Journal of Molecular Medicine

, 85:1137 | Cite as

Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy

  • Yeonjoo Jung
  • Jinah Park
  • Tai Young Kim
  • Jung-Hyun Park
  • Hyun-Soon Jong
  • Seock-Ah Im
  • Keith D. Robertson
  • Yung-Jue Bang
  • Tae-You Kim
Original Article

Abstract

The deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) has been used as a drug in a part of cancer therapy. However, because of its incorporation into DNA during DNA synthesis, 5-aza-dC can cause DNA damage, mutagenesis, and cytotoxicity. In view of the adverse effects of 5-aza-dC, DNMT-targeted inhibition may be a more effective approach than treatment with 5-aza-dC. To address the possibility of DNMT-targeted cancer therapy, we compared the effects of treatment with small interfering ribonucleic acids (siRNAs) specific for DNMT1 or DNMT3b and treatment with 5-aza-dC on transcription, cell growth, and DNA damage in gastric cancer cells. We found that DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CDKN2A, RASSF1A, HTLF, RUNX3, and AKAP12B) out of seven genes examined, and 5-aza-dC reactivated and demethylated all seven genes. In contrast, DNMT3b siRNAs did not show any effect. Furthermore, the double knockdown of DNMT1 and DNMT3b did not show a synergistic effect on gene re-expression and demethylation. In addition, DNMT1 siRNAs showed an inhibitory effect of cell proliferation in the cancer cells and the induction of cell death without evidence of DNA damage, whereas treatment with 5-aza-dC caused DNA damage as demonstrated by the comet assay. These results provide a rationale for the development of a DNMT1-targeted strategy as an effective epigenetic cancer therapy.

Keywords

Epigenetic gene silencing Promoter hypermethylation DNA methyltransferase siRNAs 5-aza-dC 

Abbreviations

DNMT

DNA methyltransferase

5-aza-dC

5-aza-2′-deoxycytidine

siRNA

small interfering RNA

Notes

Acknowledgments

This work was supported in part by grants from the Korean Ministry of Science and Technology through the National Research Laboratory Program for Cancer Epigenetics (No. M10400000336-06J0000-33610), and BK21 Project for Medicine, Dentistry and Pharmacy.

Supplementary material

109_2007_216_MOESM1_ESM.pdf (181 kb)
Supplementary Table 1 Primer sequences for RT-PCR analysis (PDF 180 KB)
109_2007_216_MOESM2_ESM.pdf (130 kb)
Supplementary Table 2 Primer sequences for MS-PCR analysis (PDF 130 KB)
109_2007_216_MOESM3_ESM.pdf (96 kb)
Supplementary Figure 1 (PDF 96.1 KB)
109_2007_216_MOESM4_ESM.pdf (23 kb)
Supplementary Figure 2 (PDF 22.8 KB)

References

  1. 1.
    Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3:415–428PubMedCrossRefGoogle Scholar
  2. 2.
    Rountree MR, Bachman KE, Herman JG, Baylin SB (2001) DNA methylation, chromatin inheritance, and cancer. Oncogene 20:3156–3165PubMedCrossRefGoogle Scholar
  3. 3.
    Robertson KD (2001) DNA methylation, methyltransferases, and cancer. Oncogene 20:3139–3155PubMedCrossRefGoogle Scholar
  4. 4.
    Rhee I, Jair KW, Yen RW, Lengauer C, Herman JG, Kinzler KW, Vogelstein B, Baylin SB, Schuebel KE (2000) CpG methylation is maintained in human cancer cells lacking DNMT1. Nature 404:1003–1007PubMedCrossRefGoogle Scholar
  5. 5.
    Rhee I, Bachman KE, Park BH, Jair KW, Yen RW, Schuebel KE, Cui H, Feinberg AP, Lengauer C, Kinzler KW, Baylin SB, Vogelstein B (2002) DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature 416:552–556PubMedCrossRefGoogle Scholar
  6. 6.
    Egger G, Jeong S, Escobar SG, Cortez CC, Li TW, Saito Y, Yoo CB, Jones PA, Liang G (2006) Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival. Proc Natl Acad Sci USA 103:14080–14085PubMedCrossRefGoogle Scholar
  7. 7.
    Robert MF, Morin S, Beaulieu N, Gauthier F, Chute IC, Barsalou A, MacLeod AR (2003) DNMT1 is required to maintain CpG methylation and aberrant gene silencing in human cancer cells. Nat Genet 33:61–65PubMedCrossRefGoogle Scholar
  8. 8.
    Suzuki M, Sunaga N, Shames DS, Toyooka S, Gazdar AF, Minna JD (2004) RNA interference-mediated knockdown of DNA methyltransferase 1 leads to promoter demethylation and gene re-expression in human lung and breast cancer cells. Cancer Res 64:3137–3143PubMedCrossRefGoogle Scholar
  9. 9.
    Ting AH, Jair KW, Suzuki H, Yen RW, Baylin SB, Schuebel KE (2004) CpG island hypermethylation is maintained in human colorectal cancer cells after RNAi-mediated depletion of DNMT1. Nat Genet 36:582–584PubMedCrossRefGoogle Scholar
  10. 10.
    Beaulieu N, Morin S, Chute IC, Robert MF, Nguyen H, MacLeod AR (2002) An essential role for DNA methyltransferase DNMT3B in cancer cell survival. J Biol Chem 277:28176–28181PubMedCrossRefGoogle Scholar
  11. 11.
    Leu YW, Rahmatpanah F, Shi H, Wei SH, Liu JC, Yan PS, Huang TH (2003) Double RNA interference of DNMT3b and DNMT1 enhances DNA demethylation and gene reactivation. Cancer Res 63:6110–6115PubMedGoogle Scholar
  12. 12.
    Juttermann R, Li E, Jaenisch R (1994) Toxicity of 5-aza-2′-deoxycytidine to mammalian cells is mediated primarily by covalent trapping of DNA methyltransferase rather than DNA demethylation. Proc Natl Acad Sci USA 91:11797–11801PubMedCrossRefGoogle Scholar
  13. 13.
    Zhu WG, Hileman T, Ke Y, Wang P, Lu S, Duan W, Dai Z, Tong T, Villalona-Calero MA, Plass C, Otterson GA (2004) 5-aza-2′-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation. J Biol Chem 279:15161–15166PubMedCrossRefGoogle Scholar
  14. 14.
    Weisenberger DJ, Velicescu M, Cheng JC, Gonzales FA, Liang G, Jones PA (2004) Role of the DNA methyltransferase variant DNMT3b3 in DNA methylation. Mol Cancer Res 2:62–72PubMedGoogle Scholar
  15. 15.
    Kang GH, Lee S, Kim JS, Jung HY (2003) Profile of aberrant CpG island methylation along multistep gastric carcinogenesis. Lab Invest 83:519–526PubMedGoogle Scholar
  16. 16.
    Kim TY, Jong HS, Jung Y, Kang GH, Bang YJ (2004) DNA hypermethylation in gastric cancer. Aliment Pharmacol Ther 20(Suppl 1):131–142PubMedCrossRefGoogle Scholar
  17. 17.
    Kim TY, Jong HS, Song SH, Dimtchev A, Jeong SJ, Lee JW, Kim NK, Jung M, Bang YJ (2003) Transcriptional silencing of the DLC-1 tumor suppressor gene by epigenetic mechanism in gastric cancer cells. Oncogene 22:3943–3951PubMedCrossRefGoogle Scholar
  18. 18.
    Choi MC, Jong HS, Kim TY, Song SH, Lee DS, Lee JW, Kim NK, Bang YJ (2004) AKAP12/Gravin is inactivated by epigenetic mechanism in human gastric carcinoma and shows growth suppressor activity. Oncogene 23:7095–7103PubMedCrossRefGoogle Scholar
  19. 19.
    Kim TY, Lee HJ, Hwang KS, Lee M, Kim JW, Bang YJ, Kang GH (2004) Methylation of RUNX3 in various types of human cancers and premalignant stages of gastric carcinoma. Lab Invest 84:479–484PubMedCrossRefGoogle Scholar
  20. 20.
    Ku JL, Park JG (2005) Biology of SNU cell lines. Cancer Res Treat 37:1–19CrossRefGoogle Scholar
  21. 21.
    Robertson KD, Uzvolgyi E, Liang G, Talmadge C, Sumegi J, Gonzales FA, Jones PA (1999) The human DNA methyltransferases (DNMTs) 1, 3a and 3b: coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res 27:2291–2298PubMedCrossRefGoogle Scholar
  22. 22.
    Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP (2000) Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet 25:315–319PubMedCrossRefGoogle Scholar
  23. 23.
    Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221PubMedCrossRefGoogle Scholar
  24. 24.
    Olive PL, Durand RE (2005) Heterogeneity in DNA damage using the comet assay. Cytometry A 66:1–8PubMedGoogle Scholar
  25. 25.
    Ting AH, Jair KW, Schuebel KE, Baylin SB (2006) Differential requirement for DNA methyltransferase 1 in maintaining human cancer cell gene promoter hypermethylation. Cancer Res 66:729–735PubMedCrossRefGoogle Scholar
  26. 26.
    Chen T, Tsujimoto N, Li E (2004) The PWWP domain of Dnmt3a and Dnmt3b is required for directing DNA methylation to the major satellite repeats at pericentric heterochromatin. Mol Cell Biol 24:9048–9058PubMedCrossRefGoogle Scholar
  27. 27.
    Gius D, Cui H, Bradbury CM, Cook J, Smart DK, Zhao S, Young L, Brandenburg SA, Hu Y, Bisht KS, Ho AS, Mattson D, Sun L, Munson PJ, Chuang EY, Mitchell JB, Feinberg AP (2004) Distinct effects on gene expression of chemical and genetic manipulation of the cancer epigenome revealed by a multimodality approach. Cancer Cell 6:361–371PubMedCrossRefGoogle Scholar
  28. 28.
    Santini V, Kantarjian HM, Issa JP (2001) Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann Intern Med 134:573–586PubMedGoogle Scholar
  29. 29.
    Leone G, Voso MT, Teofili L, Lubbert M (2003) Inhibitors of DNA methylation in the treatment of hematological malignancies and MDS. Clin Immunol 109:89–102PubMedCrossRefGoogle Scholar
  30. 30.
    Yang AS, Doshi KD, Choi SW, Mason JB, Mannari RK, Gharybian V, Luna R, Rashid A, Shen L, Estecio MR, Kantarjian HM, Garcia-Manero G, Issa JP (2006) DNA methylation changes after 5-aza-2′-deoxycytidine therapy in patients with leukemia. Cancer Res 66:5495–5503PubMedCrossRefGoogle Scholar
  31. 31.
    Brueckner B, Boy RG, Siedlecki P, Musch T, Kliem HC, Zielenkiewicz P, Suhai S, Wiessler M, Lyko F (2005) Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases. Cancer Res 65:6305–6311PubMedCrossRefGoogle Scholar
  32. 32.
    Stresemann C, Brueckner B, Musch T, Stopper H, Lyko F (2006) Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines. Cancer Res 66:2794–2800PubMedCrossRefGoogle Scholar
  33. 33.
    Szyf M (2005) DNA methylation and demethylation as targets for anticancer therapy. Biochemistry (Mosc) 70:533–549CrossRefGoogle Scholar
  34. 34.
    Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, John M, Kesavan V, Lavine G, Pandey RK, Racie T, Rajeev KG, Rohl I, Toudjarska I, Wang G, Wuschko S, Bumcrot D, Koteliansky V, Limmer S, Manoharan M, Vornlocher HP (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432:173–178PubMedCrossRefGoogle Scholar
  35. 35.
    Behlke MA (2006) Progress towards in vivo use of siRNAs. Mol Ther 13:644–670PubMedCrossRefGoogle Scholar
  36. 36.
    Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, Molema G, Lu PY, Scaria PV, Woodle MC (2004) Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res 32:e149PubMedCrossRefGoogle Scholar
  37. 37.
    Urban-Klein B, Werth S, Abuharbeid S, Czubayko F, Aigner A (2005) RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther 12:461–466PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Yeonjoo Jung
    • 1
  • Jinah Park
    • 1
  • Tai Young Kim
    • 1
  • Jung-Hyun Park
    • 1
  • Hyun-Soon Jong
    • 1
  • Seock-Ah Im
    • 1
    • 2
  • Keith D. Robertson
    • 3
  • Yung-Jue Bang
    • 1
    • 2
  • Tae-You Kim
    • 1
    • 2
  1. 1.National Research Laboratory for Cancer Epigenetics, Cancer Research InstituteSeoul National University College of MedicineSeoulSouth Korea
  2. 2.Department of Internal MedicineSeoul National University College of MedicineSeoulSouth Korea
  3. 3.Department of Biochemistry and Molecular BiologyUniversity of FloridaGainesvilleUSA

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