Hypermethylation of metallothionein-3 CpG island in gastric carcinoma

Dajun Deng1,3, Wa'el El-Rifai2, Jiafu Ji1, Budong Zhu1, Paul Trampont2, Jiyou Li1, Michael F. Smith2 and Steven M. Powel2,3

1 Peking University Health Science Center and Beijing Institute for Cancer Research, Beijing, 100034, China and
2 Division of Gastroenterology and Hepatology, University of Virginia Health Systems, Charlottesville, VA 22908-0708, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of metallothionein (MT)-3 is often markedly reduced in gastric carcinoma (GC). The molecular mechanism of this MT-3 downregulation is unknown. Transcriptional silencing of MT-3 by methylation of CpG island was investigated by nucleotide sequencing and denaturing high performance liquid chromatography (DHPLC) analyses. We found that normal brain tissue and a xenografted GC that expressed MT-3 mRNA had unmethylated regions of the CpG island in intron1 of this gene. On the other hand, gastric cancer cell lines AGS and MKN445, a xenografted GC, and a representative primary gastric cancer that had no expression of MT-3 mRNA demonstrated hypermethylation of the MT-3 intron1 CpG island. Treatment of the gastric cancer cell lines with 5-azacytidine resulted in new expression of MT-3 mRNA in these cells. A quantifying DHPLC assay was developed to determine the methylation status of this specific region of the MT-3 gene.

Fifty-eight primary GC and their corresponding normal gastric epithelial tissues, and 34 normal gastric mucosa were analyzed for MT-3 methylation by DHPLC in the region of methylation abnormalities initially identified. Our DHPLC analyses of the methylated MT-3 product demonstrated that the primary gastric cancers have an average methylation percentage of 6.3% per tumor compared with 2.4% in normal gastric tissues (P < 0.05). The MT-3 was not methylated in all of eight P53-positive GCs and hypermethylated in eight of 13 P53-negative cases by immunohistochemistry staining (P = 0.007). In conclusion, the CpG island in the MT-3 intron1 are abnormally hypermethylated in many gastric carcinomas and may account for the downregulation of MT-3 in gastric carcinogenesis.

Abbreviations: DHPLC, denaturing high performance liquid chromatography; GC, gastric carcinoma; MT, metallothionein; RMF, relative methylation factor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Metallothionein (MT)-3 was originally discovered as a growth inhibitory factor that is markedly reduced in the brain of patients with Alzheimer's disease. The MT-3 protein distinguishes itself from other MT isomers by exerting an inhibitory effect on cortical neuron growth independent of its metal binding ability (1). MT-3 is expressed in the organs of the reproductive system and kidney (2,3). Recently, MT-3 was shown to be markedly downregulated in gastric carcinoma (GC) (4). It was found that a 25 x CTG repeat in the promoter region seemed to contribute to the repression activity in a human hepatoma cell line HepG2 transfected with various deletion mutants of the MT-3 promoter, although no binding protein was detected by gel-shift and footprint analyses (5). However, it was reported that deletion of the CTG repeat or of the JC virus silencer did not promote MT-3 promoter activity in non-permissive cells, or enhance expression in permissive cells (6). Thus, the exact molecular mechanism of MT-3 expression downregulation is unclear.

Methylation of CpG islands is associated with transcription silencing and gene imprinting (7). Many tumor suppressor genes are downregulated by promoter methylation during the development and progression of cancer (8). It was reported that expression of MT-1, an isoform of MT-3, was silenced by promoter methylation (9). MT-3 contains a CpG island in the promoter and 5' region of this gene. Previously, it was reported that the CpG methylation pattern of the MT-3 promoter in non-permissive DI TNC1 and permissive C6 cells was identical, as assessed by Southern analysis using genomic DNA digested with HpaII and MspI (6). Denaturing high performance liquid chromatography (DHPLC) is a useful tool that is usually used to detect DNA point mutation based on the denaturing temperatures difference between mutant and wild type sequence (10). We developed a DHPLC assay, which differentiates CpG islands with various methylation patterns from each other after bisulfite modification of DNA (11,12). The basis of the assay is a combination of conversion of the unmethylated C, but not the methylated C, to U by bisulfite and detection of the `multi-mutations' in the target sequences by DHPLC. In this study, we investigated regulation of MT-3 expression by methylation of CpGs around the translation start site of this gene employing the DHPLC assay.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines, 5-azacytidine (azaC) treatment, and total RNA extraction
The human GC cell lines AGS and MKN45 were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% of fetal bovine serum at 37°C with 5% CO2. To inhibit DNMT activity, cells were treated by a final concentration of 2.5 µM of azaC (Sigma Chemical Co., USA) for 96 h. The total RNA was extracted with TRIzol® Reagents (Invitrogen, USA) for RT–PCR.

Gastric and brain tissue samples
Fifty-eight primary GC and their corresponding normal gastric epithelial samples were collected for analysis: 33 from the Beijing Cancer Hospital surgically (27 males and six females, 29–59 years old, the average age 47.8 years), and 25 from the University of Virginia Health System (17 males and eight females, 41–84 years old, the average age 64.0 years). Five xenografted human GC were obtained from nude mice as described (4). In addition, 34 biopsy samples of gastric epithelial tissues were collected from non-malignant patients at Beijing Institute for Cancer Research (33 males and one female, 18–47 years old, the average age 29.4 years). One normal human brain sample was obtained from the University of Virginia surgically. Immunohistochemical staining for mutant tumor suppressor p53 expression was available for 21 of 33 GC samples from Beijing Cancer Hospital (13). All clinical samples and histopathological information for each case were obtained according to approved institutional guidelines.

DNA extraction and bisulfite modification
Genomic DNA of the cultured cells at log phase and tissue samples was isolated with QIAGen DNA Purification Kits. Two µg of genomic DNA was treated with sodium bisulfite as described (14) for subsequent Wizard® DNA Clean-Up System Kit (Promega, USA) preparation prior to PCR amplification. During this modification, the unmethylated C is converted to U (T in PCR products) and the methylated C is not.

Design of primers and PCR conditions
Primers were designed according to the CpG island of the sense strand of MT-3 (GenBank accession number AC009155.3, GI: 7328722). The strand-specific primers for the bisulfite-treated CpG island in the promoter and exon1 (region-A) were MT-3AmF (5'-ggaagagaggtagggaagag-3') and MT-3AmR (5'-acaaatctcaaaatccatatc-3'); in the exon1, intron1, exon2, and intron2 (region-B), MT-3BmF (5'-ggattttgagatttgtttttgttttt-3') and MT-3BmR (5'-acccgaataattctattccaacaa-3') (Figure 1Go). These primers for the modified templates amplify both the methylated and the unmethylated sequences due to the absence of CpG in the primer sequences. There are 57 CpG sites in the strand-specific PCR (ssPCR) products of the region-A, 31 CpG sites in the region-B. Hot-start touchdown PCR (–1.0°C per cycle, total 40 cycles) was used to amplify the MT-3 sense strand templates with bisulfite-treatment (annealing temperature: 65°C->55°C for the region-A, 60°C->48°C for the region-B, –1°C per cycle). Primers for the unmodified MT-3 region-B were MT-3BwF: 5'-gacatggaccctgagacctg-3' and MT-3BwR: 5'-aggggcatctgtattttaatgtct-3' (Figure 1Go). Regular PCR (wPCR) reactions using Platinum-Taq DNA polymerase (Gibco, USA) was used to amplify the untreated template with annealing temperature 57°C for 36 cycles. The other conditions in the touchdown PCR are the same as described (12).



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Fig. 1. Schematic diagram of the 5' CpG island of human MT-3. ssPCR: strand-specific PCR for the bisulfite-modified template; wPCR: regular PCR for the wild template without modification; MSP-M and MSP-U: methylation-specific PCR for the methylated and unmethylated modified templates; -> and <- arrows: forward and reverse primers.

 
RT–PCR for MT-3 mRNA
cDNA was synthesized with the Advantage RT for PCR Kit (Clontech, USA). Regular PCR (36 cycles) was used to amplify MT-3 mRNA at the annealing temperature 57°C. The forward primer is 5'-cgtccagttgcttggaga-3' in exon1; the reverse one, 5'-agctgcacttctctgcttct-3' in exon3. GAPDH was used as the internal control. RT–PCR products (233 bp) were run on 2% agarose gel.

Sequencing of the CpG island of the modified MT-3 region-A and region-B
The ssPCR products amplified from the bisulfite treated templates with above primers were purified with QIAquick PCR Purification Kit (Qiagen, USA). The eluted DNA was mixed with the forward or reverse primer and sequenced on a Perkin-Elmer ABI PRISM 373 DNA Analyzer.

Detection of CpG methylation by DHPLC
ssPCR products of the bisulfite-modified MT-3 region-B were analyzed directly by DHPLC as described (11). Briefly, during bisulfite modification, the unmethylated C was converted to U (T in PCR product), but the methylated C was not. Therefore, the difference of methylation extent between the methylated CpG island and the unmethylated one was transformed to multi-mutations. A universal primer set was used to amplify both the methylated and unmethylated CpG islands then. The G + C content of the amplicons of the methylated CpG island was higher than that of the unmethylated one. Thus, the denaturing temperature of the methylated one is higher than that of the unmethylated one. The retention time of the methylated ssPCR products is longer than that of the unmethylated ones when they were separated by DHPLC at partial denaturing temperature. The average partial denaturing temperature (55°C) of the methylated (56°C) and unmethylated (53°C) amplicons was used to separate the target fragments with various methylation patterns. At 55°C, most unmethylated amplicons were denatured to single-strands, but most methylated ones were not denatured and remained as double-strands. wPCR products of unmodified template were detected with DHPLC at the partial denaturing temperature 65°C after hybridization to exclude point mutation in the same sequence (11).

Methylation specific PCR (MSP) (14)
MSP was used to confirm the methylation status of CpG sites in the MT-3 intron1, which were methylated in samples not expressing MT-3 and not methylated in cells expressing MT-3 in above analyses. The forward primer and reverse one for the methylated template were 5'-gtttttttggtgagttttcgttttcgttc-3' and 5'-aataccaaatctccctattctccgcg-3', respectively. For the unmethylated one, forward, 5'-ttgtttttttggtgagtttttgtttttgttt-3'; reverse, 5'-aaaataccaaatctccctattctccaca-3' (Figure 1Go). Hot-start touchdown PCR (annealing temperature: 71°C->61°C, –1.0°C per cycle; total 40 cycles) was used to amplify the methylated and unmethylated templates. The other MSP conditions were the same as described above. 3.5% of agarose gel was used to run the MSP products (the methylated one, 184 bp; the unmethylated one, 188 bp).

Immunohistochemical staining for P53
P53 immunostaining was carried out as described (13). Briefly, the formalin-fixed paraffin-embedded tissues block was cut at 4 µm for use in the staining. The primary anti-P53 antibody (1:50, D0-1, Santa C, USA) was localized using the strepto–avidin–biotin-peroxidase complex procedure (Zymed, CA, USA) and using diaminobenzidine for visualization. The sample was classified as P53-positive when >10% of target cells were stained positively in their nucleic regions. Tissue section of a P53-positive breast carcinoma was used as positive control, and those with the primary antibody omitted as negative control.

Data analysis
The DHPLC hardware control software HSM-D7000 reported the area of each DHPLC peak automatically. The detection value is considered as informative when the sample's total amount of the areas from retention time (tR) 2.5 min to tR 5.3 min is >300 (µVs). The percentage of the peak contributing to the methylated fragment from tR 4.9 min to tR 5.3 min in the total area was calculated for each sample. Relative methylation factor (RMF) of MT-3 in the GC samples was calculated to measure methylation relative to their corresponding normal tissues:

When there is a methylated peak on the chromatogram of GC but not the normal sample, the RMF is considered as >1.5. If the RMF for a GC is >1.5, the sample was classified as hypermethylated. Correlations between RMF and various histological phenotypes were analyzed with software Epi Info (6.04b version) and Microsoft Excel 2000 statistically.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression and hypermethylation of MT-3
Expression of MT-3.
MT-3 expression levels were analyzed with RT–PCR. Expression of MT-3 was observed in human normal brain and a xenografted human GC (X14). MT-3 mRNA was not detectable in gastric carcinoma cell lines AGS, MKN45 or the xenografted human GC (X20). Treatment of the gastric cancer cell lines with 2.5 µM of azaC for 96 h resulted in clear new expression of MT-3 in these cells (Figure 2Go).



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Fig. 2. RT–PCR products of MT-3 mRNA in brain tissue, xenografted human GCs (X14 and X20) and GC cell lines AGS and MKN45. GAPDH mRNA was amplified as the internal control. RT–PCR products (233 bp) were run on 2% of agarose gel.

 
Mapping of methylation patterns.
The methylation status of 57 CpG sites in the region-A (promoter and exon1), 31 CpG sites in the region-B (intron1, exon2, and intron2) were determined by DNA sequencing in several samples to map the methylated and unmethylated CpG sites. Results showed that the methylation pattern in the region-A is the same for the MT-3 expressing brain tissue and X14 GC samples and the MT-3 downregulated AGS and MKN45 cell lines and X20 GC samples with all of 57 CpG sites methylated (data not shown). However, obvious differences in methylation patterns in the MT-3 intron1 were observed between samples with and without MT-3 expression: more than half of CpGs in the intron1 were not methylated in X14 and brain tissues that expressed MT-3 mRNA; whereas most of these CpG sites in intron1 were methylated in the AGS and MKN45 cell line and X20 that did not express MT-3 mRNA (Figure 3Go). But the methylation pattern in the exon2 and intron2 was the same between samples with and without MT-3 expression. Therefore, the ssPCR products of the MT-3 region-B were used in the development of the DHPLC analysis below to quantify the ratio of the methylated and unmethylated target sequences in gastric samples. In addition, the sequence of the MT-3 intron1 was used to design MSP primers below.



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Fig. 3. Genomic sequencing data of the CpG island at the MT-3 region-B in tissue samples and GC cell line. Methylation status of 31 CpG sites of samples with the methylated peak (X20, AGS, and MKN45) and without the methylated peak (G154, N251, X14, and brain) were compared. Each row of circles represents one sample sequenced from ssPCR products generated from amplification of bisulfite-treated DNA. {circ}, Unmethylated cytosines; {odot}, hemimethylated cytosines; •, methylated cytosines.

 
Determination of methylation status by DHPLC
ssPCR products of the bisulfite-modified CpG island in region-B were obtained by the hot-start touchdown PCR, and separated directly by DHPLC at 55°C. In the X14 and brain tissue, which contain unmethylated CpGs in the MT-3 intron1 and express MT-3 mRNA, a major peak was observed at tR 4.0 min (the unmethylated peak, Figure 4AGo). On the other hand, the AGS and MKN45 cell lines and X20, which contain methylated CpGs in the MT-3 intron1 and do not express MT-3, had a tR of the major peak which was delayed to 5.3 min (the methylated peak, Figure 4AGo). A weak unmethylated peak was also observed in the MKN45 cell line. The methylated and/or unmethylated peaks were observed on chromatograms in human gastric samples.



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Fig. 4. Chromatograms of the PCR products from the methylated and unmethylated CpG island of the MT-3 region-B. (A) Detection of point mutation/methylation in ssPCR products generated from amplification of bisulfite-treated DNA at partial denaturing temperature 55°C. Open arrow, unmethylated peak; closed arrow, methylated peak; (B) detection of point mutation in wPCR products of the untreated DNA at partial denaturing temperature 64°C. The same chromatogram for each sample indicated there was no sequence difference between them. Thus, various peaks with different retention times in ssPCR products should result from the CpG island with various methylation patterns.

 
To exclude the possibility that peaks with different tR resulted from genomic point mutation, point mutation analyses were used in confirmation studies. The similar chromatogram was observed in their PCR product of the untreated genomic DNA at 64°C (Figure 4BGo).

Methylation of the CpG island of MT-3 intron1 in GC and normal gastric mucosa
The methylation pattern of the MT-3 was analyzed in 58 cases of primary GC and their corresponding normal gastric epithelium from patients using the bisulfite-DHPLC analysis that allows quantification of the methylated product. The percentage of peak area of methylated MT-3 among the total area of methylated and unmethylated MT-3 PCR products was calculated for each sample. We found that the percentage of the methylated MT-3 in GC was higher than that in their corresponding normal tissues (P < 0.05, Table IGo). The methylated MT-3 percentage in the gastric mucosa from non-cancerous patients was lower than that in the corresponding normal samples from GC patients (Table IGo).


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Table I. Average percentage of methylated MT-3
 
To study the difference of the MT-3 region-B methylation between GC and their corresponding normal gastric mucosa, RMF was calculated for each GC case when methylation of the MT-3 region-B was detectable in GC or normal tissues. Hypermethylation (RMF > 1.5) of MT-3 was observed in 22 of 52 (42.3%) GC samples.

To confirm the methylation of CpG sites in the MT-3 intron1, a MSP assay was set up. The methylated MSP product (184 bp) was observed only in the MT-3 methylated samples such as AGS and MKN45 cell lines and X20 xenograft (data not shown), but not in the MT-3 unmethylated samples such as brain, X14. Expression of MT-3 mRNA was not detectable by RT–PCR in samples in which the methylated MT-3 was detected by MSP (Figure 5Go). Therefore, the MSP assay was used to confirm the methylation status of MT-3 in gastric carcinomas and normal samples. The methylated MSP product was observed in 69 of 76 samples (91%), in which the methylated MT-3 was detectable by DHPLC. Moreover, the methylated MSP product was also observed in 35 of 55 samples (64%) without methylated MT-3 by DHPLC. The unmethylated MSP product (188 bp) was observed in all of the tested samples (Figure 5Go, MSP-U).



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Fig. 5. RT–PCR products of MT-3 mRNA in GC. G66, G69, G108, and G110: human primary GCs; X14, X26 and X37: xenografted human primary GCs; GAPDH mRNA was amplified as the internal control. MSP-M (184 bp) and MSP-U (188 bp): MSP products for the methylated and unmethylated MT-3. RT–PCR and MSP products were run on 2% and 3.5% of agarose gels, respectively.

 
Immunohistochemical staining for the mutant tumor suppressor P53 gene expression was available for 21 of 33 GC samples from Beijing Cancer Hospital: eight GC were P53 immuno-positive and 13 GC were P53 immuno-negative. The MT-3 methylation pattern in the P53-positive GCs was found to be different compared with the P53-negative GCs. The MT-3 was not methylated in all eight P53-positive GCs and hypermethylated (RMF > 1.5) in eight of 13 P53-negative cases (0/8 vs. 8/13, P = 0.007). No significant correlation was observed between the percentage of the MT-3 methylation (RMF) and other clinicopathologic parameters such as age, race, GC site within stomach, stage, differentiation grade, histology, TNM classification, and lymph node metastasis.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
MT-3 was originally discovered as a growth inhibitory factor; thus the cause of its underexpression in GC is important to elucidate. We report, for the first time, that the expression of MT-3 is correlated negatively with the hypermethylation of CpG island in the intron1 of this gene. We have compared the methylation pattern of the CpG island at the MT-3 promoter, exon1, intron1, exon2, and intron2 in the MT-3 expressing and non-expressing samples by bisulfite-DHPLC and DNA sequencing. DNA sequencing analysis demonstrated a specific region of the MT-3 intron1 that was not methylated in samples that expressed MT-3. Almost all of 31 CpG sites in the region-B were methylated in X20 and the gastric cancer cell lines AGS and MKN45 which did not express MT-3; whereas most of the 5' 17 CpGs were not methylated in X14 and brain tissue samples which expressed MT-3 (Figures 2 and 3GoGo). Moreover, DHPLC analyses showed that the tR of the chromatographic peak of the ssPCR product from the MT-3 non-expressing X20 and gastric cancer cell lines was longer than that of the MT-3 expressing samples such as X14 and brain tissue (4.0 min vs. 5.3 min, Figure 4AGo). That the same chromatograms were obtained from the wPCR products of the untreated genomic DNA indicated that the sequence of the target fragment of MT-3 is the same in these samples (Figure 4BGo). These results suggest that the CpG island of MT-3 in X20 and gastric cancer cell lines is methylated; therefore preventing the conversion of C to U by the bisulfite treatment, leading to higher C + G content in the PCR products, higher denaturing temperature, and thus delayed tR. That the demethylating agent azaC induces expression of MT-3 in the non-expressing gastric cancer cell lines also supports our findings that CpG methylation accounts for the MT-3 transcriptional silencing (Figure 2Go).

Contradictory results were reported about the role of a 25 x CTG repeat in the MT-3 promoter region in the regulation of MT-3 expression in human cells (5,6). CpG island of MT-3 covers the promoter and 5' region. We observed that all the 57 detected CpG sites in the promoter and exon1 were fully methylated in human cells whether they expressed MT-3 or not by sequencing, COBRA, and DHPLC analysis (data not shown). This is similar to the report that the CpG methylation pattern of the MT-3 promoter in non-permissive and permissive cells was identical (6). The mechanism of the promoter to regulate MT-3 expression and its relation with the intron1 CpG methylation is still unknown.

It is well recognized that the extent of hypermethylation of CpG islands in promoter and 5' region is correlated with silencing of certain genes (15). However, there was a report that only the methylation of specific key CpG sites is really correlated with repression of gene transcription (16). The present study shows also that only methylation of 17 CpG dinucleotides near the transcription start site in the intron1 is correlated with the MT-3 expression.

The methylation pattern of the CpG island of MT-3 in gastric samples from patients with and without GC was analyzed by bisulfite-DHPLC. A higher percentage of the methylated MT-3 in GC was observed compared with that in the corresponding normal gastric mucosa (Table IGo). This result may explain our previous finding that MT-3 was frequently downregulated in GCs (4). More methylated MT-3 was detected in the normal gastric tissues from GC patients than that from non-malignant patients. This might have resulted from the contamination of cancer cells from the adjacent GC and from the possible age-related methylation because the normal gastric biopsies were taken from younger patients.

DHPLC indicates overall methylation state of the sequence of interest but does not reveal methylation status of individual CpGs. In order to confirm the methylation status of 17 CpG sites in the MT-3 intron1 in gastric tissue samples, an MSP assay was developed to detect the methylation of CpG sites in the key region. Results show that the methylated MSP product was observed in almost all samples, in which the methylated MT-3 product was detectable by DHPLC. This suggests that the DHPLC used in the present study is suitable for the analysis of CpG methylation in the key region of the MT-3 intron1.

MSP is a sensitive non-quantitative method by which CpG methylation is detectable even if 0.1% of target sequence is methylated (14). In other words, result of MSP shows us only whether there is methylated template in the testing sample, but does not give us information on how many methylated templates there are. DHPLC is an assay that separates and quantifies the methylated and unmethylated target sequences. In the present study, the MSP product of the methylated MT-3 was also observed in more than half of samples, in which the methylated MT-3 was not detectable by DHPLC. Therefore, as a quantitative assay, DHPLC shows its advantage on detection of CpG methylation in heterogeneous tissue samples over non-quantitative ones, because the result of DHPLC reflects the exact methylation status of the sequence of interest.

More samples with RMF > 1.5 for MT-3 were observed in P53-immunonegative samples than P53-immunopositive ones. Generally, only mutant P53 could be detected by immunohistochemistry. This suggests that the MT-3 methylation may correlate with the expression of mutant p53 negatively and be wild p53 dependent. The expression of MT-1 is repressed by promoter methylation (9). There are a number of reports that immunohistochemical expression of P53 is correlated with the expression of MT-1 and MT-2 (17,18). The biological meaning of these correlations remains to be determined. No significant correlation was observed between the MT-3 methylation and other clinicopathologic parameters. These results suggest the MT-3 methylation might be an early event during gastric carcinogenesis. The significance of MT-3 methylation in carcinogenesis remains to be addressed.

In conclusion, we have shown that the methylation of CpG island in the intron1 might silence the transcription of MT-3. We have found that MT-3 methylation is a frequent event in gastric carcinogenesis that may explain its underexpression in gastric tumors.


    Notes
 
3 To whom correspondence should be addressed at: Peking University School of Oncology, Beijing 100036, China Email: dengdajun{at}sina.com Back


    Acknowledgments
 
This work was supported by grant 2000-A-29 from Peking University Center for Human Disease Genomics (to D.D.); by grant 0106 from Peking University Cancer Research Center (to D.D.); by grant 3171045 from National Natural Science Foundation of China (to D.D.); by NKTRDP grant 2002 BA711A06 and by NIH grant CA67900 (to S.M.P.). We thank Dr W.Davis Parker (Department of Neurology, UVa Hospital) for use of the DHPLC. We also thank the expert technical assistance of Jin Zhou, Liankun Gu, Ruming Wang, Guiguo Li, and Jeffrey C.Harper. The abstract was presented at the 7th International Human Genome Meeting (HGM2002), 14–17 April 2002, Shanghai, China.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 24, 2002; revised September 5, 2002; accepted September 13, 2002.





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