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

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite‐PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin‐1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.

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

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

[3]  N. Matsubara,et al.  Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability. , 1998, Cancer research.

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

[5]  R. Weinberg,et al.  E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

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

[7]  G. Shiota,et al.  Loss of the tumor suppressor p53 gene at the liver cirrhosis stage in Japanese patients with hepatocellular carcinoma. , 1997, Oncology.

[8]  S Srivastava,et al.  A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. , 1998, Cancer research.

[9]  N. Ahuja,et al.  Methylation and silencing of the Thrombospondin-1 promoter in human cancer , 1999, Oncogene.

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

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

[12]  W. Lau,et al.  High frequency of p16INK4A gene alterations in hepatocellular carcinoma , 1999, Oncogene.

[13]  E. Viégas-Péquignot,et al.  Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin. , 1993, Cytogenetics and cell genetics.

[14]  J. Battey,et al.  L-myc, a new myc-related gene amplified and expressed in human small cell lung cancer , 1985, Nature.

[15]  M. Loda,et al.  Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. , 1997, Cancer research.

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

[17]  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.

[18]  J. Herman,et al.  5′ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers , 1995, Nature Medicine.

[19]  P. Zimmerman,et al.  MYCL genotypes and loss of heterozygosity in non-small-cell lung cancer. , 1996, British Journal of Cancer.

[20]  S. Baylin,et al.  DNA hypermethylation is associated with 17p allelic loss in neural tumors. , 1993, Cancer research.

[21]  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.

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

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

[24]  S. Baylin,et al.  Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. , 1999, Cancer research.

[25]  P. Ganly,et al.  p53 and chromosome 3 abnormalities, characteristic of malignant lung tumours, are detectable in preinvasive lesions of the bronchus. , 1992, Oncogene.

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

[27]  J. Herman,et al.  Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G. Gyapay,et al.  A second-generation linkage map of the human genome , 1992, Nature.

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

[30]  H. Asakura,et al.  p16INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma , 1999 .

[31]  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.

[32]  M. Buendia,et al.  Comprehensive allelotyping of human hepatocellular carcinoma , 1997, Oncogene.

[33]  S. Hirohashi,et al.  Different mutations of the p53 gene in nodule-in-nodule hepatocellular carcinoma as a evidence for multistage progression. , 1994, Cancer letters.

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

[35]  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.

[36]  T. Dryja,et al.  Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. , 1991, American journal of human genetics.

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

[38]  W. Doerfler The insertion of foreign DNA into mammalian genomes and its consequences: a concept in oncogenesis. , 1995, Advances in cancer research.

[39]  J. Herman,et al.  Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. , 1995, Cancer research.

[40]  W. Doerfler,et al.  Insertion of Foreign DNA into an Established Mammalian Genome Can Alter the Methylation of Cellular DNA Sequences , 1999, Journal of Virology.

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

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

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

[44]  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.