Gastric Cancer (2001) 4: 198–205
Expression of Fas and Fas ligand in human gastric adenomas and
intestinal-type carcinomas: correlation with proliferation and apoptosis
Mitsuhiko Osaki, Satoru Kase, Isamu Kodani, Mari Watanabe, Hironobu Adachi, and Hisao Ito
First Department of Pathology, Faculty of Medicine, Tottori University, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan
Key words Fas · Fas ligand · Gastric carcinoma · Apoptosis ·
Fas gene mutation
Apoptosis is a distinct form of cell death, distinguish-
able from necrosis by morphology, biochemistry, and
molecular biology. Apoptosis is a basic biological phe-
nomenon that is of crucial importance in the regulation
of cell populations in situations as diverse as metamor-
phosis, embryogenesis and modeling, hormone-induced
organ involution, and neoplasia. Recent studies have
analyzed tumor development and progression in terms
of proliferation and apoptotic cell death (cell loss) in
a variety of human malignancies, including gastric
Our previous studies showed that apoptosis was vari-
ably detected in human gastric adenoma and carcinoma
cells. The findings are summarized as follows: (i) a
higher apoptotic index (AI; percentage of apoptotic
tumor cells) in tubular adenomas, followed by well-
differentiated (intestinal-type), and poorly differenti-
ated (diffuse-type) carcinomas [1–3], (ii) lower AI in
carcinomas expressing P53 oncoprotein [3,4], (iii) in-
crease in AI with preoperative chemotherapy , (iv)
association of thymidine phosphorylase expression with
lower AI , and (v) an inverse correlation between AI
and intratumoral microvessel density (IMVD) .
Thus, apoptosis is correlated with tumor histology,
p53 gene status, and cytotoxic agents such as anticancer
agents, as well as being correlated with blood supply.
These findings suggest the presence of variable path-
ways of signal transduction that may exert a wide spec-
trum of biologic effects, depending on the apoptosis-
inducible factors that are present.
The Fas-Fas ligand (FasL) system has been recog-
nized as the major pathway for the induction of
Background. Fas (APO-1/CD95), a member of the tumor
necrosis factor/nerve growth factor receptor superfamily,
mediates apoptosis in response to agonistic antibodies or Fas
ligand (FasL) binding. Previous reports indicated an up-
regulation of FasL in gastric carcinomas to evade host
immune attack. Fas/FasL expression, however, has not been
analyzed in terms of apoptosis and proliferation in gastric
adenoma and carcinoma.
Methods. This study was conducted on seven human gastric
carcinoma cell lines, 47 gastric adenomas, and 75 intestinal-
type adenocarcinomas (48 early and 27 advanced carcinomas).
Fas/FasL expression was examined by immunohistochemistry,
apoptosis by the terminal deoxynucleotidyl transferase-
mediated dUTP-digoxigenin nick-end labeling (TUNEL)
method, and Fas gene mutation by a reverse transcriptase
(RT) polymerase chain reaction-single-strand conformation
polymorphism (PCR-SSCP) and sequencing method.
Results. Fas and FasL expressions were noted in 18 (38.3%)
and 17 (36.2%) adenomas, in 21 (43.8%) and 33 (68.8%) early
carcinomas, and in 10 (37.0%) and 19 (70.4%) advanced car-
cinomas, respectively. The frequency of FasL expression was
significantly higher in advanced carcinomas than in the early
carcinomas and adenomas; in contrast, there was no signifi-
cant difference in Fas expression among the three groups. The
mean apoptotic index (AI) was 4.96 ? 0.51 in the adenomas,
2.96 ? 0.23 in the early carcinomas, and 1.67 ? 0.17 in the
advanced carcinomas. A significantly higher AI was noted in
the lesions with Fas expression than in those without Fas
expression in all three groups. No missense mutations of the
Fas gene were detected in any of the gastric carcinoma cell
lines, or in the gastric adenomas or carcinomas.
Conclusions. Upregulation of FasL may correlate with the
progression of gastric carcinoma. Apoptosis in gastric ad-
enoma and carcinoma cells may occur via Fas-dependent and
-independent pathways, but further clarification is needed.
Offprint requests to: M. Osaki
Received: August 6, 2001 / Accepted: October 3, 2001
M. Osaki et al.: Fas/FasL in human gastric tumors 199
apoptosis in a variety of human normal and neoplastic
cells . Fas antigen is a 45-kDa cell-surface protein
that belongs to the death receptor subfamily of the
tumor necrosis factor/nerve growth factor receptor
superfamily. Fas contains a unique cytoplasmic region,
termed the “death domain”, which is essential for the
initiation of a cytolytic response . The binding of Fas
ligand (FasL) to Fas antigen results in the transduction
of a cytolytic signal into the cell, followed by apoptosis,
a process that has been well studied, mainly in hemato-
poietic cells [9,10].
Alterations in the expression of Fas and FasL have
been demonstrated in a variety of tumor cells. To date,
several studies have demonstrated downregulation of
Fas in a number of human malignant solid tumors, in-
cluding esophageal  and breast  carcinomas. On
the other hand, upregulation of FasL has been reported
in various human malignant solid tumors, including
hepatocellular , colon , lung , and pancreatic
 carcinomas, as well as breast carcinomas .
Bennett et al.  reported a relationship between the
upregulation of FasL and apoptosis in tumor-infiltrating
lymphocytes (TILs) in gastric carcinoma. Moreover, Li
et al.  have reported Fas and FasL expression in
gastric cancer and its preneoplastic lesions in a small
number of cases. However, there have been no studies
that analyzed the relationship between Fas/FasL ex-
pression and apoptosis in gastric carcinoma cells.
In this study, we examined the expression of Fas and
FasL in human gastric adenomas and carcinomas, as
well as in seven human gastric carcinoma cell lines,
relative to tumor cell apoptosis. We also analyzed Fas
gene alterations to clarify the significance of Fas/FasL
in the progression of human gastric adenomas and
Materials and methods
Gastric carcinoma cell lines
Table 1 shows the histological types and p53 gene status
of the seven cell lines derived from human gastric carci-
nomas analyzed in this study. All the cell lines were
cultured in RPMI 1640 supplemented with 10% fetal
calf serum (FCS), 100U/ml penicillin, 100µg/ml strepto-
mycin, and 292µg/ml l-glutamine at 37°C in 5% CO2.
Studies were conducted on 47 adenomas, obtained
by endoscopic mucosal resection; and 75 surgically
removed adenocarcinomas (48 early and 27 advanced
carcinomas). The histological type of the gastric carci-
nomas in all cases was papillary and tubular adenocarci-
noma, according to the criteria of the Japanese Gastric
Cancer Association . All the specimens were taken
from the files of the Department of Pathology, Faculty
of Medicine, Tottori University, and its related teaching
hospitals. Routinely processed, formalin-fixed, paraffin-
embedded tissue blocks from the principal infiltration
of the tumoral mass were selected. Three-micrometer-
thick sections were examined by light microscopy, im-
munohistochemistry, and the terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP-digoxigenin nick-
end labeling (TUNEL) procedure.
Sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and
Western blot analysis
SDS-PAGE and Western blot analysis were performed
as described previously . To evaluate the specificity
of antibodies against human Fas and FasL, one poly-
clonal and two monoclonal anti-Fas antibodies and one
polyclonal and three monoclonal anti-FasL antibodies
were used. We selected the antibodies most specific to
each of Fas and FasL. The membranes were blotted
with nondiluted anti-Fas polyclonal antibody (Nichirei,
Tokyo, Japan), which recognized the carboxy terminus
of Fas protein, and a 1:1000 dilution of the anti-
FasL monoclonal antibody (mAb 33; Transduction
Laboratories, Lexington, KY, USA), which recognized
the carboxy terminus of FasL protein. Blots were
developed with a second peroxidase-labeled antibody
overnight at 4°C. After extensive washing had been
done, specific bands were detected, using an enhanced
chemiluminescence system (ECL Detection System;
Amersham Pharmacia Biotech, Amersham, UK).
Dewaxed paraffin sections were immunostained, using
the streptavidin-biotin peroxidase complex (SAB)
method. The following primary antibodies were used:
rabbit polyclonal antibody against Fas (Nichirei), and
mouse monoclonal antibody against FasL (mAb33;
diluted 1:400; Transduction Laboratories). Immuno-
reactions were visualized with diaminobenzidine
Table 1. Human gastric cancer cell lines
200 M. Osaki et al.: Fas/FasL in human gastric tumors
(DAB), and the sections were counterstained with 3%
We judged samples to be positive for Fas or FasL
when at least 20% of the tumor cells showed positive
To detect DNA breaks in situ, TUNEL was performed
according to the method of Gavrieli et al. , using an
Apop Tag Plus in-situ apoptosis detection kit (Intergen,
Purchase, NY, USA). Details of the method were de-
scribed previously .
To examine the specificity of the TUNEL procedure,
slides were treated with TdT buffer solution not
containing TdT or digoxigenin-11-dNTP, as negative
controls; these were invariably negative for TUNEL
signals. The TUNEL procedure was also conducted at
the same time on control slides of gastric carcinomas,
which were previously shown to have many apoptotic
cells with TUNEL signals, to obtain consistent findings.
Differences in numerical data values between two or
three groups were evaluated using the Kruskal-Wallis
test (Fas and FasL expression) and the Mann-Whitney
U-test (AI). The level of significance was set at P ? 0.05.
Fas gene mutation of coding regions in gastric cancer
Fas gene mutations of coding regions were examined in
all seven of the gastric carcinoma cell lines. Total RNA
was isolated and cDNA synthesis was performed as
described previously . The primers used in this study
and the polymerase chain reaction (PCR) procedure
used have been described previously  except that
no radioisotope was used in the PCR. All PCR prod-
ucts were analyzed by direct sequencing, using a Big
Dye Terminator Cycle Sequencing kit (Amersham
Fas gene mutations in gastric adenomas
Fas gene mutations were examined in 20 of the 47 gas-
tric adenomas and in 30 of the 75 carcinomas in the
paraffin-embedded blocks. Several 20-µm sections were
trimmed with a clean razor and placed in a 1.5-ml tube.
To avoid contamination, the microtome blade was
removed and cleaned with xylene after use with each
block. Genomic DNA was extracted using DEXPAT
(TaKaRa, Kyoto, Japan). PCR primers were synthe-
sized based on the Fas DNA sequences in exons 6 and 9,
which encode the transmembrane domain and death
domain, respectively. The sequences of the primer pairs
were: 5?-tttcatataatatgccaatgttcc-3? and 5?-cttcccccaag
ttatttcaat-3? for Fas exon 6; 5?-tgctggagtcatgacactaagt-3?
and 5?-caatgtgtcatacgcttctttc-3? for Fas exon 9A; 5?-
taattggcatcaacttcat-3? and 5?-gaatttgttgtttttcactcta-3? for
Fas exon 9B; and 5?-ggttttcactaatgggaatttcat-3? and 5?-
cttcattgacaccattctttcg-3? for Fas exon 9C [25–28]. Exon
9 was divided into three fragments. A single-strand
conformation polymorphism (SSCP) method was per-
formed by a standard procedure . All cases in which
abnormal band(s) were detected by SSCP were ana-
lyzed by direct sequencing, as described above.
Expression of Fas and FasL in human gastric
carcinoma cell lines
First, to test the specificity of the three different com-
mercially available anti-Fas antibodies and the four
anti-FasL antibodies, Western blot analysis was per-
formed on the seven human gastric carcinoma cell
lines. The anti-Fas rabbit polyclonal antibody
purchased from Nichirei and the anti-FasL mouse
monoclonal antibody (clone 33) purchased from Trans-
duction Laboratories gave almost identical results in
all cell lines. On the other hand, the other antibodies
gave rise to a faint or non-specific labeling by Western
Next, we examined the expression levels of Fas and
FasL by Western blot analysis. As shown in Fig. 1, both
Fas and FasL proteins in the gastric carcinoma cell lines
were detected specifically by the antibodies. Fas and
FasL antigen were variably expressed in all seven cell
lines examined, although there was extremely low ex-
pression in MKN-28.
Fas and FasL expression in human gastric adenoma
Fas antigen immunoreactivity was observed both on cell
membranes and in the cytoplasm of adenoma and carci-
Fig. 1. Expression of Fas and FasL in seven gastric carcinoma
cell lines, detected by Western blotting
M. Osaki et al.: Fas/FasL in human gastric tumors201
noma cells, being predominant in the cytoplasm (Fig.
2A–D). Fas antigen expression was noted in 18 (38.3%)
of the 47 gastric adenomas, in 21 (43.8%) of the 48 early
carcinomas, and in 10 (37.0%) of the 27 advanced carci-
nomas, the frequency showing no significant differences
among the three categories (Table 2).
FasL immunoreactivity was observed both on cell
membranes and in the cytoplasm of the adenoma
and carcinoma cells. In contrast to Fas antigen, immu-
noreactivity was predominant on the cell membrane
(Fig. 2E–H). FasL expression was noted in 17 (36.2%)
of the 47 gastric adenomas, in 33 (68.8%) of the 48 early
carcinomas and in 19 (70.4%) of the 27 advanced carci-
nomas. The frequency of FasL-positive cases was sig-
nificantly higher in advanced carcinomas than in early
carcinomas and adenomas (P ? 0.01; Table 2).
Correlation between AI and Fas/FasL expression in
gastric adenomas and adenocarcinomas
With the TUNEL method, apoptotic tumor cells were
clearly identified by brown nuclear signals (Fig. 3). Table
2 shows the correlations between AI and the three lesion
categories. The mean AI was 4.96 ? 0.51 in the gastric
adenomas, 2.96 ? 0.23 in the early carcinomas, and 1.67
? 0.17 in the advanced carcinomas. The mean AI was
significantly higher in the adenomas than in both the
early and the advanced carcinomas (P ? 0.01).
Next, we analyzed mean AI relative to Fas and FasL
expression (Table 3). The mean AI was significantly
higher in the Fas-positive lesions than in the Fas-
negative lesions in the adenomas (6.93 ? 1.03 versus
3.86 ? 0.42; P ? 0.015), the early carcinomas (3.80 ?
Fig. 2A–H. Fas and FasL expression in
gastric adenoma and adenocarcinoma.
Detection of Fas (B and D) and FasL (F
and H) expression by immunohistochem-
istry in gastric adenoma (B and F) and
adenocarcinoma (D and H). A, C, E, and
G are serial or semi-serial sections of B,
D, F, and H, respectively. Fas expression
is predominant in the cytoplasm rather
than on cell membranes. On the other
hand, FasL expression is predominant on
cell membranes and is higher in adenocar-
cinoma than in adenoma. A, C, E, and G:
H&E, ?140; B, D, F, and H: immunohis-
202M. Osaki et al.: Fas/FasL in human gastric tumors
0.36 versus 2.30 ? 0.23; P ? 0.002), and the advanced
carcinomas (2.30 ? 0.23 versus 1.31 ? 0.17; P ? 0.003).
FasL expression, however, was not correlated with the
AI in any of the three categories.
Fas gene mutation
Fas gene mutation was tested in the seven human gas-
tric carcinoma cell lines, and in 20 gastric adenomas and
30 gastric carcinomas. We first analyzed the mRNA
transcripts of the Fas gene by reverse transcriptase
(RT)-PCR. As shown in Fig. 4, four pairs of primers
were used to amplify four overlapping regions of Fas
cDNA (fragments A, B, C, and D), which covered the
entire coding region of Fas, along with part of the 5? and
3? untranslated regions of Fas cDNA. The 3? primer for
fragment B and the 5? primer for fragment C were
designed to localize to the transmembrane domain of
the Fas gene. The 3? primer for fragment C and the 5?
primer for fragment D were designed to localize to the
death domain of the Fas gene. Single specific bands
were detected by RT-PCR in four fragments of the
seven gastric carcinoma cell lines. Abnormal bands that
suggested spliced mutants were not detected. All frag-
ments were subsequently analyzed by direct sequencing
to detect point mutations. However, no mutations of the
Fas gene transcripts were detected in any of the cell
We also examined point mutations of the Fas gene
in the tissue specimens. None of the gastric adenoma
samples showed mutations of the gene by SSCP analy-
sis. An abnormal band was detected in one of 30 gastric
carcinomas (CTT to CTA at codon 306 in exon 9; data
not shown). Direct sequence analysis, however, showed
it to be a silent mutation (Leu to Leu).
In this study, we have clearly demonstrated both Fas
and FasL expression in human gastric adenomas and
intestinal-type carcinomas. In contrast to Fas expres-
sion, FasL expression has been shown to be restricted to
a few cells or tissues, such as cytotoxic T lymphocytes
(CTLs), Sertoli cells, and corneal epithelia, the latter
two being immunologically privileged sites, which are
able to evade a rejection reaction [30,31]. As alluded to
in the “Introduction” [13–18], FasL expression has been
analyzed in a variety of human malignancies, with re-
sults suggesting that tumor cells with FasL alterations
Table 2. Expression of Fas/FasL and apoptotic index (AI)
AI (mean ? SD)
Adenoma (n ? 47)
Early (n ? 48)
Advanced (n ? 27)
18 (38.3%)29 17 (36.2%)30 4.96 ? 0.51
152.96 ? 0.23
1.67 ? 0.178
*P ? 0.01; The frequency of FasL expression was significantly higher in the carcinomas than in the
adenomas (P ? 0.01), but there was no significant difference between FasL expression in the early
and advanced carcinomas
?, positive case; ?, negative case
Fig. 3. Terminal deoxynucleotidyl transferase (TdT)-
mediated dUTP-digoxigenin nick-end labeling (TUNEL)-
signal-positive tumor cells are indicated by arrowheads.
A TUNEL method; B H&E, ?300
M. Osaki et al.: Fas/FasL in human gastric tumors 203
may evade host immunological attack by CTLs. This
hypothesis has been questioned, however, because the
specificity of the antibodies used had not been con-
firmed . In this study, importantly, the specificity of
the commercially available antibodies for Fas and FasL
was confirmed. Western blot analysis showed that only
one antibody for Fas or FasL detected single bands of
about 45 and 37kDa, corresponding to Fas and FasL,
respectively. Moreover, Fas and FasL expressions were
variable among the human gastric carcinoma cell lines
we tested, and minimal expression of Fas was noted in
MKN-28, which has been shown to be resistant to anti-
Fas antibody-induced apoptosis . Thus, it is un-
equivocal that Fas and FasL are expressed in human
gastric tumors, although their actual roles and biological
significance are largely unknown.
Of interest is the finding that the frequency of FasL
expression was significantly higher in the carcinomas
than in the adenomas, while no significant difference
was noted in FasL expression between the early and
advanced carcinomas. On the other hand, Fas expres-
sion did not differ among the three categories. These
findings suggest that FasL is related to the carcino-
genesis of gastric adenomas and the progression of
intestinal-type gastric carcinomas. Moreover, the higher
expression of FasL could provide a preferential advan-
tage for tumor progression by allowing the tumor to
evade immunological attack by CTLs, or, in other
words, by killing the activated Fas-sensitive tumor infil-
trating lymphocytes (TILs), as has been demonstrated
in malignancies in other organs, including colon and
pancreatic carcinomas [33,34].
We also analyzed the significance of Fas/FasL expres-
sion in terms of tumor cell apoptosis. The AI was high-
est in the adenomas, followed by the early carcinomas
and advanced carcinomas. This finding supports those
Table 3. Relationship between Fas/FasL expression and AI
(mean ? SD)
6.93 ? 1.03] P ? 0.015
3.86 ? 0.42
5.44 ? 0.81]NS
4.69 ? 0.66
3.80 ? 0.36]P ? 0.002
2.30 ? 0.23
2.76 ? 0.25]NS
3.40 ? 0.48
2.30 ? 0.23]P ? 0.003
1.31 ? 0.17
1.77 ? 0.18]NS
1.45 ? 0.36
NS, Not significant
Fig. 4. Schematic presentation of Fas cDNA and the primer designed for reverse transcriptase-polymerase chain reaction-
single-strand conformation polymorphism (RT-PCR-SSCP) analysis. Nucleotide numbers are shown at the top. The 5?- and
3?- untranslated regions of the cDNA are indicated by a single line. The coding regions, which are boxed, consist of the
transmembrane domain (TM) and the death domain (DD). The sizes (in base pairs) of the products (fragments A, B, C, and D)
are shown in parentheses
204M. Osaki et al.: Fas/FasL in human gastric tumors
of our previous studies [1–3], in which we confirmed
that apoptosis occurred predominantly via a p53 gene-
independent pathway in human gastric carcinomas. In
the present study, we found that positivity for Fas ex-
pression in tumors correlated with a significantly higher
AI in all three categories, in contrast to a finding of
no association between FasL expression and AI. These
findings provide further evidence that Fas expression
may play a role in tumor cell proliferation in human
gastric adenomas and carcinomas. Moreover, there may
be some mechanism(s) for the evasion of Fas-mediated
apoptosis in gastric carcinomas, as indicated by the sig-
nificantly lower AI in the early and advanced carcino-
mas than in the adenomas, and the lack of difference
in the frequency of Fas expression among the three
Recently, somatic mutations of the Fas gene have
been demonstrated in a variety of human malignancies
(including lymphomas [35–37]), as well as in various
solid tumors (including gastric carcinoma [25–28,38]).
Park et al.  detected missense mutations in the death
domain of the Fas gene in 5 (11.6%) of 43 gastric carci-
nomas. In the present study, we investigated whether
there were alterations in the Fas gene in seven gastric
carcinoma cell lines, 20 gastric adenomas, and 30 gastric
carcinomas, targeting exons 6 and 9, which encode the
transmembrane domain and the death domain of Fas,
respectively [39,40], and which are known to show
mutations more frequently than other exons [25–28,38].
However, no mutations were detected in any samples,
except for one gastric carcinoma that showed a silent
mutation (Leu-to-Leu). This different finding in our
study may be partly explained by differences in the
methods used in our study and that of Park et al. ,
who extracted genomic DNA from microdissected tis-
sue specimens, whereas our extracted samples included
DNA not only from carcinoma cells but also from other
nontumorous cells, such as lymphocytes and endothelial
In any case, the frequency of Fas gene mutation
seems to be very low, and, thus, the lower AI in gastric
carcinomas than in adenomas cannot be explained by
mutations alone. Moreover, these findings suggest that
gastric carcinomas possess other possible mechanism(s)
of evading Fas-mediated apoptosis. The evasion of mol-
ecules related to Fas-mediated signal transduction is
needed to account for the lower AI in gastric carcino-
mas. A few reports have noted that resistance to anti-
Fas antibody-induced apoptosis may be correlated with
caspase-8 downregulation in neuroblastoma, renal cell
carcinoma, pancreatic carcinoma, and human mono-
cytes [41–44]. This downregulation is associated with
caspase-8 gene silencing through DNA methylation and
cFLIP upregulation, which inhibits the activation of
In summary, we have demonstrated Fas/FasL expres-
sion in human gastric adenomas and carcinomas. FasL
may be correlated with the progression of gastric carci-
noma by a mechanism that involves the evasion of TIL-
induced apoptosis. On the other hand, Fas expression
clearly resulted in increased apoptosis in adenoma and
carcinoma cells. Thus, the Fas/FasL system seems to be
relevant to the proliferation of gastric cancer cells.
Acknowledgments This work was supported, in part,
by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Culture, Sports, Science, and
Technology of Japan (grant number, 11470050). The
authors thank Mr. N. Itaki and Ms M. Kajimura for
their skillful technical assistance.
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