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Effect of Helicobacter pylori eradication on expression of cyclinD2 and p27 in gastric intestinal metaplasia
J . YU*, W. K. LEUNG*, E. K. W. NG , K. F. TOà , M. P. A. EBERT§, M. Y. Y. GO*, W. Y. CHANà ,
F. K. L. CHAN*, S. C. S. CHUNG , P. MALFERTHEINER§ & J. J . Y. SUNG*
*Department of Medicine & Therapeutics, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong;
Department of Surgery, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong; àDepartment of
Anatomical & Cellular Pathology, Prince of Wales Hospital, Chinese University of Hong Kong, Shatin, Hong Kong; and
§Department of Gastroenterology, Hepatology, and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany
Accepted for publication 2 April 2001
INTRODUCTION
Cyclins, cyclin dependent kinases and their inhibitors
regulate cell growth, differentiation, survival, and cell
death. Gene abnormalities and aberrant expression of
these cell cycle regulators play a pivotal role in the
pathogenesis of cancers, including gastric cancer.1
Cyclin D promotes progression through the G1 phase
of the cell cycle by regulating the activity of CDK4 and
CDK6.2 Cyclin D1 expression in malignant lesions has
been extensively studied.3±5 Nonetheless, little is
known about cyclin D2 expression in gastric cancer.
Recently, Takano et al. showed that over-expression
SUMMARY
Background and aims: Cyclins and cyclin-dependent
kinase inhibitors play a crucial role in the control of
cell cycle transitions. Enhanced expression of cyclin D2
and reduced expression of p27kip1 (p27) have been
implicated in the pathogenesis of cancer. Because
intestinal metaplasia has been regarded as a pre-
malignant lesion, we investigated the expression of
cyclin D2 and p27 in Helicobacter pylori-associated
chronic gastritis with and without intestinal metaplasia,
and followed the changes after H. pylori eradication.
Methods: Expression of cyclin D2 and p27 was studied
by immunohistochemistry in 59 patients (including 35
patients with intestinal metaplasia) and in 10 gastric
cancer patients. Among them, 29 H. pylori-infected
patients had serial gastric biopsies taken before and at
1-year after eradication of H. pylori.
Results: Expression of cyclin D2 was signi®cantly higher
in gastric cancers when compared to their adjacent
non-tumour tissues (median score 3 vs. 1, P � 0.015).
Over-expression of cyclin D2 was detected in H. pylori-
associated chronic gastritis and intestinal metaplasia,
which was reduced after eradication of the organism
(median score 2 vs. 1, P � 0.037 in chronic gastritis;
median score 2 vs. 0, P � 0.008 in intestinal metaplasia).
While the normal gastric mucosa showed strong p27
expression, ®ve of the 10 gastric cancer tissues exhibited
reduced p27 expression (P � 0.039). Diminished p27
expression was also seen in intestinal metaplasia, which
was restored 1-year after H. pylori eradication (eight out
of 16 vs. one out of 16, P � 0.018). Reduced expression
of p27 was frequently associated with increased cyclin
D2 expression in H. pylori-associated intestinal meta-
plasia (P � 0.02).
Conclusion: Over-expression of cyclin D2 and reduced
expression of p27 are closely linked to H. pylori-
associated intestinal metaplasia. Eradication of H. pylori
infection reverses the aberrant expression of cyclin D2
and p27 in intestinal metaplasia.
Correspondence to: Prof. J. J. Y. Sung, Division of Gastroenterology and
Hepatology, Department of Medicine and Therapeutics, Prince of Wales
Hospital, Shatin, N. T., Hong Kong.E-mail: [email protected]
Aliment Pharmacol Ther 2001; 15: 1505±1511.
Ó 2001 Blackwell Science Ltd 1505
of cytoplasmic cyclin D2 correlated with tumour
progression and poor survival.6 While cyclins and
cyclin dependent kinases are promoters of cell cycle
progression, cyclin dependent kinase inhibitors have
been identi®ed as potential tumour suppressors. The
cyclin dependent kinase inhibitor, p27kip1 (p27), binds
to a wide variety of cyclin/cyclin dependent kinase
complexes including CDK2 and CDK4, inhibits kinase
activity and blocks the cell cycle progression.7 Recent
research on p27 knockout mice indicates that p27
could act as a tumour suppressor gene.8 Furthermore,
decreased or absent p27 protein expression is a poor
prognostic indicator in patients with breast, colorectal
and gastric carcinomas.9±12 Nonetheless, the roles of
cyclin D2 and p27 in pre-malignant gastric lesions are
largely unknown. Helicobacter pylori infection is con-
sidered to be a major factor in gastric carcinogenesis,
yet the role of aberrant expression of cyclins and p27
has not been evaluated in H. pylori infected gastric
mucosa.13 The effects of H. pylori eradication on the
expression of these cell growth regulators have not
been studied.
In the present study, we evaluated the expression of
cyclin D2 and p27 in patients with H. pylori-associated
chronic gastritis with and without intestinal metaplasia,
and their responses to eradication therapy.
METHODS
Subjects
Endoscopic gastric biopsies were obtained from 59 non-
gastric cancer patients. H. pylori infection was diag-
nosed by rapid urease test and histology (haematoxylin
and eosin stain, and Giemsa stain). Among these
non-cancer patients, 24 were diagnosed with
H. pylori-associated intestinal metaplasia, 18 with
H. pylori-associated chronic gastritis, 11 with intestinal
metaplasia but uncon®rmed H. pylori infection, and six
were non-infected controls. All H. pylori-infected
patients were given a 1-week course of anti-Helicobacter
therapy containing omeprazole, amoxillcillin and clari-
thromycin. Serial gastric biopsies taken before and
1-year after eradication of H. pylori were available from
29 H. pylori-infected patients (16 with intestinal
metaplasia and 13 non-intestinal metaplasia). Surgical
gastrectomy specimens from 10 gastric cancer patients
that contained tumour and adjacent non-tumour were
also collected in this study.
Histology
All samples were ®xed in formalin, stained with
haematoxylin and eosin for histological evaluation.
Detection of H. pylori was carried out by examination of
the non-cancerous and non-metaplastic part of the
gastric mucosa in order to increase the sensitivity of
detection. The degree and activity of gastritis, density of
H. pylori colonization, and severity of intestinal meta-
plasia were graded according to the updated Sydney
system.14 All histological assessment was performed by
an experienced pathologist (KFT).
Immunohistochemistry
Expression of cyclin D2 and p27 protein was examined
by the avidin±biotin complex (ABC) immunoperoxidase
method. Five-micron sections were cut from the paraf®n
embedded blocks, placed on charged glass slides,
deparaf®nized, rehydrated, rinsed with distilled water
and washed with tris-buffered saline. The slides were
then treated with 3% hydrogen peroxide to block
endogenous peroxidase activity. After blocking with
5% normal serum for 20 min, the primary antibody
(polyclonal antibody against cyclin D2, SC-181, 1:100
dilution, Santa Cruz Biotechnology, Santa Cruz, CA; and
p27 polyclonal antibody, SC-528, 1:200 dilution, Santa
Cruz Biotechnology) was applied and incubated at 4 °C
overnight. After rinsing, the biotinylated secondary
antibody and ABComplex/HRP (Dako A/S, Denmark)
was applied. Peroxidase activity was visualized by the
diaminobenzidine chromogen with 0.05% hydrogen
peroxide. The sections were then counter-stained with
haematoxylin, dehydrated, cleared, and mounted.
Immunostaining conditions were the same for both
gastrectomy and endoscopic biopsy samples.
Immunohistochemical assessment
Two investigators evaluated the immunohistochemical
staining without knowledge of the clinical and patho-
logical parameters. Because metaplastic and non-meta-
plastic areas can coexist in a single biopsy, scoring was
concentrated on the immunoreactive cells within the
area of interest, such as intestinal metaplasia and
cancer, only. For cyclin D2, staining in the nucleus and
cytoplasm was evaluated by scanning of the whole
section with more than 1000 cells. A semi-quantitative
scoring system described previously was adopted with
1506 J. YU et al.
Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511
slight modi®cation:15 0 � no expression; 1 � < 5% of
cell show expression; 2 � 5±30%; 3 � 30±60%;
4 � > 60% expression. A score of 0 and 1 was
categorized as negative, whereas a score of 2±4 was
regarded as positive. For p27, samples that exhibited
50% or more immunoreactivity were de®ned as `high
expression' and those with less than 50% were de®ned
as `low expression', as described by other investiga-
tors.6, 9, 10 Lymphocytes in the same specimens were
used as internal positive controls for p27 expression,
and phosphate buffered saline alone without antibody
was used as negative controls.
Statistical analysis
Statistical analysis was performed using the Statistical
Package for the Social Science program (SPSS, version
9.0). Differences in cyclin D2 expression and gastritis
scoring were analysed by Wilcoxon test. The correlation
between p27 expression and H. pylori-associated intes-
tinal metaplasia and gastric cancer was analysed by the
v2-test. The correlation between cyclin D2 and p27
expression was analysed by the Mann±Whitney U-test.
A P-value of less than 0.05 was considered statistically
signi®cant (two-tailed).
RESULTS
Cyclin D2 expression
While cyclin D2 staining was detected in seven of the 10
gastric cancers (70%), it was only detected in two (20%)
adjacent non-tumorous gastric mucosa (P � 0.025).
The intensity of cyclin D2 expression was signi®cantly
higher in gastric cancers when compared to their paired
normal tissues (median score 3 vs. 1; P � 0.015).
Immunoreactivity of cyclin D2 was mostly detected in
the nucleus and cytoplasm of cancer cells, but was
predominantly localized in the cytoplasm of non-cancer
cells. Because of the small number of samples examined,
there was no signi®cant difference between diffuse-type
and intestinal-type tumours detected.
An increased expression of cyclin D2 was more often
detected in H. pylori infected tissues. The intensity of
cyclin D2 expression is signi®cantly higher in the 18
patients with H. pylori-associated chronic gastritis than
in the six H. pylori-negative patients (median 2 vs. 1,
P � 0.028). Similarly, the intensity of cyclin D2 stain-
ing in H. pylori-associated intestinal metaplasia is
signi®cantly higher than that of H. pylori-negative
intestinal metaplasia (median 2 vs. 0, P � 0.027).
Despite there being no signi®cant change in the severity
of intestinal metaplasia 1 year after H. pylori eradica-
tion, cyclin D2 expression was signi®cantly reduced in
both chronic gastritis (median score 2 vs. 1; P � 0.037)
and intestinal metaplasia (median score 2 vs. 0;
P � 0.008; Figures 1 and 3).
p27 expression
Normal gastric epithelium, including H. pylori-infected
gastritis, expressed strong p27 expression in both the
cytoplasm and nuclei (Figure 2A), whereas weak stain-
ing was observed in cancer cells (Figure 2B). In contrast
to adjacent non-cancerous gastric mucosa that showed
normal p27 expression, expression of p27 protein was
markedly reduced in ®ve of the 10 tumour tissues
(P � 0.039). Notably, p27 expression was also reduced
in H. pylori-associated intestinal metaplasia (Figure 2C)
but not in H. pylori-negative intestinal metaplasia. Low
p27 expression was observed in 11 (45.8%) H. pylori-
associated intestinal metaplasia patients. Among the 16
cases of H. pylori-associated intestinal metaplasia avail-
able for subsequent follow-up, eight of them showed
reduced p27 expression and seven of them were
restored to normal levels 1 year after H. pylori eradica-
tion (P � 0.018; Figure 2D).
Association between cyclin D2 and p27 expression
To elucidate whether there is any association between
cyclin D2 and p27 expression, the same specimen from
each patient with intestinal metaplasia and cancer was
evaluated. As shown in Figure 4, expression of cyclin
D2 was signi®cantly higher in intestinal metaplasia
samples with reduced p27 expression than in those with
high p27 expression (P � 0.02). Moreover, four out of
the ®ve tumour samples with low p27 expression have
increased cyclin D2 expression.
DISCUSSION
Although H. pylori has been considered a class I gastric
carcinogen, the mechanism underlying H. pylori-asso-
ciated gastric carcinogenesis remains enigmatic.13
Identi®cation of genetic alterations in pre-neoplastic
lesions such as intestinal metaplasia may help to shed
new light into this gastric carcinogenesis pathway. In
H. PYLORI ERADICATION AND EXPRESSION OF CYCLIN D2 AND P27 1507
Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511
this study, we examined the role of cyclin D2 and p27 in
both pre-malignant and malignant gastric lesions. Our
results showed that over-expression of cyclin D2 and
reduced p27 expression are detected in 70% and 50% of
gastric cancers, respectively. Interestingly, we also
demonstrated enhanced cyclin D2 expression in both
H. pylori-associated gastritis and intestinal metaplasia.
This aberrant expression could be reversed after clear-
ance of the organism. In contrast, reduced p27
expression was only recognized in H. pylori-associated
intestinal metaplasia but not in chronic gastritis. Again,
eradication of H. pylori restored the p27 protein level to
normal.
The present study demonstrated that gastric intestinal
metaplasia and cancer with reduced p27 expression
tend to have a higher level of cyclin D2 expression.
While over-expression of cyclin D2 and reduced p27
expression favour the G1 cell cycle transition, this
combination may be critical to the gastric carcinogen-
esis process. Potentially, these alterations will drive the
cell cycle transition and hence increase the oncogenic
tendency. However, this ®nding is not unexpected
because H. pylori infection is associated with active
in¯ammation, which is accompanied by increased cell
turnover and epithelial cell proliferation.16 While cell
turnover and proliferation is regulated by cyclins and
cyclin dependent kinases, aberrant expression of cyclin
D2 and p27 can be secondary to the in¯ammatory
process.2, 7 On the other hand, co-culture experiments
demonstrated that H. pylori down-regulates the expres-
sion of p27 in gastric cell lines even in the absence of
in¯ammation.17 Moreover, there are recent data to
suggest that cell turnover, in particular cellular apop-
tosis, is reduced in gastric intestinal metaplasia.18 Thus,
other factors are likely involved in the aberrant
expression of cyclin D2 and p27 in gastric intestinal
Figure 1. Representative samples of cyclin D2 expression. (A1) H. pylori associated intestinal metaplasia with positive cytoplasmic
immunostaining (´400). (A2) Cyclin D2 antibody binding was undetectable 1 year after H. pylori eradication (´400). (B1) H. pylori-
positive chronic gastritis with positive cyclin D2 antibody binding (´200). (B2) Absent cyclin D2 binding 1 year after H. pylori eradication
(´200).
1508 J. YU et al.
Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511
metaplasia. Similar observations between p27 and
cyclin D1 had been reported in gastric cancer.5
However, a recent study failed to show any direct link
among cyclin D2, cyclin E and p27 in gastric cancer.6
This discrepancy may be partly accounted for by the
different stages of gastric cancer examined and by the
different methodologies employed. Further studies are
needed to clarify this uncertainty.
Intestinal metaplasia is a well-recognized pre-malig-
nant gastric lesion, which is closely linked to H. pylori
infection. Conceivably, H. pylori eradication may result
in regression of intestinal metaplasia by the resolution
of in¯ammation and the removal of a potentially
unknown stimulant. Past studies had yielded con¯icting
results which was partly related to the small sample
size, short duration of follow-up and even sampling
error in obtaining gastric biopsy.19±21 Two large-scale
randomized controlled studies published recently sug-
gested a bene®cial effect of H. pylori eradication in the
regression of gastric intestinal metaplasia.22, 23 The
present study may offer a biological explanation for
these ®ndings and further support the role of H. pylori
Figure 2. Representative samples of p27 antibody binding. (A) p27 expression in normal gastric tissue was detected in the nucleus
as well as in the cytoplasm of 90% of gastric surface epithelium (´400). (B) Decreased p27 staining in poorly differentiated gastric
cancer cells (´400). (C) Decreased p27 binding in H. pylori associated intestinal metaplasia. (D) Normal p27 staining in intestinal
metaplasia 1 year after H. pylori eradication (´400).
Figure 3. Cyclin D2 expression before and 1 year after H. pylori
eradication. Data expressed as mean � s.d. CG: chronic gastritis;
IM: intestinal metaplasia.
H. PYLORI ERADICATION AND EXPRESSION OF CYCLIN D2 AND P27 1509
Ó 2001 Blackwell Science Ltd, Aliment Pharmacol Ther 15, 1505±1511
eradication in the management of these pre-malignant
gastric lesions.
Taken together, we conclude that over-expression of
cyclin D2 and reduced expression of p27 is intimately
linked to H. pylori-associated intestinal metaplasia and
gastric cancer. Eradication of H. pylori infection reverses
the aberrant expression of cyclin D2 and p27 in gastric
intestinal metaplasia and may possibly halt the gastric
carcinogenesis cascade. It may be interesting to deter-
mine the predictive value of these genetic markers in the
histological progression of pre-malignant gastric lesions
in the future.
ACKNOWLEDGEMENT
This study was partly supported by the German/Hong
Kong Joint Research Scheme (GHK 00/03).
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H. PYLORI ERADICATION AND EXPRESSION OF CYCLIN D2 AND P27 1511
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