Molecular Biology Reports

, Volume 40, Issue 5, pp 3851–3857 | Cite as

Different patterns of DNA methylation of the two distinct O6-methylguanine-DNA methyltransferase (O6-MGMT) promoter regions in colorectal cancer

  • P. Mokarram
  • M. Zamani
  • S. Kavousipour
  • F. Naghibalhossaini
  • C. Irajie
  • M. Moradi Sarabi
  • S. V. Hosseini


Colorectal cancer (CRC) is the third most common cancer worldwide. Colorectal cancer incidence differs widely among different geographic regions. In addition to mutational changes, epigenetic mechanisms also play important roles in the pathogenesis of CRCs. O6-methylguanine-DNA methyltransferase (O 6 -MGMT) is a DNA repair protein and in the absence of MGMT activity, G-to-A transition may accumulate in the specific genes such as K-ras and p53. To identify which CpG sites are critical for its downregulation, we analyzed the methylation status of the MGMT gene promoter in two sites in CRC patients. Then we compared the frequency of their methylation changes with the results of our previously reported K-ras gene mutation, APC2 and p16 methylation. MGMT methylation was examined in 92 tumor samples. A methylation specific PCR (MSP) method was performed for two loci of MGMT gene which described as MGMT-A and MGMT-B. The prevalence of MGMT-A, and MGMT-B methylation was 49/91 (53.8 %), and 83/92 (90.2 %), respectively. We detected high frequency of MGMT-B but not MGMT-A methylation in tumor tissues with APC2 methylation. Our results showed that MGMT-B methylation is significantly associated with K-ras gene mutation rather than MGMT-A (p = 0.04). Simultaneously, an inverse correlation was found between p16 and MGMT-B methylation simultaneously (p = 0.02). Our study indicated that hypermethylation of the specific locus near the MGMT start codon is critical for cancer progression. MGMT-B assessment that is associated with K-ras mutation can have a prognostic value in patients with CRC.


MGMT K-ras APC2 p16 Colorectal cancer 



This study was supported by Grant Number 5084 from Vice-chancellor for Research Affairs of Shiraz University of Medical Sciences. We acknowledge the cooperation of Dr.alizadeh from gastroenterohepatology research center, Shiraz University of Medical Sciences, Shiraz-Iran.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Parkin DM, Bray F, Ferlay J, Pisani P (2001) Estimating the world cancer burden: globocan 2000. Int J Cancer 94:153–156. doi: 10.1002/ijc.1440 PubMedCrossRefGoogle Scholar
  2. 2.
    Yazdizadeh B, Jarrahi AM, Mortazavi H, Mohagheghi MA, Tahmasebi S, Nahvijo A (2005) Time trends in the occurrence of major GI cancers in Iran. Asian Pac J Cancer Prev 6:130–134PubMedGoogle Scholar
  3. 3.
    Hosseini SV, Izadpanah A, Yarmohammadi H (2004) Epidemiological changes in colorectal cancer in Shiraz, Iran: 1980–2000. ANZ J Surg 74:547–549. doi: 10.1111/j.1445-2197.2004.03064.x PubMedCrossRefGoogle Scholar
  4. 4.
    Higashidani Y, Tamura S, Morita T, Tadokoro T, Yokoyama Y, Miyazaki J, Yang Y, Takeuchi S, Taguchi H, Onishi S (2003) Analysis of K-ras codon 12 mutation in flat and nodular variants of serrated adenoma in the colon. Dis Colon Rectum 46:327–332. doi: 10.1007/s10350-004-6551-z PubMedCrossRefGoogle Scholar
  5. 5.
    Smith G, Carey FA, Beattie J, Wilkie MJ, Lightfoot TJ, Coxhead J, Garner RC, Steele RJ, Wolf CR (2002) Mutations in APC, Kirsten-ras, and p53–alternative genetic pathways to colorectal cancer. Proc Natl Acad Sci U S A 99:9433–9438. doi: 10.1073/pnas.122612899 PubMedCrossRefGoogle Scholar
  6. 6.
    Fearon ER, Vogelstein B (1990) A genetic model for colorectal tumorigenesis. Cell 61:759–767. doi: 10.1016/0092-8674(90)90186-I PubMedCrossRefGoogle Scholar
  7. 7.
    Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K, Herman JG (2001) Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 10:687–692. doi: 10.1093/hmg/10.7.687 PubMedCrossRefGoogle Scholar
  8. 8.
    Baylin SB, Herman JG, Graff JR, Vertino PM, Issa JP (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv Cancer Res 72:141–196. doi: 10.1016/S0065-230X(08)60702-2 PubMedCrossRefGoogle Scholar
  9. 9.
    Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP (1999) CpG island methylator phenotype in colorectal cancer. Proc Natl Acad Sci U S A 96:8681–8686. doi: 10.1073/pnas.96.15.8681 PubMedCrossRefGoogle Scholar
  10. 10.
    Krtolica K, Krajnovic M, Usaj-Knezevic S, Babic D, Jovanovic D, Dimitrijevic B (2007) Comethylation of p16 and MGMT genes in colorectal carcinoma: correlation with clinicopathological features and prognostic value. World J Gastroenterol 13:1187–1194. doi: 1007-9327/13/1187.asp PubMedGoogle Scholar
  11. 11.
    Taghavi N, Biramijamal F, Sotoudeh M, Khademi H, Malekzadeh R, Moaven O, Memar B, A’Rabi A, Abbaszadegan MR (2010) p16INK4a hypermethylation and p53, p16 and MDM2 protein expression in esophageal squamous cell carcinoma. BMC Cancer 10:138. doi: 10.1186/1471-2407-10-138 PubMedCrossRefGoogle Scholar
  12. 12.
    Georgiou E, Kouidou S (2011) Epigenetically-targeted therapies for the treatment of hematological malignancies. Curr Med Chem 18:1757–1764. doi: 10.2174/092986711795496791 PubMedCrossRefGoogle Scholar
  13. 13.
    Zhong S, Fields CR, Su N, Pan YX, Robertson KD (2007) Pharmacologic inhibition of epigenetic modifications, coupled with gene expression profiling, reveals novel targets of aberrant DNA methylation and histone deacetylation in lung cancer. Oncogene 26:2621–2634. doi: 10.1038/sj.onc.1210041 PubMedCrossRefGoogle Scholar
  14. 14.
    Gonzalo V, Lozano JJ, Munoz J, Balaguer F, Pellise M, Rodriguez de Miguel C, Andreu M, Jover R, Llor X, Giraldez MD, Ocana T, Serradesanferm A, Alonso-Espinaco V, Jimeno M, Cuatrecasas M, Sendino O, Castellvi-Bel S, Castells A (2010) Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer. PLoS ONE 5:e8777. doi: 10.1371/journal.pone.0008777 PubMedCrossRefGoogle Scholar
  15. 15.
    Costello JF, Futscher BW, Tano K, Graunke DM, Pieper RO (1994) Graded methylation in the promoter and body of the O6-methylguanine DNA methyltransferase (MGMT) gene correlates with MGMT expression in human glioma cells. J Biol Chem 269:17228–17237PubMedGoogle Scholar
  16. 16.
    Halford S, Rowan A, Sawyer E, Talbot I, Tomlinson I (2005) O(6)-methylguanine methyltransferase in colorectal cancers: detection of mutations, loss of expression, and weak association with G:c > A:t transitions. Gut 54:797–802. doi: 10.1136/gut.2004.059535 PubMedCrossRefGoogle Scholar
  17. 17.
    Nakagawachi T, Soejima H, Urano T, Zhao W, Higashimoto K, Satoh Y, Matsukura S, Kudo S, Kitajima Y, Harada H, Furukawa K, Matsuzaki H, Emi M, Nakabeppu Y, Miyazaki K, Sekiguchi M, Mukai T (2003) Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer. Oncogene 22:8835–8844. doi: 10.1038/sj.onc.1207183 PubMedGoogle Scholar
  18. 18.
    Naghibalhossaini F, Hosseini HM, Mokarram P, Zamani M (2011) High frequency of genes’ promoter methylation, but lack of braf v600e mutation among iranian colorectal cancer patients. Pathol Oncol Res. doi: 10.1007/s12253-011-9388-5 PubMedGoogle Scholar
  19. 19.
    Mokarram P, Naghibalhossaini F, Saberi Firoozi M, Hosseini SV, Izadpanah A, Salahi H, Malek-Hosseini SA, Talei A, Mojallal M (2008) Methylenetetrahydrofolate reductase C677T genotype affects promoter methylation of tumor-specific genes in sporadic colorectal cancer through an interaction with folate/vitamin B12 status. World J Gastroenterol 14:3662–3671. doi: 10.3748/wjg.14.3662 PubMedCrossRefGoogle Scholar
  20. 20.
    Blin N, Stafford DW (1976) A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res 3:2303–2308. doi: 10.1093/nar/3.9.2303 PubMedCrossRefGoogle Scholar
  21. 21.
    Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821–9826PubMedCrossRefGoogle Scholar
  22. 22.
    Sidhu S, Deep JS, Sobti RC, Sharma VL, Thaku H (2010) Methylation pattern of MGMT gene in relation to age, smoking, drinking and dietary habits as epigenetic biomarker in prostate cancer patients. Genet Eng Biotechnol J 2010(8):1–11Google Scholar
  23. 23.
    Wang J, Sasco AJ, Fu C, Xue H, Guo G, Hua Z, Zhou Q, Jiang Q, Xu B (2008) Aberrant DNA methylation of p16, MGMT, and hMLH1 genes in combination with MTHFR C677T genetic polymorphism in esophageal squamous cell carcinoma. Cancer Epidemiol Biomarkers Prev 17:118–125. doi: 10.1158/1055-9965.EPI-07-0733 PubMedCrossRefGoogle Scholar
  24. 24.
    Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG (1999) Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59:793–797PubMedGoogle Scholar
  25. 25.
    Silber JR, Bobola MS, Ghatan S, Blank A, Kolstoe DD, Berger MS (1998) O6-methylguanine-DNA methyltransferase activity in adult gliomas: relation to patient and tumor characteristics. Cancer Res 58:1068–1073PubMedGoogle Scholar
  26. 26.
    Xiao-fang L, Xian-chun S, Cui-sheng Z, Zheng X, Shao-jun L, Xian-ting Z, Shi-jie S (2010) Promoter hypermethylation of O6-methylguanine-DNA methyltransferase gene in cholangiocarcinoma. J Chin Clin Med 5(8):463–468Google Scholar
  27. 27.
    Löf-Ohlin ZM, Nilsson TK (2009) Pyrosequencing assays to study promoter CpG site methylation of the O6-MGMT, hMLH1, p14ARF, p16INK4a, RASSF1A, and APC1A genes. Oncol Rep 21:721–729. doi: 10.3892/or_00000277 PubMedGoogle Scholar
  28. 28.
    Sciuscio D, Diserens AC, van Dommelen K, Martinet D, Jones G, Janzer RC, Pollo C, Hamou MF, Kaina B, Stupp R, Levivier M, Hegi ME (2011) Extent and patterns of MGMT promoter methylation in glioblastoma- and respective glioblastoma-derived spheres. Clin Cancer Res 17:255–266. doi: 10.1158/1078-0432.CCR-10-1931 PubMedCrossRefGoogle Scholar
  29. 29.
    Ye C, Shrubsole MJ, Cai Q, Ness R, Grady WM, Smalley W, Cai H, Washington K, Zheng W (2006) Promoter methylation status of the MGMT, hMLH1, and CDKN2A/p16 genes in non-neoplastic mucosa of patients with and without colorectal adenomas. Oncol Rep 16:429–435PubMedGoogle Scholar
  30. 30.
    Liu Y, Lan Q, Siegfried JM, Luketich JD, Keohavong P (2006) Aberrant promoter methylation of p16 and MGMT genes in lung tumors from smoking and never-smoking lung cancer patients. Neoplasia 8:46–51. doi: 10.1593/neo.05586 PubMedCrossRefGoogle Scholar
  31. 31.
    Servomaa K, Kiuru A, Kosma VM, Hirvikoski P, Rytomaa T (2000) p53 and K-ras gene mutations in carcinoma of the rectum among Finnish women. Mol Pathol 53:24–30. doi: 10.1136/mp.53.1.24 PubMedCrossRefGoogle Scholar
  32. 32.
    Mokarram P, Kumar K, Brim H, Naghibalhossaini F, Saberi-firoozi M, Nouraie M, Green R, Lee E, Smoot DT, Ashktorab H (2009) Distinct high-profile methylated genes in colorectal cancer. PLoS ONE 4:e7012. doi: 10.1371/journal.pone.0007012 PubMedCrossRefGoogle Scholar
  33. 33.
    De Vogel S, Weijenberg MP, Herman JG, Wouters KA, De Goeij AF, Van den Brandt PA, De Bruine AP, Van Engeland M (2009) MGMT and MLH1 promoter methylation versus APC, K-ras and BRAF gene mutations in colorectal cancer: indications for distinct pathways and sequence of events. Ann Oncol 20:1216–1222. doi: 10.1093/annonc/mdn782 PubMedCrossRefGoogle Scholar
  34. 34.
    Fuchikami M, Morinobu S, Segawa M, Okamoto Y, Yamawaki S, Ozaki N, Inoue T, Kusumi I, Koyama T, Tsuchiyama K, Terao T (2011) DNA methylation profiles of the brain-derived neurotrophic factor (BDNF) gene as a potent diagnostic biomarker in major depression. PLoS ONE 6:e23881. doi: 10.1371/journal.pone.0023881 PubMedCrossRefGoogle Scholar
  35. 35.
    Shacham-Shmueli E, Beny A, Geva R, Blachar A, Figer A, Aderka D (2011) Response to temozolomide in patients with metastatic colorectal cancer with loss of MGMT expression: a new approach in the era of personalized medicine? J Clin Oncol. doi: 10.1200/JCO.2010.32.0242 PubMedGoogle Scholar
  36. 36.
    Shen L, Kondo Y, Rosner GL, Xiao L, Hernandez NS, Vilaythong J, Houlihan PS, Krouse RS, Prasad AR, Einspahr JG, Buckmeier J, Alberts DS, Hamilton SR, Issa JP (2005) MGMT promoter methylation and field defect in sporadic colorectal cancer. J Natl Cancer Inst 97:1330–1338. doi: 10.1093/jnci/dji275 PubMedCrossRefGoogle Scholar
  37. 37.
    Kumar K, Brim H, Giardiello F, Smoot DT, Nouraie M, Lee EL, Ashktorab H (2009) Distinct BRAF (V600E) and K-ras mutations in high microsatellite instability sporadic colorectal cancer in African Americans. Clin Cancer Res 15:1155–1161. doi: 10.1158/1078-0432.CCR-08-1029 PubMedCrossRefGoogle Scholar
  38. 38.
    Brim H, Mokarram P, Naghibalhossaini F, Saberi-Firoozi M, Al-Mandhari M, Al-Mawaly K, Al-Mjeni R, Al-Sayegh A, Raeburn S, Lee E, Giardiello F, Smoot DT, Vilkin A, Boland CR, Goel A, Hafezi M, Nouraie M, Ashktorab H (2008) Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study. Mol Cancer 7:68. doi: 10.1186/1476-4598-7-68 PubMedCrossRefGoogle Scholar
  39. 39.
    Haydon AM, Jass JR (2002) Emerging pathways in colorectal-cancer development. Lancet Oncol 3:83–88. doi: 10.1016/S1470-2045(02)00649-6 PubMedCrossRefGoogle Scholar
  40. 40.
    Bauer KM, Hummon AB, Buechler S (2011) Right-side and left-side colon cancer follow different pathways to relapse. Mol Carcinog. doi: 10.1002/mc.20804 PubMedGoogle Scholar
  41. 41.
    Sugai T, Habano W, Jiao YF, Tsukahara M, Takeda Y, Otsuka K, Nakamura S (2006) Analysis of molecular alterations in left- and right-sided colorectal carcinomas reveals distinct pathways of carcinogenesis: proposal for new molecular profile of colorectal carcinomas. J Mol Diagn 8:193–201. doi: 10.2353/jmoldx.2006.050052 PubMedCrossRefGoogle Scholar
  42. 42.
    Jass JR (2007) Classification of colorectal cancer based on correlation of clinical, morphological and molecular features. Histopathology 50:113–130. doi: 10.1111/j.1365-2559.2006.02549.x PubMedCrossRefGoogle Scholar
  43. 43.
    Cai FF, Kohler C, Zhang B, Wang MH, Chen WJ, Zhong XY (2011) Epigenetic therapy for breast cancer. Int J Mol Sci 12:4465–4487. doi: 10.3390/ijms12074465 PubMedCrossRefGoogle Scholar
  44. 44.
    Santini V, Kantarjian HM, Issa JP (2001) Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med 134:573–586PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • P. Mokarram
    • 1
    • 2
  • M. Zamani
    • 1
  • S. Kavousipour
    • 1
  • F. Naghibalhossaini
    • 1
  • C. Irajie
    • 3
  • M. Moradi Sarabi
    • 1
  • S. V. Hosseini
    • 2
    • 4
  1. 1.Department of BiochemistryShiraz University of Medical Sciences, School of MedicineShirazIran
  2. 2.Gastroenterohepatology Research Center, School of MedicineShiraz University of Medical SciencesShirazIran
  3. 3.Department of Resource Development and ManagementShiraz University of Medical SciencesShirazIran
  4. 4.Department of Surgery, School of MedicineShiraz University of Medical SciencesShirazIran

Personalised recommendations