Progression of Hypermethylation of the p16 INK4A Gene from Normal Liver to Nontumorous Liver and Hepatocellular Carcinoma: An Evaluation Using Quantitative PCR Analysis

  • So Kurita
  • Shogo Ohkoshi
  • Masahiko Yano
  • Kazuhide Yamazaki
  • Kenta Suzuki
  • Yo-hei Aoki
  • Yasunobu Matsuda
  • Toshifumi Wakai
  • Yoshio Shirai
  • Takafumi Ichida
  • Yutaka Aoyagi
Original Article


The aim of this study was to determine to what extent hypermethylation of the p16 INK4A (p16) gene promoter is increased in nontumorous liver tissues compared with in normal liver, using two quantitative methylation-specific polymerase chain reaction (MS-PCR) methods and a bisulfite sequencing method. Methylation of the p16 gene was detected more frequently in nontumorous liver than in normal liver using the TaqMan PCR method. Methylation indices also were significantly higher in nontumorous than in normal liver. However, the bisulfite sequencing method did not detect significantly more methylation of the p16 gene in nontumorous than normal liver, nor was there a significant difference in the level of p16 mRNA. There may be a greater proportion of cells which contain methylated p16 in nontumorous than in normal liver. However, the difference was so small that the functional relevance to hepatocarcinogenesis remains elusive.


p16INK4A Methylation-specific PCR (MS-PCR) Real-time quantitative PCR Bisulfite sequencing Nontumorous liver Normal liver 


  1. 1.
    Okuda K. Hepatocellular carcinoma: recent progress. Hepatology. 1992;15:948–963.PubMedCrossRefGoogle Scholar
  2. 2.
    Shiratori Y, Yoshida H, Omata M. Different clinicopathological features of hepatocellular carcinoma in relation to causative agents. J Gastroenterol. 2001;36:73–78.PubMedCrossRefGoogle Scholar
  3. 3.
    Feitelson MA, Sun B, Satiroglu Tufan NL, Liu J, Pan J, Lian Z. Genetic mechanisms of hepatocarcinogenesis. Oncogene. 2002;21:2593–2604.PubMedCrossRefGoogle Scholar
  4. 4.
    Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer. 2006;6:674–687.PubMedCrossRefGoogle Scholar
  5. 5.
    Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res. 1998;72:141–196.PubMedCrossRefGoogle Scholar
  6. 6.
    Jones PA, Baylin SB. The fundamental role of epigenetic events in cancer. Nat Rev Genet. 2002;3:415–428.PubMedCrossRefGoogle Scholar
  7. 7.
    Bird A. The essentials of DNA methylation. Cell. 1992;70:5–8.PubMedCrossRefGoogle Scholar
  8. 8.
    Belinsky SA, Nikula KJ, Palmisano WA, et al. Aberrant methylation of p16 INK4a is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci USA. 1998;95:11891–11896.PubMedCrossRefGoogle Scholar
  9. 9.
    Hsieh CJ, Klump B, Holzmann K, Brochard F, Gregor M, Porschen R. Hypermethylation of the p16INK4a promoter in colectomy specimens of patients with long-standing and extensive ulcerative colitis. Cancer Res. 1998;58:3942–3945.PubMedGoogle Scholar
  10. 10.
    Klump B, Hsieh CJ, Holzmann K, Gregor M, Porschen R. Hypermethylation of the CDKN2/p16 promoter during neoplastic progression in Barrett’s esophagus. Gastroenterology. 1998;115:1381–1386.PubMedCrossRefGoogle Scholar
  11. 11.
    Wilentz RE, Geradts J, Maynard R, et al. Inactivation of the p16 (INK4A) tumor-suppressor gene in pancreatic duct lesions: loss of intranuclear expression. Cancer Res. 1998;58:4740–4744.PubMedGoogle Scholar
  12. 12.
    Lee JH, Park SJ, Abraham SC, et al. Frequent CpG island methylation in precursor lesions and early gastric adenocarcinomas. Oncogene. 2004;23:4646–4654.PubMedCrossRefGoogle Scholar
  13. 13.
    Kaneto H, Sasaki S, Yamamoto H, et al. Detection of hypermethylation of the p16 INK4A gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut. 2001;48:372–377.PubMedCrossRefGoogle Scholar
  14. 14.
    Lee S, Lee HJ, Kim JH, Lee HS, Jang JJ, Kang GH. Aberrant CpG island hypermethylation along multistep hepatocarcinogenesis. Am J Pathol. 2003;163:1371–1378.PubMedGoogle Scholar
  15. 15.
    Di Gioia S, Bianchi P, Destro A, et al. Quantitative evaluation of RASSF1A methylation in the non-lesional, regenerative and neoplastic liver. BMC Cancer. 2006;6:89–100.PubMedCrossRefGoogle Scholar
  16. 16.
    Lo YM, Wong IH, Zhang J, Tein MS, Ng MH, Hjelm NM. Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction. Cancer Res. 1999;59:3899–3903.PubMedGoogle Scholar
  17. 17.
    Hoque MO, Begum S, Topaloglu O, et al. Quantitative detection of promoter hypermethylation of multiple genes in the tumor, urine, and serum DNA of patients with renal cancer. Cancer Res. 2004;64:5511–5517.PubMedCrossRefGoogle Scholar
  18. 18.
    Lehmann U, Berg-Ribbe I, Wingen LU, et al. Distinct methylation patterns of benign and malignant liver tumors revealed by quantitative methylation profiling. Clin Cancer Res. 2005;11:3654–3660.PubMedCrossRefGoogle Scholar
  19. 19.
    Harden SV, Tokumaru Y, Westra WH, et al. Gene promoter hypermethylation in tumors and lymph nodes of stage I lung cancer patients. Clin Cancer Res. 2003;9:1370–1375.PubMedGoogle Scholar
  20. 20.
    Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson DA. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. Nature. 1994;368:753–756.PubMedCrossRefGoogle Scholar
  21. 21.
    Serrano M, Hannon GJ, Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993;366:704–707.PubMedCrossRefGoogle Scholar
  22. 22.
    Baek MJ, Piao Z, Kim NG, et al. p16 is a major inactivation target in hepatocellular carcinoma. Cancer. 2000;89:60–68.CrossRefGoogle Scholar
  23. 23.
    Matsuda Y, Ichida T, Matsuzawa J, Sugimura K, Asakura H. p16 INK4A is inactivated by extensive CpG methylation in human hepatocellular carcinoma. Gastroenterology. 1999;116:394–400.PubMedCrossRefGoogle Scholar
  24. 24.
    Kondo Y, Kanai Y, Sakamoto M, Mizokami M, Ueda R, Hirohashi S. 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.PubMedCrossRefGoogle Scholar
  25. 25.
    Roncalli M, Bianchi P, Bruni B, et al. Methylation framework of cell cycle gene inhibitors in cirrhosis and associated hepatocellular carcinoma. Hepatology. 2002;36:427–432.PubMedCrossRefGoogle Scholar
  26. 26.
    Li X, Hui AM, Sun L, et al. p16INK4A hypermethylation is associated with hepatitis virus infection, age, and gender in hepatocellular carcinoma. Clin Cancer Res. 2004;10:7484–7489.PubMedCrossRefGoogle Scholar
  27. 27.
    Qin Y, Liu JY, Li B, Sun ZL, Sun ZF. Association of low p16INK4a and p15INK4b mRNAs expression with their CpG islands methylation with human hepatocellular carcinogenesis. World J Gastroenterol. 2004;10:1276–1280.PubMedGoogle Scholar
  28. 28.
    Desmet VJ, Gerber M, Hoofnagle JH, Manns M, Sheuer PJ. Classification of chronic hepatitis: diagnosis, grading, and staging. Hepatology. 1994;19:1513–1520.PubMedCrossRefGoogle Scholar
  29. 29.
    Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996;93:9821–9826.PubMedCrossRefGoogle Scholar
  30. 30.
    Kim BN, Yamamoto H, Ikeda K, et al. Methylation and expression of p16INK4A tumor suppressor gene in primary colorectal cancer tissues. Int J Oncol. 2005;26:1217–1226.PubMedGoogle Scholar
  31. 31.
    Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994;22:2990–2997.PubMedCrossRefGoogle Scholar
  32. 32.
    Issa JP. CpG-island methylation in aging and cancer. Curr Top Microbiol Immunol. 2000;249:101–118.PubMedGoogle Scholar
  33. 33.
    Waki T, Tamura G, Sato M, Motoyama T. Age-related methylation of tumor suppressor and tumor-related genes: an analysis of autopsy samples. Oncogene. 2003;22:4128–4133.PubMedCrossRefGoogle Scholar
  34. 34.
    Narimatsu T, Tamori A, Koh N, et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology. 2004;47:26–31.PubMedCrossRefGoogle Scholar
  35. 35.
    Muhlisch J, Bajanowski T, Rickert CH, et al. Frequent but borderline methylation of p16 INK4A and TIMP3 in medulloblastoma and sPNET revealed by quantitative analyses. J Neurooncol. 2007;83:17–29. (online first).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • So Kurita
    • 1
  • Shogo Ohkoshi
    • 1
  • Masahiko Yano
    • 1
  • Kazuhide Yamazaki
    • 1
  • Kenta Suzuki
    • 1
  • Yo-hei Aoki
    • 1
  • Yasunobu Matsuda
    • 1
  • Toshifumi Wakai
    • 2
  • Yoshio Shirai
    • 2
  • Takafumi Ichida
    • 3
  • Yutaka Aoyagi
    • 1
  1. 1.Division of Gastroenterology and HepatologyGraduate School of Medical and Dental Sciences of Niigata UniversityNiigata-cityJapan
  2. 2.First Department of SurgeryGraduate School of Medical and Dental Sciences of Niigata UniversityNiigata-cityJapan
  3. 3.Division of Gastroenterology and HepatologyJuntendo University School of Medicine and Juntendo University Hospital of ShizuokaShizuokaJapan

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