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Cancer Chemotherapy and Pharmacology

, Volume 74, Issue 2, pp 291–302 | Cite as

Novel DNA methyltransferase-1 (DNMT1) depleting anticancer nucleosides, 4′-thio-2′-deoxycytidine and 5-aza-4′-thio-2′-deoxycytidine

  • Jaideep V. ThottasseryEmail author
  • Vijaya Sambandam
  • Paula W. Allan
  • Joseph A. Maddry
  • Yulia Y. Maxuitenko
  • Kamal Tiwari
  • Melinda Hollingshead
  • William B. ParkerEmail author
Original Article

Abstract

Purpose

Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2′-deoxycytidine (dCyd) analogs, 4′-thio-2′-deoxycytidine (T-dCyd) and 5-aza-4′-thio-2′-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth.

Methods

DNMT1 protein levels in in vitro and in vivo cancer models were determined by Western blotting and antitumor efficacy was evaluated using xenografts. Effects on CpG methylation were evaluated using methylation-specific PCR. T-dCyd metabolism was evaluated using radiolabeled substrate.

Results

T-dCyd markedly depleted DNMT1 in CCRF-CEM and KG1a leukemia and NCI-H23 lung carcinoma cell lines, while it was ineffective in the HCT-116 colon or IGROV-1 ovarian tumor lines. On the other hand, aza-T-dCyd potently depleted DNMT1 in all of these lines indicating that dCyd analogs with minor structural dissimilarities induce different DNMT1 turnover mechanisms. Although T-dCyd was deaminated to 4′-thio-2′-deoxyuridine, very little was converted to 4′-thio-thymidine nucleotides, suggesting that inhibition of thymidylate synthase would be minimal with 4′-thio dCyd analogs. Both T-dCyd and aza-T-dCyd also depleted DNMT1 in human tumor xenografts and markedly reduced in vivo tumor growth. Interestingly, the selectivity index of aza-T-dCyd was at least tenfold greater than that of decitabine.

Conclusions

Collectively, these data show that 4′-thio modified dCyd analogs, such as T-dCyd or aza-T-dCyd, could be a new source of clinically effective DNMT1 depleting anticancer compounds with less toxicity.

Keywords

DNMT1 DNA methyltransferase Decitabine Azacytidine Zebularine Deoxycytidine 

Notes

Acknowledgments

We thank Dr. Mark Suto for careful review of the manuscript. We also thank Dr. Joel Morris for T-dCyd synthesis and Dr. Robert J. Kinders for providing tumor lysates from T-dCyd-treated mice. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. NCI-Frederick and Southern Research Institute are accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 2010; National Academy Press; Washington, D.C.). Work described in this report was funded by NIH Grant # P01 CA34200, NCI contract N01-CO-12400, the ADDA (Alabama Drug Discovery Alliance) and the UAB Center for Clinical and Translational Science under 5UL1 RR025777.

Conflict of interest

None.

References

  1. 1.
    Brown KD, Robertson KD (2007) DNMT1 knockout delivers a strong blow to genome stability and cell viability. Nat Genet 39:289–290PubMedCrossRefGoogle Scholar
  2. 2.
    Esteller M, Corn PG, Baylin SB, Herman JG (2001) A gene hypermethylation profile of human cancer. Cancer Res 61:3225–3229PubMedGoogle Scholar
  3. 3.
    Jones PA, Baylin SB (2007) The epigenomics of cancer. Cell 128:683–692PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Baylin SB, Ohm JE (2006) Epigenetic gene silencing in cancer—a mechanism for early oncogenic pathway addiction? Nat Rev Cancer 6:107–116PubMedCrossRefGoogle Scholar
  5. 5.
    Ooi SK, O’Donnell AH, Bestor TH (2009) Mammalian cytosine methylation at a glance. J Cell Sci 122:2787–2791PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Wu J, Issa JP, Herman J, Bassett DE Jr, Nelkin BD, Baylin SB (1993) Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells. Proc Natl Acad Sci USA 90:8891–8895PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Vertino PM, Issa JP, Pereira-Smith OM, Baylin SB (1994) Stabilization of DNA methyltransferase levels and CpG island hypermethylation precede SV40-induced immortalization of human fibroblasts. Cell Growth Differ 5:1395–1402PubMedGoogle Scholar
  8. 8.
    Bakin AV, Curran T (1999) Role of DNA 5-methylcytosine transferase in cell transformation by fos. Science 283:387–390PubMedCrossRefGoogle Scholar
  9. 9.
    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
  10. 10.
    Laird PW, Jackson-Grusby L, Fazeli A, Dickinson SL, Jung WE, Li E, Weinberg RA, Jaenisch R (1995) Suppression of intestinal neoplasia by DNA hypomethylation. Cell 81:197–205PubMedCrossRefGoogle Scholar
  11. 11.
    Belinsky SA, Klinge DM, Stidley CA, Issa JP, Herman JG, March TH, Baylin SB (2003) Inhibition of DNA methylation and histone deacetylation prevents murine lung cancer. Cancer Res 63:7089–7093PubMedGoogle Scholar
  12. 12.
    Milutinovic S, Knox JD, Szyf M (2000) DNA methyltransferase inhibition induces the transcription of the tumor suppressor p21(WAF1/CIP1). J Biol Chem 275:6353–6359PubMedCrossRefGoogle Scholar
  13. 13.
    Milutinovic S, Brown SE, Zhuang Q, Szyf M (2004) DNA methyltransferase 1 knock down induces gene expression by a mechanism independent of DNA methylation and histone deacetylation. J Biol Chem 279:27915–27927PubMedCrossRefGoogle Scholar
  14. 14.
    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 103:14080–14085PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Agoston AT, Argani P, Yegnasubramanian S, De Marzo AM, Ansari-Lari MA, Hicks JL, Davidson NE, Nelson WG (2005) Increased protein stability causes DNA methyltransferase 1 dysregulation in breast cancer. J Biol Chem 280:18302–18310PubMedCrossRefGoogle Scholar
  16. 16.
    Zhou Q, Agoston AT, Atadja P, Nelson WG, Davidson NE (2008) Inhibition of histone deacetylases promotes ubiquitin-dependent proteasomal degradation of DNA methyltransferase 1 in human breast cancer cells. Mol Cancer Res 6:873–883PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Ghoshal K, Datta J, Majumder S, Bai S, Kutay H, Motiwala T, Jacob ST (2005) 5-Aza-deoxycytidine induces selective degradation of DNA methyltransferase 1 by a proteasomal pathway that requires the KEN box, bromo-adjacent homology domain, and nuclear localization signal. Mol Cell Biol 25:4727–4741PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Patel K, Dickson J, Din S, Macleod K, Jodrell D, Ramsahoye B (2010) Targeting of 5-aza-2′-deoxycytidine residues by chromatin-associated DNMT1 induces proteasomal degradation of the free enzyme. Nucleic Acids Res 38:4313–4324PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Santi DV, Norment A, Garrett CE (1984) Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci USA 81:6993–6997PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Beumer JH, Parise RA, Newman EM, Doroshow JH, Synold TW, Lenz HJ, Egorin MJ (2008) Concentrations of the DNA methyltransferase inhibitor 5-fluoro-2′-deoxycytidine (FdCyd) and its cytotoxic metabolites in plasma of patients treated with FdCyd and tetrahydrouridine. Cancer Chemother Pharmacol 62:363–368PubMedCrossRefGoogle Scholar
  21. 21.
    Marquez VE, Barchi JJ Jr, Kelley JA, Rao KV, Agbaria R, Ben-Kasus T, Cheng JC, Yoo CB, Jones PA (2005) Zebularine: a unique molecule for an epigenetically based strategy in cancer chemotherapy. The magic of its chemistry and biology. Nucleosides, Nucleotides Nucleic Acids 24:305–318PubMedCrossRefGoogle Scholar
  22. 22.
    Parker WB, Shaddix SC, Rose LM, Waud WR, Shewach DS, Tiwari KN, Secrist JA III (2000) Metabolism of 4′-thio-beta-D-arabinofuranosylcytosine in CEM cells. Biochem Pharmacol 60:1925–1932PubMedCrossRefGoogle Scholar
  23. 23.
    Thottassery JV, Tiwari KN, Westbrook L, Secrist JA III, Parker WB (2011) Novel 2′-deoxycytidine analogs as DNA demethylation agents. Proc Am Assoc Cancer Res 71:2537. doi: 10.1158/1538-7445.AM2011-2537 Google Scholar
  24. 24.
    Secrist JA III, Tiwari KN, Riordan JM, Montgomery JA (1991) Synthesis and biological activity of 2′-deoxy-4′-thio pyrimidine nucleosides. J Med Chem 34:2361–2366PubMedCrossRefGoogle Scholar
  25. 25.
    Tiwari KN, Cappellacci L, Montgomery JA, Secrist JA III (2003) Synthesis and anti-cancer activity of some novel 5-azacytosine nucleosides. Nucleosides, Nucleotides Nucleic Acids 22:2161–2170PubMedCrossRefGoogle Scholar
  26. 26.
    Someya H, Shaddix SC, Tiwari KN, Secrist JA III, Parker WB (2003) Phosphorylation of 4′-thio-beta-D-arabinofuranosylcytosine and its analogs by human deoxycytidine kinase. J Pharmacol Exp Ther 304:1314–1322PubMedCrossRefGoogle Scholar
  27. 27.
    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 USA 93:9821–9826PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Dodge JE, Munson C, List AF (2001) KG-1 and KG-1a model the p15 CpG island methylation observed in acute myeloid leukemia patients. Leuk Res 25:917–925PubMedCrossRefGoogle Scholar
  29. 29.
    Kumar S, Horton JR, Jones GD, Walker RT, Roberts RJ, Cheng X (1997) DNA containing 4′-thio-2′-deoxycytidine inhibits methylation by HhaI methyltransferase. Nucleic Acids Res 25:2773–2783PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Herman JG, Civin CI, Issa JP, Collector MI, Sharkis SJ, Baylin SB (1997) Distinct patterns of inactivation of p15INK4B and p16INK4A characterize the major types of hematological malignancies. Cancer Res 57:837–841PubMedGoogle Scholar
  31. 31.
    Dodge JE, List AF, Futscher BW (1998) Selective variegated methylation of the p15 CpG island in acute myeloid leukemia. Int J Cancer 78:561–567PubMedCrossRefGoogle Scholar
  32. 32.
    Verri A, Focher F, Duncombe RJ, Basnak I, Walker RT, Coe PL, de Clercq E, Andrei G, Snoeck R, Balzarini J, Spadari S (2000) Anti-(herpes simplex virus) activity of 4′-thio-2′-deoxyuridines: a biochemical investigation for viral and cellular target enzymes. Biochem J 351(Pt 2):319–326PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Rogstad DK, Herring JL, Theruvathu JA, Burdzy A, Perry CC, Neidigh JW, Sowers LC (2009) Chemical decomposition of 5-aza-2′-deoxycytidine (Decitabine): kinetic analyses and identification of products by NMR, HPLC, and mass spectrometry. Chem Res Toxicol 22:1194–1204PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Esteve PO, Chin HG, Benner J, Feehery GR, Samaranayake M, Horwitz GA, Jacobsen SE, Pradhan S (2009) Regulation of DNMT1 stability through SET7-mediated lysine methylation in mammalian cells. Proc Natl Acad Sci USA 106:5076–5081PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Parker WB, Shaddix SC, Rose LM, Tiwari KN, Montogmery JA, Secrist JA III, Bennett LL Jr (1995) Metabolism and metabolic actions of 4′-thiothymidine in L1210 cells. Biochem Pharmacol 50:687–695PubMedCrossRefGoogle Scholar
  36. 36.
    Someya H, Waud WR, Parker WB (2006) Long intracellular retention of 4′-thio-arabinofuranosylcytosine 5′-triphosphate as a critical factor for the anti-solid tumor activity of 4′-thio-arabinofuranosylcytosine. Cancer Chemother Pharmacol 57:772–780PubMedCrossRefGoogle Scholar
  37. 37.
    Richardson KA, Vega TP, Richardson FC, Moore CL, Rohloff JC, Tomkinson B, Bendele RA, Kutcha RD (2004) Polymerization of the triphosphates of araC, 2′,2′-difluorodeoxycytidine (dFdC) and OSI-7836 (T-araC)by human DNA polymerase alpha and DNA primase. Biochem Pharmacol 68:2337–2346PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jaideep V. Thottassery
    • 1
    • 3
    Email author
  • Vijaya Sambandam
    • 1
  • Paula W. Allan
    • 1
  • Joseph A. Maddry
    • 1
  • Yulia Y. Maxuitenko
    • 1
  • Kamal Tiwari
    • 1
  • Melinda Hollingshead
    • 2
  • William B. Parker
    • 1
    • 3
    Email author
  1. 1.Drug Discovery DivisionSouthern Research InstituteBirminghamUSA
  2. 2.Biological Testing BranchNCI at FrederickFrederickUSA
  3. 3.University of Alabama at Birmingham, Comprehensive Cancer CenterBirminghamUSA

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