Annals of Surgical Oncology

, Volume 12, Issue 5, pp 354–363 | Cite as

Tumor Progression Through Epigenetic Gene Silencing of O6−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers

  • Yasuo Koga
  • Yoshihiko Kitajima
  • Atsushi Miyoshi
  • Ken Sato
  • Kenji Kitahara
  • Hidenobu Soejima
  • Kohji Miyazaki



We previously demonstrated in an immunohistochemical study that reduced expression of O6−methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer.


We examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry.


Frequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry (P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation (P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, β-catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation (P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations.


Reduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms.


DNA alkylation DNA repair gene MGMT Biliary tract cancer Multigene methylation Gene mutation 


  1. 1.
    Okusaka, T 2002Chemotherapy for biliary tract cancer in JapanSemin Oncol29513CrossRefPubMedGoogle Scholar
  2. 2.
    Nimura, Y 2002Extended surgery in bilio-pancreatic cancer: the Japanese experienceSemin Oncol291722CrossRefPubMedGoogle Scholar
  3. 3.
    Paimela, H, Karppinen, A, Hockerstedt, K, Perhoniemi, V, Kivilaakso, E 1997Poor prognosis of gallbladder cancer persist regardless of improved diagnostic methods. Incidence and results of surgery during 20 years in HelsinkiAnn Chir Gynaecol861317PubMedGoogle Scholar
  4. 4.
    Rashid, A 2002Cellular and molecular biology of biliary tract cancersSurg Oncol Clin North Am119951009CrossRefGoogle Scholar
  5. 5.
    Loveless, A 1969Possible relevance of O-6 alkylation of deoxyguanosine to the mutagency and carcinogencity of nitrosamines and nitrosamidesNature132067CrossRefGoogle Scholar
  6. 6.
    Pegg, AE 1990Mammalian O6-alkylguanine-DNA alkyltransferase: regulation and importance in response to alkylating carcinogenic and therapeutic agentsCancer Res50611929PubMedGoogle Scholar
  7. 7.
    Demple, B, Jacobsson, A, Olsson, M, Robins, P, Lindahl, T 1982Repair of alkylated DNA in Escherichia coli: physical properties of O6−methylguanine-DNA methyltransferaseJ Biol Chem2571377680PubMedGoogle Scholar
  8. 8.
    Sakumi, K, Shiraishi, A, Shimizu, S, Tsuzuki, T, Ishikawa, T, Sekiguchi, M 1997Methylnitrosourea-induced tumorigenesis in MGMT gene knockout miceCancer Res1524158Google Scholar
  9. 9.
    Gerson, SL, Trey, JE, Miller, K, Berger, NA 1986Comparison of O6−alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissuesCarcinogenesis77459PubMedCrossRefGoogle Scholar
  10. 10.
    Kohya, N, Miyazaki, K, Matsukura, S,  et al. 2002Deficient expression of O6−methylguanine-DNA methyltransferase combined with mismatch-repair proteins hMLH1 and MSH2 is related to poor prognosis in human biliary tract carcinomaAnn Surg Oncol93719CrossRefPubMedGoogle Scholar
  11. 11.
    Esteller, M 2003Relevance of DNA methylation in the management of cancerLancet Oncol43518CrossRefPubMedGoogle Scholar
  12. 12.
    Hanada, K, Itoh, M, Fujii, K, Tsuchida, A, Ooishi, H, Kajiyama, G 1996K-ras and p53 mutations in stag I gallbladder carcinoma with an anomalous junction of the pancreaticobiliary ductCancer774528CrossRefPubMedGoogle Scholar
  13. 13.
    Itoi, T, Watanabe, H, Ajioka, Y,  et al. 1996APC, K-ras codon 12 mutations and p53 gene expression in carcinoma and adenoma of the gallbladder suggest two genetic pathways in gallbladder carcinogenesisPathol Int4633340PubMedCrossRefGoogle Scholar
  14. 14.
    Yoshida, S, Todoroki, T, Ichikawa, Y,  et al. 1995Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancersCancer Res55275660PubMedGoogle Scholar
  15. 15.
    Watanabe, H, Date, K, Itoi, T,  et al. 1999Histological and genetic changes in malignant transformation of gallbladder adenomaAnn Oncol101369CrossRefPubMedGoogle Scholar
  16. 16.
    Wistuba, II, Albores-Saavedra, J 1999Genetic abnormalities involved in the pathogeneisis of gallbladder carcinomaJ Hepatobiliary Pancreat Surg623744CrossRefPubMedGoogle Scholar
  17. 17.
    Kohya, N, Kitajima, Y, Kitahara, K, Miyazaki, K 2003Mutation analysis of K-ras and β-catenin genes related to O6-methylguanine-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinomaInt J Mol Med11659PubMedGoogle Scholar
  18. 18.
    Baylin, SB, Herman, JG 2000DNA hypermethylation in tumorigenesis: epigenetics joins geneticsTrends Genet1616874CrossRefPubMedGoogle Scholar
  19. 19.
    Matsukura, S, Soejima, H, Nakagawachi, T,  et al. 2003CpG methylation of MGMT and MLH1 promoter in hepatocellular carcinoma associated with hepatitis viral infectionBm J Cancer885219CrossRefGoogle Scholar
  20. 20.
    Esteller, M, Hamilton, SR, Burger, PC, Baylin, SB, Herman, JG 1999Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasiaCancer Res597937PubMedGoogle Scholar
  21. 21.
    Esteller, M, Sanchez-Cespedes, M, Rosell, R, Sidransky, D, Baylin, SB, Herman, JG 1999Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patientsCancer Res596770PubMedGoogle Scholar
  22. 22.
    Brabender, J, Usadel, H, Metzger, R,  et al. 2003Quantitative O6-methylguanine-DNA methyltransferase analysis in curatively resected non-small cell lung cancer; association with clinical outcomeClin Cancer Res92237PubMedGoogle Scholar
  23. 23.
    Wong, DJ, Barrett, MT, Stoger, R, Emond, MJ, Reid, BJ 1997p16/INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomasCancer Res57261922PubMedGoogle Scholar
  24. 24.
    Tsao, SW, Liu, Y, Wang, X,  et al. 2003The association of E-cadherin expression and the methylation status of the E-cadherin gene in nasopharyngeal carcinoma cellsEur J Cancer3952431CrossRefPubMedGoogle Scholar
  25. 25.
    Fleisher, AS, Esteller, M, Wang, S,  et al. 1999Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instabilityCancer Res5910905PubMedGoogle Scholar
  26. 26.
    Tang, X, Khuri, FR, Lee, JJ,  et al. 2000Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small cell lung cancerJ Natl Cancer Inst9215116CrossRefPubMedGoogle Scholar
  27. 27.
    Toyota, M, Ahuja, N, Ohe-Toyota, M, Herman, JG, Baylin, SB, Issa, J-PJ 1999CpG island methylator phenotype in colorectal cancerProc Natl Acad Sci9686816CrossRefPubMedGoogle Scholar
  28. 28.
    Toyota, M, Ohe-Toyota, M, Ahuja, N, Issa, J-PJ 2000Distinct genetic profiles in colorectal tumors with or without the CpG island methylator phenotypeProc Natl Acad Sci USA977106CrossRefPubMedGoogle Scholar
  29. 29.
    Brock, MV, Gou, M, Akiyama, Y,  et al. 2003Prognostic importance of promoter hypermethylation of multiple genes in esophageal adenocarcinomaClin Cancer Res929129PubMedGoogle Scholar
  30. 30.
    House, MG, Herman, JG, Guo, MZ,  et al. 2003Aberrant hypermethylation of tumor suppressor genes in pancreatic endocrine neoplasmsAnn Surg Oncol23842332Google Scholar
  31. 31.
    House, MG, Wistuba, II, Argani, P,  et al. 2003Progression of gene hypermethylation in gallstone disease leading to gallbladder cancerAnn Surg Oncol108829CrossRefPubMedGoogle Scholar
  32. 32.
    Lee, S, Kim, WH, Jung, H-Y, Yang, MH, Kang, GH 2002Aberrant CpG island methylation of multiple genes in intrahepatic cholangiocarcinomaAm J Patho161101522Google Scholar
  33. 33.
    Herman, JG, Graff, R, Myohanen, S, Nelkin, BD, Baylin, SB 1996Methylation-specific PCR: a novel PCR assay for methylation status of CpG islandsProc Natl Acad Sci USA93 98216PubMedCrossRefGoogle Scholar
  34. 34.
    Paulin, R, Grigg, GW, Davey, MW, Piper, AA 1998Urea improves efficiency of bisulfite-mediated sequencing of 5′-methylcytosine in genomic DNANucleric Acids Res26500910CrossRefGoogle Scholar
  35. 35.
    Scarpa, A, Capelli, P, Villaneuva, A,  et al. 1994Pancreatic cancer in Europe: Ki-ras gene mutation pattern shows geographical differencesInt J Cancer5716771PubMedCrossRefGoogle Scholar
  36. 36.
    Scarpa, A, Capelli, P, Mukai, K,  et al. 1993Pancreatic adenocarcinomas frequently show p53 mutationsAm J Pathol 142153443PubMedGoogle Scholar
  37. 37.
    Zhang, SY, Klein-Szanto, AJ, Sauter, ER,  et al. 1994Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neckCancer Res5450503PubMedGoogle Scholar
  38. 38.
    Matsukura, S, Miyazaki, K, Yakushiji, H,  et al. 2001Expression and prognostic significance of O6-methylguanine-DNA methyltransferase in hepatocellular, gastric, and breast cancersAnn Surg Oncol880716PubMedCrossRefGoogle Scholar
  39. 39.
    Kitajima, Y, Miyazaki, K, Matsukura, S, Tanaka, M, Sekiguchi, M. 2003Loss of expression of DNA repair enzymes MGMT, hMLH1, and MSH2 during tumor progression in gastric cancerGastric Cancer68695PubMedGoogle Scholar
  40. 40.
    Esteller, M, Garcia-Foncillas, J, Andion, E,  et al. 2000Inactivation of the DNA repair gene MGMT and clinical response of gliomas to alkylating agentsN Engl J Med34313504CrossRefPubMedGoogle Scholar
  41. 41.
    Esteller, M, Gaidano, G, Goodman, SN,  et al. 2002Hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase and survival of patients with diffuse large B-cell lymphomaJ Natl Cancer Inst942632PubMedGoogle Scholar
  42. 42.
    Esteller, M, Toyota, M, Sanchez-Cespedes, M,  et al. 2000Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesisCancer Res60236871PubMedGoogle Scholar
  43. 43.
    Esteller, M, Risques, R-A, Toyota, M,  et al. 2001Promoter hypermethylation of the DNA repair gene O6-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutation in p53 in human colorectal tumorigenesisCancer Res61468992PubMedGoogle Scholar
  44. 44.
    Wolf, P, Hu, YC, Doffek, K, Sidransky, D, Ahrendt, SA 2001O6-Methylguanine-DNA methyltransferase promoter hypermethylation shifts the p53 mutational spectrum in non-small cell lung cancerCancer Res6181127Google Scholar

Copyright information

© The Society of Surgical Oncology, Inc. 2005

Authors and Affiliations

  • Yasuo Koga
    • 1
  • Yoshihiko Kitajima
    • 1
  • Atsushi Miyoshi
    • 1
  • Ken Sato
    • 1
  • Kenji Kitahara
    • 1
  • Hidenobu Soejima
    • 2
  • Kohji Miyazaki
    • 1
  1. 1.Department of SurgerySaga University Faculty of MedicineJapan
  2. 2.Department of Biomolecular Sciences, Division of Molecular Biology & GeneticsSaga University Faculty of MedicineJapan

Personalised recommendations