Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

A “combination oligonucleotide” antisense strategy to downregulate thymidylate synthase and decrease tumor cell growth and drug resistance

Cancer Gene Therapy volume 10, pages 278–286 (2003)Cite this article

Abstract

Thymidylate synthase (TS) catalyzes de novo production of thymidylate for DNA synthesis and cell proliferation. As such, TS has been a target of antitumor chemotherapy for many years. Our laboratory has identified several antisense oligodeoxynucleotides (ODNs) that downregulate TS mRNA and protein, inhibit cell proliferation, and sensitize cells to TS-directed chemotherapeutic drugs. Based on our observation that targeting distinct regions of the TS mRNA with a variety of antisense molecules resulted in differential effects on TS mRNA levels, it was hypothesized that use of multiple ODNs targeting distinct noncontiguous regions would result in synergistic or antagonistic interactions. In this study, we report that some combinations of TS antisense ODNs were more effective at reducing TS mRNA abundance and inhibiting cell proliferation than the individual ODNs used alone. However, in contrast to the effects on cell proliferation, the enhanced sensitivity to anti-TS chemotherapeutic drugs (i.e., raltitrexed and 5-fluorodeoxyuridine) that is achieved by treatment with individual ODNs was not further augmented by combined ODN treatment. This suggests that ODNs targeting TS mRNA inhibit an alternative function of TS mRNA or protein, distinct from thymidylate production. The results are evidence that the novel use of multiple antisense ODNs that target different regions of the same mRNA represents a general strategy to improve antisense effectiveness.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  1. Carreras CW, Santi DV . The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem. 1995;64:721–762.

    Article  CAS  Google Scholar 

  2. Danenberg PV . Thymidylate synthetase—a target enzyme in cancer chemotherapy. Biochim Biophys Acta. 1977;473:73–92.

    CAS  PubMed  Google Scholar 

  3. Danenberg PV, Malli H, Swenson S . Thymidylate synthase inhibitors. Semin Oncol. 1999;26:621–631.

    CAS  PubMed  Google Scholar 

  4. Papamichael D . The use of thymidylate synthase inhibitors in the treatment of advanced colorectal cancer: current status. Oncologist. 1999;4:478–487.

    CAS  PubMed  Google Scholar 

  5. Smith JB, Wickstrom E . Preclinical antisense DNA therapy of cancer in mice. Methods Enzymol. 2000;314:537–580.

    Article  CAS  Google Scholar 

  6. Gewirtz AM . Oligonucleotide therapeutics: a step forward. J Clin Oncol. 2000;18:1809–1811.

    Article  CAS  Google Scholar 

  7. Ferguson PJ, Collins O, Dean NM, et al. Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells. Br J Pharmacol. 1999;127:1777–1786.

    Article  CAS  Google Scholar 

  8. Berg RW, Werner M, Ferguson PJ, et al. Tumor growth inhibition in vivo and G2/M cell cycle arrest induced by antisense oligodeoxynucleotide targeting thymidylate synthase. J Pharmacol Exp Ther. 2001;298:477–484.

    CAS  PubMed  Google Scholar 

  9. Sato N, Mizumoto K, Maehara N, et al. Enhancement of drug-induced apoptosis by antisense oligodeoxynucleotides targeted against Mdm2 and p21WAF1/CIP1. Anticancer Res. 2000;20:837–842.

    CAS  PubMed  Google Scholar 

  10. Traidej M, Chen L, Yu D, Agrawal S, Chen J . The roles of E6-AP and MDM2 in p53 regulation in human papillomavirus-positive cervical cancer cells. Antisense Nucleic Acid Drug Dev. 2000;10:17–27.

    Article  CAS  Google Scholar 

  11. Normanno N, Bianco C, Damiano V, et al. Growth inhibition of human colon carcinoma cells by combinations of anti-epidermal growth factor-related growth factor antisense oligonucleotides. Clin Cancer Res. 1996;2:601–609.

    CAS  PubMed  Google Scholar 

  12. Skorski T, Nieborowska-Skorska M, Campbell K, et al. Leukemia treatment in severe combined immunodeficiency mice by antisense oligodeoxynucleotides targeting cooperating oncogenes. J Exp Med. 1995;182:1645–1653.

    Article  CAS  Google Scholar 

  13. Skorski T, Nieborowska-Skorska M, Wlodarski P, et al. Antisense oligodeoxynucleotide combination therapy of primary chronic myelogenous leukemia blast crisis in SCID mice. Blood. 1996;88:1005–1012.

    CAS  PubMed  Google Scholar 

  14. DeMoor JM, Vincent MD, Collins OM, Koropatnick J . Antisense nucleic acids targeted to the thymidylate synthase (TS) mRNA translation start site stimulate TS gene transcription. Exp Cell Res. 1998;243:11–21.

    Article  CAS  Google Scholar 

  15. Spears CP, Gustavsson BG . Methods for thymidylate synthase pharmacodynamics: serial biopsy, free and total TS, FdUMP and dUMP, and H4PteGlu and CH2-H4PteGlu assays. Adv Exp Med Biol. 1988;244:97–106.

    Article  CAS  Google Scholar 

  16. Hoganson DK, Williams AW, Berger SH . Isolation and characterization of a thymidylate synthase-deficient human colon tumor cell line. Biochem Pharmacol. 1999;58:1529–1537.

    Article  CAS  Google Scholar 

  17. Omura K, Morishita M, Kawakami K, et al. Quantification of thymidylate synthase gene expression in human gastrointestinal carcinoma tissues using competitive PCR. Hepatogastroenterology. 1999;46:985–990.

    CAS  PubMed  Google Scholar 

  18. Chu E, Koeller DM, Casey JL, et al. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proc Natl Acad Sci USA. 1991;88:8977–8981.

    Article  CAS  Google Scholar 

  19. Chu E, Voeller DM, Jones KL, et al. Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells. Mol Cell Biol. 1994;14:207–213.

    Article  CAS  Google Scholar 

  20. Mathews DH, Sabina J, Zuker M, et al. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J Mol Biol. 1999;288:911–940.

    Article  CAS  Google Scholar 

  21. Zuker M, Mathews DH, Turner DH . Algorithms and thermodynamics for RNA secondary structure prediction: a practical guide. In: Barciszewski J, Clark BFC, (eds). RNA Biochemistry and Biotechnology. New York, NY: Kluwer Academic Publishers;1999:pp. 11–43.

    Chapter  Google Scholar 

  22. Ho SP, Scully MS, Krauthauser CM, et al. Antisense oligonucleotides selectively regulate aspartyl beta-hydroxylase and its truncated protein isoform in vitro but distribute poorly into A549 tumors in vivo. J Pharmacol Exp Ther. 2002;302:795–803.

    Article  CAS  Google Scholar 

  23. Kandimalla ER, Manning A, Lathan C, Byrn RA, Agrawal S . Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides. Nucl Acids Res. 1995;23:3578–3584.

    Article  CAS  Google Scholar 

  24. Gewirtz AM, Stein CA, Glazer PM . Facilitating oligonucleotide delivery: helping antisense deliver on its promise. Proc Natl Acad Sci USA. 1996;93:3161–3163.

    Article  CAS  Google Scholar 

  25. Lebedeva I, Stein C . Antisense oligonucleotides: promise and reality. Annu Rev Pharmacol Toxicol. 2001;41:403–419.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by funds awarded to MV and JK by Zeneca Pharma Canada, Ltd, and Imperial Oil, Ltd. We thank Charlene Stirling, Helen Guenther, and Natalia Naraine for excellent technical assistance, and Mike Keeney (London Health Sciences Centre) for flow cytometry analysis. We also thank Dr Nicholas Dean (ISIS Pharmaceuticals, Carlsbad, CA) for supplying ODNs, and Astra Zeneca, Inc. (Macclesfield, UK) for supplying raltitrexed (Tomudex®).

Author information

Authors and Affiliations

  1. Cancer Research Laboratories, London Regional Cancer Centre, London, Ontario, Canada

    Randal W Berg, Peter J Ferguson, Mark D Vincent & D James Koropatnick

  2. Department of Pharmacology and Physiology, The University of Western Ontario, London, Ontario, Canada

    Peter J Ferguson & D James Koropatnick

  3. Department of Oncology, The University of Western Ontario, London, Ontario, Canada

    Mark D Vincent & D James Koropatnick

  4. Department of Pathology, The University of Western Ontario, London, Ontario, Canada

    D James Koropatnick

  5. Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada

    D James Koropatnick

Authors
  1. Randal W Berg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter J Ferguson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark D Vincent
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D James Koropatnick
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D James Koropatnick.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Berg, R., Ferguson, P., Vincent, M. et al. A “combination oligonucleotide” antisense strategy to downregulate thymidylate synthase and decrease tumor cell growth and drug resistance. Cancer Gene Ther 10, 278–286 (2003). https://doi.org/10.1038/sj.cgt.7700566

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700566

Keywords

Search

Advanced search

Quick links