter S phase. Thus, the results of this in vivo study are supportive of the hypothesis that, in H. pylori-associated gastritis, increased Skp2 may be the mechanism common to both downregulation of p27 and increased c-Myc, the net result of which is increased cell proliferation. However, further dissection of the responsible molecular mechanisms will be necessary because of the evidence that Skp2 is not involved in the downregulation of p27 by H. pylori in vitro.28

In this study we used LCM to extract RNA, thus allowing us to examine in parallel the expression of protein and mRNA for p27, skp2, and c-myc. As p27 is highly expressed in inflammatory cells that accumulate in the lamina propria during H. pylori infection, LCM allowed us to specifically examine the regulation of p27 in gastric epithelial cells in the absence of contaminating nonepithelial cells. While changes in p27, Skp2, and c-Myc were all highly statistically significant at the protein level, their mRNA expression was not significantly influenced by H. pylori eradication. To confirm that this was not an artifact of the LCM itself, we also performed in parallel evaluation of the mRNA expression of IL-8, known to be regulated transcriptionally by H. pylori in gastric epithelial cells.44, 52 IL-8 mRNA was significantly lower following the eradication of H. pylori confirming these previous reports, providing evidence that the relative stability of p27, Skp 2, and c-Myc transcripts following the eradication of H. pylori in gastric epithelial cells is an authentic finding. Apart from its effects on p27, post-translational regulation of epithelial cell gene expression by H. pylori has not been previously emphasized. H. pylori does not generally upregulate proteasomal activity, as determined by fluorogenic substrate assays,28 but it may have specific effects on proteasomal function or eukaryotic protein targeting, as has been demonstrated for some other pathogenic or commensal bacteria.53, 54

A limitation of our study was that relatively few patients were included and only two endoscopic antral biopsies were available at each time point for each patient, thus allowing us to sample only a small percentage of the gastric mucosa. Sampling variability might be responsible for some of the outlying data points evident in mRNA analysis. However, a strength of the study design was the ability to use each patient as their control in paired analysis and the fact that the study population was ethnically relatively homogenous. While the acid-secretory status of the subjects was not determined in this study, it is likely that, because almost all the subjects had histological evidence of intestinal metaplasia or atrophic gastritis, some may have already developed hypochlorhydria with the subsequent overgrowth of non-Helicobacter bacteria and gastric nitrosamine generation.55 Variability in acid-secretory status may have conceivably influenced our results—the effects of acid secretion or nitrosamine generation on the expression of gastric p27, Skp2, or c-Myc are not known.

In conclusion, H. pylori downregulates p27 through increasing p27 protein degradation in vivo and this is reversed following H. pylori eradication. Our study indicates that the possible pathways mediating this effect may involve both Skp2 and c-Myc. The ability of H. pylori to downregulate p27 may provide a mechanistic link between H. pylori, gastric epithelial cell hyperproliferation, and gastric carcinogenesis.