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Carcinogenesis Advance Access originally published online on September 24, 2007
Carcinogenesis 2007 28(12):2434-2442; doi:10.1093/carcin/bgm206
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Alterations of DNA methylation associated with abnormalities of DNA methyltransferases in human cancers during transition from a precancerous to a malignant state

Yae Kanai* and Setsuo Hirohashi

Pathology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan

* To whom correspondence should be addressed. Tel: +81 3 3542 2511; Fax: +81 3 3248 2463; Email: ykanai{at}ncc.go.jp


   Abstract
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 Abstract
Introduction
 Perspectives
 Funding
 References
 
Alterations of DNA methylation are one of the most consistent epigenetic changes in human cancers. Human cancers generally show global DNA hypomethylation accompanied by region-specific hypermethylation. Alterations of DNA methylation may result in chromosomal instability as a result of changes in chromatin structure. DNA hypermethylation of CpG islands silences various tumor-related genes. Alterations of DNA methylation are frequently observed in cancers associated with chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, such as hepatitis B or C viruses, Epstein–Barr virus, human papillomavirus and Helicobacter pylori, or with cigarette smoking. Accumulating evidence suggests that alterations of DNA methylation are involved even in the early and precancerous stages. On the other hand, in patients with cancers, aberrant DNA methylation is significantly associated with poorer tumor differentiation, tumor aggressiveness and poor prognosis. Precancerous conditions showing alterations of DNA methylation may progress rapidly and generate more malignant cancers. DNA methyltransferase (DNMT) 1 over-expression is not a secondary result of increased cell proliferative activity but is significantly correlated with the CpG island methylator phenotype, which is defined as frequent DNA hypermethylation of C-type CpG islands that are usually methylated in a cancer-specific (not age-dependent) manner. Splicing alteration of DNMT3b may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alteration of DNA methylation may become an indicator for carcinogenetic risk estimation and early diagnosis of cancers and a biological predictor of poor prognosis in patients with cancers. Correction of DNA methylation status may offer a new strategy for prevention and therapy of cancers.

Abbreviations: CIMP, CpG island methylator phenotype; DNMT, DNA methyltransferase; HCC, hepatocellular carcinoma; LOH, loss of heterozygosity; MBD, methyl-CpG-binding protein; mRNA, messenger RNA; PanIN, pancreatic intra-epithelial neoplasia; PCR, polymerase chain reaction; PCNA, proliferating cell nuclear antigen; RCC, renal cell carcinoma; STAT, signal transducer and activator of transcription; TCC, transitional cell carcinoma


   Introduction
Top
 Abstract
 Introduction
Perspectives
 Funding
 References
 
In the earlier days of cancer research, stepwise and orderly progression of genetic alterations causing activation of oncogenes and inactivation of tumor suppressor genes was considered to be the molecular framework responsible for multistage carcinogenesis in humans. However, genetic events alone may not explain the entire process of carcinogenesis: only a few genetic alterations are known to be responsible, especially in the earlier, precancerous stages. Moreover, microscopic observation of cancers frequently reveals histological heterogeneity (e.g. well, moderately or poorly differentiated carcinoma components are simultaneously observed even in tissue sections from any single patient), reflecting complexity of the biological characteristics of tumors. In addition to genetic events, epigenetic events such as alterations of DNA methylation, which can be reversible and underlie the histological heterogeneity of cancers, are another leading player in multistage carcinogenesis.

DNA methylation, a covalent chemical modification resulting in addition of a methyl (CH3) group at the carbon 5 position of the cytosine ring in CpG dinucleotides, plays important roles in chromatin structure modulation, transcriptional regulation and genomic stability, and is essential for the development of mammals (1). The C-terminal catalytic domain of DNA methyltransferases (DNMTs), the major and best known of which is DNMT1, transfers methyl groups from S-adenosylmethionine to cytosines (2). DNMT1's preference for hemimethylated over unmethylated substrates in vitro and its targeting of replication foci are believed to allow copying of the methylation pattern of the parental strand to the newly synthesized daughter DNA strand (3). Thus, DNMT1 has been recognized as the ‘maintenance’ DNMT. DNMT1 can interact with the DNMT1-associated protein 1, histone deacetylase 1 and 2 and Rb and can repress gene transcription (4–6). Since DNMT1–/– embryonic stem cells are able to methylate viral DNA de novo (7), independently encoded DNMTs have been sought. Among the subsequently identified DNMTs 2 (8), 3a and 3b (9), DNMT activity of DNMT2 has never been demonstrated (10), whereas DNMT3a and DNMT3b do show de novo DNA methylation activity in vitro (11).

In comparison with normal cells, human cancer cells show a drastic change in DNA methylation status, generally exhibiting global DNA hypomethylation as well as accompanying region-specific hypermethylation (12,13). As 5-methylcytosine is deaminated to thymine, DNA hypermethylation facilitates gene mutation in human cancers. DNA methylation normally promotes a highly condensed chromatin structure through recruitment of DNA-organizing proteins, and DNA hypomethylation in cancer cells causes chromatin decondensation and chromosomal rearrangements that may result in chromosomal instability. Moreover, DNA hypermethylation of CpG islands near gene-regulatory regions silences specific genes, in cooperation with histone modification, including tumor suppressor genes (14).

Association of DNA methylation alterations with both the precancerous stage and malignant progression
In general, candidate tumor suppressor genes, which are located in the commonly deleted chromosomal regions revealed by genomic structural analysis in human cancers, are frequently silenced by alternative two-hit mechanisms consisting of loss of heterozygosity (LOH) and DNA hypermethylation, rather than gene mutation. Thus, DNA hypermethylation of some chromosomal loci is frequently associated with LOH at the same chromosomal loci in human cancers. LOH on chromosome 16 has been frequently detected by classical restriction fragment length polymorphism analysis using Southern blotting in hepatocellular carcinomas (HCCs) which are poorly differentiated, large in size and associated with metastasis (15). Therefore, LOH on chromosome 16 seems to be a late event during multistage hepatocarcinogenesis. At the time of these discoveries, only a few molecular events in the earlier stage of hepatocarcinogenesis were known. Therefore, we first examined DNA methylation status on chromosome 16 in surgically resected tissue specimens.

Classical Southern blotting showed that the digestion patterns obtained using HpaII, a DNA methylation-sensitive restriction enzyme, were similar to those obtained using MspI, a DNA methylation non-sensitive restriction enzyme, at the D16S32 (16 pter to p13), TAT (16q22.2) and D16S7 (16q24.3) loci in normal liver tissue obtained from patients with liver metastases from primary colon cancer, indicating that genomic DNA is normally unmethylated in these regions. Surprisingly, DNA hypermethylation at the D16S32, TAT and D16S7 loci, compared with normal liver tissues, was frequently detected even in non-cancerous liver tissues showing chronic hepatitis or liver cirrhosis, which are widely considered to be precancerous conditions, indicating that alterations of DNA methylation are a very early event during multistage hepatocarcinogenesis (16). This was one of the earliest reports of alterations of DNA methylation in the precancerous stage.

Since the molecular weight of HpaII-digested DNA fragments in HCCs was higher than that in precancerous conditions and the intensity of larger sized bands was increased in HCCs in comparison with precancerous conditions, the numbers of methylated CpG dinucleotides and cells showing DNA hypermethylation may increase progressively as precancerous conditions develop into HCCs. The incidence of DNA hypermethylation at any of the D16S32, TAT or D16S7 loci in progressed HCCs was significantly higher than that in early HCCs and significantly correlated with higher histological grade (16). DNA hypermethylation at any of the D16S32, TAT or D16S7 loci was detected more frequently in HCCs showing associated involvement of the portal vein and intrahepatic metastasis than in HCCs without these features (16). The presence of DNA hypermethylation in both precancerous conditions and progressed HCCs suggests that precancerous conditions with aberrant DNA methylation might generate HCCs rapidly and that the HCCs thus generated might already be at a progressed stage when diagnosed.

Silencing of tumor suppressor genes by DNA hypermethylation
The E-cadherin gene is located on 16q22.1 near to the above-mentioned hot spots of both DNA hypermethylation and LOH in HCCs. E-cadherin acts as a Ca2+-dependent cell–cell adhesion molecule in the adherens junctions of epithelial cells (17). Interactions between E-cadherin and cytoskeletal actin proteins through {alpha}- and β-catenins confer stability on the adherens junctions. Cell–cell adhesion determines cell polarity and participates in histogenesis. The mutual adhesiveness of cancer cells is significantly weaker than that of normal cells, and this allows cancer cells to disobey the social order, resulting in destruction of histological architecture, which is a morphological hallmark of malignant tumors (18). In signet-ring cell carcinoma of the stomach (19) and lobular carcinoma of the breast (20), in which cancer cells completely lose their mutual adhesiveness even in the in situ carcinoma stage, the E-cadherin gene is silenced by a two-hit mechanism comprising LOH and gene mutation. A large kindred study of early-onset, diffuse-type stomach cancers in New Zealand revealed a germ line mutation (21), indicating that the E-cadherin gene actually satisfies the criteria for a tumor suppressor gene.

On the other hand, suppression of E-cadherin activity is believed to trigger the release of cancer cells from primary cancer nests, resulting in cancer invasion and metastasis. In fact, non-invasive epithelial cells acquired the ability to invade into collagen gels upon addition of antibodies against E-cadherin (22) or plasmids encoding E-cadherin-specific anti-sense RNA (23). Generally, E-cadherin expression is reduced in poorly differentiated cancers that have lost their cell–cell adhesion and show a strong invasive tendency (18). Significant correlations between reduced E-cadherin expression and poor prognosis have been reported in patients with cancers (18). In order to clarify the mechanism responsible for regulation of E-cadherin expression in cancers, we cloned the promoter region of the human E-cadherin gene and demonstrated that it showed DNA methylation in human cancer cell lines lacking E-cadherin expression (24). We also observed induction of E-cadherin expression after treatment with the DNMT inhibitor 5-azacytidine in such cell lines (24). Thus, following the RB and VHL genes, the E-cadherin gene became the third example of a tumor suppressor gene that is silenced by DNA hypermethylation.

When assessed by Southern blotting analysis, DNA hypermethylation around the promoter region of the E-cadherin gene was detected in 46% of examined non-cancerous liver tissues showing chronic hepatitis or cirrhosis and in 67% of examined HCCs (25). Immunohistochemical examination revealed that hepatocytes in normal liver tissues showed strong E-cadherin immunoreactivity at their cell–cell borders. We found a significant correlation between DNA hypermethylation around the promoter region and reduced E-cadherin expression in primary HCCs (25). This was the first demonstration of a significant correlation between DNA hypermethylation and reduced expression in clinical tissue samples. Heterogeneous E-cadherin expression in non-cancerous liver tissues showing chronic hepatitis or cirrhosis, which is associated with small focal areas of hepatocytes showing only slight E-cadherin immunoreactivity and is not observed in normal liver tissues, might be due, at least partly, to DNA hypermethylation (25). DNA hypermethylation around the promoter region, which increases during progression from precancerous conditions to HCCs, may participate in hepatocarcinogenesis through reduction of E-cadherin expression, resulting in loss of intercellular adhesiveness and destruction of tissue morphology.

DNA hypermethylation of NotI sites at the D17S5 locus has been detected in various human cancers (26). The hypermethylated-in-cancer-1 gene at this locus (17q13.3) was the first tumor suppressor gene to be identified in commonly methylated chromosomal loci in human cancers (27); human cancer cells transfected with the hypermethylated-in-cancer-1 gene grew slowly (27) and mice with germ line disruption of one allele of Hic1 developed different spontaneous malignant tumors (28). DNA methylation at the D17S5 locus was never detected in normal liver tissues but was detected in 44% of examined non-cancerous liver tissues showing chronic hepatitis or cirrhosis and in 90% of examined HCCs (29). In almost all the paired samples showing DNA hypermethylation, the molecular weight of NotI-digested DNA fragments in HCCs was higher than that in precancerous conditions and the intensity of the larger sized bands was higher in HCCs than in precancerous conditions, indicating that the degree of DNA methylation seems to further increase during progression from a precancerous condition to an HCC (29). The level of hypermethylated-in-cancer-1 messenger RNA (mRNA) expression in non-cancerous liver tissues showing chronic hepatitis or cirrhosis was significantly lower than that in normal liver tissues and was further decreased in HCCs (29).

It is now recognized that numerous tumor-related genes, such as p16, hMLH1, BRCA1, MGMT, GSTP1, TIMP-3 and DAPK-1, are silenced by regional DNA hypermethylation around their promoter regions in human cancers (14). Several techniques, such as restriction landmark genomic scanning (30), methylation-sensitive representational difference analysis (31) and methylated CpG islands amplification (32), have been developed for cloning genes that are differentially methylated between cancer cells and normal cells (33), and the list of tumor-related genes silenced by DNA hypermethylation is being expanded (14).

Alterations of DNA methylation precede chromosomal instability during multistage carcinogenesis
The hot spot for DNA hypermethylation in HCCs corresponds to a previously reported hot spot of LOH on chromosome 16. It remains to be examined whether alterations of DNA methylation might predispose the locus to allelic loss or whether common or different causes facilitate both alterations of DNA methylation and LOH at certain loci. However, it is at least clear that DNA hypermethylation precedes LOH at the same chromosomal loci during hepatocarcinogenesis: even classical Southern blotting has detected DNA hypermethylation in bulk non-cancerous liver tissues showing chronic hepatitis or cirrhosis, in which LOH has never been detected using the same method (16).

Recently, microdissection techniques and polymerase chain reaction (PCR) using microsatellite markers have been developed for detecting LOH in small numbers of cells from paraffin-embedded tissues. LOH has been reported even in microdissected specimens from non-cancerous lesions, e.g. hyperplastic or dysplastic lesions accompanying non-small cell lung cancers (34) and proliferative lesions adjacent to breast cancers (35). In order to re-examine whether aberrant DNA methylation precedes chromosomal instability during hepatocarcinogenesis, we obtained 308 microdissected specimens and examined LOH and microsatellite instability by PCR using 39 microsatellite markers and DNA methylation status of eight C-type CpG islands that are known to be methylated in a cancer-specific, but not age-dependent, manner, as shown in Table I, by methylation-specific PCR and combined bisulfite restriction enzyme analysis.


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  		Table I. DNA methylation status on C-type CpG islands in tissue samples of various organs

 
In non-cancerous liver tissues showing chronic hepatitis, LOH for at least one marker was found in 20% of informative microdissected specimens, and LOH in at least one microdissected specimen was found in 45% of informative cases (36). In non-cancerous liver tissues showing cirrhosis, LOH for at least one marker was found in 15% of informative microdissected specimens, and LOH in at least one microdissected specimen was found in 40% of informative cases (36). LOH was never detected in normal liver tissues obtained from patients with liver metastases from primary colon cancer and in non-cancerous liver tissue showing no remarkable histological findings from patients with HCCs. Although no degree of DNA methylation of any of the examined CpG islands was ever detected in normal liver tissues obtained from patients with liver metastases from primary colon cancer, DNA hypermethylation was found on at least one CpG island even in 58% of examined microdissected specimens of non-cancerous liver tissue showing no remarkable histological features obtained from patients with HCCs, in which LOH was never detected (36). Thus, aberrant DNA methylation is an earlier event preceding chromosomal instability during hepatocarcinogenesis, even when examined using microdissection techniques (41). The low incidence of microsatellite instability in Japanese patients (42) was compatible with absence of silencing of the hMLH1 gene by DNA hypermethylation during hepatocarcinogenesis (36).

Etiologic backgrounds of carcinogenesis and regional DNA hypermethylation
Alterations of DNA methylation are frequently associated with carcinogenesis related to chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, such as chronic hepatitis associated with hepatitis B virus or hepatitis C virus infection. As mentioned above, alterations of DNA methylation occur even in non-cancerous liver tissues showing no remarkable histological findings obtained from patients with HCCs, i.e. even before inflammation has become histologically obvious. This phenomenon might be at least partly attributable to hepatitis viral infection. Hepatitis B virus DNA is integrated into the cellular genome, and the integrated viral DNA is known to alter the DNA methylation status in several adjacent cellular genes and DNA segments (43). Epstein–Barr virus infection in stomach cancers is significantly associated with marked accumulation of DNA hypermethylation of C-type CpG islands (37). Induction of latent membrane protein 1 of Epstein–Barr virus has been reported to induce DNMT1 over-expression in cultured cancer cells (44). Helicobacter pylori infection, another etiologic factor that is believed to be involved in stomach carcinogenesis, has also been reported to strongly promote regional DNA hypermethylation (45), although the molecular mechanisms by which H.pylori infection alters DNA methylation are still unclear and warrant further investigation. Cervical intra-epithelial neoplasia is a precursor lesion for squamous cell carcinoma of the uterine cervix closely associated with human papillomavirus infection. DNMT1 protein expression is increased even in low-grade cervical intra-epithelial neoplasias compared with normal squamous epithelium and further increased in higher-grade cervical intra-epithelial neoplasias and squamous cell carcinomas of the uterine cervix (46). Human papillomavirus-16 E7 protein has been reported to associate directly with DNMT1 and stimulate the methyltransferase activity of DNMT1 in vitro (47), and accumulation of DNA hypermethylation on tumor-related genes has also been observed during cervical carcinogenesis (48).

In the same way that HCCs are preceded by chronic hepatitis, ductal carcinomas frequently emerge in pancreases damaged by chronic pancreatitis. Therefore, at least a proportion of peripheral pancreatic duct epithelia with an inflammatory background may be at the precancerous stage. When the DNA methylation status of the p14, p15, p16, p73, APC, hMLH1, MGMT, BRCA1, GSTP1, TIMP-3, CDH1 and DAPK-1 tumor-related genes was examined, the incidence of DNA hypermethylation of at least one of the genes and the average number of methylated genes were significantly higher in microdissected specimens of peripheral pancreatic duct epithelia with an inflammatory background and in another precancerous lesion, pancreatic intra-epithelial neoplasia (PanIN), compared with that in peripheral pancreatic duct epithelia without an inflammatory background, and was further increased in ductal carcinomas (Figure 1; 50). The BRCA1, APC, p16 and TIMP-3 genes are frequently methylated in ductal carcinomas of the pancreas (50). With respect to inflammation-related carcinogenesis, cytokine interleukin-6 treatment has been reported to induce DNMT1 over-expression in cultured cells (51), though the significance of cytokine signaling in alterations of DNA methylation has never been confirmed in chronic pancreatitis in vivo.


Figure 1
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  		Fig. 1. DNMT1 protein expression and DNA methylation status of CpG islands in tumor-related genes during multistage carcinogenesis of the pancreas. Immunohistochemical examination for DNMT1 was performed in peripheral pancreatic duct epithelia without an inflammatory background, peripheral pancreatic duct epithelia with an inflammatory background (arrows), PanINs and ductal carcinomas (49). Infiltrating lymphocytes (asterisk) were used as an internal positive control for DNMT1 immunoreactivity. Tissue samples were microdissected from surgically resected materials, embedded in agarose beads and subjected to methylation-specific PCR to evaluate the DNA methylation status of the p14, p15, p16, p73, APC, hMLH1, MGMT, BRCA1, GSTP1, TIMP-3, CDH1 and DAPK-1 genes (50). The incidence of DNMT1 nuclear immunoreactivity, the incidence of DNA methylation of at least one of the 12 genes and the average number of methylated genes increased progressively during multistage carcinogenesis of the pancreas. The average number of methylated genes in ductal carcinomas was significantly correlated with DNMT1 protein expression level (P = 0.0093).

 
Cigarette smoking is another background factor associated with alterations of DNA methylation during multistage carcinogenesis. DNA hypermethylation at the D17S5 locus was observed in 31% of examined non-cancerous lung tissues, which may contain progenitor cells for cancers, obtained from patients with non-small cell lung cancers and in 33% of corresponding non-small cell lung cancers (52). The incidence of DNA hypermethylation at the D17S5 locus was significantly associated with poorer differentiation of lung adenocarcinomas (52). The incidence of DNA hypermethylation in both non-cancerous lung tissues and non-small cell lung cancers of patients who were current smokers was significantly higher than in patients who had never smoked (52). The incidence of DNA hypermethylation in non-cancerous lung tissues obtained from patients with non-small cell lung cancers was significantly correlated with the extent of pulmonary anthracosis, as an index for the cumulative effects of smoking (Figure 2; K. Eguchi, Y. Kanai, K. Kobayashi and S. Hirohashi, unpublished data). Cigarette smoking may participate in alteration of DNA methylation during the development of non-small cell lung cancers. The molecular mechanisms by which carcinogens related to cigarette smoking affect DNA methylation status are still unclear and warrant further investigation.


Figure 2
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  		Fig. 2. Correlations between DNA hypermethylation at the D17S5 locus and anthracosis in non-cancerous lung tissues from patients with non-small cell lung cancers. The extent of pulmonary anthracosis in each resected lung was graded macroscopically: grade 1, slight accumulation of charcoal particles in the intra-lobular lymphatics forming a fine reticular pattern scattered in the visceral pleura (A); grade 2, the reticular pattern due to charcoal particle accumulation was denser and showed fusion in places (B) and grade 3, dense accumulation of charcoal particles was present throughout most of the visceral pleura (C). The incidence of DNA hypermethylation at the D17S5 locus (IM) analyzed by Southern blotting using a DNA methylation-sensitive restriction enzyme (NotI) in non-cancerous lung tissues showing grade 3 anthracosis (62.5%) was higher than that in those showing grade 2 (44.0%) or 1 (0%) anthracosis (P = 0.0011).

 
The incidence of DNA hypermethylation at multiple C-type CpG islands in non-cancerous tissues and cancers from various organs is summarized in Table I. For example, the methylated in tumor-25 clone is methylated in normal renal tissues obtained from patients without renal cancers as frequently as in non-cancerous renal tissues showing no marked histological findings obtained from patients with renal cancers or in renal cancers (39), although it is never methylated in normal liver tissues. DNA methylation profiles of normal tissues tend to be organ specific. Moreover, hot spots of DNA hypermethylation vary among cancers arising in different organs and may reflect the influence of various carcinogenetic factors. The molecular mechanisms responsible for determination of target genes of the CpG island methylator phenotype (CIMP), defined by frequent DNA hypermethylation of C-type CpG islands (53), should be further clarified.

Alterations of DNA methylation are a hallmark of precancerous conditions even in histologically normal tissues
Alterations of DNA methylation are considered to participate in the precancerous stage in various organs, in association with obvious etiological factors, e.g. chronic inflammation, persistent infection with viruses or other pathogenic microorganisms, as mentioned above. Unlike cancers derived from such organs, precancerous conditions in the kidney have been rarely described: pathologists hardly ever observe histological changes in non-cancerous renal tissues obtained from patients with renal cancers. Surprisingly, even in non-cancerous renal tissues showing no marked histological findings obtained from patients with renal cancers, the average number of methylated CpG islands was significantly higher than that in normal renal tissues obtained from patients without renal cancers, regardless of patient age and smoking history (39). The average number of methylated CpG islands was even higher in renal cancers. From the viewpoint of alterations of DNA methylation, the presence of precancerous conditions can be recognized even in the kidney. In other words, regional DNA hypermethylation participates in the early and precancerous stage of multistage renal carcinogenesis. More surprisingly, the average number of methylated CpG islands in non-cancerous renal tissues showing no marked histological change obtained from patients with conventional renal cell carcinomas (RCCs) was significantly correlated with a higher histological grade of corresponding RCCs developing in individual patients (Figure 3; 39), indicating that precancerous conditions showing accumulation of DNA methylation may generate more malignant RCCs.


Figure 3
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  		Fig. 3. DNA methylation status in non-cancerous renal tissues obtained from patients with RCC (arrowheads). The average number of methylated CpG islands in non-cancerous renal tissues obtained from patients with histological grade 3 RCCs was significantly higher than that in equivalent tissues obtained from patients with histological grade 1 or 2 RCCs (39): accumulation of DNA methylation of CpG islands in non-cancerous renal tissues was significantly correlated with a higher histological grade of the corresponding RCCs, indicating that precancerous conditions showing regional DNA hypermethylation may generate more malignant RCCs.

 
Regional DNA hypermethylation has a prognostic impact on patients with cancers
Accumulation of DNA methylation at CpG islands in conventional RCCs is significantly correlated with higher histological grade, an infiltrating growth pattern and vascular involvement (39), suggesting that regional DNA hypermethylation is continuously involved in multistage renal carcinogenesis from precancerous conditions to malignant progression. The recurrence-free survival rate of patients with RCCs showing accumulated DNA methylation of CpG islands was significantly lower than that of patients with RCCs not showing this feature (39).

The incidence of increased DNMT1 protein expression in HCCs is significantly correlated with poorer tumor differentiation and portal vein involvement (54). Moreover, the recurrence-free and overall survival rates of patients with HCCs showing increased DNMT1 protein expression are significantly lower than those of patients with HCCs that do not (54). Increased DNMT1 protein expression in ductal carcinomas of the pancreas is significantly correlated with the extent of cancer invasion to the anterior pancreatic capsule, retroperitoneal tissue and other surrounding organs and with advanced stage (49), suggesting that DNMT1 over-expression is associated with aggressiveness of pancreatic cancers. Moreover, patients with ductal carcinomas of the pancreas showing increased DNMT1 protein expression have a poorer prognosis (49).

Regional DNA hypermethylation and increased DNMT1 protein expression participate not only in the precancerous stage but also in malignant progression, and have a prognostic impact on patients with cancers. Analysis of DNA methylation status at multiple CpG islands and/or immunohistochemical examination for DNMT1 in biopsy specimens obtained for histological diagnostic purposes and/or surgically resected materials may become a useful tool for prognostication in individual clinical cases.

DNMT1 over-expression is not always a secondary result of increased cell proliferative activity but is significantly correlated with regional DNA hypermethylation
We focused on abnormalities of DNMTs underlying alterations of DNA methylation in human cancers. The expression level of DNMT1 mRNA was first examined by quantitative reverse transcription–PCR analysis, and found to be significantly higher even in non-cancerous liver tissues showing chronic hepatitis or cirrhosis than in normal liver tissues and was even higher in HCCs (55,56). With respect to multistage carcinogenesis of the pancreas, the incidence of nuclear DNMT1 immunoreactivity was significantly elevated in peripheral pancreatic ductal epithelia with an inflammatory background and PanINs than in peripheral pancreatic ductal epithelia without an inflammatory background (Figure 1; 49). The incidence of nuclear DNMT1 immunoreactivity was significantly associated with the degree of PanIN dysplasia. The incidence of nuclear DNMT1 immunoreactivity was significantly higher in invasive ductal carcinomas of the pancreas than in PanINs (Figure 1; 49). The average number of methylated tumor-related genes in microdissected specimens of ductal carcinomas of the pancreas was significantly correlated with the expression level of DNMT1 protein examined immunohistochemically in the precisely microdissected areas (50).

The level of DNMT1 mRNA expression was higher in colorectal and stomach cancers than in the corresponding non-cancerous mucosae (38). The average ratio of DNMT1 expression in tumor tissue relative to the corresponding non-cancerous mucosa was significantly higher in CIMP-positive colorectal and stomach cancers than in CIMP-negative colorectal and stomach cancers, but no such association was observed for the expression of DNMT2, DNMT3a or DNMT3b (38). Immunohistochemical examination showed that increased DNMT1 protein expression was significantly associated with poorer differentiation of stomach cancers, whereas none of the examined non-cancerous stomach epithelia exhibited DNMT1 immunoreactivity (except in the proliferative zones) (37). Significant correlation between DNMT1 over-expression and CIMP in stomach cancers was also confirmed by immunohistochemistry at the protein level (37). The hMLH1, THBS-1 and E-cadherin genes may be targets for over-expressed DNMT1 in stomach cancers (37).

Thus, DNMT1 may be responsible for de novo methylation of CpG islands during multistage carcinogenesis. The maintenance activities of DNMT1 are related to its preference for hemimethylated substrates in vitro. A theoretical explanation for the role of DNMT1 in de novo DNA methylation in human cancers with dysfunction of p21WAF1, which competes with DNMT1 for binding with proliferating cell nuclear antigen (PCNA), has been proposed (57). Moreover, it has recently been suggested that DNMT1 is capable of de novo DNA-methylating activity in vivo as well as having a maintenance function: de novo methylation of CpG islands has actually been observed in human fibroblasts over-expressing DNMT1 (58,59). Therefore, it is feasible that, in cancers, DNMT1 over-expression participates in regional DNA hypermethylation.

Transitional cell carcinomas (TCCs) of the urinary bladder are clinically remarkable because of their multicentricity and tendency to recur: synchronously or metachronously multifocal TCCs often develop in individual patients. A possible mechanism for such multiplicity is the ‘field effect’, whereby carcinogenic agents in the urine cause malignant transformation of multiple urothelial cells. Even non-cancerous urothelia showing no remarkable histological features obtained from patients with urinary bladder cancers can be considered precancerous because they may be exposed to carcinogens in the urine. On the other hand, DNMT1 mRNA is expressed mainly during S phase and because tumor tissues of various organs generally contain a greater proportion of dividing cells than do normal tissues, it has been debatable whether increased DNMT1 expression is due to an increase in the proportion of dividing cells or to an acute increase of DNMT1 expression per individual cancer cell. This uncertainty prompted us to compare DMNT1 immunoreactivity and the PCNA-labeling index during urothelial carcinogenesis. The incidence of nuclear DNMT1 immunoreactivity had already increased independently of cell proliferative activity in non-cancerous urothelia showing no marked histological features obtained from patients with urinary bladder cancers, where the PCNA-labeling index had not yet increased, compared with that in normal urothelia obtained from patients without urinary bladder cancers, indicating that DNMT1 over-expression preceded increased cell proliferative activity during multistage urothelial carcinogenesis (60). A similar discrepancy between DNMT1 immunoreactivity and the PCNA-labeling index was also observed in liver (54), stomach (37) and uterine cervix cancers (46). Excessive amounts of DNMT1 compared with PCNA, which targets DNMT1 to replication foci, may participate in de novo methylation of CpG islands. Further examinations are required to clarify whether or not unknown functional protein complexes recruit DNMT1 to specific DNA sequences during carcinogenesis. The incidence of nuclear DNMT1 immunoreactivity was even higher in dysplastic urothelia and TCCs than in non-cancerous urothelia showing no marked histological features obtained from patients with urinary bladder cancers (60). Among all examined microdissected specimens of non-cancerous urothelia showing no marked histological features obtained from patients with urinary bladder cancers, dysplastic urothelia and TCCs, concurrent DNA hypermethylation of three or more examined C-type CpG islands was significantly correlated with increased DNMT1 protein expression (40).

Splicing alteration of DNMT3b may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions
Satellite regions are abundant in pericentromeric heterochromatin DNA on chromosomes 1, 9 and 16 and are heavily methylated in normal cells. DNA hypomethylation of these regions is known to result in centromeric decondensation and enhanced chromosome recombination. In 18% of examined non-cancerous liver tissues showing chronic hepatitis or cirrhosis and 67% of examined HCCs, satellites 2 and 3 were hypomethylated (56). Frequent chromosome 1q copy gain with a pericentromeric breakpoint has been reported in HCCs showing DNA hypomethylation of satellite 2 (61). In TCCs of the urinary bladder, the ureter or the renal pelvis, DNA hypomethylation of satellites 2 and 3 was significantly correlated with LOH on chromosome 9 (62). Therefore, DNA hypomethylation of pericentromeric satellite regions may induce chromosomal instability during multistage carcinogenesis.

DNMT3b is specifically required for DNA methylation of pericentromeric satellite regions in embryonic stem cells and early mouse embryos (11). Germ line mutations of the DNMT3b gene have been reported in patients with immunodeficiency, centromeric instability and facial anomalies syndrome, a rare recessive autosomal disorder characterized by DNA hypomethylation of pericentromeric satellite regions (63). However, no mutation of any coding exon of the DNMT3b gene was detected in examined HCCs (64). When examined by quantitative reverse transcription–PCR analysis using a primer set not discriminating splice variants of DNMT3b, the total level of DNMT3b mRNA was higher in HCCs than in the corresponding non-cancerous liver tissues (64). Thus, it is unlikely that reduced expression of DNMT3b simply causes DNA hypomethylation of these regions during hepatocarcinogenesis.

There are four splice variants in the C-terminal catalytic domain of DNMT3b. DNMT3b3 possesses the N-terminal region and conserved methyltransferase motifs I, IV, VI, IX and X. DNMT activity of human DNMT3b3 has been confirmed in vitro (65), and DNMT3b3 is expressed ubiquitously in normal human tissues. Our data obtained by splice variant-specific quantitative reverse transcription–PCR has also indicated that the major variant in normal liver tissues is DNMT3b3 (64). On the other hand, DNMT3b4 probably does not show DNMT activity because it lacks the conserved methyltransferase motifs IX and X, although it retains the N-terminal domain required for targeting to heterochromatin sites through binding to RP58. We have confirmed that normal liver tissues show only a trace level of DNMT3b4 expression (64). The level of DNMT3b4 mRNA in non-cancerous liver tissues obtained from patients with HCCs and in HCCs was significantly correlated with the degree of DNA hypomethylation of pericentromeric satellite regions (64). In addition, the ratio of DNMT3b4 mRNA to DNMT3b3 mRNA in non-cancerous liver tissues obtained from patients with HCCs and in HCCs was also significantly correlated with the degree of DNA hypomethylation of pericentromeric satellite regions (64). DNMT3b4 lacking DNMT activity may compete with the major variant, DNMT3b3, for targeting to pericentromeric satellite regions. This may be the reason why DNMT3b4 over-expression results in DNA hypomethylation of pericentromeric satellite regions in precancerous conditions and HCCs. To confirm this possibility, we introduced DNMT3b4 into human epithelial 293 cells, which express a significant level of DNMT3b3 mRNA and a trace level of endogenous DNMT3b4 mRNA. DNA demethylation on satellite 2 was observed in DNMT3b4 transfectants, depending on the expression level of myc-tagged DNMT3b4 (64). DNMT3b4 over-expression may lead to chromosomal instability through induction of DNA hypomethylation of pericentromeric satellite regions during hepatocarcinogenesis.

The growth rate of DNMT3b4 transfectants was approximately double that of mock transfectants soon after the introduction of DNMT3b4, when chromosomal instability may not yet have accumulated (66). We assumed that this change was caused by altered gene expression. A majority of the genes that were up-regulated in DNMT3b4 transfectants but not in mock transfectants were implicated in interferon signaling (66). Although genes that encoded interferons themselves were not up-regulated, signal transducer and activator of transcription (STAT) 1, which acts as an effector of interferon signaling, has been listed as one of the up-regulated genes in DNMT3b4 transfectants (66). It had been reported previously that inhibition of DNA methylation in cultured human cancer cells by 5-aza-2'-deoxycytidine induces a set of genes implicated in interferon signaling primarily via over-expression of STAT1, 2 and 3 (67). DNMT3b may act to maintain the DNA methylation status of not only pericentromeric satellite regions but also specific genes, probably in cooperation with DNMT1, in cancer cells, and this may explain why inhibition of DNMT3b activity by induction of DNMT3b4 produced a similar result to the general inhibition of DNA methylation obtained with 5-aza-2'-deoxycytidine. There is a significant correlation between the mRNA expression levels of DNMT3b4 and STAT1 in HCCs (66). Over-expression of DNMT3b4 is involved in multistage carcinogenesis not only by inducing chromosomal instability but also by affecting the expression of specific genes.

Significance of DNA hypomethylation in human cancers
In addition to the above-mentioned satellites 2 and 3, DNA hypomethylation of other tandem repeats such as NBL2 and D4Z4 and retrotransposons such as LINE-1 and Alu was frequently observed in various human cancers (68). Hypomethylation of repeated DNA sequences can disrupt the functions of neighboring genes through transcriptional interference by either sense or anti-sense transcripts (68). Activation of transposable elements can potentially lead to insertional mutagenesis (68). Although global DNA hypomethylation, i.e. reduction of the total level of 5-methylcytosine examined by high-performance liquid chromatography of DNA digested to mononucleotides, is considered to mainly reflect the above-mentioned hypomethylation of repeated DNA sequences, individual genes are also hypomethylated in human cancers. In earlier days, DNA hypomethylation of oncogenes such as c-myc in human cancers was frequently reported (69). However, such DNA hypomethylation affects the body or 3' end of genes whose DNA methylation status is usually not related to gene expression. Recently, significant correlation between over-expression and DNA hypomethylation of tumor-related genes such as maspin (70) and synuclein {gamma} (71) and cancer/testis antigens such as melanoma and germ cell-expressed genes (72) has been reported in human cancers.

We focused on the possibility that functional disruption of DNMT1 due to gene mutations might induce hypomethylation of repeated DNA sequences and individual genes in human cancers. Mutations of the DNMT1 gene, including a one-base deletion resulting in deletion of the whole catalytic domain due to a premature stop codon, were detected in 7% of examined colorectal cancers (73). This was the first evidence of DNMT1 gene mutations in human cancers. However, no stomach cancers or HCCs showed mutations in any of the 40 coding exons of the DNMT1 gene (73). Mutational inactivation of the DNMT1 gene may be a rare event during human carcinogenesis. Critical dietary components leading to synthesis of the methyl group donor, S-adenosylmethionine, include folate, vitamins B6 and B12, methionine and choline. Diets devoid of folate and choline and low in methionine are sufficient to independently induce hepatocarcinogenesis in rats (74). Non-coding RNAs such as anti-sense transcripts of individual genes may be involved in demethylation of the genes (75).

Although global DNA hypomethylation as an early event during colorectal carcinogenesis was noticed in the early days of molecular cancer research (76), the relative timing of global DNA hypomethylation has been revealed to differ between cancers derived from various organs (68). Researchers focusing on cancer epigenetics generally pay more attention to DNA hypermethylation than to DNA hypomethylation, and the clinicopathological significance of DNA hypomethylation during multistage carcinogenesis is, if anything, less well understood. DNA hypomethylation around the promoter regions of individual genes is usually correlated with global DNA hypomethylation (77), but not with DNA hypermethylation of C-type CpG islands in human cancers (78). Hypomethylation of both repeat DNA sequences and individual genes and regional DNA hypermethylation seem to be distinct consequences and are not mutually exclusive in individual patients with cancer.

Altered expression of methyl-CpG-binding proteins in human cancers
Until 1998, MeCP2 had been the only functionally defined methyl-CpG-binding protein (MBD). When MeCP2 binds to methylated CpG dinucleotide, its transcriptional repression domain recruits a co-repressor complex containing Sin 3A and histone deacetylases, resulting in compaction of the chromatin and stable repression of the target gene (79,80). Later, MBD1, MBD2, MBD3 and MBD4 were identified. MBD2 is a transcriptional repressor involved in the MeCP1 complex, identified in mammalian nuclear extracts, and represses transcription from the methylated promoter (81). MBD3 is involved in another histone deacetylase complex, Mi-2/NuRD (82). MBD4 is thought to act as a thymine DNA glycosylase, repairing G:T or G:U mismatches at CpG sites (83).

The expression level of MeCP2 mRNA in HCCs with portal vein involvement is significantly lower than that in HCCs without such involvement, suggesting that reduced expression of MeCP2 may be associated with malignant progression of HCCs (56). Reduced MBD2 mRNA expression has been observed in HCCs (56), colorectal and stomach cancers (84), suggesting that reduced MBD2 expression may be associated with a particular step in human carcinogenesis. The expression level of MBD4 mRNA in HCCs is significantly lower than that in the corresponding non-cancerous liver tissues and is significantly correlated with poorer tumor differentiation and involvement of the portal vein (56). Reduced MBD4 expression may result in frequent C–T transitions in tumor suppressor genes. Although many researchers have focused on crosstalk between DNA methylation and histone modification, abnormalities of MBDs in human cancers do not seem to have attracted much attention, and the implications of these proteins in carcinogenesis need to be further clarified.


   Perspectives
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 Abstract
 Introduction
 Perspectives
Funding
 References
 
Alterations of DNA methylation are associated with multistage carcinogenesis from precancerous conditions to malignant progression. Therefore, estimation of carcinogenetic risk and early diagnosis of cancers using alterations of DNA methylation as indicators are promising approaches for mass screening of clinical samples. For such purposes, non- or less invasive methodologies for detecting subtle alterations of DNA methylation have been developed for serum, urine, sputum and other body fluid samples. Moreover, recently developed array-based technology for accessing genome-wide DNA methylation status (85) will be useful for identifying the DNA methylation profile that is the optimum indicator for risk estimation and early diagnosis. Analysis of DNA methylation status in biopsy specimens and/or surgically resected materials may also become a useful tool for prognostication. Clinical trials of DNA demethylation agents are underway and many DNA demethylation agents are now being developed (86). However, global DNA hypomethylation and regional DNA hypermethylation are commonly observed during multistage carcinogenesis. In some patients, global DNA hypomethylation will have greater significance than regional DNA hypermethylation during carcinogenesis. Therefore, before using DNA demethylation agents for prevention or therapy of cancers, it will be necessary to carefully identify patients who might benefit most from this type of demethylation strategy. It may also be necessary to develop sequence-specific demethylation agents to reduce any severe side effects. In order to apply correction of DNA methylation status to practical prevention and therapy of cancers, the full picture and molecular mechanisms of DNA methylation alterations corresponding to specific carcinogenetic factors should be further clarified for each organ.


   Funding
Top
 Abstract
 Introduction
 Perspectives
 Funding
References
 
Third Term Comprehensive 10-Year Strategy for Cancer Control (H19-002); Cancer Research from the Ministry of Health, Labor and Welfare of Japan (15-20); a program for promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (05-31).


   Acknowledgments
 
Conflict of Interest Statement: None declared.


   References
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 Abstract
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 References
 

  1. Li E. Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. (2002) 3:662–673.[CrossRef][Web of Science][Medline]
  2. Bestor TH. The DNA methyltransferases of mammals. Hum. Mol. Genet. (2000) 9:2395–2402.[Abstract/Free Full Text]
  3. Chuang LS, et al. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science (1997) 277:1996–2000.[Abstract/Free Full Text]
  4. Rountree MR, et al. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat. Genet. (2000) 25:269–277.[CrossRef][Web of Science][Medline]
  5. Fuks F, et al. DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat. Genet. (2000) 24:88–91.[CrossRef][Web of Science][Medline]
  6. Robertson KD, et al. DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters. Nat. Genet. (2000) 25:338–342.[CrossRef][Web of Science][Medline]
  7. Lei H, et al. De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. Development (1996) 122:3195–3205.[Abstract]
  8. Yoder JA, et al. A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast. Hum. Mol. Genet. (1998) 7:279–284.[Abstract/Free Full Text]
  9. Okano M, et al. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat. Genet. (1998) 19:219–220.[CrossRef][Web of Science][Medline]
  10. Okano M, et al. Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. Nucleic Acids Res. (1998) 26:2536–2540.[Abstract/Free Full Text]
  11. Okano M, et al. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell (1999) 99:245–257.
  12. Jones PA, et al. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. (2002) 3:415–428.[Web of Science][Medline]
  13. Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat. Rev. Cancer (2005) 5:223–231.[CrossRef][Web of Science][Medline]
  14. Baylin SB, et al. Epigenetic gene silencing in cancer—a mechanism for early oncogenic pathway addiction? Nat. Rev. Cancer (2006) 6:107–116.[CrossRef][Web of Science][Medline]
  15. Tsuda H, et al. Allele loss on chromosome 16 associated with progression of human hepatocellular carcinoma. Proc. Natl Acad. Sci. USA (1990) 87:6791–6794.[Abstract/Free Full Text]
  16. Kanai Y, et al. Aberrant DNA methylation on chromosome 16 is an early event in hepatocarcinogenesis. Jpn. J. Cancer Res. (1996) 87:1210–1217.[CrossRef][Web of Science]
  17. Takeichi M. Cadherin cell adhesion receptor as a morphogenetic regulator. Science (1991) 251:1451–1455.[Abstract/Free Full Text]
  18. Hirohashi S, et al. Cell adhesion system and human cancer morphogenesis. Cancer Sci. (2003) 94:575–581.[CrossRef]
  19. Becker KF, et al. E-cadherin gene mutations provide clues to diffuse type gastric carcinomas. Cancer Res. (1994) 54:3845–3852.[Abstract/Free Full Text]
  20. Kanai Y, et al. Point mutation of the E-cadherin gene in invasive lobular carcinoma of the breast. Jpn. J. Cancer Res. (1994) 85:1035–1039.[CrossRef][Web of Science]
  21. Guilford P, et al. E-cadherin germline mutations in familial gastric cancer. Nature (1998) 392:402–405.[CrossRef][Medline]
  22. Behrens J, et al. Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell-cell adhesion. J. Cell Biol. (1989) 108:2435–2447.[Abstract/Free Full Text]
  23. Vleminckx K, et al. Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell (1991) 66:107–119.[CrossRef][Web of Science][Medline]
  24. Yoshiura K, et al. Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. Proc. Natl Acad. Sci. USA (1995) 92:7416–7419.[Abstract/Free Full Text]
  25. Kanai Y, et al. The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas. Int. J. Cancer (1997) 71:355–359.[CrossRef][Web of Science][Medline]
  26. Kanai Y, et al. DNA hypermethylation at the D17S5 locus is associated with gastric carcinogenesis. Cancer Lett. (1998) 122:135–141.[CrossRef][Web of Science][Medline]
  27. Wales MM, et al. p53 activates expression of HIC-1, a new candidate tumour suppressor gene on 17p13.3. Nat. Med. (1995) 1:570–577.[CrossRef][Web of Science][Medline]
  28. Chen WY, et al. Heterozygous disruption of Hic1 predisposes mice to a gender-dependent spectrum of malignant tumors. Nat. Genet. (2003) 33:197–202.[CrossRef][Web of Science][Medline]
  29. Kanai Y, et al. DNA hypermethylation at the D17S5 locus and reduced HIC-1 mRNA expression are associated with hepatocarcinogenesis. Hepatology (1999) 29:703–709.[CrossRef][Web of Science]
  30. Hayashizaki Y, et al. Restriction landmark genomic scanning method and its various applications. Electrophoresis (1993) 14:251–258.[CrossRef][Web of Science][Medline]
  31. Ushijima T, et al. Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors. Proc. Natl Acad. Sci. USA (1997) 94:2284–2289.[Abstract/Free Full Text]
  32. Toyota M, et al. Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Res. (1999) 59:2307–2312.[Abstract/Free Full Text]
  33. Kanai Y, et al. mRNA expression of genes altered by 5-azacytidine treatment in cancer cell lines is associated with clinicopathological parameters of human cancers. J. Cancer Res. Clin. Oncol. (2001) 127:697–706.[Web of Science][Medline]
  34. Sundaresan V, et al. p53 and chromosome 3 abnormalities, characteristic of malignant lung tumours, are detectable in preinvasive lesions of the bronchus. Oncogene (1992) 7:1989–1997.[Web of Science][Medline]
  35. O'Connell P, et al. Molecular genetic studies of early breast cancer evolution. Breast Cancer Res. Treat. (1994) 32:5–12.[Web of Science][Medline]
  36. Kondo Y, et al. Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis—a comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma. Hepatology (2000) 32:970–979.[CrossRef][Web of Science]
  37. Etoh T, et al. Increased DNA methyltransferase 1 (DNMT1) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpG islands in gastric cancers. Am. J. Pathol. (2004) 164:689–699.[Abstract/Free Full Text]
  38. Kanai Y, et al. DNA methyltransferase expression and DNA methylation of CpG islands and peri-centromeric satellite regions in human colorectal and stomach cancers. Int. J. Cancer (2001) 91:205–212.[CrossRef][Web of Science][Medline]
  39. Arai E, et al. Regional DNA hypermethylation and DNA methyltransferase (DNMT) 1 protein overexpression in both renal tumors and corresponding nontumorous renal tissues. Int. J. Cancer (2006) 119:288–296.[CrossRef][Web of Science][Medline]
  40. Nakagawa T, et al. DNA hypermethylation on multiple CpG islands associated with increased DNA methyltransferase DNMT1 protein expression during multistage urothelial carcinogenesis. J. Urol. (2005) 173:1767–1771.[CrossRef][Web of Science][Medline]
  41. Kanai Y, et al. Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis. Cancer Lett. (2000) 148:73–80.[CrossRef][Web of Science][Medline]
  42. Kondo Y, et al. Microsatellite instability associated with hepatocarcinogenesis. J. Hepatol. (1999) 31:529–536.[CrossRef][Web of Science][Medline]
  43. Muller K, et al. Foreign DNA integration. Genome-wide perturbations of methylation and transcription in the recipient genomes. J. Biol. Chem. (2001) 276:14271–14278.[Abstract/Free Full Text]
  44. Tsai CN, et al. The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases. Proc. Natl Acad. Sci. USA (2002) 99:10084–10089.[Abstract/Free Full Text]
  45. Maekita T, et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin. Cancer Res. (2006) 12:989–995.[Abstract/Free Full Text]
  46. Sawada M, et al. Increased expression of DNA methyltransferase 1 (DNMT1) protein in uterine cervix squamous cell carcinoma and its precursor lesion. Cancer Lett. (2007) 251:211–219.[CrossRef][Web of Science][Medline]
  47. Burgers WA, et al. Viral oncoproteins target the DNA methyltransferases. Oncogene (2007) 26:1650–1655.[CrossRef][Web of Science][Medline]
  48. Dong SM, et al. Promoter hypermethylation of multiple genes in carcinoma of the uterine cervix. Clin. Cancer Res. (2001) 7:1982–1986.[Abstract/Free Full Text]
  49. Peng DF, et al. Increased DNA methyltransferase 1 (DNMT1) protein expression in precancerous conditions and ductal carcinomas of the pancreas. Cancer Sci. (2005) 96:403–408.[CrossRef][Medline]
  50. Peng DF, et al. DNA methylation of multiple tumor-related genes in association with overexpression of DNA methyltransferase 1 (DNMT1) during multistage carcinogenesis of the pancreas. Carcinogenesis (2006) 27:1160–1168.[Abstract/Free Full Text]
  51. Hodge DR, et al. Interleukin 6 supports the maintenance of p53 tumor suppressor gene promoter methylation. Cancer Res. (2005) 65:4673–4682.[Abstract/Free Full Text]
  52. Eguchi K, et al. DNA hypermethylation at the D17S5 locus in non-small cell lung cancers: its association with smoking history. Cancer Res. (1997) 57:4913–4915.[Abstract/Free Full Text]
  53. Issa JP. CpG island methylator phenotype in cancer. Nat. Rev. Cancer (2004) 4:988–993.[CrossRef][Web of Science][Medline]
  54. Saito Y, et al. Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas. Int. J. Cancer (2003) 105:527–532.[CrossRef][Web of Science][Medline]
  55. Sun L, et al. Increased DNA methyltransferase expression is associated with an early stage of human hepatocarcinogenesis. Jpn. J. Cancer Res. (1997) 88:1165–1170.[CrossRef][Web of Science]
  56. Saito Y, et al. Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis. Hepatology (2001) 33:561–568.[CrossRef][Web of Science]
  57. Baylin SB. Tying it all together: epigenetics, genetics, cell cycle, and cancer. Science (1997) 277:1948–1949.[Free Full Text]
  58. Vertino PM, et al. De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase. Mol. Cell. Biol. (1996) 16:4555–4565.[Abstract]
  59. Rhee I, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature (2002) 416:552–556.[CrossRef][Medline]
  60. Nakagawa T, et al. Increased DNA methyltransferase 1 protein expression in human transitional cell carcinoma of the bladder. J. Urol. (2003) 170:2463–2466.[CrossRef][Web of Science][Medline]
  61. Wong N, et al. Hypomethylation of chromosome 1 heterochromatin DNA correlates with q-arm copy gain in human hepatocellular carcinoma. Am. J. Pathol. (2001) 159:465–471.[Abstract/Free Full Text]
  62. Nakagawa T, et al. DNA hypomethylation on pericentromeric satellite regions significantly correlates with loss of heterozygosity on chromosome 9 in urothelial carcinomas. J. Urol. (2005) 173:243–246.[Web of Science][Medline]
  63. Hansen RS, et al. The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc. Natl Acad. Sci. USA (1999) 96:14412–14417.[Abstract/Free Full Text]
  64. Saito Y, et al. Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis. Proc. Natl Acad. Sci. USA (2002) 99:10060–10065.[Abstract/Free Full Text]
  65. Soejima K, et al. DNA methyltransferase 3b contributes to oncogenic transformation induced by SV40T antigen and activated Ras. Oncogene (2003) 22:4723–4733.[CrossRef][Web of Science][Medline]
  66. Kanai Y, et al. Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis. J. Cancer Res. Clin. Oncol. (2004) 130:636–644.[CrossRef][Web of Science][Medline]
  67. Karpf AR, et al. Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells. Proc. Natl Acad. Sci. USA (1999) 96:14007–14012.[Abstract/Free Full Text]
  68. Wilson A, et al. DNA hypomethylation and human diseases. Biochim. Biophys. Acta (2007) 1775:138–162.[Medline]
  69. Cheah M, et al. Hypomethylation of DNA in human cancer cells: a site-specific change in the c-myc oncogene. J. Natl Cancer Inst. (1984) 73:1057–1065.[Web of Science][Medline]
  70. Akiyama Y, et al. Cell-type-specific repression of the maspin gene is disrupted frequently by demethylation at the promoter region in gastric intestinal metaplasia and cancer cells. Am. J. Pathol. (2003) 163:1911–1919.[Abstract/Free Full Text]
  71. Gupta A, et al. Hypomethylation of the synuclein gamma gene CpG island promotes its aberrant expression in breast carcinoma and ovarian carcinoma. Cancer Res. (2003) 63:664–673.[Abstract/Free Full Text]
  72. Jang S, et al. Activation of melanoma antigen tumor antigens occurs early in lung carcinogenesis. Cancer Res. (2001) 61:7959–7963.[Abstract/Free Full Text]
  73. Kanai Y, et al. Mutation of the DNA methyltransferase (DNMT) 1 gene in human colorectal cancers. Cancer Lett. (2003) 192:75–82.[CrossRef][Web of Science][Medline]
  74. James S, et al. Mechanisms of DNA damage, DNA hypomethylation, and tumor progression in the folate/methyl-deficient rat model of hepatocarcinogenesis. J. Nutr. (2003) 133:3740S–3747S.[Abstract/Free Full Text]
  75. Imamura T, et al. Non-coding RNA directed DNA demethylation of Sphk1 CpG island. Biochem. Biophys. Res. Commun. (2004) 322:593–600.[CrossRef][Web of Science][Medline]
  76. Goelz S, et al. Hypomethylation of DNA from benign and malignant human colon neoplasms. Science (1985) 228:187–190.[Abstract/Free Full Text]
  77. De Smet C, et al. The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc. Natl Acad. Sci. USA (1996) 93:7149–7153.[Abstract/Free Full Text]
  78. Kaneda A, et al. Frequent hypomethylation in multiple promoter CpG islands is associated with global hypomethylation, but not with frequent promoter hypermethylation. Cancer Sci. (2004) 95:58–64.[CrossRef]
  79. Jones PL, et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. (1998) 19:187–191.[CrossRef][Web of Science][Medline]
  80. Nan X, et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature (1998) 393:386–389.[CrossRef][Medline]
  81. Ng HH, et al. MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex. Nat. Genet. (1999) 23:58–61.[Web of Science][Medline]
  82. Zhang Y, et al. Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev. (1999) 13:1924–1935.[Abstract/Free Full Text]
  83. Hendrich B, et al. The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites. Nature (1999) 401:301–304.[CrossRef][Medline]
  84. Kanai Y, et al. Reduced mRNA expression of the DNA demethylase, MBD2, in human colorectal and stomach cancers. Biochem. Biophys. Res. Commun. (1999) 264:962–966.[CrossRef][Web of Science][Medline]
  85. Inazawa J, et al. Comparative genomic hybridization (CGH)-arrays pave the way for identification of novel cancer-related genes. Cancer Sci. (2004) 95:559–563.[CrossRef][Medline]
  86. Mack GS. Epigenetic cancer therapy makes headway. J. Natl Cancer Inst. (2006) 98:1443–1444.[Free Full Text]
Received May 2, 2007; revised September 5, 2007; accepted September 12, 2007.


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[Abstract] [Full Text] [PDF]

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