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Keywords:

  • chromosomal instability;
  • chronic inflammation;
  • DNA methylation;
  • DNMT1;
  • DNMT3B;
  • hepatocellular carcinoma;
  • multistage carcinogenesis;
  • precancerous condition;
  • renal cell carcinoma;
  • urothelial carcinoma

Abstract

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

Alterations of DNA methylation can account for the histological heterogeneity, reflected in the stepwise progression and complex biological characteristics of human cancers, that genetic alterations alone cannot explain. Analysis of DNA methylation status in tissue samples can be an aid to understanding the molecular mechanisms of multistage carcinogenesis. Human cancer cells show a drastic change in DNA methylation status, that is, overall DNA hypomethylation and regional DNA hypermethylation, which results in chromosomal instability and silencing of tumor-suppressor genes. Overexpression of DNA methyltransferase (DNMT) 1 is not a secondary result of increased cell proliferative activity but may underline the CpG island methylator phenotype of cancers. Splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alterations of DNA methylation are observed even in the precancerous stage frequently associated with chronic inflammation and/or persistent viral infection or with cigarette smoking. Precancerous conditions showing alterations of DNA methylation may generate more malignant cancers. Aberrant DNA methylation is significantly associated with aggressiveness of cancers and poorer outcome of cancer patients. Genome-wide analysis of DNA methylation status based on array-based technology may identify DNA methylation profiles that can be used as appropriate indicators for carcinogenetic risk estimation and prognostication.

Microscopy of human cancers, which are considered to be genetically clonal lesions, frequently indicates histological heterogeneity (e.g. well, moderately or poorly differentiated carcinoma components are simultaneously observed even in tissue sections from a single patient). Such histological heterogeneity reflects the stepwise progression and complex biological characteristics of each tumor. Genetic alterations causing activation of oncogenes and inactivation of tumor suppressor genes cannot solely explain such histological heterogeneity of human cancers. Epigenetics has been defined as ‘heritable changes in gene expression that are not due to any alteration in the DNA sequence’1 and normally accounts for the diversity of phenotypes within cloned animals, monozygotic twins and single populations that genetics alone cannot explain.2 Analysis of epigenetic alterations in tissue samples, in connection with the histological features of each cancer, may aid understanding of the molecular background of clinocopathological diversity in human cancers. DNA methylation is one of the most consistent and best-known epigenetic events in human cancers.

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 (Fig. 1a), plays important roles in chromatin organization and gene expression.3 DNA methylation can directly impede the binding of transcription factors to their target sites, thus prohibiting the transcription of specific genes. Moreover, DNA methylation normally promotes a highly condensed heterochromatin structure, where active transcription does not occur, through recruitment of DNA-organizing proteins (Fig. 1b). DNA methylation is a stable modification that is inherited throughout cell divisions (Fig. 1c). When found within the promoter regions, DNA methylation prevents the reactivation of silent genes. This allows the daughter cells to retain the same expression pattern as the parent cells and is important for inactivation of the X chromosome and imprinting. Transposons and other parasitic elements have been acquired in the mammalian genome over time, and make up the repetitive sequences in the intergenic and intragenic regions of DNA. The activation of these parasitic elements can allow for the movement of these elements within the genome. To preserve the integrity of the genome, DNA methylation persistently silences such parasitic elements.4

image

Figure 1. DNA methylation in mammals. (a) DNA methylation is a covalent chemical modification resulting in addition of a methyl (CH3) group at the carbon 5 position of the cytosine ring in CpG dinucleotides. DNA methyltransferases (DNMT) transfer methyl groups from S-adenosyl-l-methionine to cytosines. (b) DNA methylation normally promotes a highly condensed heterochromatin structure, in which active transcription does not occur, through recruitment of DNA-organizing proteins including histone deacetylase complex and methyl CpG binding proteins (MBD). The repressed regions are associated with deacetylation of histones H3 and H4 and gain of histone H3, lysine 9 (H3K9) methylation. H, histone octamer; (○) unmethylated CpG dinucleotides; (●) methylated CpG dinucleotides. (c) DNA methylation is a stable modification that is inherited throughout cell divisions (maintenance methylation). The preference of maintenance DNMT for hemi-methylated over unmethylated substrates 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. De novo methylation occurs by de novo DNMT during the development of mammals and carcinogenesis. (d) Structure of DNMT. The C-terminal catalytic domain is characterized by the presence of conserved motifs I, IV, VI, IX and X. The N-terminal regulatory domain of DNMT1 contains a proliferating cell nuclear antigen-binding domain (PBD), a nuclear localization signal (NLS), a cysteine-rich alpha thalassemia and retardation on the X (ATRX) zinc finger DNA-binding motif, and a polybromo homology domain (PHD) targeting DNMT1 to the replication foci.

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Murine DNA methyltransferase 1 (Dnmt 1) cDNA was cloned in 1988 and its C-terminal domain was found to show striking similarities to the catalytic methyltransferase domain of bacterial type II DNA cytosine methyltransferases.5 Homologs of DNMT1 have been found in nearly all eukaryotes that have DNA bearing 5-methylcytosine, but not in those that lack it. Until the identification of DNA methyltransferase (DNMT) 2,6 DNMT3A and DNMT3B7 in 1998, DNMT1 (EC2.1.1.37) had been the only known DNMT, and is currently the major and best-known of this enzyme family. Embryos of Dnmt1 −/− mice, which have genome-wide DNA hypomethylation, are stunted, show delayed development, and do not survive past mid-gestation,8 indicating that DNA methylation is essential for the development of mammals.

The C-terminal catalytic domain of DNMT transfers methyl groups from S-adenosyl-l-methionine (AdoMet) to cytosines (Fig. 1a).9 Critical dietary components leading to synthesis of AdoMet include folate, vitamins B6 and B12, methionine and choline. The C-terminal catalytic domain of DNMT is characterized by the presence of five conserved amino acid motifs, namely I, IV, VI, IX and X (Fig. 1d).9 Motifs I and X are filed together to form most of the binding site for AdoMet. Motif IV contains the prolylcysteinyl dipeptide that provides the thiolate at the active site. Motif VI contains the glutamyl residue that protonates the 3 position of the target cytosine. Motif IX has a role in maintaining the structure of the target recognition domain.

The N-terminal regulatory domain of DNMT1 contains a proliferating cell nuclear antigen (PCNA)-binding domain, a nuclear localization signal, a cysteine-rich alpha thalassemia and retardation on the X (ATRX) zinc finger DNA-binding motif, and a polybromo homology domain targeting DNMT1 to the replication foci (Fig. 1d).10 Thus DNMT1 forms the core of the DNA replication machinery complex. In addition to methyltransferase activity, interaction with DNMT1-associated protein (DMAP) 1,11 E2F1,12 histone deacetylase (HDAC) 1 and 2 and methyl CpG binding proteins (MBD)13 through the N-terminal regulatory domain makes DNMT1 a crucial element of the transcription suppression complex.

The preference of DNMT1 for hemi-methylated over unmethylated substrates in vitro14 and its targeting of replication foci15 are believed to allow copying of the methylation pattern of the parental strand to the newly synthesized daughter DNA strand. Thus, DNMT1 has been recognized as the ‘maintenance’ DNMT (Fig. 1c). Although DNMT2 contains the full set of conserved motifs of the C-terminal catalytic domain, it lacks the N-terminal regulatory domain characteristic of eukaryotic DNMT (Fig. 1d). The methyltransferase activity of the recombinant DNMT2 protein is weak in vitro and in vivo. DNMT3A and DNMT3B also contain the full set of conserved motifs of the C-terminal catalytic domain, and their N-terminal regulatory domains are divergent on the N-terminal side of the cysteine-rich ATRX zinc finger DNA-binding motif (Fig. 1d). DNMT3A and DNMT3B show de novo DNA methylation activity (Fig. 1c) in vitro.16 Pericentromeric satellite regions are considered to be one of the specific targets of DNMT3B, because Dnmt3B−/− mice lack DNA methylation in such regions and die in utero.16 Germline mutations of the DNMT3B gene have been reported in patients with immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome, a rare recessive autosomal disorder characterized by DNA hypomethylation on pericentromeric satellite regions.17 Because de novo methylation of CpG islands has actually been observed in human fibroblasts overexpressing DNMT1,18 DNMT1 is capable of de novo DNA methylation activity in vivo as well as having a maintenance function, and DNA methylation status may be determined on the basis of cooperation between DNMT1 and the DNMT3 family in vivo.19 DNMT3L lacks conserved motifs of the catalytic domain but is otherwise closely related to the N-terminal regulatory domain of DNMT3A and DNMT3B (Fig. 1d) and cooperates with the DNMT3 family to establish an imprinting pattern.20

MBD are one of mediators of cross-talk between DNA methylation and another major epigenetic event, histone modification. Until 1998, MeCP2 had been the only functionally defined MBD. When MeCP2 binds to methylated CpG dinucleotide, its transcriptional repression domain recruits a co-repressor complex containing Sin 3A and HDAC, resulting in compaction of the chromatin and stable repression of the target gene.21,22 Later, MBD1, MBD2, MBD3 and MBD4 were identified. MBD1 interacts with histone H3 methyltransferase SETDB1.23 MBD224 and MBD325 are involved in another HDAC complex, Mi-2/NuRD. MBD4 is thought to act as a thymine DNA glycosylase, repairing G:T or G:U mismatches at CpG sites.26

DNA METHYLATION AND HUMAN CANCERS

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

In comparison with normal cells, human cancer cells show a drastic change in DNA methylation status, generally exhibiting global DNA hypomethylation and accompanying region-specific hypermethylation.27–30 Because 5-methylcytosine is deaminated to thymine, DNA hypermethylation facilitates gene mutation in human cancers. DNA hypomethylation in cancer cells causes chromatin decondensation and chromosomal rearrangements that may result in chromosomal instability. Moreover, DNA hypermethylation of CpG islands near the promoter regions silences specific genes including tumor suppressor genes in cooperation with histone modification:31 hypermethylation of CpG islands in the promoter regions of tumor-suppressor genes in cancer cells is associated withdeacetylation of histones H3 and H4, loss of histone H3, lysine 4 (H3K4) methylation, and gain of H3K9 methylation (Fig. 1b).

A reduction of DNMT1 activity in ApcMin mice due to heterozygosity of the Dnmt1 gene, in conjunction with treatment using the DNMT inhibitor 5-aza-deoxycytidine, reduces the average number of intestinal adenomas.32 In contrast, genomic hypomethylation in Nf1+/− p53+/− (NPcis) mice due to the introduction of a hypomorphic allele of Dnmt 1 (Dnmt1Chip/–) induces sarcomas at an earlier age in comparison with NPcis littermates possessing normal levels of DNA methylation (Dnmt1Chip/+).33 The loss of heterozygosity (LOH) rate is increased in hypomethylated cells in Dnmt1Chip/− mice. Chromosomal instability accompanied by activation of endogenous retroviral elements has also been observed in Dnmt1Chip/− mice.34 These observations in genetically engineered animals clearly demonstrate a causal relationship between alterations of DNA methylation and human cancers. Correlation between the etiological backgrounds of human cancers and alterations of DNA methylation, however, can be clarified only by analysis of clinical samples.

In order to determine the significance of DNA methylation alterations during multistage carcinogenesis, DNA methylation status should be analyzed in a range of tissue samples from precancerous conditions to malignant states (Fig. 2). Such empirical data are indispensable for clinical application of DNA methylation to carcinogenetic risk estimation, early diagnosis, prognostication, prevention and therapy. Therefore the following sections describe the results obtained by analysis of DNA methylation status in tissue samples for which the clinicopathological characteristics have been strictly determined.

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Figure 2. Analysis of DNA methylation status in tissue specimens. Tissue sample of carcinoma in situ (a) before and (d) during microdissection, and that of invasive carcinoma (b) before and (c) after microdissection. (e) Microdissected specimens were subjected to agarose bead-embedded methylation-specific polymerase chain reaction (MSP),35 which was originally developed for analysis of DNA methylation in tiny tissue samples. (f) Examples of the results of MSP for the p16 gene and combined bisulfite restriction enzyme analysis (COBRA) for MINT 2 and 12 clones. Polymerase chain reaction products yielded by primer sets for methylated (M), unmethylated (U) and unmodified wild-type (W) DNA and digested (D) and non-digested (N) DNA fragments using methylation-sensitive restriction enzymes are shown. Arrows, methylated DNA fragments.

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HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

Alterations of DNA methylation in precancerous conditions

The majority of hepatocellular carcinomas (HCC) are associated with HBV or HCV infection. Clonal expansion of hepatocytes is initiated during the regeneration process in damaged livers; a clonal integration pattern of HBV is evident in each cirrhotic nodule. Therefore, chronic hepatitis and liver cirrhosis are considered to be precancerous conditions. Small nodular lesions of early-stage HCC first develop in livers with chronic hepatitis and cirrhosis, and then progressed HCC often emerge within early-stage HCC nodules (nodule-in-nodule-type HCC). Thus, macro- and microscopically, HCC represent a typical scenario of multistage carcinogenesis.36

The LOH on chromosome 16 has been frequently detected on classic restriction fragment length polymorphism using Southern blot in HCC that are poorly differentiated, large in size, and associated with metastasis.37 Therefore, this 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. But studies using classic Southern blot with a DNA methylation-sensitive restriction enzyme frequently showed alterations of DNA methylation at multiple loci on chromosome 16 even in non-cancerous liver tissues with chronic hepatitis or cirrhosis, unlike normal liver tissues obtained from patients with liver metastases from primary colon cancer.38 This was one of the earliest reports of alterations of DNA methylation in the precancerous stage. Because the molecular weight of DNA fragments digested using a DNA methylation-sensitive restriction enzyme in HCC was higher than that in precancerous conditions, and the intensity of larger-sized bands was increased in HCC in comparison with precancerous conditions, the numbers of methylated CpG dinucleotides and cells having DNA hypermethylation may increase progressively as precancerous conditions develop into HCC.38 The incidence of DNA hypermethylation on chromosome 16 was significantly correlated with higher histological grade, portal vein involvement and intrahepatic metastasis of HCC.38 The presence of DNA hypermethylation in both precancerous conditions and progressed HCC suggests that aberrant DNA methylation is one of the earliest molecular events during hepatocarcinogenesis and also participates in malignant progression.

Silencing of tumor suppressor genes

The E-cadherin gene is located on 16q22.1 near the aforementioned hot spots of both DNA hypermethylation and LOH in HCC. E-cadherin acts as a Ca2+-dependent cell–cell adhesion molecule in the adherens junctions of epithelial cells.39 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. The E-cadherin gene is a tumor suppressor gene that can be silenced by a two-hit mechanism consisting of LOH and gene mutation in cancers such as signet-ring cell carcinoma of the stomach40 and lobular carcinoma of the breast,41 in which cancer cells completely lose their mutual adhesiveness even in the in situ carcinoma stage. In contrast, reduced expression of E-cadherin is believed to trigger the release of cancer cells from primary cancer nests, resulting in cancer invasion and metastasis.42 Significant correlations between reduced expression of E-cadherin and poor prognosis have been reported in patients with cancers.42 The promoter region of the E-cadherin gene contained DNA methylation in human cancer cell lines lacking E-cadherin expression, and E-cadherin expression was induced after treatment with the DNMT inhibitor 5-azacytidine in such cell lines.43 Thus, following the retinoblastoma (RB) and von Hippel-Lindau (VHL) genes, the E-cadherin gene became the third example of a tumor suppressor gene that is silenced by DNA hypermethylation.43 When assessed on Southern blot, DNA hypermethylation around the promoter region of the E-cadherin gene can be frequently detected even in non-cancerous liver tissues showing chronic hepatitis or cirrhosis.44 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, might be due, at least partly, to DNA hypermethylation.44 A significant correlation between DNA hypermethylation around the promoter region and reduced expression of E-cadherin was found in HCC.44 This was the first demonstration of a significant correlation between DNA hypermethylation and reduced expression in a cohort of clinical tissue samples. DNA hypermethylation around the promoter region may participate in hepatocarcinogenesis through reduction of E-cadherin expression, resulting in loss of intercellular adhesiveness and destruction of tissue morphology.

The hypermethylated in cancer (HIC)-1 gene at the D17S5 locus (17q13.3) was the first tumor suppressor gene to be identified in commonly methylated chromosomal loci in human cancers;45 mice with germ line disruption of one allele of Hic1 developed spontaneous malignant tumors.46 DNA hypermethylation at the D17S5 locus was frequently detectable in non-cancerous liver tissues showing chronic hepatitis or cirrhosis,47 as well as in stomach mucosa showing intestinal metaplasia.48 Southern blot using a DNA methylation-sensitive restriction enzyme suggested that the numbers of methylated CpG dinucleotides and cells showing DNA hypermethylation might increase progressively as precancerous conditions develop into HCC.47 The expression level of HIC-1 mRNA in non-cancerous liver that had chronic hepatitis or cirrhosis was significantly lower than that in normal liver tissues, and was further decreased in HCC.47

Alterations of DNA methylation and chromosomal instability

The hot spot for DNA hypermethylation in HCC corresponds to a previously reported hot spot of LOH on chromosome 16.38 It remains to be clarified 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. But classic Southern blot clearly showed that DNA hypermethylation precedes LOH at the same chromosomal loci during hepatocarcinogenesis: DNA hypermethylation was detected in bulk non-cancerous liver tissues showing chronic hepatitis or cirrhosis, in which LOH has never been detected using the same method.

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 dysplastic lesions adjacent to cancers. In order to re-examine whether aberrant DNA methylation precedes chromosomal instability during hepatocarcinogenesis, in microdissected specimens obtained from pseudo-lobules and regenerative nodules in non-cancerous liver tissues having chronic hepatitis or cirrhosis and HCC, LOH and microsatellite instability were examined using multiple microsatellite markers, and the DNA methylation status of multiple C-type CpG islands49 that are known to be methylated in a cancer-specific, but not age-dependent manner, was examined on methylation-specific PCR and combined bisulfite restriction enzyme analysis.50,51

LOH was never detected in normal liver tissues obtained from patients with liver metastases from primary colon cancers or in non-cancerous liver tissues having no remarkable histology from patients with HCC (Fig. 3a).51 The incidence of LOH in chronic hepatitis or liver cirrhosis stages was almost the same (Fig. 3a). 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 cancers, DNA hypermethylation was found even in non-cancerous liver tissues having no remarkable histological features obtained from patients with HCC, in which LOH was never detected (Fig. 3a).51 This phenomenon might be at least partly attributable to hepatitis viral infection. HBV-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.52 The incidence of DNA hypermethylation on CpG islands overwhelmed that of LOH at all stages of chronic hepatitis, liver cirrhosis and HCC. Thus aberrant DNA methylation is an earlier event preceding chromosomal instability during hepatocarcinogenesis, even when examined using PCR-LOH and microdissection. The low incidence of microsatellite instability in Japanese patients with HCC53 (Fig. 3a) was compatible with absence of silencing of the human MutL homologue 1 (hMLH1) gene by DNA hypermethylation during hepatocarcinogenesis.51

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Figure 3. Alterations of DNA methylation during hepatocarcinogenesis. (a) Loss of heterozygosity (LOH; arrow) and microsatellite instability (MSI; arrowhead) were examined using 39 microsatellite markers; and DNA methylation status on 8 C-type CpG islands was examined on methylation-specific polymerase chain reaction and combined bisulfite restriction enzyme analysis of microdissected tissue specimens.51 (b) The expression level of DNA methyltransferase 1 (DNMT1) mRNA was significantly higher even in non-cancerous liver tissues that had chronic hepatitis or cirrhosis than in normal liver tissues, and was even higher in hepatocellular carcinomas.55,56 (c) DNMT1 protein expression in hepatocellular carcinomas was significantly correlated with poorer tumor differentiation and portal vein involvement.57 (d) Recurrence-free survival rate of patients whose hepatocellular carcinomas had protein overexpression of DNMT1 was significantly lower than that of patients whose hepatocellular carcinomas did not.57

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Overexpression of DNMT1

Abnormalities of DNMT underlying alterations of DNA methylation was examined during hepatocarcinogenesis. Mutational inactivation of the DNMT1 gene that can potentially cause genome-wide alterations of DNA methylation was never detected in HCC or in stomach cancers, whereas colorectal cancers infrequently had mutations of the DNMT1 gene, including a mutation resulting in deletion of the whole catalytic domain due to a premature stop codon.54 Mutational inactivation of the DNMT1 gene may be a rare event during human carcinogenesis. In contrast, the expression level of DNMT1 mRNA was significantly higher even in non-cancerous liver tissues having chronic hepatitis or cirrhosis than in normal liver tissues, and was even higher in HCC (Fig. 3b).55,56 The incidence of DNMT1 protein overexpression in HCC is significantly correlated with poorer tumor differentiation and portal vein involvement (Fig. 3c).57 Moreover, the recurrence-free and overall survival rates of patients with HCC that has overexpression of DNMT1 protein are significantly lower than those of patients with HCC that do not (Fig. 3d).57 Immunohistochemistry of DNMT1 in liver biopsy specimens obtained for histological diagnostic purposes and/or hepatectomy specimens may become a useful tool for prognostication in individual clinical cases.

Aberrant splicing of DNMT3B

Although DNA hypomethylation on pericentromeric satellite regions, such as satellites 2 and 3, was frequently detected in both non-cancerous liver tissues having chronic hepatitis or cirrhosis and HCC (Fig. 4a),56 and such regions are one of the target sequences of DNMT3B, no mutation of any coding exon of the DNMT3B gene was detected in the examined HCC.58 The total level of DNMT3B mRNA was higher in HCC than in the corresponding non-cancerous liver tissues (Fig. 4b).56 Thus, it is unlikely that reduced expression of DNMT3B simply causes DNA hypomethylation in 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 (Fig. 4c) and its DNMT activity has been confirmed in vitro.59 Data obtained on splice-variant-specific quantitative reverse transcription–PCR have indicated that the major variant in normal liver tissues is DNMT3B3.58 In contrast, DNMT3B4 probably does not show DNMT activity because it lacks the conserved methyltransferase motifs IX and X (Fig. 4c), although it retains the N-terminal domain required for targeting to pericentromeric satellite regions. Normal liver tissues showed only a trace level of DNMT3B4 expression.58 The level of DNMT3B4 mRNA in non-cancerous liver tissues obtained from patients with HCC and in HCC was significantly correlated with the degree of DNA hypomethylation in pericentromeric satellite regions (Fig. 4d).58 In addition, the ratio of DNMT3B4 mRNA to DNMT3B3 mRNA in non-cancerous liver tissues obtained from patients with HCC and in HCC was also significantly correlated with the degree of DNA hypomethylation in pericentromeric satellite regions (Fig. 4d).58 DNMT3B4 lacking DNMT activity may compete with the major variant, DNMT3B3, for targeting to pericentromeric satellite regions. Then DNMT3B4 was introduced 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 the DNMT3B4 transfectants, depending on the expression level of myc-tagged DNMT3B4 (Fig. 4e).58 Satellite regions are abundant in pericentromeric heterochromatin DNA on chromosomes 1, 9 and 16. In fact, frequent chromosome 1q copy gain with a pericentromeric breakpoint has been reported in HCC having DNA hypomethylation on satellite 2.60 DNMT3B4 overexpression may lead to chromosomal instability through induction of DNA hypomethylation in pericentromeric satellite regions during hepatocarcinogenesis.

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Figure 4. Overexpression of DNA methyltransferase 3B4 (DNMT3B4) associated with DNA hypomethylation in pericentromeric satellite regions during hepatocarcinogenesis. (a) Although satellites 2 and 3 were fully methylated in normal liver tissue obtained from a patient with liver metastasis from primary colon cancer (C, arrowheads), DNA hypomethylation on satellites 2 and 3 was frequently detected in both non-cancerous liver tissues that had chronic hepatitis or cirrhosis (N) and hepatocellular carcinomas (T).56 H, methylation-sensitive restriction enzyme HpaII; M, methylation-non-sensitive restriction enzyme Msp I. Arrows, DNA hypomethylation. (b) The total level of DNMT3B mRNA was higher in hepatocellular carcinomas than in the corresponding non-cancerous liver tissue.56 Thus, it is unlikely that reduced expression of DNMT3B causes DNA hypomethylation in these regions during hepatocarcinogenesis. (c) Structure of splice variants of DNMT3B, DNMT3B3 and DNMT3B4. (d) The level of DNMT3B4 mRNA and the ratio of DNMT3B4 mRNA to DNMT3B3 mRNA in non-cancerous liver tissues obtained from patients with hepatocellular carcinomas and in hepatocellular carcinomas were significantly correlated with the degree of DNA hypomethylation in pericentromeric satellite regions.58+, smaller fragments detected in the Hpa II digest compared with the Hpa II digest of normal liver tissues; ++, Hpa II digest had the same hybridization pattern as the Msp I digest of its own and normal liver tissues. (e) DNA hypomethylation on satellite 2 was observed in transfection of human epithelial 293 cells with DNMT3B4 cDNA (clones 3B4-2 and 3B4-5) compared to mock transfectant (Mock) and parent 293 cells (Parent).58 H; Hpa II digest; M, Msp I digest. (f) The growth rate of DNMT3B4 transfectants (clones DNMT3B4-4 and 3B4-5) was approximately double that of mock-transfectants (clones Mock-1 and Mock-2) soon after the introduction of DNMT3B4,61 when chromosomal instability may not yet have accumulated.

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The growth rate of DNMT3B4 transfectants was approximately double that of mock-transfectants soon after the introduction of DNMT3B4 (Fig. 4f),61 when chromosomal instability may not yet have accumulated. It was assumed that this change was caused by altered gene expression. Although the majority of the genes that were upregulated in DNMT3B4 transfectants were implicated in interferon signaling,61 genes that encoded interferons themselves were not upregulated. Signal transducer and activator of transcription (STAT) 1,61 which acts as an effector of interferon signaling, has been listed as one of the upregulated genes in DNMT3B4 transfectants. A significant correlation between the expression levels of DNMT3B4 and STAT1 mRNA was confirmed in tissue specimens of HCC.61 Overexpression of DNMT3B4 is involved in multistage carcinogenesis not only by inducing chromosomal instability but also by affecting the expression of specific genes.

Altered expression of methyl CpG binding proteins

Although many researchers have focused on cross-talk between DNA methylation and histone modification, abnormalities of MBD in human cancers do not seem to have attracted much attention. The expression level of MeCP2 mRNA in HCC with portal vein involvement is significantly lower than that in HCC without such involvement,56 suggesting that reduced expression of MeCP2 may be associated with malignant progression of HCC. Reduced MBD2 mRNA expression has been observed in HCC,56 as well as in colorectal and stomach cancers,62 suggesting that reduced MBD2 expression may be associated with a particular step in human carcinogenesis. The expression level of MBD4 mRNA in HCC is significantly lower than that in the corresponding non-cancerous liver tissues and is significantly correlated with poorer tumor differentiation and portal vein involvement.56 Reduced MBD4 expression may result in frequent C-T transitions in tumor suppressor genes.

VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

In immunohistochemistry for DNMT1, nuclear immunoreactivity was not detected in any of the non-cancerous epithelia, except in proliferative zones, but was frequently found in stomach cancers.63 DNMT1 overexpression, at both the mRNA64 and protein63 levels, correlated significantly with poorer tumor differentiation and with CpG island methylator phenotype (CIMP), defined by frequent DNA hypermethylation of C-type CpG islands,65 in stomach cancers. The hMLH1, thrombospondin-1 (THBS-1) and E-cadherin genes may be targets for overexpressed DNMT1 in stomach cancers.63 Four percent of the examined patients with stomach cancers had EBV infection, a potential etiological factor in gastric carcinogenesis, in their cancer cells, and all cancers with EBV infection had DNMT1 protein overexpression.63 Induction of latent membrane protein 1 of EBV has been reported to induce overexpression of DNMT1 in cultured cancer cells.66 EBV infection in stomach cancers was associated with marked accumulation of DNA hypermethylation of C-type CpG islands.63 With respect to stomach carcinogenesis, Helicobacter pylori infection, another etiological factor, is known to strongly promote regional DNA hypermethylation.67

Cervical intra-epithelial neoplasia (CIN) is a precursor lesion for squamous cell carcinoma of the uterine cervix closely associated with HPV infection. DNMT1 protein expression is increased even in low-grade CIN relative to normal squamous epithelium, and further increased in higher-grade CIN and squamous cell carcinomas of the uterine cervix.68 HPV-16 E7 protein has been reported to associate directly with DNMT1 and stimulate the enzyme activity of DNMT1 in vitro,69 and accumulation of DNA hypermethylation on tumor-related genes has also been observed during cervical carcinogenesis.

PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

Cumulative DNA methylation of tumor-related genes

In the same way that HCC 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. DNA methylation status of the p14, p15, p16, p73, adenomatous polyposis coli (APC), hMLH1, O-6-methylguanine-DNA methyltransferase (MGMT), breast cancer 1 (BRCA1), glutathione S-transferase pi (GSTP1), tissue inhibitor of metalloproteinase 3 (TIMP-3), E-cadherin, and death-associated protein kinase 1 (DAPK-1) tumor-related genes was examined on agarose bead-embedded methylation-specific PCR, which had been developed for DNA methylation analysis of tiny microdissected tissue specimens (Fig. 2e).35 The incidence of DNA hypermethylation of at least one of the genes and the average number of methylated genes were significantly higher in peripheral pancreatic duct epithelia with an inflammatory background and in another precancerous lesion, pancreatic intra-epithelial neoplasia (PanIN), than in peripheral pancreatic duct epithelia without an inflammatory background, and was further increased in ductal carcinomas (Fig. 5a).70 The BRCA1, APC, p16 and TIMP-3 genes are frequently methylated in ductal carcinomas of the pancreas.70

image

Figure 5. (a) Alterations of DNA methylation during pancreatic carcinogenesis. When DNA methylation status of the p14, p15, p16, p73, APC, hMLH1, MGMT, BRCA1, GSTP1, TIMP-3, E-cadherin and DAPK-1 tumor-related genes was examined in microdissected specimens, the incidence of DNA hypermethylation of at least one of the genes and the average number of methylated genes were significantly higher in peripheral pancreatic duct epithelia with an inflammatory background (DEI) and pancreatic intra-epithelial neoplasia (PanIN) than in peripheral pancreatic duct epithelia without an inflammatory background (DE), and were further increased in ductal carcinomas (Ca).70 (b) Incidence of nuclear DNA methyltransferase 1 (DNMT1) immunoreactivity was significantly elevated in DEI and PanIN than in DE.71 The incidence of nuclear DNMT1 immunoreactivity was significantly associated with the degree of PanIN dysplasia (PanIN I vs Pan IN II). The incidence of nuclear DNMT1 immunoreactivity was significantly higher in ductal carcinomas than in PanIN, and was associated with poorer differentiation of ductal carcinomas (MD, moderately differentiated adenocarcinoma; PD, poorly differentiated adenocarcinoma; WD, well-differentiated adenocarcinoma). Heterogeneity of DNMT1 protein expression among components having different grades of histological differentiation was observed in a representative cancer from a single patient:71 (d) Moderately and (e) poorly differentiated adenocarcinoma components had a higher incidence of DNMT1 immunoreactivity than (c) the well-differentiated adenocarcinoma component. (f) The average number of methylated tumor-related genes in microdissected specimens of ductal carcinomas was significantly correlated with the expression level of DNMT1 protein examined on immunohistochemistry in the precisely microdissected areas.70

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Overexpression of DNMT1

When examined on immunohistochemistry, the incidence of nuclear DNMT1 immunoreactivity was significantly elevated in peripheral pancreatic ductal epithelia with an inflammatory background and PanIN than in peripheral pancreatic ductal epithelia without an inflammatory background (Fig. 5b).71 With respect to inflammation-related carcinogenesis,72 treatment with the cytokine interleukin-6 has been reported to induce overexpression of DNMT1 in cultured cells.73 The incidence of nuclear DNMT1 immunoreactivity was significantly associated with the degree of PanIN dysplasia (Fig. 5b), being significantly higher in invasive ductal carcinomas than in PanIN, and was associated with poorer differentiation of invasive ductal carcinomas (Fig. 5b–e).71 Protein overexpression of DNMT1 in ductal carcinomas is significantly correlated with the extent of cancer invasion to surrounding organs and with advanced stage,71 suggesting that overexpression of DNMT1 is associated with aggressiveness of pancreatic cancers. Moreover, patients with ductal carcinomas of the pancreas having overexpression of DNMT1 protein have a poorer outcome.71

The average number of methylated tumor-related genes in microdissected specimens of invasive ductal carcinoma was significantly correlated with the expression level of DNMT1 protein examined on immunohistochemistry in the precisely microdissected areas (Fig. 5f).70 Thus DNMT1 may be responsible for de novo methylation of CpG islands during pancreatic carcinogenesis. A theoretical explanation for the role of DNMT1 in de novo DNA methylation in human cancers with dysfunction of p21WAF1,74 which competes with DNMT1 for binding with PCNA, has been proposed.15 Moreover, although maintenance activities of DNMT1 have been noticed in vitro in relation to its preference for hemi-methylated substrates, it has recently been suggested that DNMT1 is capable of de novo DNA methylating activity in vivo.18,19 Therefore, it is feasible that, in cancers, overexpression of DNMT1 participates in regional DNA hypermethylation.

LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

In addition to chronic inflammation and/or persistent infection with pathogenic microorganisms, cigarette smoking is another background factor associated with alterations of DNA methylation during multistage carcinogenesis. DNA hypermethylation at the D17S5 locus has been frequently observed in non-cancerous lung tissues, which may contain progenitor cells for cancers, obtained from patients with non-small-cell lung cancers, and in corresponding non-small-cell lung cancers.75 The incidence of DNA hypermethylation at the D17S5 locus is significantly associated with poorer differentiation of lung adenocarcinomas.75 The incidence of DNA hypermethylation in both non-cancerous lung tissues and non-small-cell lung cancers of patients who are current smokers is significantly higher than in patients who have never smoked.75 The extent of pulmonary anthracosis is an index for the cumulative effects of smoking. The extent of pulmonary anthracosis in each resected lung has been graded macroscopically: grade 1, slight accumulation of charcoal particles in the intra-lobular lymphatics forming a fine reticular pattern scattered in the visceral pleura; grade 2, reticular pattern due to charcoal particle accumulation is denser and shows fusion in places; and grade 3, dense accumulation of charcoal particles is present throughout most of the visceral pleura. The incidence of DNA hypermethylation at the D17S5 locus analyzed on Southern blot using a DNA methylation-sensitive restriction enzyme in non-cancerous lung tissues showing grade 3 anthracosis obtained from patients with non-small-cell lung cancers was higher than that in patients with grade 2 or 1 anthracosis.30 The molecular mechanisms by which carcinogens related to cigarette smoking affect DNA methylation status should be investigated.

UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

DNMT1 overexpression is not always a secondary result of increased cell proliferative activity but correlated with regional DNA hypermethylation

Urothelial carcinomas of the urinary bladder are clinically remarkable because of their multicentricity and tendency to recur: synchronously or metachronously multifocal urothelial carcinomas 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 with no remarkable histology obtained from patients with urinary bladder cancers can be considered precancerous, because they may be exposed to carcinogens in the urine. DNMT1 protein expression is significantly higher in non-cancerous urothelia having no remarkable histology obtained from patients with urinary bladder cancers than in normal urothelia obtained from patients without urinary bladder cancers, and further increases from dysplastic urothelia to urothelial carcinoma.76 Thus progressively increasing DNMT1 protein expression is associated with multistage urothelial carcinogenesis from precancerous stages.

In contrast, DNMT1 mRNA is expressed mainly during the 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 in non-cancerous urothelia having no remarkable histology obtained from patients with urinary bladder cancers, for which the PCNA labeling index had not yet increased, indicating that overexpression of DNMT1 is not a secondary result of increased cell proliferative activity but precedes increased cell proliferative activity during multistage urothelial carcinogenesis.76 Excessive amounts of DNMT1 compared to PCNA, which targets DNMT1 to replication foci, may participate in de novo methylation of CpG islands. Indeed, among all examined microdissected specimens of non-cancerous urothelia having no remarkable histology obtained from patients with urinary bladder cancers, dysplastic urothelia and urothelial carcinomas, concurrent DNA hypermethylation of three or more examined C-type CpG islands was significantly correlated with overexpression of DNMT1 protein.77 Further examination is required to clarify the molecular mechanisms of recruitment of DNMT1 in a sequence-specific manner during carcinogenesis.

Alterations of DNA methylation participate particularly in the development of nodular invasive carcinomas via widely spreading carcinomas in situ

Although the incidence and intensity of nuclear DNMT1 immunoreactivity are correlated significantly with the histological grade of urothelial carcinomas, they appear not to be simply correlated with the depth of invasion.76 Therefore, the morphological structures of urothelial carcinomas were examined (Fig. 6a). Urinary bladder cancers are classified as papillary or nodular according to their macroscopic configuration. Papillary carcinomas usually remain non-invasive, although patients need to undergo repeated cystoscopic resections because of recurrences. In contrast, the clinical outcome of nodular invasive carcinomas is poor. Flat carcinomas in situ, which frequently spread widely and are sometimes scattered all over the urinary tract, are considered to be a precursor lesion of nodular invasive carcinomas.78 In flat carcinomas in situ and nodular invasive carcinomas, the incidence and intensity of nuclear DNMT1 immunoreactivity were significantly higher than those in papillary tumors (Fig. 6a).76 The incidence of CIMP in flat carcinomas in situ and nodular invasive carcinomas was significantly higher than that in papillary tumors.77 These data suggest that DNMT1 protein overexpression resulting in regional DNA hypermethylation may be associated with the distinct pathway leading to the development of nodular invasive carcinomas with aggressive clinical courses via widely spreading carcinomas in situ. Furthermore, even in an invasive carcinoma, cancer cells occasionally have particularly strong nuclear DNMT1 immunoreactivity at the invading front (Fig. 6b) or in involved lymphatic vessels (Fig. 6c).76 Therefore, there appears to be a mechanism regulating DNMT1 protein expression, possibly one that depends on cancer–stromal interactions and/or the microenvironment of cancer cells, other than the mechanism associated with the sequence of progression of flat carcinomas in situ to nodular invasive carcinomas.

image

Figure 6. Immunohistochemistry of DNA methyltransferase 1 (DNMT1) in urothelial carcinomas. (a) The incidence and intensity of nuclear DNMT1 immunoreactivity were significantly higher in nodular invasive carcinomas with aggressive clinical courses and carcinomas in situ, which are considered to be precursor lesions of nodular invasive carcinoma, than in papillary carcinomas, which usually remain non-invasive.76+, >30% of the cells had the same nuclear staining intensity as the positive internal control lymphocytes; ++, >30% of the cells had stronger intensity. (b) In invasive carcinomas, cancer cells occasionally have particularly strong nuclear DNMT1 immunoreactivity at the invading front (arrows) or (c) in involved lymphatic vessels.76 (*) Center of the cancer nest.

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DNA hypomethylation in pericentromeric satellite regions significantly correlates with chromosomal instability

DNA hypomethylation on satellites 2 and 3 was frequently detected in urothelial carcinomas of the urinary bladder, the ureter or the renal pelvis.79 In almost all the carcinoma samples in which DNA hypomethylation was detected, hypomethylation occurred on both satellites 2 and 3. The incidence of DNA hypomethylation in pericentromeric satellite regions was significantly correlated with histological grade of urothelial carcinomas and was higher in nodular invasive carcinomas than in papillary tumors.79 Satellites 2 and 3 are abundant in pericentromeric heterochromatin regions on chromosome 9. The incidence of LOH on chromosome 9 was significantly correlated with the histological grade of urothelial carcinomas and was higher in nodular invasive carcinomas than in papillary tumors.79 DNA hypomethylation in pericentromeric satellite regions was significantly correlated with the presence of LOH on chromosome 9 in urothelial carcinomas,79 suggesting that DNA hypomethylation inducing chromosomal instability may participate in urothelial carcinogenesis.

RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

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 association with chronic inflammation and persistent infection with viruses or other pathogenic microorganisms, as described for the liver, stomach, uterine cervix and pancreas. Unlike cancers derived from these organs, precancerous conditions in the kidney have been rarely described. Surprisingly, even in non-cancerous renal tissues having no remarkable histology obtained from patients with renal cell carcinomas (RCC), the average number of methylated CpG islands was significantly higher than in normal renal tissues obtained from patients without renal tumors, and was even higher in RCC (Fig. 7a).80 From the viewpoint of alterations of DNA methylation, the presence of precancerous conditions can be recognized even in the kidney. Regional DNA hypermethylation participates in the early and precancerous stage of multistage renal carcinogenesis. In addition, accumulation of DNA methylation at CpG islands in conventional RCC is significantly correlated with higher histological grade, an infiltrating growth pattern, and vascular involvement,80 suggesting that regional DNA hypermethylation is continuously involved in malignant progression. The recurrence-free survival rate of patients with conventional RCC having accumulated DNA methylation of CpG islands was significantly lower than that of patients with conventional RCC lacking this feature.80

image

Figure 7. Precancerous conditions with alterations of DNA methylation generate more malignant renal cell carcinomas (RCC). (a) Even though non-cancerous renal tissues obtained from patients with renal cell carcinomas lacked remarkable histology (N), the average number of methylated CpG islands was significantly higher in N than in normal renal tissues obtained from patients without renal tumors (C).80 The average number of methylated CpG islands was even higher in renal cell carcinomas (T).80 (b) The average number of methylated CpG islands in N was significantly correlated with a higher histological grade of corresponding renal cell carcinomas developing in individual patients,80 suggesting that regional DNA hypermethylation in precancerous conditions generates more malignant renal cell carcinomas. (c) Example of hybridization in the bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA) method using MCG Whole Genome Array-4500 constructed by Dr Johji Inazawa, Tokyo Medical and Dental University, Tokyo, Japan and Dr Misao Ohki and Dr Fumie Hosoda, National Cancer Center Research Institute, Tokyo, Japan. This technique allows high resolution and genome-wide analysis of DNA methylation status, and suggests that the future development of more malignant RCC is determined by the genome-wide DNA methylation profile at the precancerous stage.

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Genome-wide analysis indicates the significance of alterations of DNA methylation in precancerous conditions

The average number of methylated CpG islands in non-cancerous renal tissues lacking remarkable histology obtained from patients with conventional RCC was significantly correlated with a higher histological grade of corresponding RCC developing in individual patients (Fig. 7b).80 These are striking data suggesting the possibility that regional DNA hypermethylation in precancerous conditions generates more malignant RCC. Examination of the DNA methylation status of only a restricted number of C-type CpG islands, however, cannot clarify whether DNA methylation status in restricted regions is simply altered in the precancerous stage or whether the genome-wide DNA methylation profile in the precancerous stage determines the clinicopathological characteristics of developing cancers. Recently, several techniques such as restriction landmark genomic scanning, methylation-sensitive representational difference analysis, methylated CpG island amplification and array-based technology have been developed for genome-wide analysis of DNA methylation status.81–85 For DNA methylation profiling during multistage renal carcinogenesis, we used bacterial artificial chromosome (BAC) array-based methylated CpG island amplification (BAMCA, Fig. 7c).86 Many researchers use arrays in which the promoter regions are enriched, as probes for cloning genes that are differentially methylated between cancer cells and normal cells. But the promoter regions of specific genes are not the only target of alterations of DNA methylation in human cancers. DNA methylation status in genomic regions not affecting the expression of specific genes may be altered before the alterations of the promoter regions themselves at the precancerous stage. Genomic regions in which DNA methylation status affects chromosomal instability are not contained in promoter-arrays. To obtain a high-resolution genome-wide overview of DNA methylation status, the BAMCA method was used, focusing not only on the promoter regions. In non-cancerous renal tissues lacking remarkable histology, obtained from patients with conventional RCC, the number of BAC clones having DNA hypermethylation or hypomethylation exceeding individual variation was significantly correlated with the recurrence-free survival rate of patients after nephrectomy for established RCC (Arai E. et al., unpubl. data, 2008). These data suggest that the future development of more malignant RCC is determined by the genome-wide DNA methylation profile at the precancerous stage.

PERSPECTIVES

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES

In earlier days it was thought, mistakenly, that alterations of DNA methylation occurred only as a result of cancerization. But because alterations of DNA methylation occur even in the precancerous stage before the establishment of cancer and determine the clinicopathological characteristics of the developing malignancies, it is clear that they are not a secondary result of cancerization. Precancerous conditions involving alterations of DNA methylation may generate more malignant cancers. Alterations of DNA methylation are frequently observed in precancerous conditions associated with chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, such as HBV or HCV, EBV, HPV and Helicobacter pylori, or with cigarette smoking. When estimating carcinogenetic risk using DNA methylation profiles as indicators, elimination of such etiological factors may be efficient for cancer prevention. Moreover, DNA methylation is reversible, thus differing from genetic events during multistage carcinogenesis, and regional DNA hypermethylation can be corrected by passive demethylation with demethylating agents. Therefore, carcinogenetic risk estimation may pave the way to chemoprevention of precancerous conditions characterized by alterations of DNA methylation.

Early diagnosis of cancers using DNA methylation profiles as indicators is also a promising avenue. For this purpose, less invasive methodologies for detecting subtle alterations of DNA methylation should be developed for serum, urine, sputum and other body fluid samples. Because even subtle alterations of DNA methylation in the early stage are stably preserved on DNA double strands by covalent bonds, alterations of DNA methylation on appropriate genes and/or DNA fragments may be better indicators for early diagnosis than levels of mRNA and protein expression of specific genes that can be easily affected by the microenvironment of precursor cells or cancer cells.

Alterations of DNA methylation seem to continuously participate in the malignant progression of cancers by inducing chromosomal instability and silencing tumor-suppressor genes. Overexpression of DNMT1 is not a secondary result of increased cell proliferative activity but is significantly correlated with CIMP. DNA methylation profiles of not only normal tissues but also cancers tend to be organ-specific, and hot spots of DNA hypermethylation may reflect the diversity of carcinogenetic factors. The molecular mechanisms responsible for determination of the target genes of CIMP should be further clarified. Because alterations of DNA methylation and overexpression of DNMT1 are significantly associated with poorer tumor differentiation, tumor aggressiveness, and poorer patient outcome, estimation of DNA methylation status or immunohistochemistry of DNMT1 in biopsy specimens and/or surgically resected materials may also become a useful tool for prognostication. In contrast, splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation in pericentromeric satellite regions.

Recently developed technologies for accessing genome-wide DNA methylation status will be useful for identifying the DNA methylation profile, which is the optimum indicator of prognosis. Reactivation of tumor-suppressor genes by demethylating agents can provide a strategy of cancer therapy.87 But overall DNA hypomethylation and regional DNA hypermethylation are commonly observed during multistage carcinogenesis, and they do not seem mutually exclusive in an individual patient. Therefore, before using DNA demethylation agents for prevention or therapy of cancers, it will be necessary to carefully identify patients who might benefit from this type of demethylation strategy. Accessing genome-wide DNA methylation status again seems indispensable for identification of patients whose cancers have a demethylating agent-sensitive DNA methylation profile.

REFERENCES

  1. Top of page
  2. Abstract
  3. DNA METHYLATION AND HUMAN CANCERS
  4. HEPATOCARCINOGENESIS IN LIVERS DAMAGED BY HEPATITIS VIRUS INFECTION
  5. VIRUS INFECTION-ASSOCIATED CARCINOGENESIS IN THE STOMACH AND THE UTERINE CERVIX
  6. PANCREATIC CARCINOGENESIS ASSOCIATED WITH PERSISTENT INFLAMMATION
  7. LUNG CARCINOGENESIS ASSOCIATED WITH CIGARETTE SMOKING
  8. UROTHELIAL CARCINOGENESIS SHOWING MULTICENTRICITY AND TENDENCY TO RECUR
  9. RENAL CARCINOGENESIS: PRECANCEROUS CONDITIONS WITH ALTERATIONS OF DNA METHYLATION GENERATE MORE MALIGNANT CANCERS
  10. PERSPECTIVES
  11. ACKNOWLEDGMENTS
  12. REFERENCES