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179: 3 9 4 2 (1996)
Departments of Pathology* and Medicine?, Tohoku University School of Medicine, Sendai, Japan
In situ tissue dynamics were studied in 12 cases of human gastric mucosa, including normal gastric body mucosa and gastric glands
with intestinal metaplasia, obtained from gastrectomy specimens of adenocarcinoma. Cell proliferation was determined by Ki67
immunoreactivity. DNA fragmentation was studied in sits by TdT-mediated dUTP-biotin nick end labelling (TUNEL). In addition, p53
expression was examined by both immunohistochemistry and mRNA in situ hybridization. In the oxyntic gastric glands, Ki67
immunoreactivity was observed exclusively in the proliferative zone and TUNEL-positive cells were present predominantly in the surface
foveolar epithelium. In the gastric glands with complete intestinal metaplasia, Ki67-positive cells were present in the lower portion of the
glands and TUNEL-positive cells in the superficial epithelium. In the gastric glands with incomplete intestinal metaplasia,
TUNEL-positive cells were detected in the lower gastric glands adjacent to cells immunoreactivefor Ki67; the proportion of these gastric
glands with TUNEL-positive cells (40 out of 108 glands) was significantly higher than for oxyntic glands (94 out of 620 glands) or for
glands with complete metaplasia (31 out of 254 glands). Relatively strong p53 immunoreactivity and mRNA hybridization were also
observed in the proliferative and apoptotic areas of gastric glands with incomplete intestinal metaplasia. These results indicate that
incomplete intestinal metaplasia is associated with increased cell turnover and p53 overexpression, possibly in response to various
noxious or DNA-damaging stimuli.
woms-stomach; cell proliferation; Ki67; apoptosis; p53
A multifocal gastritis associated with diminution in
the number of glands and intestinal metaplasia is the
most common type worldwide and is endemic in some
parts of the world, including Japan.' In addition, an
increased incidence of this type of chronic gastritis
correlates with the geographical distribution of populations at increased risk for gastric carcinoma. 1,2 Although
controversy exist^,^ several groups of investigators have
proposed that this type of chronic atrophic gastritis,
especially involving the presence of intestinal metaplasia, is a precursor of the intestinal type of adenocarcinoma of the stomach.'.* Little is known, however,
about the biological features of this type of gastritis, and
especially about the in situ tissue dynamics of the lesion.
This study, therefore, initially examined cell proliferation by immunoreactivity for Ki67, one of the cell
cycle-related antigens, and DNA fragmentation, which
is associated with apoptosis or programmed cell death,
by the 3'-OH nick end labelling technique, or TdTmediated dUTP-biotin nick end labelling (TUNEL)
m e t h ~ dThe
. ~ aim of the study was to elucidate the tissue
topography of these markers in human gastric mucosa,
including gastric mucosa with complete and/or incomplete intestinal metaplasia. p53 protein and mRNA
expression were then examined in order to study the
possible involvement of either mutated or overexpressed
p53 in these lesions.
Addressee for correspondence: Hironobu Sasano, MD, Department
of Pathology, Tohoku University School of Medicine, 2-1 Seiryoumachi, Aobaku, Sendai, Japan 980.
CCC 0022-341 7/96/050039-04
01996 by John Wiley & Sons, Ltd.
Tissue source and preparation
Gastric mucosa was obtained from the gastric body in
12 patients (eight men and four women, mean age 61
years) undergoing total or partial gastrectomy for
intestinal-type adenocarcinoma at Tohoku University
Hospital and Sendai City Hospital, Sendai, Japan.
Tissue strips 3-5mm in width were made from the
non-pathological gastric body along the line of the
incision. They were fixed in 8 per cent paraformaldehyde
(pH 7-4) for 18 h at 4°C and paraffin-embedded following fixation. Sections 3pm thick were then cut and
placed on glass slides.
Immunohistochemistry was performed by a modified
strept avidin biotin method using a Histofine kit
(Nichirei, Tokyo, Japan), as previously described by the
author^.^ The primary antibodies employed were monoclonal antibodies MIB-1 for Ki67 (Immunotech SA,
Marseille, France) and DO-7 for p53 (Novocastra Ltd.,
Newcastle upon Tyne, U.K.). The negative control of
immunostaining was 0.01 M PBS and normal mouse IgG
instead of primary antibodies; immunoreactivity was not
observed in these tissue sections.
TUNEL (3'-0H nick end labelling)
DNA fragmentation associated with apoptosis was
detected in situ by nick end labelling (transfer of biotinylated nucleotide to the 3'-OH end) according to the
Received 3 April 1995
Accepted 4 December 1995
Fig. l-(A) Ki67 immunohistochemistry in the gastric mucosa without atrophy or inflammation. Nuclear immunoreactivity is present in the
proliferative zone. x 40. (B) TUNEL or 3'-OH nick end labelling in the same field as (B). Reactive cells can be observed predominantly in the
superficial foveolar epithelium (arrow-heads). x 40
Fig. 2--(A) Ki67 immunohistochemistry in the gastric mucosa with incomplete intestinal metaplasia. x 40. (B) TUNEL or 3'-OH nick end labelling
in the same case as (A). TUNEL-reactive cells (arrow-heads) are not present in the areas occupied by Ki67-immunoreactive cells. x 40
method of Gavrieli et ~ 1with
. ~some m~difications.~
the positive control, tissue sections were treated with
DNase I (Stratagene Co., La Jolla, CA, U.S.A.) in
0.7pglml potassium cacodylate buffer (pH 7.2) for 10
min prior to treatment with TdT. Tissue sections incubated with TdT buffer without DNase I were used as the
negative control. We defined TUNEL-positive glands as
those which were longitudinally sectioned from surface
to base and which contained at least one TUNELpositive cell. We then obtained the TUNEL index, by
examining the percentage of glands containing TUNELpositive cells among gastric glands, including 620 oxyntic glands, 108 glands with incomplete intestinal
metaplasia, and 254 glands with complete intestinal
metaplasia. An overall comparison was performed with
a one-way analysis of variance (ANOVA) by the
Kruskal-Wallis rank-sum test, and the Bonferroni
method was applied to the significance of simultaneous
multiple comparisons."
We classified gastric glands from the gastric body into
oxyntic glands and glands with intestinal metaplasia
by histological examination. We further subclassified
gastric oxyntic glands with intestinal metaplasia into
incomplete and complete metaplasia, by the presence or
absence of a brush border and Paneth cells.3
Both Ki67 and p53 immunoreactivity were observed
in the nuclei of the cells in normal gastric mucosa and
chronic atrophic gastritis with complete or incomplete
intestinal metaplasia (Figs lA, 2A, 3, and 4A).
In situ hybridization
DNA fragmentation in situ detected by the TUNEL
method was confined to the nucleus (Figs I B and 2B).
Pretreatment with DNase I caused intense staining of all
nuclei in the preparation. Tissue sections incubated in
the absence of TdT did not demonstrate nuclear staining.
The in situ hybridization procedures for p53 mRNA
used in this study, including the oligonucleotide probes
employed, have been previously described by Sasano
et a1.7
In situ hybridization
p53 hybridization signals were observed as black
silver dots on autoradiography (Fig. 4B). Sections
Table I-The
percentage of TUNEL glands in human gastric
Oxyntic glands
Incomplete metaplasia
Complete metaplasia
Fig. 3-Ki67 immunohistochemistry in the gastric rnucosa with complete intestinal metaplasia. Nuclear immunoreactivity is observed in
the lower part of the glands. x 40
hybridized with a sense oligonucleotide did not yield
accumulation of hybridization signal.
Oxyntic glands
Ki67 immunoreactivity was observed in all the oxyntic glands and was confined to the neck or the proliferative zone (Fig. 1A). TUNEL-positive cells were observed
at the tip of the foveolar glands, among the superficial
epithelial cells and exfoliated cells in the lumen, and very
sporadically at the base of the glands, in 94 of 620
oxyntic glands observed (Fig. 1B). Very weak nuclear
p53 immunoreactivity and accumulation of p53 mRNA
hybridization signal were sporadically observed in the
proliferative zone of oxyntic glands.
Gastric glands with incomplete intestinal metapfasia
Ki67 immunoreactivity was observed in almost all of
the cells in the areas below the goblet cells and above the
base of the gland (Fig. 2A). Ki67-immunoreactive cells
in the glands with incomplete intestinal metaplasia
appeared generally to be more numerous than those in
oxyntic glands and glands with complete metaplasia.
TUNEL-reactive cells were observed among the superficial cells and more frequently in the cells at the base of
the glands (Fig. 2B). TUNEL-reactive cells included
cells with apoptotic bodies. TUNEL-reactive glands
No. of
*There are statistically significant differences between oxyntic glands
and incomplete metaplasia (P<0.01) and between complete and
incomplete metaplasia (P<0.05) by the Bonferroni multiple comparison test.
comprised 40 out of 108 glands with incomplete intestinal metaplasia (Table I). The proportion of TUNEL
reactive glands in incomplete intestinal metaplasia was
significantly higher than for oxyntic glands and glands
with complete intestinal metaplasia (Table I).
Relatively strong p53 nuclear immunoreactivity
was observed in 28 out of 96 glands with incomplete
intestinal metaplasia (Fig. 4A). Some of the p53immunoreactive cells demonstrated mild to moderate
degrees of dysplasia, but none was severely dysplastic.
The gastric glands containing p53-positive cells were
associated generally with marked inflammatory cell infiltration, including lymphocytes and neutrophils. Accumulation of p53 mRNA was also observed in these
glandular cells (Fig. 4B). Serial tissue sections revealed
that cells immunoreactive for p53 protein product were
distributed between Ki67-immunoreactive and TUNELreactive cells, but cells with p53 mRNA accumulation
were observed generally among the latter cell type. The
cells with accumulation of p53 mRNA included both
p53-positive and p53-negative cells.
Gastric glands with complete intestinal metaplasia
Ki67 immunoreactivity was confined to the lower part
of the glands (Fig. 3). Ki67 immunoreactivity was
observed in all the glands which had a complete longitudinal profile on histological section. TUNEL-reactive
cells were observed predominantly in the superficial
Fig. &(A) Imrnunohistochemistry of p53 in the gastric glands with incomplete intestinal metaplasia. x 45. (B) In situ hybridization of p53 mRNA
in the gastric glands with incomplete intestinal metaplasia in the same field as (A). Accumulation of hybridization signal is demonstrated (arrows).
Signals are observed in both p53-immunopositive and -negative cells
layer and among exfoliated cells in the lumen. TUNELreactive cells were observed in 31 out of 254 glands.
Weak pS3 nuclear immunoreactivity and mRNA
hybridization were observed sporadically in the lower
part of the glands where Ki67-positive cells were located.
There has been controversy as to whether intestinal
metaplasia, especially incomplete intestinal metaplasia,
represents a precancerous lesion for gastric carcin ~ m a . ~On, ~ the other hand, intestinal metaplasia
appears to represent a reaction to a more advanced
degree of chronic gastritis and possibly an adaptation to
long-standing damage to gastric mucosa.8 In our study,
patterns of the intramucosal distribution of proliferating
and apoptotic cells in gastric glands with complete
intestinal metaplasia were not markedly different from
those in the non-metaplastic oxyntic glands; Ki67positive cells were located beneath the area of goblet
cells, corresponding to the proliferative zone, and
TUNEL-reactive cells were present predominantly in the
superficial epithelium. However, in oxyntic glands with
incomplete metaplasia, cells with DNA fragmentation
were present adjacent to the proliferating areas. In
addition, the proportion of glands with cells positive for
nick end labelling was significantly higher among glands
with incomplete intestinal metaplasia than in complete
intestinal metaplasia and oxyntic glands. The proliferative zones in the oxyntic glands with incomplete intestinal metaplasia were also much larger than those in
non-metaplastic oxyntic glands and glands with complete intestinal metaplasia. These results all indicate
that the oxyntic glands with incomplete intestinal metaplasia are associated with rapid cell turnover, with high
proliferative and apoptotic activities.
Abnormalities of p53 are considered to represent the
most common molecular change in human cancer.
Abnormalities have been reported not only in cancer,
but also in precancerous gastric lesions, including gastric
dysplasia.’,1° In our present study, relatively strong pS3
immunoreactivity and mRNA hybridization signals
were observed only in oxyntic glands with incomplete
intestinal metaplasia, especially in the areas of active cell
proliferation and apoptosis. Wild-type p53 protein is
involved in normal cellular proliferation and transcription of its mRNA increases by 10- to 20-fold late in the
G1 phase.” In addition, wild-type p53 has also been
considered to play a critical role in the process of
apoptosis,I2 its gene product being required to induce
apoptosis following exposure to various DNAdamaging agents; l 3 such agents, in turn, have been
demonstrated to induce nuclear accumulation of
wild-type p53 protein. l4 Apparent abnormalities in the
development and differentiation of the stomach have not
been reported in studies of pS3 null mice.IS The low
background level of apoptosis in the intestine of these
mice appeared to be p53-independent, but pS3 was
required for the induction of apoptosis following DNA
damage. l 6 Increased wild-type p53 expression may,
therefore, also represent a cellular response to DNA
Marked inflammatory cell infiltration of the gastric
mucosa has been considered to be one of the sources of
nitric oxide-related mutagens or DNA-damaging agents
in gastric carcinogenesis. Infiltration of inflammatory
cells, including lymphocytes and neutrophils, was
observed around the glands which contained p53reactive cells. p53 expression observed in human oxyntic
glands with incomplete intestinal metaplasia is therefore
considered to reflect adaptation of the gastric mucosa to
various noxious stimuli. Further studies, including
analysis of the DNA damage and of pS3 mutations in
these cells, will be necessary to clarify the relationship
between noxious stimuli to gastric mucosa, p53 expression, and increased cell turnover. It remains a possibility
that the overexpressed p53 in gastric glands with incomplete intestinal metaplasia examined in this study could
represent a mutant form of the protein. Even if this were
so, various DNA-damaging agents which result in
increased cell turnover, possibly associated with elevated
wild-type p53, can still be considered to play an important role in the development of genetic instability,
including p53 mutations.
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