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.

  • Research Article
  • Published:

Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer

Gene Therapy volume 12, pages 1417–1423 (2005)Cite this article

Abstract

Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase (iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (<5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine (PE). CMViNOS transfection also reduced response to PE, but by only 52%. A single injection of 25 μg of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMV-driven construct. Our data suggest that generation of NO•, as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO•. This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.

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

Similar content being viewed by others

References

  1. Kramer MG et al. In vitro and in vivo comparative study of chimeric liver-specific promoters. Mol Ther 2003; 7: 375–385.

    Article  CAS  PubMed  Google Scholar 

  2. McArt JA et al. Development of a melanoma-specific adenovirus. Mol Ther 2002; 6: 471–480.

    Article  Google Scholar 

  3. Liu BH et al. CMV enhancer/human PDGF-beta promoter for neuron-specific transgene expression. Gene Therapy 2004; 11: 52–60.

    Article  PubMed  Google Scholar 

  4. Abdul-Ghani R et al. Use of transcriptional regulatory sequences of telomerase (hTER and hTERT) for selective killing of cancer cells. Mol Ther 2000; 2: 539–544.

    Article  CAS  PubMed  Google Scholar 

  5. Cao G et al. Effective and safe gene therapy for colorectal carcinoma using the cytosine deaminase gene directed by the carcinoembryonic antigen promoter. Gene Therapy 1999; 6: 83–90.

    Article  CAS  PubMed  Google Scholar 

  6. Cowen RL et al. Hypoxia targeted gene therapy to increase the efficacy of tirapazamine as an adjuvant to radiotherapy: reversing tumor radioresistance and effecting cure. Cancer Res 2004; 64: 1396–1402.

    Article  CAS  PubMed  Google Scholar 

  7. Guilhon E et al. Image-guided control of transgene expression based on local hyperthermia. Mol Imaging 2003; 2: 1–7.

    Article  Google Scholar 

  8. Li GC et al. Adenovirus-mediated heat-activated antisense Ku70 expression radiosensitizes tumor cells in vitro and in vivo. Cancer Res 2003; 63: 3268–3274.

    CAS  PubMed  Google Scholar 

  9. Kufe D, Weichselbaum R . Radiation therapy: activation for gene transcription and the development of genetic radiotherapy-therapeutic strategies in oncology. Cancer Biol Ther 2003; 2: 326–329.

    Article  CAS  PubMed  Google Scholar 

  10. Chtarto A et al. Minocycline-induced activation of tetracycline-responsive promoter. Neurosci Lett 2003; 352: 155–158.

    Article  CAS  PubMed  Google Scholar 

  11. Worthington J et al. Modification of vascular tone using iNOS under the control of a radiation-inducible promoter. Gene Therapy 2000; 7: 1126–1131.

    Article  CAS  PubMed  Google Scholar 

  12. Worthington J, Robson T, O'Keefe M, Hirst DG . Tumour cell radiosensitization using constitutive (CMV) and radiation inducible (WAF1) promoters to drive the iNOS gene: a novel suicide gene therapy. Gene Therapy 2002; 9: 263–269.

    Article  CAS  PubMed  Google Scholar 

  13. Worthington J et al. Use of the radiation-inducible WAF1 promoter to drive iNOS gene therapy as a novel anti-cancer treatment. J Gene Med 2004; 6: 673–680.

    Article  CAS  PubMed  Google Scholar 

  14. Datta R et al. Ionizing radiation activates transcription of the EGR1 gene via CArG elements. Proc Natl Acad Sci USA 1992; 89: 10149–11053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Takahashi T, Namiki Y, Ohno T . Induction of the suicide HSV-TK gene by activation of the Egr-1 promoter with radioisotopes. Human Gene Ther 1997; 8: 827–833.

    Article  CAS  Google Scholar 

  16. Hallahan D et al. Spatial and temporal control of gene therapy using ionizing radiation. Nat Med 1995; 1: 786–791.

    Article  CAS  PubMed  Google Scholar 

  17. Seung LP et al. Genetic radiotherapy overcomes tumour resistance to cytotoxic agents. Cancer Res 1995; 55: 5561–5565.

    CAS  PubMed  Google Scholar 

  18. Marples B et al. Development of synthetic promoters for radiation-mediated gene therapy. Gene Therapy 2000; 7: 511–517.

    Article  CAS  PubMed  Google Scholar 

  19. Marples B, Greco O, Joiner M, Scott S . Molecular approaches to chemo-radiotherapy. Eur J Cancer 2002; 38: 231–239.

    Article  CAS  PubMed  Google Scholar 

  20. Scott S, Joiner M, Marples B . Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene Therapy 2002; 9: 1396–1402.

    Article  CAS  PubMed  Google Scholar 

  21. Greco O et al. Novel chimeric gene promoters responsive to hypoxia and ionizing radiation. Gene Therapy 2002; 9: 1403–1411.

    Article  CAS  PubMed  Google Scholar 

  22. Datta R et al. Reactive oxygen intermediates target CC(A/T)6GG sequences to mediate activation of the early growth response 1 transcription factor gene by ionizing radiation. Proc Natl Acad Sci USA 1993; 90: 2419–2422.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Mitchell JB et al. Hypoxic mammalian cell radiosensitization by nitric oxide. Cancer Res 1993; 53: 5845–5848.

    CAS  PubMed  Google Scholar 

  24. Griffin RJ, Makepeace CM, Hur WJ, Song CW . Radiosensitization of hypoxic tumor cells in vitro by nitric oxide. Int J Radiat Oncol Biol Phys 1996; 36: 377–383.

    Article  CAS  PubMed  Google Scholar 

  25. Kurimoto M et al. Growth inhibition and radiosensitization of cultured glioma cells by nitric oxide generating agents. J Neurooncol 1999; 42: 35–44.

    Article  CAS  PubMed  Google Scholar 

  26. Chung HT et al. Nitric oxide as a bioregulator of apoptosis. Biochem Biophys Res Commun 2001; 282: 1075–1079.

    Article  CAS  PubMed  Google Scholar 

  27. Van de Casteele M, Hosli M, Sagesser H, Reichen J . Intraportal administration of glyceryl trinitrate or nitroprusside exerts more systemic than intrahepatic effects in anaesthetised cirrhotic rats. J Hepatol 1999; 31: 300–305.

    Article  CAS  PubMed  Google Scholar 

  28. Robson T, Hirst DG . Transcriptional targeting in cancer gene therapy. J Biomed Biotech 2003; 2: 110–137.

    Article  Google Scholar 

  29. Greco O, Marples B, Joiner M, Scott S . How to overcome (and exploit) tumor hypoxia for targeted gene therapy. J Cell Physiol 2003; 197: 312–325.

    Article  CAS  PubMed  Google Scholar 

  30. Weichselbaum RR, Hallahan D, Fuks Z, Kufe D . Radiation induction of immediate early genes: effectors of the radiation-stress response. Int J Radiat Oncol Biol Phys 1994; 30: 229–234.

    Article  CAS  PubMed  Google Scholar 

  31. Wang Z et al. Adenoviral gene transfer of the human inducible nitric oxide synthase gene enhances the radiation response of human colorectal cancer associated with alterations in tumor vascularity. Cancer Res 2004; 64: 1386–1395.

    Article  CAS  PubMed  Google Scholar 

  32. Karihtala P, Kinnula VL, Soini Y . Antioxidative response for nitric oxide production in breast carcinoma. Oncol Rep 2004; 12: 755–759.

    CAS  PubMed  Google Scholar 

  33. Kao CL et al. Elevated nitric oxide levels in childhood brain tumors. Childs Nerv Syst 2003; 19: 744–749.

    Article  PubMed  Google Scholar 

  34. Wang J et al. Expression of inducible nitric oxide synthase in paired neoplastic and non-neoplastic primary prostate cell cultures and prostatectomy specimen. Urol Oncol 2003; 21: 117–122.

    Article  PubMed  Google Scholar 

  35. Gallo O et al. Down-regulation of nitric oxide synthase-2 and cyclooxygenase-2 pathways by p53 in squamous cell carcinoma. Am J Pathol 2003; 163: 723–732.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Chung P et al. Overexpression of the human inducible nitric oxide synthase gene enhances radiation-induced apoptosis in colorectal cancer cells via a caspase-dependent mechanism. Nitric Oxide 2003; 8: 119–126.

    Article  CAS  PubMed  Google Scholar 

  37. Khare PD et al. Tumor growth suppression by a retroviral vector displaying scFv antibody to CEA and carrying the iNOS gene. Anticancer Res 2002; 22: 2443–2446.

    CAS  PubMed  Google Scholar 

  38. Heigold S et al. Nitric oxide mediates apoptosis induction selectively in transformed fibroblasts compared to nontransformed fibroblasts. Carcinogenesis 2002; 23: 929–941.

    Article  CAS  PubMed  Google Scholar 

  39. Hemmrich K, Suschek CV, Lerzynski G, Kolb-Bachofen V . iNOS activity is essential for endothelial stress gene expression protecting against oxidative damage. J Appl Physiol 2003; 95: 1937–1946.

    Article  CAS  PubMed  Google Scholar 

  40. Suschek CV et al. Even after UVA-exposure will nitric oxide protect cells from reactive oxygen intermediate-mediated apoptosis and necrosis. Cell Death Differ 2001; 8: 515–527.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was funded by project Grants C1278/A3608 (DGH, JW, TR) and SP2490/0101 (SDS) from Cancer Research UK.

Author information

Authors and Affiliations

  1. School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, UK

    J Worthington

  2. School of Pharmacy, Queen's University Belfast, Medical Biology Centre, Belfast, UK

    T Robson & D Hirst

  3. Division of Genomic Medicine, Academic Unit of Pathology, University of Sheffield Medical School, Sheffield, UK

    S Scott

Authors
  1. J Worthington
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T Robson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D Hirst
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Worthington, J., Robson, T., Scott, S. et al. Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer. Gene Ther 12, 1417–1423 (2005). https://doi.org/10.1038/sj.gt.3302552

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302552

Keywords

This article is cited by

Search

Advanced search

Quick links