Download Promoter methylation of p16, Runx3, DAPK and

Tumori, 96: 726-733, 2010
Promoter methylation of p16, Runx3, DAPK and
CHFR genes is frequent in gastric carcinoma
Shi-Lian Hu1, Xiang-Yong Kong1, Zhao-Dong Cheng1, Yu-Bei Sun2, Gan Shen3,
Wei-Ping Xu4, Lei Wu4, Xiu-Cai Xu5, Xiao-Dong Jiang1, and Da-Bing Huang1
1
Department of Geriatrics, Affiliated Anhui Province Hospital, Anhui Medical University, Hefei;
Department of Oncology, Anhui Province Hospital, Hefei; 3Cadre’s Ward of Anhui PPC Hospital, Hefei;
4
Anhui Evidence-based Medicine Center, Hefei; 5Anhui Provincial Hospital Center Lab, Hefei, China
2
ABSTRACT
Aims and background. Transcriptional silencing induced by hypermethylation of
CpG islands in the promoter regions of genes is believed to be an important mechanism of carcinogenesis in human cancers including gastric cancer. A number of reports on methylation of various genes in gastric cancer have been published, but
most of these studies focused on cancer tissues or only a single gene. In this study, we
determined the promoter hypermethylation status and mRNA expression of 4 genes:
p16, Runx3, DAPK and CHFR.
Methods. Methylation-specific polymerase chain reaction (MSP) was used to determine the methylation status of p16, Runx3, DAPK and CHFR gene promoters in cancer and adjacent normal gastric mucosa specimens from 70 patients with gastric cancer, as well as normal gastric biopsy samples from 30 people without cancer serving
as controls. In addition, the mRNA expression of p16, Runx3, DAPK and CHFR was investigated in 34 gastric cancer patients by RT-PCR. Bisulfite DNA sequence analysis
was applied to check the positive samples detected by MSP.
Results. When carcinoma specimens were compared with adjacent normal gastric
mucosa samples, a significant increase in promoter methylation of p16, Runx3, DAPK
and CHFR was observed, while all 30 histologically normal gastric specimens were
methylation free for all 4 genes. The methylation rate of the 4 genes increased from
normal stomach tissue to tumor-adjacent gastric mucosa to gastric cancer tissue.
Concurrent methylation in 2 or more genes was found in 22.9% of tumor-adjacent
normal gastric mucosa and 75.7% of cancer tissues. No correlation was found between hypermethylation and other clinicopathological parameters such as sex, age,
and tumor location. However, the frequency of DAPK and CHFR methylation in cancer tissues was significantly associated with the extent of differentiation and lymph
node metastasis (P <0.05) and the frequency of Runx3 methylation was significantly
associated with tumor size (P <0.05). Weak expression and loss of expression of the 4
genes was observed in cancer tissues and was significantly associated with promoter
hypermethylation (P <0.05).
Conclusions. Promoter hypermethylation of p16, Runx3, DAPK and CHFR is frequent
in gastric cancer. DAPK and CHFR promoter hypermethylation may be an important
help in evaluating the differentiation grade and lymph node status of gastric cancer.
Weak gene expression and loss of gene expression due to promoter hypermethylation
may be a cancer-specific event. Free full text available at www.tumorionline.it
Introduction
Gastric cancer is one of the most frequent tumor types worldwide and the major
cause of cancer-related deaths in China1. Its development is associated with genetic
changes in the host, such as loss of heterozygosity and several types of mutations in
tumor suppressor genes2.
Key words: gastric carcinoma, p16,
Runx3, DAPK, CHFR, DNA methylation
Acknowledgments: This work was
supported by the National Natural Science Foundation of China, item number 30672383.
Correspondence to: Professor Shi-lian
Hu, Department of Geriatrics, Anhui
Province Hospital, Anhui Medical University, Hefei 230001, China.
Tel +86-0551-2283589;
e-mail [email protected]
Received November 19, 2009;
accepted April 8, 2010.
PROMOTER METHYLATION OF GENES AND GASTRIC CARCINOMA
Epigenetic alterations are now also regarded as key
mechanisms of the development and progression of
gastric carcinoma3,4. Methylation of CpG islands is one
of the crucial epigenetic mechanisms: it can lead to
changes in chromosome structure, DNA constitution,
and DNA stability5,6. CpG islands are DNA segments of
at least 0.5 kb with a high G:C and CpG content which
are often located in the promoter or 5’-exon sequences
of genes7. Aberrant methylation of CpG islands is tumor
specific, and may result in inactivation of tumor suppressor genes8-11. Recently, in addition to its potential
role in gene inactivation in human cancers, CpG island
hypermethylation has been gaining attention as a molecular marker for tumor detection and prediction of
cancer development.
Aberrant methylation of the promoters of tumor suppressor genes such as p16, Runx3, DAPK and CHFR has
been reported in many kinds of cancer12,13. p16 is a cell
cycle regulator involved in the inhibition of G1 phase
progression14. Loss of function of p16 results in higher
cyclin D-dependent protein kinase activity and thus
leads to aberrant phosphorylation of retinoblastoma
protein, which accelerates cell growth15. Inactivation of
p16 by homozygous deletion, point mutation or hypermethylation is one of the most commonly observed
aberrations in tumors16. CHFR (checkpoint with forkhead-associated [FHA] and RING finger) encodes a protein with FHA and RING finger domains that functions
in the mitotic checkpoint pathway, which governs the
transition from prophase to prometaphase. It is localized at chromosome 12q24.3317,18. Runt-related transcription factor 3 gene (Runx3) was originally cloned as
AML2 and localized on human chromosome 1p36.119,20.
Death-associated protein kinase (DAPK), an actin-associated calcium/calmodulin-dependent enzyme with
serine/threonine kinase activity21, is involved in apoptosis induced by tumor necrosis factor-alpha, Fas, and
IFN-γ22. Recent studies have shown that silencing of
p16, Runx3, DAPK and CHFR by hypermethylation of
the CpG-rich promoter region occurred frequently in
non-small cell lung cancer23, colorectal carcinomas24,
esophageal carcinomas, and gastric carcinomas25,26.
However, most of these studies were focused on cancer
tissues or only a single gene, while there have been few
studies on the relationship between clinicopathological
prognostic factors and the methylation status of multiple genes in gastric cancer.
In the present study we examined the promoter hypermethylation status of the human p16, Runx3, DAPK
and CHFR genes in the DNA of primary gastric tumor
tissues and adjacent normal gastric mucosa. We analyzed the relationship between the methylation of these
genes and clinicopathological parameters. In addition,
we determined the mRNA expression of p16, Runx3,
DAPK and CHFR in 34 primary gastric cancers to analyze the potential relationship between loss of mRNA
expression and promoter hypermethylation.
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Material and methods
Patients and tissue samples
Tumor specimens were obtained from 70 patients. All
of them gave their informed consent before collection
of the samples according to institutional guidelines.
They underwent surgical resection of primary gastric
cancer at the Department of General Surgery, Anhui
Provincial Hospital and the Department of General Surgery, the Third Affiliated Hospital of Anhui Medical University, between September 2007 and March 2009. For
all these tumors, adjacent normal gastric mucosa (located at least 5 cm from the primary tumor) was also
obtained. In addition, 30 normal gastric biopsy specimens serving as controls were obtained from people
without any evidence of cancer. These samples were immediately frozen after resection and stored at -80 . Of
the 70 gastric cancer patients, 31 had clinical stage I and
II while 39 had clinical stage III and IV; the mean age of
the patients was 62.11 years (range, 32-85 years).
DNA extraction and bisulfite modification
Genomic DNA was obtained from samples using a QIAamp DNA Micro Kit (QIAGEN, Germany) according to
the manufacturer’s instructions. Extracted DNA was
identified by agarose gel electrophoresis and then
quantified by calculating the A260/280 ratio. Genomic
DNA was modified with sodium bisulfite as described
previously27. Briefly, 1 µg of genomic DNA was denatured with 2M NaOH for 10 minutes at 37 °C. 10 mM hydroquinone and 3 µM sodium bisulfite at PH 5.0 were
added and mixed. Samples were incubated at 50 for 16
hours. DNA was purified using the Wizard DNA purification kit (Promega, Madison, WI, USA), treated again
with NaOH, precipitated with ethanol, and resuspended
in water. This procedure converts unmethylated cytosine residues to uracil that is recognized as thymine by
Taq polymerase, whereas the methylated cytosine remains unchanged. The modified DNA was either used
immediately as a template for PCR or stored at -20 °C.
Methylation-specific PCR
For detection of promoter methylation status, methylation-specific polymerase chain reaction (MSP) was
performed as described by Herman et al.28. Briefly, modified DNA was subjected to MSP using specific primers
(Table 1). The PCR was carried out in a volume of 25 µL
with 2 µL of bisulfite-modified DNA, 10x PCR buffer, 25
mM MgCl2, 10 pmol of each primer, 200 µM dNTPs, and
1 U of Hot-Goldstar Taq polymerase (Takara Biochemical, Japan). PCR conditions were as follows: after initial
denaturing for 10 minutes at 95 °C, 35 cycles at 95 °C for
30 seconds, 30 seconds at different annealing temperatures with the primers (Table 1), and 72 °C for 30 seconds
were followed by a final extension step at 72 °C for 10
minutes. Plasma DNA from a healthy individual was
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SL HU, XY KONG, ZD CHENG ET AL
Table 1 - Primers and conditions used for MSP and BSP
Primers
p16 (MSP)
p16 (BSP)
Runx3 (MSP)
Runx3 (BSP)
DAPK (MSP)
DAPK (BSP)
CHFR (MSP)
CHFR (BSP)
M
U
M
U
M
U
M
U
Forward sequence (5’-3’)
Reverse sequence (5’-3’)
Base pairs
Temperature (ºC)
ATAATAGCGGTCGTTAGGGCGTCG
TTATGAGGGGTGGTTGTATGTGGG
TTAATATGAGAATTGGTTAAAATTTATATT
ATAATAGCGGTCGTTAGGGCGTCG
TTATGAGGGGTGGTTGTATGTGGG
TAGGGTTTTTAGGAGATTTTTTTT
GGATAGTCGGATCGAGTTAACGTC
GGAGGATAGTTGGATTGAGTTAATGTT
TTTTTATTTATTTTTTAGTTGTGTTTT
TTTCGTGATTCGTAGGCGAC
TTTTGTGATTTGTAGGTGAT
TGTTTATTAAGAGYGGTAGTTAAAG
GCTTCTACTTTCCCGCTTCTCGCG
AAAACAACCAACACAAACACCTCC
AAAAAAAACACCAAACAATATTTAC
GCTTCTACTTTCCCGCTTCTCGCG
AAAACAACCAACACAAACACCTCC
AACTTCCCCAACTCTTACTACTTC
CCCTCCCAAACGCCGA
CAAATCCCTCCCAAACACCAA
CCTTAACCTTCCCAATTACTC
GCGATTAACTAACGACGACG
ACAATTAACTAACAACAACA
AAAATCCTTAAAACTTCCAATCC
234
234
207
115
234
295
98
106
237
155
155
197
65
60
56
55
61
60
55
55
53
58
53
55
MSP, methylation-specific polymerase chain reaction; BSP, bisulfite DNA sequencing PCR; M, methylated specific primers; U, unmethylated specific primers.
used as the positive control for unmethylation, the same
plasma DNA was methylated with excess Sss I methyltransferase (New England Biolabs Inc, Beverly, MA, USA)
to generate completely methylated DNA at all CpG islands and used as the positive control for methylation;
distilled water without DNA was used as negative control. The PCR products were separated in 1.5% agarose
gels, stained with ethidium bromide, and visualized directly under ultraviolet illumination (BioSpectrum Imaging System; Ultra-Violet Products Inc, Upland, CA, USA).
Bisulfite DNA sequencing PCR (BSP)
We sequenced the bisulfite-PCR products of the
methylation-positive gastric cancer tissue as detected
by MSP. Genomic DNA was modified with sodium bisulfite as described previously23. The bisulfite-modified
DNA was used for PCR amplification. The PCR was performed as mentioned previously. The primer sequences
and PCR annealing temperatures used for bisulfite DNA
sequence analysis are shown in Table 1. PCR products
were purified by E.Z.N.A.Cycle-Pure Kit (Omega, USA)
as described previously. The sequence of PCR products
was analyzed by Takara Biotechnology (Dalian, China).
Reverse transcription-PCR (RT-PCR)
Germany), then amplified by primer sets specific for the
p16 and DAPK genes (Table 2). PCR was performed in a
total volume of 25 µL with 1 µL of Taq, 2.5 µL of PCR
buffer, 2 µL of MgCl2, 0.5 µL of dNTP, 1 µL of primers, 4 µL
of cDNA, and 13 µL DNase-free water. PCR amplification
consisted of 1 cycle at 95 °C for 5 minutes, 35 cycles at 95
°C for 30 seconds, at different annealing temperatures
with the primers (Table 2) for 30 seconds, and at 72 °C for
30 seconds, followed by a final extension at 72 °C for 10
minutes. The PCR products were separated in 2%
agarose gels, stained with ethidium bromide, and visualized directly under ultraviolet transillumination.
Statistical analysis
The data were processed by the SPSS 16.0 statistical
software. The associations between p16, Runx3, DAPK
and CHFR methylation and clinicopathological parameters were statistically analyzed using the chi-square
test. Correlations between p16, Runx3, DAPK and CHFR
methylation status and mRNA expression were analyzed using Fisher’s exact test. Differences were considered statistically significant at P values <0.05.
Results
Expression of p16, Runx3, DAPK and CHFR mRNA was
analyzed by RT-PCR. Total RNA was extracted with TRIzol
reagent (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer’s instructions. First-strand cDNA was generated using a first-strand cDNA synthesis kit (Qiagen,
Promoter hypermethylation of p16, Runx3, DAPK and
CHFR in gastric cancer
As shown in Figure 1, only methylated and unmethylated primers achieved the expected amplified
Table 2 - Primers and conditions used for RT-PCR
Primers
Forward sequence (5’-3’)
Reverse sequence (5’-3’)
Size (bp)
p16
Runx3
DAPK
CHFR
GAPDH
AGCCTTCGGCTGACTGGCTGG
GAGTTTCACCCTGACCATCACTGTG
AACCCATCATCCATGCCATC
TAA AGGAAGTGGTCCCTC TGTG
CTGCACCACCAACTGCTTAG
CTGCCCATCATCATGACCTGG
GCCCATCACTGGTCTTGAAGGTTGT
TCTCTCCTTCTCGGTTCTTGA
GGTTTGGGCATTTCTACGC
TGAAGTCAGAGGAGACCACC
150
198
200
205
407
Annealing temperature
60
55
51
58
60
°C
°C
°C
°C
°C
PROMOTER METHYLATION OF GENES AND GASTRIC CARCINOMA
p16
Runx3
DAPK
CHFR
H2O N2 T2 N1 T1 UP MP
U M U M U M U M U M U M U M Ma
H2O N2 T2 N1 T1 UP MP
U M U M U M U M U M U M U M Ma
H2O N2
T2 N1
T1
UP MP
200bp
100bp
200bp
100bp
p16, Runx3, DAPK and CHFR were significantly different between gastric cancer tissues and adjacent normal gastric mucosa (P <0.05). All 30 histologically normal gastric biopsy specimens were methylation free
for all 4 genes. The methylation rate of the 4 genes increased from gastric biopsy specimens to adjacent
gastric mucosa to gastric cancer tissue. Concurrent
methylation in 2 or more genes was found in 22.9% of
adjacent normal gastric mucosa and 75.7% of cancer
tissues (Figure 3).
The results of bisulfite DNA sequence analysis
H2O N2 T2 N1 T1 UP MP
U M U M U M U M U M U M U M Ma
200bp
100bp
200bp
100bp
Figure 1 - Methylation-specific PCR analysis of p16, Runx3, DAPK and
CHFR methylation status. H2O: negative control; T1, T2: tumor tissues; N1, N2: adjacent normal tissues; Ma: 100-bp DNA ladder; MP:
methylated positive control; UP: unmethylated positive control; M:
methylated; U: unmethylated.
fragment size in methylation-positive control and unmethylation-positive control samples, respectively,
while the blank control sample did not amplify any
fragment. This suggested that the experimental technique and the use of primers and reagents was correct, and the experimental result credible. As shown in
Figure 2, the frequency of promoter methylation in tumor tissues was 68.6% (48 of 70) in p16, 60.0% (42 of
70) in Runx3, 60.0% (42 of 70) in DAPK, and 48.6% (34
of 70) in CHFR. In adjacent normal gastric mucosa,
hypermethylation was found with the following frequency: 12.9% (9 of 70) in p16, 27.1% (19 of 70) in
Runx3, 14.3% (10 of 70) in DAPK, and 22.9% (16 of 70)
in CHFR. The promoter methylation frequencies for
50
45
40
35
30
25
20
15
10
5
0
729
Gastric cancer
Adjacent gastric
mucosa
Normal contral
*P <0.05
We carried out direct sequence analysis in all the positive samples for 4 genes as determined by MSP, and discovered that all positive samples showed different degrees of methylation. Representative results of bisulfite
sequence analysis for gene promoters in gastric cancer
samples are shown in Figure 4.
Association between promoter methylation and
clinicopathological parameters
The relationships between the methylation status of
p16, Runx3, DAPK and CHFR promoters and established clinicopathological parameters in gastric cancer
are summarized in Table 3. No correlation was found
between hypermethylation and other clinicopathological parameters such as age, sex, and tumor location (P
>0.05). However, we observed a significant association
between hypermethylation of Runx3 and tumor size (P
<0.05). A statistically significant correlation was also
observed between the methylation frequency of DAPK
and CHFR and differentiation grade and lymph node
metastasis in gastric cancer. Poorly differentiated cancers exhibited higher promoter methylation frequency
for DAPK and CHFR than well differentiated cancers,
while cancers metastatic to lymph nodes showed 70.2%
(33/47) and 57.4% (27/47) promoter methylation for
DAPK and CHFR, respectively, which was significantly
higher than the 39.1% (9/23) and 30.4% (7/23) found in
cancers without lymph node involvement (both P
<0.05). In addition, a significant association was ob-
g0
40
35
30
g2
20
p 16
Runx 3
DAPX
CHFR
Figure 2 - Methylation frequency of gastric cancer, adjacent normal
gastric mucosa, and normal gastric biopsy samples. Methylation of
p16, Runx3, DAPK and CHFR was more frequently detected in gastric
cancer than in adjacent gastric mucosa and normal gastric samples (P
<0.05).
15
10
0 gene
1 gene
2 gene
3 gene
4 gene
g3
25
g4
g1
g2
g0
g1
g3
g4
5
0
Gastric cancer
Adjacent gastric mucosa
Figure 3 - Different levels and numbers of gene methylation in gastric cancer and adjacent normal gastric mucosa. g, gene.
730
SL HU, XY KONG, ZD CHENG ET AL
T
C C
20
G A C C C G
30
GT T A C G A T T C G
served between hypermethylation of DAPK and clinical
stage (P <0.05). We failed to find a significant correlation between hypermethylation of p16 and clinicopathological parameters including sex, age, clinical
stage, differentiation grade, and lymph node involvement (P >0.05).
Expression of p16, Runx3, DAPK and CHFR mRNA in
gastric cancer
120
T C G G C G T
p16
In the 34 gastric cancer tissues examined in this
study, the expression of mRNA was 26.5% in p16,
20.6% in Runx3, 29.4% in DAPK, and 32.4% in CHFR,
which was lower than in adjacent normal gastric mucosa with 64.7% in p16, 67.6% in Runx3, 76.5% in
DAPK, and 58.8% in CHFR. The levels of p16, Runx3
and DAPK mRNA expression were significantly different between cancer and adjacent normal gastric mucosa (P <0.05) (Figure 5). Moreover, as shown in Table
4, among the 34 gastric carcinoma samples available
for RT-PCR analysis, a significant association was observed between the mRNA expression of the 4 genes
and their hypermethylation (P <0.05). In the samples
with hypermethylation of the 4 genes, we found only 1
sample with DAPK expression, while the remaining 3
genes showed loss of expression. Representative results of RT-PCR in human gastric cancer samples are
shown in Figure 6.
Runx3
90
G C G G TC G G CG T T A
DAPK
CHFR
Figure 4 - Representative results of bisulfite DNA sequence analysis
of p16, Runx3, DAPK and CHFR genes. Sodium bisulfite treatment of
DNA converted all unmethylated cytosines to uracils, but the level of
methylated cytosines was unaffected.
Table 3 - Relationship between promoter hypermethylation and patients’ clinicopathological parameters
Parameters
No.
p16 gene
Runx3 gene
DAPK gene
CHFR gene
M (%)
P value
M (%)
P value
M (%)
P value
M (%)
P value
0.408
Gender
Male
Female
53
17
37 (69.8)
11 (64.7)
0.767
32 (60.4)
10 (58.8)
1.000
33 (62.3)
9 (52.9)
0.574
24 (45.3)
10 (58.8)
Age (years)
≥60
<60
43
27
30 (69.8)
18 (66.7)
0.797
26 (60.5)
16 (59.3)
1.000
24 (55.8)
18 (66.7)
0.455
19 (44.2)
15 (55.6)
0.462
Clinical stage
I/II
III/IV
31
39
18 (58.1)
30 (76.9)
0.122
16 (51.6)
26 (66.7)
0.228
14 (45.2)
28 (71.8)
0.029
11 (35.5)
23 (59.0)
0.059
Differentiation
High/
intermediate
Low
25
45
16 (64.0)
32 (71.1)
0.597
15 (60.0)
27 (60.0)
1.000
10 (40.0)
32 (71.1)
0.021
8 (32.0)
26 (57.8)
0.048
Tumor size
≥5 cm
<5 cm
43
27
31 (72.1)
17 (63.0)
0.441
30 (69.8)
12 (44.4)
0.046
29 (67.4)
13 (48.1)
0.136
24 (55.8)
10 (37.0)
0.147
Tumor location
Cardia
Gastric body
Antrum
29
20
21
22 (75.9)
14 (70.0)
12 (57.1)
0.359
20 (69.0)
10 (50.0)
12 (57.1)
0.239
17 (58.6)
14 (70.0)
11 (52.4)
0.755
10 (34.5)
11 (55.0)
13 (61.9)
0.152
Lymph node metastasis
N0
N1/N2/N3
23
47
14 (60.9)
34 (72.3)
0.413
14 (60.9)
28 (59.6)
1.000
9 (39.1)
33 (70.2)
0.019
7 (30.4)
27 (57.4)
0.043
PROMOTER METHYLATION OF GENES AND GASTRIC CARCINOMA
Gastric cancer
Adjacent gastric
mucosa
30
25
*P <0.05
20
15
10
5
0
p 16
Runx 3
DAPX
CHFR
Figure 5 - mRNA expression of 4 genes in gastric cancer and adjacent
normal gastric mucosa. mRNA expression of p16, Runx3 and DAPK
was more frequently detected in gastric cancer than adjacent normal gastric mucosa (P<0.05).
Table 4 - Relationship between promoter hypermethylation
and mRNA expression of 4 genes in gastric cancer
mRNA expression
p16
mRNA expression
P value Runx3
positive negative
M
U
0
9
P value
positive negative
25
0
0.000
M
U
0
7
mRNA expression
DAPK
27
0
0.000
mRNA expression
P value CHFR
positive negative
M
U
1
9
p16
GAPDH
DAPK
GAPDH
22
2
1
1
2
2
0.000
3
3
P value
positive negative
4
4
M
U
Runx3
GAPDH
CHFR
GAPDH
0
11
20
3
1
2
1
0.000
3
2
4
3
4
Figure 6 - Representative results of RT-PCR in human gastric cancer
tissue samples.
GAPDH was used as an internal control. 1, 2: tumor tissues. 3, 4: adjacent normal gastric mucosa.
Discussion
Altered gene silencing due to DNA hypermethylation
is common in human malignancies. This affects multiple genes in cancer cells and the affected genes vary in
chemical and disease-dependent aspects29,30. It is therefore necessary to analyze the methylation status of a
panel of representative genes in each cancer type. For
this purpose CpG island methylator phenotype (CIMP)
was introduced by Toyota et al.31 In gastric cancer, hy-
731
permethylation of CpG islands in promoter regions often occurs in important tumor suppressor genes such as
hMLH1, p16, CDH1 and APC32-34. In particular, aberrant
methylation of the normally unmethylated CpG islands
of many tumor suppressor genes is associated with transcriptional inactivation and loss of expression. In this
study we examined the methylation status and mRNA
expression of the p16, Runx3, DAPK and CHFR genes in
primary gastric cancer both using MSP and RT-PCR.
In previous studies, the frequencies of the promoter
methylation of p16, Runx3, DAPK and CHFR in gastric
cancer were 32-43%35-38, 50-64%39-41, 30-90%42-47 and 3052%48-50, respectively. Our results were similar, and the
methylation rate of the 4 genes increased from gastric
biopsy specimens to adjacent gastric mucosa to gastric
cancer tissue. Moreover, the hypermethylation rates for
the p16, Runx3, DAPK and CHFR promoters were significantly higher in gastric cancer tissues than in adjacent
normal gastric mucosa and control gastric specimens (P
<0.05). The overall findings suggest that promoter hypermethylation of the p16, Runx3, DAPK and CHFR
genes is frequent in gastric cancer and may be an early
molecular event in gastric carcinogenesis. Concurrent
methylation of multiple genes appears to be a tumorspecific phenomenon.
We found that hypermethylation of Runx3 was significantly associated with tumor size (P <0.05), and that
promoter hypermethylation of DAPK and CHFR was associated with differentiation grade and lymph node
metastasis in gastric cancer (P <0.05) but not significantly associated with other clinicopathological factors
such as sex, age, and tumor location. In contrast to previous reports45,49-51, our data indicated that the hypermethylation rate of DAPK and CHFR was significantly
higher in poorly differentiated than well-differentiated
gastric cancer (PDAPK = 0.021, PCHFR = 0.048). This suggests that hypermethylation of the DAPK and CHFR
genes may play a role in the histological differentiation
of gastric cancer and is associated with tumor behavior.
The hypermethylation rate of DAPK and CHFR was significantly higher in gastric cancer metastatic to lymph
nodes than gastric cancer without lymph node involvement (PDAPK = 0.019, PCHFR = 0.043). In addition, we observed a significant association between hypermethylation of DAPK and clinical stage (P <0.05). Our results
suggest that hypermethylation of DAPK and CHFR promoters occurs more often in poorly differentiated tumors and tumors with lymph node metastasis, as the
hypermethylation rate of DAPK and CHFR increased
with the degree of malignancy of the tumor cells. Hypermethylation of DAPK and CHFR may therefore be an
important indicator of molecular biology for evaluating
the degree of malignancy and lymph node status of gastric cancer.
We determined the expression of p16, Runx3, DAPK
and CHFR mRNA by RT-PCR in tissues of 34 patients included in this study, and investigated the correlation be-
732
tween hypermethylation of the 4 genes and mRNA expression. We found a higher level of p16, Runx3, DAPK
and CHFR mRNA expression in adjacent normal gastric
mucosa than in cancer tissues. Moreover, the levels of
p16, Runx3 and DAPK mRNA expression were significantly different between cancer tissues and adjacent
normal gastric mucosa. According to Kim et al.40 and
Kato et al.52, Runx3 and DAPK mRNA expression is absent in methylated cell lines but present in unmethylated cell lines, and loss of Runx3 and DAPK mRNA expression was related to the aberrant methylation of
their CpG islands. Our data in the present study are similar. In gastric cancer tissues with hypermethylation of 4
genes, we found only 1 sample presenting DAPK expression, while the other 3 genes showed loss of expression.
Furthermore, a significant association was observed between mRNA expression of the 4 genes and their hypermethylation status (P <0.05). This suggests that promoter hypermethylation of the 4 genes is possibly the main
cause of downregulation or loss of mRNA expression.
Weak expression and loss of expression due to promoter hypermethylation may play an important role in the
pathogenesis of gastric cancer. These results support
our initial hypothesis and further studies are warranted
to determine whether promoter hypermethylation in
precancerous gastric lesions is associated with a higher
risk of subsequent cancer development and how to interrupt the malignant transition by developing genetargeting therapies that may reverse aberrant methylation.
In conclusion, hypermethylation of tumor-associated
genes may be a reaction that damaged cells adopt to
survive, which plays an important role in tumor initiation, promotion and progression. CpG island hypermethylation of p16, Runx3, DAPK and CHFR promoters is
frequent in gastric cancer and may be an early molecular event in its development. Weak expression and loss
of expression due to promoter hypermethylation may
play an important role in the pathogenesis of gastric
cancer. Analysis of DNA methylation could be used in
tumor diagnosis, evaluation of chemosensitivity, and
prognosis. Tumors in which DNA methylation occurred
are easier to correct than tumors with DNA sequence
mutations and genetic damage. How to resume DNA expression by developing gene-targeting therapies that
may reverse aberrant methylation is currently considered a promising new target of gastric cancer treatment.
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