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Tumor Biology |
Department of Gastroenterology, Hepatology and Infectious Diseases [M. P. A. E., J. H., J. Y., P. M.], Institute of Pathology [T. G., A. R.], and Department of General Surgery [H. U. S.], Otto-von-Guericke University of Magdeburg, D-39120 Magdeburg, Germany; Medical Department I, Technical University of Dresden, D-01307 Dresden, Germany [S. M.]; and Division of Endocrinology, Diabetes and Metabolism, University of California Irvine, Irvine, California 92697 [M. K.]
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ABSTRACT |
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INTRODUCTION |
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Differential display presents a novel method for the identification of aberrantly expressed genes in various biological states, such as carcinogenesis or development (4 , 5) . Generally, this method has proven to be highly effective for the identification of differentially expressed genes in the process of malignant transformation (4 , 5) . Furthermore, compared with other cloning methods, such as subtraction hybridization, this method is advantageous because of its high reproducibility and the identification of mRNAs with a low copy-number per cell. We used this method to search for differentially expressed genes in gastric cancer.
MTs3 constitute a family of several intracellular, low-molecular-weight MTs with a high affinity to various heavy metals, such as zinc and copper. Four different sets of MT genes, named MT I-IV, clustered on chromosome 16, have been described. MT I genes encode seven different transcripts, named MT IA, MT IB, MT IE, MT IF, MT IG, MT IH, and MT IX (6 , 7) . The MT II gene encodes only one transcript, MT IIa, similar to MT III, also known as growth-inhibiting factor (7) . These proteins are thought to contribute to the detoxification and metabolism of compounds such as heavy metal ions, and further interest in these proteins has been raised after demonstrating their potential role in the generation of cisplatin resistance in patients with malignancies undergoing cisplatin chemotherapy (8, 9, 10) . Furthermore, the overexpression of MTs in brain tumors and in gastric and breast cancers is associated with poor survival (8 , 9 , 11 , 12) . Other groups have also demonstrated the expression of MTs in proliferating normal cells and regenerating cells apart from cancer cells (13) .
In our aim to assess the role of MT in the multistage process of gastric carcinogenesis, we used Northern blot analysis, immunohistochemical analysis, and RT-PCR analysis to explore the cellular expression and localization of this gene in the normal gastric mucosa, in premalignant lesions, and in gastric cancer. Furthermore, we assessed the expression of MT in the histologically normal gastric mucosa of individuals with an increased risk of developing gastric cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gastric cancer tissues for differential display analysis were obtained from two male patients, ages 61 and 69 years, who were undergoing gastric resection for gastric adenocarcinoma of the intestinal type. In addition, a liver metastasis was resected in the older patient.
For immunohistochemical analysis, tumor tissue was obtained from 34
patients who underwent gastric cancer surgery or endoscopy for the
diagnosis of gastric carcinoma. The characteristics and
histomorphological data for these patients are given in Table 1
. In addition, we collected gastric biopsies from eight patients (five
male, three female) without any gastric disease. The median age of this
group of patients was 51 years (range, 32–81 years). Another group, 12
patients with IM in the stomach, was studied. Apart from the IM, all of
the gastric biopsies from this group of patients revealed gastritis
(Table 2)
.
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The individuals in all of the groups gave informed consent to participate in this study. Immediately after removal, all of the tissues for molecular analysis were put in liquid nitrogen and stored at -80°C until use. This study was approved by the Ethics Committee of the University of Magdeburg, Germany.
Histology.
Formalin-fixed tissues were processed as previously described
(14)
, and sections were stained with H&E for histological
evaluation and with Warthin-Starry stain to detect Helicobacter
pylori colonization. For the histological tumor typing, we used
Lauren’s classification; tumor stage was determined according to the
TNM classification (15
, 16)
. There were 31 carcinomas of
the intestinal type, 19 carcinomas of the diffuse type, and 3
carcinomas of the mixed type. Tumor stage and grade of differentiation
are given in Table 1
. Gastritis was histologically classified according
to the updated Sydney system (Table 2
; Ref. 17
). Subtyping
of IM was performed with the use of Gomori’s aldehyde fuchsin
(GAF)-alcian blue (AB) staining (18
, 19)
, and three
types of IM were identified as previously described by Jass and Felipe
(20)
.
Cell Line.
The gastric cancer cell line AGS (ATCC-No. CRL-1739) was obtained from
the ATCC (Rockville, MD), and cells were grown in DMEM. Cells were
grown to 80–90% confluency and subcultured, the cells were harvested
after passages 6–8.
mRNA Isolation.
Total cellular RNA was isolated from 100–300 mg of tissue and cells by
using the guanidinium thiocyanate method and ultracentrifugation. The
chromosomal DNA was removed by the incubation of 50–100 µg of RNA
for 15 min at 37°C with 10 units DNaseI (Promega, Mannheim, Germany)
and 20 units RNasin (Promega) in 40 mM Tris-HCl (pH 8.3), 6
mM MgCl2, 10 mM NaCl, 2
mM spermidine, and 10 mM DTT. After
phenol/chloroform extraction, the RNA was precipitated and dissolved in
diethyl pyrocarbonate and water (21)
.
RT-PCR Analysis.
One µg of total RNA was reverse transcribed using dNTPs (1
mM), DTT (5 mM), reverse transcription buffer
[50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl2, and 1 mM DTT],
and 40 units of RNasin; 6 units of Superscript plus were added to a
total volume of 20 µl. After incubation at 37°C for 1 h, the
reaction was terminated by incubating the mixture at 95°C for 10 min.
Three different pairs of primers were designed to amplify the coding
region of MT Ia, MT IIa and MT III mRNA. Primer sequences were as
reported by Blaauwgeers et al. (22)
and were
chosen outside the coding regions to avoid amplification of pseudogenes
as follows: (a) MT Ia sense: 5'-TTCCACGTGCGCCTTATAGCC-3';
(b) MT Ia antisense: 5'-ATGGGTCAGGGTTGTATGGAA-3';
(c) MT IIa sense: 5'-AACCTGTCCCGACTCTAGCCG-3';
(d) MT IIa antisense: 5'-TATAGCAAACGGTCACGGTCA-3';
(e) MT III sense: 5'-TTGGAGAAGCCCGTTCAC-3'; and
(f) MT III antisense: 5'-TGGGATTTATTGTCATTCCTCC-3'. The 30
µl of reaction mixture was incubated for 4 min at 95°C. The samples
were denatured at 95°C for 30 s, annealed at 59°C for 60 s, and then extended at 72°C for 2 min. After 35 cycles, there was a
final elongation for 5 min at 72°C, and the samples were stored at
4°C until further use. Eight µl of the PCR product was then
electrophoresed on a 1.25% agarose gel together with size
markers. The integrity of the cDNAs was confirmed by RT-PCR analysis
using primers specific for ß-actin, as previously described
(21
, 23)
.
Differential Display.
For mRNA differential display, 8 µl of total RNA (3 µg) of each
sample was denatured for 10 min at 65°C and then reverse transcribed
for 1 h at 37°C in a 20-µl reaction volume using 2.5
mM MgCl2, 25 µM each
dNTP (TaKaRa, Otsu, Japan), 2.5 µM
T12CG oligonucleotide as anchor primer, 20 units
RNasin (Promega), and 4.5 units AMV-RT with 4 µl of 5x buffer
(Promega). Six µl of the resulting cDNA, after a 1:37 dilution, was
included as template in a 20-µl PCR reaction that contained 0.5
µM arbitrary 10mer primer (5'-AGGTGACCGT-3'), 2.5
µM T12CG primer, 2.5 mM
MgCl2, 25 µM each dNTP (TaKaRa), 2
µl of 10x buffer, and 1 unit of recombinant Taq-DNA-polymerase (Life
Technologies, Inc., Eggenstein, Germany). Forty-three cycles of PCR
were performed with cycle times of 30 s at 94°C, 1 min at
42°C, and 30 s at 72°C. After completion of the PCR reaction,
14 µl of sequencing stop solution (95% formamide, 20 mM
Na2EDTA, 0.05% w/v bromphenol blue, and 0.05%
w/v xylene cyanol) were added to each resulting PCR product. The
samples (8 µl) were then loaded on a horizontal 6% polyacrylamide
gel and were visualized by silver staining (24)
. The band
of interest (Fig. 1A
, arrowhead) was cut from the gel and then
transferred into a PCR tube with 100 µl of sterile double distilled
H2O. The gel slice was minced with a fine sterile
needle and then boiled for 15 min. After centrifugation, the
supernatant was transferred in a new tube to precipitate the cDNA
fragment with 10 µl of 3 M sodium acetate (pH
6.0) and 450 µl of ethanol at -80°C for at least 30 min. The cDNA
was pelleted by centrifugation and dissolved in 10 µl of sterile
double distilled H2O. For re-amplification, 2
µl of the dissolved cDNA fragment was included as template for a
further PCR reaction using the initial primer set and the same
conditions described above, with two changes: 6 units of Stoffel
fragment of AmpliTaq DNA polymerase (Perkin-Elmer, Langen, Germany) and
a 2.5-µM concentration of both primers were
used for successful reamplification (24
, 25)
.
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Northern Blot Analysis.
For Northern blot analysis, 20 µg of total RNA of each sample was
denatured with glyoxal and DMSO at 50°C for 1 h and fractionated
on a 1.2% agarose/4-morpholinepropanesulfonic acid gel. After
running at 90 V for 3 h, the RNA was transferred onto a nylon
membrane (Hybond-N+, Amersham, Braunschweig, Germany) using 20x SSC.
The 99-bp PCR fragment of MT II was purified with QIAquick PCR
Purification Kit (Qiagen, Hilden, Germany) and labeled with
[
-32P]dCTP using the Oligolabeling Kit
(Pharmacia, Freiburg, Germany). The hybridization of the probe was
performed at 63°C for 16 h as described previously (21
, 23)
. After hybridization, the filter was washed in 2x SSC/0.1%
SDS and was evaluated after autoradiography. To verify mRNA integrity
and equal loading, a 1.1-kb EcoRI ß-actin cDNA fragment
(ATCC) was hybridized to the same filters after removal of the first
probe by boiling in 0.1x SSC/0.1% SDS. The blot was exposed at
-80°C to Kodak XAR-5 film with intensifying screens, and the
intensity of the radiographic bands was quantified by laser
densitometry (Ultrascan XL, Pharmacia, Uppsala, Sweden).
Immunohistochemistry.
The presence of MT was assessed using formalin-fixed, paraffin-embedded
gastric tissue sections. Sections (4-µm) were deparaffinized in
graded alcohol (21
, 23)
. After rinsing with Tris buffer,
the sections were incubated with anti-MT antibody. Highly specific
monoclonal antibodies for MT were used at a dilution of 1:50 and
incubated at 37°C for 60 min. The MT antibody E9 (DAKO, Carpinteria,
CA) is a monoclonal mouse antibody derived from the ascites of
immunized mice. Horse self-polymerized MT-1 and MT-2 were used as
immunogen. Further analysis revealed the specific inhibition of the
antibody by glutaraldehyde-polymerized human, horse, sheep, and rat
MT-1 and MT-2, pointing to a single and highly conserved epitope
(12
, 26) . Detection of the bound primary antibody was
performed using the avidin-biotin-complex method (Vectastain ABC-AP
KIT, Vector Laboratories, Burlingame, CA). The sections were
incubated with diluted biotinylated secondary antibody (1:200 in RPMI)
at 22°C for 30 min, then rinsed with Tris buffer for 5 min, and
finally incubated with Vectastain ABC-AP reagent (1:100 in RPMI) for 30
min. The sections were rinsed with Tris buffer again and incubated with
Fast Red Chromogen System (ImmunoTech, Marseille, France). Finally, the
slides were counterstained and mounted with aqueous mounting medium
(21
, 23)
. All of the immunohistochemical studies were
performed using a number of negative and positive controls. As positive
controls, breast cancer samples that had previously been shown to
express abundant MT by immunohistochemistry were used in our
immunohistochemical analysis (12
, 26)
. Furthermore, to
ensure the specificity of immunostaining, we performed
immunohistochemistry using consecutive sections in the absence of the
primary antibody and with preimmune serum. In all of these cases, no
immunostaining was detected (not shown). Immunohistochemical data were
reviewed by an experienced pathologist (T. G.) who was blinded to the
clinical data of the patients. The number of positive cells was
counted, and the immunoreactivity was graded as absent, low (<5%),
moderate (5–20%), or strong.
Statistical Analysis.
Whenever indicated, the Fisher’s exact test and
2 test were used to determine statistical
analysis, with P < 0.05 taken as the level
of significance (27)
.
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RESULTS |
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, arrowhead) and in the cell
line. In the matched noncancerous gastric tissues of the cancer
patients, this band was only faintly present. After an additional round
of PCR using the primers as outlined in "Materials and Methods," we
obtained the identical picture, thus confirming the differential
expression of this cDNA fragment (not shown). After the cloning of the
PCR product, sequencing of the cDNA fragment revealed a cDNA fragment
corresponding to the 3' end of the human MT IIa mRNA. To confirm
overexpression of MT IIa, we used this fragment for the generation of a
randomly primed labeled cDNA probe for Northern blot analysis.
The Northern blot analysis demonstrates enhanced expression of MT IIa
mRNA in the tumor tissue (Fig. 1B, T)
as compared
with the normal counterpart (Fig. 1B, N)
. This
case is identical to the first pair of gastric tissues that were used
for the initial differential display analysis (Fig. 1A)
.
Thus, Northern blot analysis confirmed the overexpression of MT IIa in
gastric cancer as compared with the noncancerous gastric mucosa.
In Gastric Cancers, MT Expression Is Present in IM and Dysplasia
and in the Cancer Cells.
To determine the cellular site of MT expression in gastric cancers and
to assess the expression of MT in preneoplastic lesions of the gastric
mucosa, we performed immunohistochemical analysis using a panel of 34
paraffin-embedded gastric cancer tissues. MT immunoreactivity was
observed in all 10 gastric cancers of the diffuse type, in 19 (90%) of
the 21 intestinal type, and in all of the 3 mixed type (Table 1)
. Immunoreactivity was present in the cytoplasm and the
surface of the cancer cells (Fig. 2)
. The degree of MT immunoreactivity in the gastric cancers was
classified as absent or low in 19 cases, whereas moderate to intense
immunoreactivity was observed in 15 cases (Table 1)
. However, neither
the tumor stage, nor the grade of differentiation, nor the histological
tumor type correlated with the degree of MT expression in the gastric
carcinomas. In addition, we also observed MT immunostaining in
dysplasia adjacent to the cancer cells in five cases of gastric cancer
(Table 1)
. In addition, 16 of the 34 gastric cancers exhibited abundant
MT immunostaining in areas of IM (Fig. 3)
.
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. Gastric biopsies of eight healthy
patients without IM and without histological signs of gastritis that
were negative for H. pylori exhibited MT immunoreactivity
only at the foveolar neck of the gastric glands but not in the
superficial gastric epithelium (Fig. 4)
. In all cases, goblet cells exhibited intense MT
immunoreactivity.
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. In all of
the cases, MT expression was found in areas of IM and at the foveolar
neck of the gastric glands but, again, was not observed at the
superficial gastric epithelium. In summary, neither group of patients—who differed with respect to the presence of IM and H. pylori colonization of the gastric mucosa—expressed MT at the superficial gastric epithelium, but both groups exhibited MT expression at the foveolar neck of the gastric glands.
First-Degree Relatives of Patients with Gastric Cancer Express MT
mRNA in the Gastric Mucosa.
A total of 18 first-degree relatives were studied for the presence of
MT expression in biopsies taken from the antrum and the corpus. Using
specific primers for MT Ia, IIa, and III, we performed RT-PCR analyses.
Whereas individuals with a documented history of gastric cancer in
their family exhibited MT Ia and III mRNA in 16 and 5 of 18 cases,
respectively, individuals without a familial history of gastric
cancer exhibited MT Ia mRNA transcripts in only 3 cases
(P < 0.01). MT III was not detected at all
in the biopsies of healthy individuals. In gastric cancers, the
expression of MT Ia and MT III was observed in 13 and 5 of the 19
cases, respectively (Table 3)
. MT IIa mRNA was observed in all of the cases; however, due to the
limited amount of RNA available, quantification of mRNA levels was not
performed (not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our immunohistochemical analysis revealed that MT is expressed in at least 90% of gastric cancers and is present in premalignant lesions such as dysplasia and IM. Although other groups have demonstrated MT expression in various cancers and found an association between the overexpression of MT and poor survival in astrocytic and pancreatic tumors (8 , 9) , we could not find an association of MT expression with tumor stage or grade of differentiation in gastric cancer. In addition, MT expression was independent of the type of gastric cancer. Although survival data were not available for these patients, an association seems highly unlikely, inasmuch as the above mentioned parameters seemed to be independent of MT expression. Whereas areas of dysplasia and IM exhibited abundant MT expression, the adjacent noncancerous gastric mucosa as well as the gastric mucosa of healthy individuals did not appear to exhibit MT expression except for cells of the foveolar neck, which is known to be a zone of high proliferative activity (32) . When we compared our findings in the cancers with noncancerous gastric tissues, we found expression of MT in areas of IM in gastric cancer patients and in H. pylori-infected patients without gastric cancer. However, the superficial epithelium of the gastric mucosa in healthy individuals did not exhibit MT expression, no matter whether these patients were infected with H. pylori or not. The lack of association between MT expression and the stage of the tumor according to the TNM classification—together with the presence of MT in preneoplastic lesions of gastric cancer and the localized expression of MT in areas of high proliferative activity and regeneration in the normal gastric mucosa—support the hypothesis that the alteration of MT expression may be an early event in the process of gastric carcinogenesis.
In general, normal tissues usually do not exhibit MT expression.
However, after treatment with cytokines, metal ions, or UV irradiation,
expression of MT is induced (6
, 7)
. In addition to its
potential beneficial effect via detoxification of metal ions, MT may
also exert an antiapoptotic and mitogenic effect. Abdel-Mageed and
Agrawal (33)
have demonstrated that the down-regulation of
MT inhibited cell growth and initiated apoptosis in MCF-7 cells. This
effect was associated with the induction of p53 and
c-fos expression, whereas c-myc and
bcl-2 transcripts were down-regulated. Additional evidence
for an antiapoptotic role for MT was derived from studies demonstrating
a specific interaction between MT and the p50 subunit of NF-
B
in MCF-7 cells, which leads to the transactivation of this
transcription factor by MT, thus supporting the hypothesis that NF-B
may mediate the antiapoptotic effects of MT (34)
.
Together, these findings support the hypothesis that MT is a
growth-promoting and antiapoptotic intracellular protein that may
contribute to the pathogenesis of human malignancies.
Epidemiological analysis has revealed that individuals with a family history of gastric cancer have a 3-fold increased risk of developing gastric cancer as compared with the unaffected population (30 , 31) . Furthermore, it has recently been shown that cell proliferation in the gastric mucosa of these individuals increases independent of H. pylori infection (35) . MT Ia mRNA transcripts were found in gastric biopsies obtained from first-degree relatives and cancer patients in 16 of 18 and 13 of 19 cases, respectively. In contrast, healthy controls exhibited MT Ia in only 3 cases. MT IIa mRNA was present in all of the tissue samples. However, we did not quantitate the mRNA levels of MT IIa in these specimens because the amount of RNA was very limited. Inasmuch as the expression of MT III (also known as growth-inhibiting factor; Ref. 7 ) was not significantly altered in the relatives of cancer patients nor in gastric cancer patients as compared with healthy controls, the fact that the growth-promoting factors MT Ia and MT IIa are aberrantly expressed in the gastric mucosa raises the hypothesis that these two factors may contribute to increased cell proliferation in the gastric mucosa.
The mechanisms leading to overexpression of MT in tumor tissues are largely unknown. It has been demonstrated by several groups that MT expression is induced by growth factors, cytokines, and UV irradiation (6 , 7) . Overexpression of MT resulting from MT gene amplification has been demonstrated in cell lines chronically exposed to cadmium. Furthermore, Ha-ras mutation may also lead to enhanced MT transcription (6 , 7) . Clonal overexpression of MT in the colon of mice treated with the mutagen dimethylhydrazine was linked to somatic mutations in the morphologically normal mucosa (36) . In this model, it has been proposed that MT overexpression may be the consequence of cis-activating mutations of the MT gene or trans-activating mutations of regulatory genes (36) . In our study, we found expression of MT in the normal mucosa of the stomach that was confined to the foveolar neck. Because the number of cases was limited, the presence of H. pylori and its role in the expression of MT in the areas of high proliferative activity of the gastric mucosa was not addressed in this study. Thus, additional studies are necessary to elucidate the molecular mechanisms that lead to the aberrant expression of MT in the normal-appearing gastric mucosa.
In conclusion, we have demonstrated the overexpression of MT IIa in gastric cancer and the expression of MT in intestinal metaplasia and dysplasia. Together with the presence of MT Ia isoforms in gastric biopsies of individuals with an increased risk of developing gastric cancer, our findings point to a role for MT in the early phases of gastric carcinogenesis.
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FOOTNOTES |
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1 Supported by the Deutsche Forschungsgemeinschaft
(Eb 187/1-1, 1-2) and the Land-Sachsen-Anhalt (2775A/0087H; to
M. E.). 
2 To whom requests for reprints should be
addressed, at Department of Gastroenterology, Otto-von-Guericke
Universität Magdeburg, D-39120 Magdeburg, Germany.
Phone: 49-391-6713156; Fax: 49-391-67190054; E-mail: matthias.ebert{at}medizin.uni-magdeburg.de
3 The abbreviations used are: MT,
metallothionein; IM, intestinal metaplasia; RT, reverse transcription;
TNM, tumor-node-metastasis.
Received 8/27/99. Accepted 2/ 2/00.
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REFERENCES |
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, and P-glycoprotein in curatively resected gastric cancer. Oncology (Basel), 54: 391-399, 1997.[Medline]
. Gut, 45: 105-111, 1999.B: potential role in metallothionein-mediated mitogenic response. Cancer Res., 58: 2335-2338, 1998.This article has been cited by other articles:
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  C. P. Giacomini, S. Y. Leung, X. Chen, S. T. Yuen, Y. H. Kim, E. Bair, and J. R. Pollack A Gene Expression Signature of Genetic Instability in Colon Cancer Cancer Res., October 15, 2005; 65(20): 9200 - 9205. [Abstract] [Full Text] [PDF]
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 A. M. L. Janssen, W. van Duijn, F. J. G. M. Kubben, G. Griffioen, C. B. H. W. Lamers, J. H. J. M. van Krieken, C. J. H. van de Velde, and H. W. Verspaget Prognostic Significance of Metallothionein in Human Gastrointestinal Cancer Clin. Cancer Res., June 1, 2002; 8(6): 1889 - 1896. [Abstract] [Full Text] [PDF]
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