Medical Oncology

, 31:784 | Cite as

Promoter methylation status of MGMT, hMSH2, and hMLH1 and its relationship to corresponding protein expression and TP53 mutations in human esophageal squamous cell carcinoma

  • Yaoyao Su
  • Lihong Yin
  • Ran Liu
  • JingYi Sheng
  • Miao Yang
  • Yi Wang
  • Enchun Pan
  • Wei Guo
  • Yuepu Pu
  • Juan Zhang
  • Geyu Liang
Original Paper


To determine the relevance of O-6-methylguanine-DNA methyltransferase (MGMT), human mutS homolog 2 (hMSH2), and human mutL homolog 1 (hMLH1) in TP53 mutations in esophageal squamous cell carcinoma, we employed methylation-sensitive high-resolution melting technology and methylation-specific polymerase chain reaction (PCR) to analyze promoter hypermethylation of MGMT, hMSH2, and hMLH1, respectively, in 51 paired tumors and their adjacent normal tissues. The protein expression of the three proteins was also evaluated by Western blot analysis, and the PCR products of TP53, from exon 5 to exon 8, were directly sequenced to measure the mutation spectrum. Esophageal tumor tissues embraced statistically higher MGMT and hMSH2 promoter methylation level than normal tissue. The promoter methylation status of MGMT and hMSH2 corresponds positively with the protein expression level of MGMT and hMSH2. However, such relevance was not found for hMLH1. Furthermore, TP53 mutation status was well associated with MGMT and hMSH2 promoter methylation status, indicating that silencing of the two genes could lead to TP53 mutation in ESCC.


Promoter methylation TP53 mutation Human esophageal squamous cell carcinoma 



This study was funded by the National Natural Science Foundation of China (Nos. 81072259, 30671732, 30800891), Research Fund for the Doctoral Program of Higher Education of China (No. 200802861045), Natural Science Foundation of JiangSu province of China (No. BK2010407), and Research and Teaching Fund for the Excellent Youth Scholars of Southeast University (2009).

Conflict of interest



  1. 1.
    Blin N, Stafford DW. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976;3:2303–8.PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Brock MV, Gou M, Akiyama Y, et al. Prognostic importance of promoter hypermethylation of multiple genes in esophageal adenocarcinoma. Clin Cancer Res. 2003;9:2912–9.PubMedGoogle Scholar
  3. 3.
    de la Chapelle A. Genetic predisposition to colorectal cancer. Nat Rev Cancer. 2004;4:769–80.PubMedCrossRefGoogle Scholar
  4. 4.
    Duckett DR, Drummond JT, Murchie AI, et al. Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d (GpG) adduct. Proc Natl Acad Sci USA. 1996;93:6443–7.PubMedCrossRefGoogle Scholar
  5. 5.
    Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457–63.PubMedCrossRefGoogle Scholar
  6. 6.
    Esteller M. CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene. 2002;21:5427–40.PubMedCrossRefGoogle Scholar
  7. 7.
    Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res. 1999;59:793–7.PubMedGoogle Scholar
  8. 8.
    Fearon ER, Vogelstein BA. A genetic model for colorectal tumorigenesis. Cell. 1990;61:759–67.PubMedCrossRefGoogle Scholar
  9. 9.
    Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer. 2004;4:143–53.PubMedCrossRefGoogle Scholar
  10. 10.
    Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet. 2005;7:21–33.CrossRefGoogle Scholar
  11. 11.
    Fox EJ, Leahy DT, Geraghty R, et al. Mutually exclusive promoter hypermethylation patterns of hMLH1 and O6-methylguanine DNA methyltransferase in colorectal cancer. J Mol Diagn. 2006;8:68–74.PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Galloway SM, Greenwood SK, Hill RB, Bradt CI, Bean CL. A role for mismatch repair in production of chromosome aberrations by methylating agents in human cells. Mutat Res. 1995;346:231–45.PubMedCrossRefGoogle Scholar
  13. 13.
    Herman JG. Hypermethylation of tumor suppressor genes in cancer. Semin Cancer Biol. 1999;9:359–67.PubMedCrossRefGoogle Scholar
  14. 14.
    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;93:9821–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Herman JG, Umar A, Polyak K, et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc Natl Acad Sci USA. 1998;95:6870–5.PubMedCrossRefGoogle Scholar
  16. 16.
    Kaina B, Christmann M, Naumann S, Roos WP. MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents. DNA Repair. 2007;6:1079–99.PubMedCrossRefGoogle Scholar
  17. 17.
    Karran P, Bignami M. DNA damage tolerance, mismatch repair and genome instability. BioEssays. 1994;16:833–9.PubMedCrossRefGoogle Scholar
  18. 18.
    Kawate H, Sakumi K, Tsuzuki T, et al. Separation of killing and tumorigenic effects of an alkylating agent in mice defective in two of the DNA repair genes. Proc Natl Acad Sci USA. 1998;95:5116–20.PubMedCrossRefGoogle Scholar
  19. 19.
    Kuester D, El-Rifai W, Peng DF, et al. Silencing of MGMT expression by promoter hypermethylation in the metaplasia–dysplasia–carcinoma sequence of Barrett’s esophagus. Cancer Lett. 2009;275:117–26.PubMedCrossRefGoogle Scholar
  20. 20.
    Leung SY, Yuen ST, Chung LP, Chu KM, Chan AY, Joana CH. hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability. Cancer Res. 1999;59:159–64.PubMedGoogle Scholar
  21. 21.
    Liu R. Study on expression characteristics of susceptibility genes and genetic susceptibility markers for esophageal carcinomas. Dissertation for the Academic Degree of Doctor of Medicine in Southeast University. 2007.Google Scholar
  22. 22.
    Lu SH, Chui S, Yang WX, Hu XN, Guo LP, Li FM. Relevance of N-nitrosamines to oesophageal cancer in China. IARC Sci Publ. 1991;84:538–43.Google Scholar
  23. 23.
    Margison GP, Koref MF, Povey AC. Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine. Mutagenesis. 2002;17:483–7.PubMedCrossRefGoogle Scholar
  24. 24.
    Martin P, SantÓn A, GarcÍa-Cosio M, Bellas C. hMLH1 and MGMT inactivation as a mechanism of tumorigenesis in monoclonal gammopathies. Mod Pathol. 2006;19:914–21.PubMedCrossRefGoogle Scholar
  25. 25.
    Mukai T, Sekiguchi M. Gene silencing in phenomena related to DNA repair. Oncogene. 2002;21:9033–42.PubMedCrossRefGoogle Scholar
  26. 26.
    Murata H, Khattar NH, Gu L, Li GM. Roles of mismatch repair proteins hMSH2 and hMLH1 in the development of sporadic breast cancer. Cancer Lett. 2005;223:143–50.PubMedCrossRefGoogle Scholar
  27. 27.
    Nakagawachi T, Soejima H, Urano T, et al. Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer. Oncogene. 2003;22:8835–44.PubMedGoogle Scholar
  28. 28.
    Naumann SC, Roos WP, Jöst E. Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53. Br J Cancer. 2009;100:322–33.PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Nie Y, Liao J, Zhao X, et al. Detection of multiple gene hypermethylation in the development of esophageal squamous cell carcinoma. Carcinogenesis. 2002;23:1713–20.PubMedCrossRefGoogle Scholar
  30. 30.
    Ochs K, Kaina B. Apoptosis induced by DNA damage O6-methylguanine is Bcl-2 and caspase-9/3 regulated and Fas/caspase-8 independent. Cancer Res. 2000;60:5815–24.PubMedGoogle Scholar
  31. 31.
    Poynter JN, Siegmund KD, Weisenberger DJ, et al. Molecular characterization of MSI-H colorectal cancer by MLH1 promoter methylation, immunohistochemistry, and mismatch repair germline mutation screening. Cancer Epidemiol Biomarkers Prev. 2008;17:3208–15.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Rowley JD. The Philadelphia chromosome translocation: a paradigm for understanding leukemia. Cancer. 1990;65:2178–84.PubMedCrossRefGoogle Scholar
  33. 33.
    Takagi Y, Takahashi M, Sanada M. Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis. DNA Repair. 2003;2:1135–46.PubMedCrossRefGoogle Scholar
  34. 34.
    Toorchen D, Topal MD. Mechanisms of chemical mutagenesis and carcinogenesis: effects on DNA replication of methylation at the O6-guanine position of dGTP. Carcinogenesis. 1983;4:1591–7.PubMedCrossRefGoogle Scholar
  35. 35.
    Tzao C, Hsu HS, Sun GH, et al. Promoter methylation of the hMLH1 gene and protein expression of human mutL homolog 1 and human mutS homolog 2 in resected esophageal squamous cell carcinoma. J Thorac Cardiovasc Surg. 2005;130:1371–7.PubMedCrossRefGoogle Scholar
  36. 36.
    Wojdacz TK, Dobrovic A, Hansen LL. Methylation-sensitive high-resolution melting. Nat Protoc. 2008;3:1903–8.PubMedCrossRefGoogle Scholar
  37. 37.
    Wojdacz TK, Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive an high-throughput assessment of methylation. Nucleic Acids Res. 2007;35:1–7.CrossRefGoogle Scholar
  38. 38.
    Wu Y, Chen J, Ohshima H, et al. Geographic association between urinary excretion of N-nitroso compounds and oesophageal cancer mortality in china. Int J Cancer. 1993;54:713–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Yaoyao Su
    • 1
  • Lihong Yin
    • 1
  • Ran Liu
    • 1
  • JingYi Sheng
    • 1
  • Miao Yang
    • 1
  • Yi Wang
    • 2
  • Enchun Pan
    • 2
  • Wei Guo
    • 3
  • Yuepu Pu
    • 1
  • Juan Zhang
    • 1
  • Geyu Liang
    • 1
  1. 1.Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public HealthSoutheast UniversityNanjingChina
  2. 2.Disease Control and Prevention Center of HuaianHuaianChina
  3. 3.The First People’s Hospital of HuaianHuaianChina

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