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

To evaluate the significance of alterations in DNA methylation during human hepatocarcinogenesis, we examined levels of mRNA for DNA methyltransferases and methyl‐CpG‐binding proteins and the DNA methylation status in 67 hepatocellular carcinomas (HCCs). The average level of mRNA for DNMT1 and DNMT3a was significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis than in histologically normal liver tissues, and was even higher in HCCs. Significant overexpression of DNMT3b and reduced expression of DNMT2 were observed in HCCs compared with the corresponding noncancerous liver tissues. DNA hypermethylation on CpG islands of the p16 (8% and 66%) and hMLH1 (0% and 0%) genes and methylated in tumor (MINT) 1 (6% and 34%), 2 (24% and 58%), 12 (21% and 33%), 25 (0% and 5%), and 31 (0% and 23%) clones, and DNA hypomethylation on satellites 2 and 3 (18% and 67%), were detected in noncancerous liver tissues and HCCs, respectively. There was no significant correlation between the expression level of any DNA methyltransferase and DNA methylation status. Reduced expression of DNA repair protein, MBD4, was significantly correlated with poorer tumor differentiation and involvement of portal vein. Slightly reduced expression of MBD2 was detected in HCCs, and the expression of MeCP2 was particularly reduced in HCCs with portal vein involvement. These data suggest that overexpression of DNMT1 and DNMT3a, DNA hypermethylation on CpG islands, and DNA hypomethylation on pericentromeric satellite regions are early events during hepatocarcinogenesis, and that reduced expression of MBD4 may play a role in malignant progression of HCC.

[1]  S. Hirohashi,et al.  p53 gene mutation spectrum in hepatocellular carcinoma. , 1992, Cancer research.

[2]  S. Baylin,et al.  Regional DNA hypermethylation at D17S5 precedes 17p structural changes in the progression of renal tumors. , 1993, Cancer research.

[3]  E. Ballestar,et al.  Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation , 1999, Nature Genetics.

[4]  S. Baylin,et al.  Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. , 1999, Cancer research.

[5]  D. Haber,et al.  DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development , 1999, Cell.

[6]  S. Hirohashi,et al.  DNA hypermethylation at the D17S5 locus and reduced HIC‐1mRNA expression are associated with hepatocarcinogenesis , 1999, Hepatology.

[7]  H. Ng,et al.  Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. , 1997, Science.

[8]  C. Wijmenga,et al.  The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Hirohashi,et al.  Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis. , 2000, Cancer letters.

[10]  O. Hino,et al.  Clonal growth of hepatitis B virus-integrated hepatocytes in cirrhotic liver nodules. , 1992, Cancer research.

[11]  P. S. Neumaier,et al.  Recognition sequences of restriction endonucleases and methylases--a review. , 1985, Gene.

[12]  A. Bird,et al.  The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites , 1999, Nature.

[13]  Z. Siegfried,et al.  Spl elements protect a CpG island from de novo methylation , 1994, Nature.

[14]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[15]  Peter A. Jones,et al.  Cancer-epigenetics comes of age , 1999, Nature Genetics.

[16]  Christine Pourcel,et al.  Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma , 1980, Nature.

[17]  Rudolf Jaenisch,et al.  DNA hypomethylation leads to elevated mutation rates , 1998, Nature.

[18]  M. Nakao,et al.  Methylation-Mediated Transcriptional Silencing in Euchromatin by Methyl-CpG Binding Protein MBD1 Isoforms , 1999, Molecular and Cellular Biology.

[19]  S. Hirohashi,et al.  The E‐cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas , 1997, International journal of cancer.

[20]  Peter A. Jones,et al.  High frequency mutagenesis by a DNA methyltransferase , 1992, Cell.

[21]  E. Li,et al.  Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells. , 1998, Nucleic acids research.

[22]  H. Novotná,et al.  Hypomethylation of CCGG sites in the 3' region of H-ras protooncogene is frequent and is associated with H-ras allele loss in non-small cell lung cancer. , 1994, Cancer research.

[23]  S. Hirohashi,et al.  Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[24]  S. Hirohashi,et al.  DNA hypermethylation at the D17S5 locus in non-small cell lung cancers: its association with smoking history. , 1997, Cancer research.

[25]  A. Bird,et al.  Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. , 1999, Genes & development.

[26]  W. Doerfler,et al.  Chromosomal insertion of foreign (adenovirus type 12, plasmid, or bacteriophage lambda) DNA is associated with enhanced methylation of cellular DNA segments. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Colin A. Johnson,et al.  Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex , 1998, Nature.

[28]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[29]  H. Hasegawa,et al.  Clonal origin of atypical adenomatous hyperplasia of the liver and clonal identity with hepatocellular carcinoma. , 1988, Gastroenterology.

[30]  J. Herman,et al.  Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  S. Hirohashi,et al.  Microsatellite instability associated with hepatocarcinogenesis. , 1999, Journal of hepatology.

[32]  Paul Tempst,et al.  MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex , 1999, Nature Genetics.

[33]  M. Ehrlich,et al.  The 5-methylcytosine content of DNA from human tumors. , 1983, Nucleic acids research.

[34]  S. Hirohashi,et al.  DNA hypermethylation at the D17S5 locus is associated with gastric carcinogenesis. , 1998, Cancer letters.

[35]  S. Hirohashi,et al.  Increased DNA Methyltransferase Expression Is Associated with an Early Stage of Human Hepatocarcinogenesis , 1997, Japanese journal of cancer research : Gann.

[36]  A. Bird,et al.  Purification, sequence, and cellular localization of a novel chromosomal protein that binds to Methylated DNA , 1992, Cell.

[37]  J. Herman,et al.  CpG island methylator phenotype in colorectal cancer. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[38]  T. Bestor,et al.  Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain. , 1992, The EMBO journal.

[39]  S. Hirohashi,et al.  DNA methyltransferase expression and DNA methylation of CPG islands and peri‐centromeric satellite regions in human colorectal and stomach cancers , 2001, International journal of cancer.

[40]  J. Strouboulis,et al.  Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription , 1998, Nature Genetics.

[41]  S. Hirohashi,et al.  Aberrant DNA Methylation on Chromosome 16 Is an Early Event in Hepatocarcinogenesis , 1996, Japanese journal of cancer research : Gann.

[42]  S. Hirohashi,et al.  Reduced mRNA expression of the DNA demethylase, MBD2, in human colorectal and stomach cancers. , 1999, Biochemical and biophysical research communications.

[43]  E W Geddes,et al.  Establishment of a continuously growing cell line from primary carcinoma of the liver. , 1976, South African medical journal = Suid-Afrikaanse tydskrif vir geneeskunde.

[44]  P. Laird,et al.  COBRA: a sensitive and quantitative DNA methylation assay. , 1997, Nucleic acids research.

[45]  T. Bestor,et al.  A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast. , 1998, Human molecular genetics.

[46]  M. Szyf,et al.  A mammalian protein with specific demethylase activity for mCpG DNA , 1999, Nature.

[47]  P. Laird,et al.  CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression. , 1999, Cancer research.

[48]  J. Herman,et al.  Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[49]  S. Hirohashi,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 , 2000, Hepatology.

[50]  K. Robertson,et al.  The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. , 1999, Nucleic acids research.

[51]  E. Li,et al.  Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases , 1998, Nature Genetics.

[52]  B. Trask,et al.  Isolation and characterization of the cDNA encoding human DNA methyltransferase. , 1992, Nucleic acids research.

[53]  N. Tommerup,et al.  Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene , 1999, Nature.

[54]  Y. Wataya,et al.  Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. , 1970, Biochemistry.

[55]  A. Bird,et al.  Identification and Characterization of a Family of Mammalian Methyl-CpG Binding Proteins , 1998, Molecular and Cellular Biology.

[56]  S. Baylin Tying It All Together: Epigenetics, Genetics, Cell Cycle, and Cancer , 1997, Science.

[57]  R. Jaenisch,et al.  De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells. , 1996, Development.

[58]  Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs , 1989, Cell.