p16(INK4) is inactivated by extensive CpG methylation in human hepatocellular carcinoma

Gastroenterology. 1999 Feb;116(2):394-400. doi: 10.1016/s0016-5085(99)70137-x.


Background & aims: The molecular status of the p16(INK4) tumor-suppressor gene has not been fully elucidated in hepatocellular carcinoma. The aim of this study was to clarify the mechanism that gives rise to inactivation of p16(INK4) in hepatocellular carcinoma.

Methods: The status of p16(INK4) was evaluated in 60 hepatocellular carcinomas by immunohistochemical staining, differential polymerase chain reaction, single-strand conformational polymorphism, methylation-specific polymerase chain reaction, and methylation-sensitive single nucleotide primer extension.

Results: Immunohistochemical staining showed that 29 of the 60 tumors exhibited complete loss of p16(INK4) expression. High levels of DNA methylation were detected in 24 of 29 cases of hepatocellular carcinoma with negative p16(INK4) expression, with methylation of 60%-85% of the CpG islands. In contrast, the level of methylation was <25% in tumors with faint p16(INK4) staining, and no methylation was detected in tumors with positive immunostaining. Intragenic alteration of p16(INK4) was detected in 4 cases.

Conclusions: A strong correlation was found between the extent of methylation and the degree of expression of p16(INK4) in tumor tissues, indicating that epigenetic change due to extensive CpG methylation is the main cause of inactivation of p16(INK4) in hepatocellular carcinoma.

MeSH terms

  • Adult
  • Aged
  • Carcinoma, Hepatocellular / genetics
  • Carcinoma, Hepatocellular / metabolism*
  • CpG Islands*
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism*
  • DNA Primers
  • DNA, Neoplasm / analysis
  • Female
  • Gene Expression Regulation, Neoplastic
  • Genes, p16*
  • Humans
  • Immunohistochemistry
  • Liver Neoplasms / genetics
  • Liver Neoplasms / metabolism*
  • Male
  • Methylation
  • Middle Aged
  • Polymerase Chain Reaction
  • Polymorphism, Single-Stranded Conformational


  • Cyclin-Dependent Kinase Inhibitor p16
  • DNA Primers
  • DNA, Neoplasm