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:

Novel chimeric gene promoters responsive to hypoxia and ionizing radiation

Gene Therapy volume 9, pages 1403–1411 (2002)Cite this article

Abstract

Despite being an adverse prognostic factor in radiotherapy, hypoxia represents a physiological difference that can be exploited for selective cancer gene therapy. In this study gene therapy vectors responsive to both hypoxia and ionizing radiation (IR) were developed. Gene expression was regulated by novel, synthetic promoters containing hypoxia responsive elements (HREs) from the erythropoietin (Epo), the phosphoglycerate kinase 1 (PGK1) and the vascular endothelial growth factor (VEGF) genes, and IR-responsive CArG elements from the early growth response (Egr) 1 gene. All chimeric promoters could be activated by hypoxia and/or IR-treatment, and selectively control marker gene expression in human T24 bladder carcinoma and MCF-7 mammary carcinoma cells. Importantly, enhancers containing combinations of HREs and CArG elements were able to respond to both triggering treatments, with the Epo HRE/CArG combination proving to be the most responsive and robust. The Epo HRE/CArG enhancer could effectively control a suicide gene therapy strategy by selectively sensitizing hypoxic and/or irradiated cells expressing the enzyme horseradish peroxidase (HRP) to the prodrug indole-3-acetic acid (IAA). These data indicate that the use of such chimeric promoters may effectively regulate therapeutic gene expression within the tumor microenvironment in gene therapy strategies aimed at addressing the problem of hypoxia in radiotherapy.

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. Brown JM, Giaccia AJ . The unique physiology of solid tumors: opportunities (and problems) for cancer therapy Cancer Res 1998 58: 1408–1416

    CAS  PubMed  Google Scholar 

  2. Dachs GU et al. Targeting gene expression to hypoxic tumor cells Nat Med 1997 3: 515–520

    Article  CAS  PubMed  Google Scholar 

  3. Ratcliffe PJ, O'Rourke JF, Maxwell PH, Pugh CW . Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression J Exp Biol 1998 201: 1153–1162

    CAS  PubMed  Google Scholar 

  4. Semenza GL, Wang GL . A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation Mol Cell Biol 1992 12: 5447–5454

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wang GL, Jiang BH, Rue EA, Semenza GL . Hypoxia-inducible factor 1 is a basic helix-loop-helix-PAS heterodimer regulated by cellular O2 tension Proc Natl Acad Sci USA 1995 92: 5510–5514

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. O'Rourke JF et al. Hypoxia response elements Oncol Res 1997 9: 327–332

    CAS  PubMed  Google Scholar 

  7. Zhong H et al. Overexpression of hypoxia-inducible factor 1 alpha in common human cancers and their metastases Cancer Res 1999 59: 5830–5835

    CAS  PubMed  Google Scholar 

  8. Talks KL et al. The expression and distribution of the hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha in normal human tissues, cancers, and tumor-associated macrophages Am J Pathol 2000 157: 411–421

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Greco O, Patterson AV, Dachs GU . Can gene therapy overcome the problem of hypoxia in radiotherapy? J Radiat Res (Tokyo) 2000 41: 201–212

    Article  CAS  Google Scholar 

  10. Greco O, Tozer GM, Dachs GU . Oxic and anoxic enhancement of radiation-mediated toxicity by horseradish peroxidase/indole-3-acetic acid gene therapy Int J Radiat Biol 2002 78: 178–181

    Article  Google Scholar 

  11. Greco O . Horseradish peroxidase-mediated gene therapy: choice of prodrugs in oxic and anoxic tumour conditions Mol Cancer Ther 2001 1: 151–160

    CAS  PubMed  Google Scholar 

  12. Greco O et al. Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid Cancer Gene Ther 2000 7: 1414–1420

    Article  CAS  PubMed  Google Scholar 

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

    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: 10149–10153

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. Scott SD et al. A radiation-controlled molecular switch for use in gene therapy of cancer Gene Therapy 2000 7: 1121–1125

    Article  CAS  PubMed  Google Scholar 

  17. Nettelbeck DM, Jerome V, Muller R . Gene therapy: designer promoters for tumour targeting Trends Genet 2000 16: 174–181

    Article  CAS  PubMed  Google Scholar 

  18. Stackhouse MA, Buchsbaum DJ . Radiation to control gene expression Gene Therapy 2000 7: 1085–1086

    Article  CAS  PubMed  Google Scholar 

  19. Nabel GJ . Development of optimized vectors for gene therapy Proc Natl Acad Sci USA 1999 96: 324–326

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hernandez-Alcoceba R, Pihalja M, Nunez G, Clarke MF . Evaluation of a new dual-specificity promoter for selective induction of apoptosis in breast cancer cells Cancer Gene Ther 2001 8: 298–307

    Article  CAS  PubMed  Google Scholar 

  21. Modlich U, Pugh CW, Bicknell R . Increasing endothelial cell specific expression by the use of heterologous hypoxic and cytokine-inducible enhancers Gene Therapy 2000 7: 896–902

    Article  CAS  PubMed  Google Scholar 

  22. Ido A et al. Gene therapy targeting for hepatocellular carcinoma: selective and enhanced suicide gene expression regulated by a hypoxia-inducible enhancer linked to a human alpha-fetoprotein promoter Cancer Res 2001 61: 3016–3021

    CAS  PubMed  Google Scholar 

  23. Wosikowski K et al. Normal p53 status and function despite the development of drug resistance in human breast cancer cells Cell Growth Differ 1995 6: 1395–1403

    CAS  PubMed  Google Scholar 

  24. Kawasaki T et al. Abrogation of apoptosis induced by DNA-damaging agents in human bladder- cancer cell lines with p21/WAF1/CIP1 and/or p53 gene alterations Int J Cancer 1996 68: 501–505

    Article  CAS  PubMed  Google Scholar 

  25. Ebert BL, Firth JD, Ratcliffe PJ . Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct Cis-acting sequences J Biol Chem 1995 270: 29083–29089

    Article  CAS  PubMed  Google Scholar 

  26. Muller JM et al. Hypoxia induces c-fos transcription via a mitogen-activated protein kinase-dependent pathway J Biol Chem 1997 272: 23435–23439

    Article  CAS  PubMed  Google Scholar 

  27. Yan SF et al. Hypoxia-associated induction of early growth response-1 gene expression J Biol Chem 1999 274: 15030–15040

    Article  CAS  PubMed  Google Scholar 

  28. Lo LW et al. Endothelial exposure to hypoxia induces Egr-1 expression involving PKCalpha-mediated Ras/Raf-1/ERK1/2 pathway J Cell Physiol 2001 188: 304–312

    Article  CAS  PubMed  Google Scholar 

  29. Yan SF et al. Protein kinase C-beta and oxygen deprivation. A novel Egr-1-dependent pathway for fibrin deposition in hypoxemic vasculature J Biol Chem 2000 275: 11921–11928

    Article  CAS  PubMed  Google Scholar 

  30. 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 

  31. Gonzalez-Flecha B et al. Oxidative stress produced by suprahepatic occlusion and reperfusion Hepatology 1993 18: 881–889

    Article  CAS  PubMed  Google Scholar 

  32. Chandel NS et al. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription Proc Natl Acad Sci USA 1998 95: 11715–11720

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chandel NS et al. Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing J Biol Chem 2000 275: 25130–25138

    Article  CAS  PubMed  Google Scholar 

  34. Bonventre JV et al. Localization of the protein product of the immediate early growth response gene, Egr-1, in the kidney after ischemia and reperfusion Cell Regul 1991 2: 251–260

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Shibata T, Giaccia AJ, Brown JM . Development of a hypoxia-responsive vector for tumor-specific gene therapy Gene Therapy 2000 7: 493–498

    Article  CAS  PubMed  Google Scholar 

  36. Ruan H et al. A hypoxia-regulated adeno-associated virus vector for cancer-specific gene therapy Neoplasia 2001 3: 255–263

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gorski DH et al. Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation Cancer Res 1999 59: 3374–3378

    CAS  PubMed  Google Scholar 

  38. Kuroki M et al. Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo J Clin Invest 1996 98: 1667–1675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Chua CC, Hamdy RC, Chua BH . Upregulation of vascular endothelial growth factor by H2O2 in rat heart endothelial cells Free Radic Biol Med 1998 25: 891–897

    Article  CAS  PubMed  Google Scholar 

  40. Kosmidou I, Xagorari A, Roussos C, Papapetropoulos A . Reactive oxygen species stimulate VEGF production from C(2)C(12) skeletal myotubes through a PI3K/Akt pathway Am J Physiol Lung Cell Mol Physiol 2001 280: L585–L592

    Article  CAS  PubMed  Google Scholar 

  41. Gleadle JM, Ebert BL, Ratcliffe PJ . Diphenylene iodonium inhibits the induction of erythropoietin and other mammalian genes by hypoxia. Implications for the mechanism of oxygen sensing Eur J Biochem 1995 234: 92–99

    Article  CAS  PubMed  Google Scholar 

  42. Huang LE, Arany Z, Livingston DM, Bunn HF . Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit J Biol Chem 1996 271: 32253–32259

    Article  CAS  PubMed  Google Scholar 

  43. Fandrey J et al. Cobalt chloride and desferrioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species Kidney Int 1997 51: 492–496

    Article  CAS  PubMed  Google Scholar 

  44. Binley K et al. An adenoviral vector regulated by hypoxia for the treatment of ischaemic disease and cancer Gene Therapy 1999 6: 1721–1727

    Article  CAS  PubMed  Google Scholar 

  45. Boast K et al. Characterization of physiologically regulated vectors for the treatment of ischemic disease Hum Gene Ther 1999 10: 2197–2208

    Article  CAS  PubMed  Google Scholar 

  46. An WG et al. Stabilization of wild-type p53 by hypoxia-inducible factor 1alpha Nature 1998 392: 405–408

    Article  CAS  PubMed  Google Scholar 

  47. Firth JD, Ebert BL, Pugh CW, Ratcliffe PJ . Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3’ enhancer Proc Natl Acad Sci USA 1994 91: 6496–6500

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Wang GL, Semenza GL . General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia Proc Natl Acad Sci USA 1993 90: 4304–4308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Connolly CN et al. Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase J Cell Biol 1994 127: 641–652

    Article  CAS  PubMed  Google Scholar 

  50. Hart SL et al. Lipid-mediated enhancement of transfection by a nonviral integrin- targeting vector Hum Gene Ther 1998 9: 575–585

    Article  CAS  PubMed  Google Scholar 

  51. Coralli C et al. Limitations of the reporter green fluorescent protein under simulated tumor conditions Cancer Res 2001 61: 4784–4790

    CAS  PubMed  Google Scholar 

  52. Coralli C et al. Limitations of the reporter green fluorescent protein under simulated tumor conditions Scott SD, Joiner MC, Marples B. Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene Therapy 2002; 3: 1396–1402.

Download references

Acknowledgements

We are very grateful to Dr Gill Tozer, Professor Mike Joiner and Professor Ian Stratford for helpful discussion, and to Mrs Claudia Coralli and Mrs Sara Bourne for excellent technical support. This work was funded by the Gray Cancer Institute (OG), the Cancer Research Campaign (SS, BM, GD) and the Medical Research Council (KW, AP).

Author information

Author notes

  1. B Marples & S D Scott

    Present address: Karmanos Cancer Institute, Wayne State University, Hudson-Webber CRC (Room 823), 4100 John R, Detroit, 48201, MI, USA

Authors and Affiliations

  1. Gray Cancer Institute, Mount Vernon Hospital, Northwood, UK

    O Greco, B Marples, G U Dachs & S D Scott

  2. University of Manchester, School of Pharmacy and Pharmaceutical Sciences, Manchester, UK

    K J Williams

  3. Experimental Oncology, ACSRC, The University of Auckland, Auckland, New Zealand

    A V Patterson

Authors
  1. O Greco
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B Marples
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. G U Dachs
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K J Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A V Patterson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. S D Scott
    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

Greco, O., Marples, B., Dachs, G. et al. Novel chimeric gene promoters responsive to hypoxia and ionizing radiation. Gene Ther 9, 1403–1411 (2002). https://doi.org/10.1038/sj.gt.3301823

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301823

Keywords

This article is cited by

Search

Advanced search

Quick links