Digestive Diseases and Sciences

, Volume 55, Issue 12, pp 3449–3457 | Cite as

Association Between Cyclin D1 Polymorphism with CpG Island Promoter Methylation Status of Tumor Suppressor Genes in Gastric Cancer

  • Tomomitsu Tahara
  • Tomoyuki Shibata
  • Masakatsu Nakamura
  • Hiromi Yamashita
  • Daisuke Yoshioka
  • Masaaki Okubo
  • Joh Yonemura
  • Yoshiteru Maeda
  • Naoko Maruyama
  • Toshiaki Kamano
  • Yoshio Kamiya
  • Hiroshi Fujita
  • Yoshihito Nakagawa
  • Mitsuo Nagasaka
  • Masami Iwata
  • Ichiro Hirata
  • Tomiyasu Arisawa
Original Article



CpG island hypermethylation of tumor suppressor genes is highly involved in gastric carcinogenesis, and enhanced cell proliferation could accelerate this process. Cyclin D1 regulates cell cycle function and may play a role in methylation-related carcinogenesis.


We investigated the association between Cyclin D1 gene G870A polymorphism and the methylation status of tumor suppressor genes in gastric cancer.


Polymorphisms at G870A in the Cyclin D1 gene were genotyped, and methylation status of the p14, p16, DAP-kinase, and CDH1 genes were determined by methylation-specific-polymerase chain reaction in 139 gastric cancer tissues. CIHM high was defined as three or more methylated CpG islands.


Although no association was found between methylation status and different stages and Lauren’s subtypes, patients with CIHM of DAP-kinase showed significantly worse survival than those without (p = 0.017). In addition, the number of methylated sites was also associated with survival curves (p = 0.0397). The 870G carrier a significantly lower prevalence of CIHM high compared to the AA genotype in advanced-stage gastric cancer (adjusted OR = 0.32, p = 0.047). A weak correlation between the same genotypes and CIHM of p14 were found in the same subtype (adjusted OR = 0.32, p = 0.052). The mean methylation number was significantly lower in G carriers than in AA genotypes in advanced-stage gastric cancer (p = 0.017).


Genetic polymorphism of CCND1 is associated with CIHM status in gastric cancer, especially in the advanced stage, but is independent of clinico-pathological features.


CpG island hypermethylation Gastric cancer CCND1 Polymorphism G870A Methylation-specific-polymerase chain reaction 



CpG island hypermethylation


cyclin D1

H. pylori

Helicobacter pylori


Methylation-specific PCR


  1. 1.
    Rizos H, Darmanian AP, Mann GJ, Kefford RF. Two arginine rich domains in the p14ARF tumour suppressor mediate nucleolar localization. Oncogene. 2000;19:2978–2985.CrossRefPubMedGoogle Scholar
  2. 2.
    Tannapfel A, Busse C, Weinans L, et al. INK4a-ARF alterations and p53 mutations in hepatocellular carcinomas. Oncogene. 2001;20:7104–7109.CrossRefPubMedGoogle Scholar
  3. 3.
    Toyota M, Ahuja N, Suzuki H, et al. Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res. 1999;59:5438–5442.PubMedGoogle Scholar
  4. 4.
    Kang GH, Shim YH, Jung HY, Kim WH, Ro JY, Rhyu MG. CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res. 2001;61:2847–2851.PubMedGoogle Scholar
  5. 5.
    Chan AO, Lam SK, Wong BC, et al. Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer. Gut. 2003;52:502–506.CrossRefPubMedGoogle Scholar
  6. 6.
    Raveh T, Kimchi A. DAP kinase—a proapoptotic gene that functions as a tumor suppressor. Exp Cell Res. 2001;264:185–192.CrossRefPubMedGoogle Scholar
  7. 7.
    Schildhaus HU, Krockel I, Lippert H, Malfertheiner P, Roessner A, Schneider-Stock R. Promoter hypermethylation of p16INK4a, E-cadherin, O6-MGMT, DAPK and FHIT in adenocarcinomas of the esophagus, esophagogastric junction and proximal stomach. Int J Oncol. 2005;26:1493–1500.PubMedGoogle Scholar
  8. 8.
    Waki T, Tamura G, Sato M, Terashima M, Nishizuka S, Motoyama T. Promoter methylation status of DAP-kinase and RUNX3 genes in neoplastic and non-neoplastic gastric epithelia. Cancer Sci. 2003;94:360–364.CrossRefPubMedGoogle Scholar
  9. 9.
    Issa PJ, Ahuja N, Toyota M, Bronner P, Brentnall TA. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res. 2001;61:3573–3577.PubMedGoogle Scholar
  10. 10.
    Velicescu M, Weisenberger DJ, Gonzales FA, Tsai YC, Nguyen CT, Jones PA. Cell division is required for de novo methylation of CpG islands in bladder cancer cells. Cancer Res. 2002;62:2378–2384.PubMedGoogle Scholar
  11. 11.
    Sherr CJ. Cancer cell cycles. Science. 1996;274:1672–1677.CrossRefPubMedGoogle Scholar
  12. 12.
    Jiang W, Kahn SM, Zhou P, et al. Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression. Oncogene. 1993;8:3447–3457.PubMedGoogle Scholar
  13. 13.
    Quelle DE, Ashmun RA, Shurtleff SA, et al. Overexpression of mouse D-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev. 1993;7:1559–1571.CrossRefPubMedGoogle Scholar
  14. 14.
    Resnitzky D. Reed SI Different roles for cyclins D1 and E in regulation of the G1-to-S transition. Mol Cell Biol. 1995;15:3463–3469.PubMedGoogle Scholar
  15. 15.
    Robles AI, Larcher F, Whalin RB, et al. Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia. Proc Natl Acad Sci USA. 1996;93:7634–7638.CrossRefPubMedGoogle Scholar
  16. 16.
    Sauter ER, Yeo UC, von Stemm A, et al. Cyclin D1 is a candidate oncogene in cutaneous melanoma. Cancer Res. 2002;1:3200–3206.Google Scholar
  17. 17.
    Lung JC, Chu JS, Yu JC, et al. Aberrant expression of cell-cycle regulator cyclin D1 in breast cancer is related to chromosomal genomic instability. Genes Chromosomes Cancer. 2002;34:276–284.CrossRefPubMedGoogle Scholar
  18. 18.
    Moonen L, Ong F, Gallee M, et al. Apoptosis, proliferation and p53, cyclin D1, and retinoblastoma gene expression in relation to radiation response in transitional cell carcinoma of the bladder. Int J Radiat Oncol Biol Phys. 2001;49:1305–1310.PubMedGoogle Scholar
  19. 19.
    Sepulveda AR, Tao H, Carloni E, Sepulveda J, Graham DY, Peterson LE. Screening of gene expression profiles in gastric epithelial cells induced by Helicobacter pylori using microarray analysis. Aliment Pharmacol Ther. 2002;16(Suppl 2):145–157.CrossRefPubMedGoogle Scholar
  20. 20.
    Hirata Y, Maeda S, Mitsuno Y, et al. Helicobacter pylori activates the cyclin D1 gene through mitogen-activated protein kinase pathway in gastric cancer cells. Infect Immun. 2001;69:3965–3971.CrossRefPubMedGoogle Scholar
  21. 21.
    Arber N, Gammon MD, Hibshoosh H, et al. Overexpression of cyclin D1 occurs in both squamous carcinomas and adenocarcinomas of the esophagus and in adenocarcinomas of the stomach. Hum Pathol. 1999;30:1087–1092.CrossRefPubMedGoogle Scholar
  22. 22.
    Udhayakumar G, Jayanthi V, Devaraj N, Devaraj H. Interaction of MUC1 with beta-catenin modulates the Wnt target gene cyclin D1 in H. pylori-induced gastric cancer. Mol Carcinog. 2007;46:807–817.CrossRefPubMedGoogle Scholar
  23. 23.
    Yang GF, Deng CS, Xiong YY, Gong LL, Wang BC, Luo J. Expression of nuclear factor-kappa B and target genes in gastric precancerous lesions and adenocarcinoma: association with Helicobacter pylori cagA (+) infection. World J Gastroenterol. 2004;10:491–496.PubMedGoogle Scholar
  24. 24.
    Betticher DC, Thatcher N, Altermatt HJ, Hoban P, Ryder WD, Heighway J. Alternate splicing produces a novel cyclin D1 transcript. Oncogene. 1995;11:1005–1011.PubMedGoogle Scholar
  25. 25.
    Simpson DJ, Fryer AA, Grossman AB, et al. Cyclin D1 (CCND1) genotype is associated with tumour grade in sporadic pituitary adenomas. Carcinogenesis. 2001;22:1801–1807.CrossRefPubMedGoogle Scholar
  26. 26.
    Holley SL, Parkes G, Matthias C, et al. Cyclin D1 polymorphism and expression in patients with squamous cell carcinoma of the head and neck. Am J Pathol. 2001;159:1917–1924.PubMedGoogle Scholar
  27. 27.
    Kong S, Amos CI, Luthra R, Lynch PM, Levin B, Frazier ML. Effects of cyclin D1 polymorphism on age of onset of hereditary nonpolyposis colorectal cancer. Cancer Res. 2000;60:249–251.PubMedGoogle Scholar
  28. 28.
    Wang L, Habuchi T, Takahashi T, et al. Cyclin D1 gene polymorphism is associated with an increased risk of urinary bladder cancer. Carcinogenesis. 2002;23:257–264.CrossRefPubMedGoogle Scholar
  29. 29.
    Dhar KK, Branigan K, Howells RE, et al. Prognostic significance of cyclin D1 gene (CCND1) polymorphism in epithelial ovarian cancer. Int J Gynecol Cancer. 1999;9:342–347.CrossRefPubMedGoogle Scholar
  30. 30.
    Guo W, Dong Z, Chen Z, et al. Aberrant CpG island hypermethylation of RASSF1A in gastric cardia adenocarcinoma. Cancer Invest. 2009;27:459–465.CrossRefPubMedGoogle Scholar
  31. 31.
    Ogino S, Nosho K, Irahara N, et al. A cohort study of cyclin D1 expression and prognosis in 602 colon cancer cases. Clin Cancer Res. 2009;15:4431–4438.CrossRefPubMedGoogle Scholar
  32. 32.
    Liu T, Niu Y, Feng Y, et al. Methylation of CpG islands of p16(INK4a) and cyclin D1 overexpression associated with progression of intraductal proliferative lesions of the breast. Hum Pathol. 2008;39:1637–1646.CrossRefPubMedGoogle Scholar
  33. 33.
    Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand. 1965;64:31–49.PubMedGoogle Scholar
  34. 34.
    Esteller M, Tortola S, Toyota M, et al. Hypermethylation-associated inactivation of p14(ARF) is independent of p16(INK4a) methylation and p53 mutational status. Cancer Res. 2000;60:129–133.PubMedGoogle Scholar
  35. 35.
    Herman JG, Graff JR, Myöhänen 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.CrossRefPubMedGoogle Scholar
  36. 36.
    Katzenellenbogen RA, Baylin SB, Herman JG. Hypermethylation of the DAP-kinase CpG island is a common alteration in B-cell malignancies. Blood. 1999;93:4347–4353.PubMedGoogle Scholar
  37. 37.
    McKay JA, Douglas JJ, Ross VG, et al. Cyclin D1 protein expression and gene polymorphism in colorectal cancer. Aberdeen colorectal initiative. Int J Cancer. 2000;88:77–81.CrossRefPubMedGoogle Scholar
  38. 38.
    Kang GH, Lee HJ, Hwang KS, Lee S, Kim JH, Kim JS. Aberrant CpG island hypermethylation of chronic gastritis, in relation to aging, gender, intestinal metaplasia, and chronic inflammation. Am J Pathol. 2003;163:1551–1556.PubMedGoogle Scholar
  39. 39.
    Maekita T, Nakazawa K, Mihara M, et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res. 2006;12:989–995.CrossRefPubMedGoogle Scholar
  40. 40.
    Tahara T, Arisawa T, Shibata T, et al. Increased number of methylated CpG islands correlates with Helicobacter pylori infection, histological and serological severity of chronic gastritis. Eur J Gastroenterol Hepatol. 2009;21:613–619.CrossRefGoogle Scholar
  41. 41.
    Issa JP. GpG-island methylation in aging and cancer. Curr Top Microbiol Immunol. 2000;249:101–118.PubMedGoogle Scholar
  42. 42.
    Meyer-ter-Vehn T, Covacci A, Kist M, Pahl HL. Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun. J Biol Chem. 2000;275:16064–16072.CrossRefPubMedGoogle Scholar
  43. 43.
    Zhang J, Li Y, Wang R, et al. Association of cyclin D1 (G870A) polymorphism with susceptibility to esophageal and gastric cardiac carcinoma in a northern Chinese population. Int J Cancer. 2003;105:281–284.CrossRefPubMedGoogle Scholar
  44. 44.
    Song JH, Kim CJ, Cho YG, et al. Association of cyclin D1 G870A polymorphism with susceptibility to gastric cancers in Korean male patients. Neoplasma. 2007;54:235–239.PubMedGoogle Scholar
  45. 45.
    Tahara T, Shibata T, Yamashita H, Hirata I, Arisawa T. Effect of cyclin D1 (CCND1) polymorphism on gastric premalignant condition. Clin Chem Lab Med. 2008;46:1696–1701.CrossRefPubMedGoogle Scholar
  46. 46.
    Sawa H, Ohshima TA, Ukita H, et al. Alternatively spliced forms of cyclin D1 modulate entry into the cell cycle in an inverse manner. Oncogene. 1998;16:1701–1712.CrossRefPubMedGoogle Scholar
  47. 47.
    Hosokawa Y, Gadd M, Smith AP, Koerner FC, Schmidt EV, Arnold A. Cyclin D1 (PRAD1) alternative transcript b: full-length cDNA cloning and expression in breast cancers. Cancer Lett. 1997;113:123–130.CrossRefPubMedGoogle Scholar
  48. 48.
    Catarino R, Pereira D, Breda E, et al. Oncogenic virus-associated neoplasia: a role for cyclin D1 genotypes influencing the age of onset of disease? Biochem Biophys Res Commun. 2008;370:118–122.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Tomomitsu Tahara
    • 1
  • Tomoyuki Shibata
    • 1
  • Masakatsu Nakamura
    • 1
  • Hiromi Yamashita
    • 1
  • Daisuke Yoshioka
    • 1
  • Masaaki Okubo
    • 1
  • Joh Yonemura
    • 1
  • Yoshiteru Maeda
    • 1
  • Naoko Maruyama
    • 1
  • Toshiaki Kamano
    • 1
  • Yoshio Kamiya
    • 1
  • Hiroshi Fujita
    • 1
  • Yoshihito Nakagawa
    • 1
  • Mitsuo Nagasaka
    • 1
  • Masami Iwata
    • 1
  • Ichiro Hirata
    • 1
  • Tomiyasu Arisawa
    • 2
  1. 1.Department of GastroenterologyFujita Health University School of MedicineToyoakeJapan
  2. 2.Department of GastroenterologyKanazawa Medical UniversityUchinadamachiJapan

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