Skip to main content

Advertisement

SpringerLink
Log in

Demonstration of Tightly Radiation-Controlled Molecular Switch Based on CArG Repeats by In Vivo Molecular Imaging

  • Research Article
  • Published:
Molecular Imaging and Biology Aims and scope Submit manuscript

Abstract

Purpose

Promoters developed for radiogene therapy always show non-negligible transcriptional activities, even when cells are not irradiated. This study developed a tightly radiation-controlled molecular switch based on radiation responsive element (CArG) repeats for in vivo molecular imaging using the Cre/loxP system.

Procedures

Different numbers of CArG repeats were cloned as a basal promoter directly, and its pre- and postirradiation transcriptional activities were analyzed by luciferase assay. Nine CArG repeats (E9) were chosen for use as a radiation-controlled molecular switch for the Cre/loxP system, and the feasibility of the switch in vitro and in vivo was demonstrated by luciferase assay and bioluminescence imaging, respectively.

Results

The E9 promoter, which exhibits extremely low transcriptional activity, showed a 1.8-fold enhancement after irradiation with a clinical dose of 2 Gy. Both in vitro and in vivo results indicated that E9 is relatively inert but sufficient to trigger the Cre/loxP system. The luciferase activity of stable H1299/pSTOP-FLuc cells transfected with pE9-NLSCre and exposed to 2-Gy radiation can reach 44 % of that of the same cells transfected with pCMV-NLSCre and not subjected to irradiation. By contrast, no appreciable difference was observed in reporter gene expression in both H1299/pSTOPFluc cells and tumors transfected with pE4Pcmv-NLSCre before and after irradiation, because the strong basal transcriptional activity of the CMV promoter, which acts as a copartner of E4, masked the response of E4 to radiation.

Conclusions

Our results provide detailed insight into CArG elements as a radiation-controlled molecular switch that can facilitate the development of radiogene therapy.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Hellman S (2001) Principles of cancer management: radiation therapy. In: DeVita Jr VT, Hellman S, Rosenberg SA (eds) Cancer: principlesand practice of oncology, 6th edn. Lippincott Williams & Wilkins, Philadelphia, pp 265–288

  2. Mundt AJ, Roeske JC, Chung TD, Weichselbaum RR (2003) Principles of radiation oncology. In: Kufe DW, Pollock RE, Weichselbaum RR, et al. (eds) Holland–Frei cancer medicine, 6th edn. BC Decker, Hamilton, pp 585–604

  3. Provencio M, Sanchez A, Garrido P, Valcarcel F (2010) New molecular targeted therapies integrated with radiation therapy in lung cancer. Clin Lung Cancer 11:91–97

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  5. Valerie K, Yacoub A, Hagan MP et al (2007) Radiation-induced cell signaling: inside-out and outside-in. Mol Cancer Ther 6:789–801

    Article  CAS  PubMed  Google Scholar 

  6. Gottesman MM (2003) Cancer gene therapy: an awkward adolescence. Cancer Gene Ther 10:501–508

    Article  CAS  PubMed  Google Scholar 

  7. Danda R, Krishnan G, Ganapathy K et al (2013) Targeted expression of suicide gene by tissue-specific promoter and microRNA regulation for cancer gene therapy. PLoS One 8:e83398

    Article  PubMed Central  PubMed  Google Scholar 

  8. Wang HW, Breslin MB, Chen C et al (2009) INSM1 promoter-driven adenoviral herpes simplex virus thymidine kinase cancer gene therapy for the treatment of primitive neuroectodermal tumors. Hum Gene Ther 20:1308–1318

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Hsieh YJ, Chen FD, Ke CC et al (2012) The EIIAPA chimeric promoter for tumor specific gene therapy of hepatoma. Mol Imaging Biol 14:452–461

    Article  PubMed  Google Scholar 

  10. Robson T, Worthington J, McKeown SR, Hirst DG (2005) Radiogenic therapy: novel approaches for enhancing tumor radiosensitivity. Technol Cancer Res Treat 4:343–361

    Article  CAS  PubMed  Google Scholar 

  11. Rodningen OK, Overgaard J, Alsner J et al (2005) Microarray analysis of the transcriptional response to single or multiple doses of ionizing radiation in human subcutaneous fibroblasts. Radiother Oncol 77:231–240

    Article  PubMed  Google Scholar 

  12. Snyder AR, Morgan WF (2004) Gene expression profiling after irradiation: clues to understanding acute and persistent responses? Cancer Metastasis Rev 23:259–268

    Article  CAS  PubMed  Google Scholar 

  13. Rodemann HP, Dittmann K, Toulany M (2007) Radiation-induced EGFR-signaling and control of DNA-damage repair. Int J Radiat Biol 83:781–791

    Article  CAS  PubMed  Google Scholar 

  14. Dittmann K, Mayer C, Kehlbach R, Rodemann HP (2008) Radiation-induced caveolin-1 associated EGFR internalization is linked with nuclear EGFR transport and activation of DNA-PK. Mol Cancer. doi:10.1186/1476-4598-7-69

    PubMed Central  PubMed  Google Scholar 

  15. Shore P, Sharrocks AD (1994) The transcription factors Elk-1 and serum response factor interact by direct protein-protein contacts mediated by a short region of Elk-1. Mol Cell Biol 14:3283–3291

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Murai K, Treisman R (2002) Interaction of serum response factor (SRF) with the Elk-1 B box inhibits RhoA-actin signaling to SRF and potentiates transcriptional activation by Elk-1. Mol Cell Biol 22:7083–7092

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Osborn L, Kunkel S, Nabel GJ (1989) Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc Natl Acad Sci U S A 86:2336–2340

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Janknecht R (1995) Regulation of the c-fos promoter. Immunobiology 193:137–142

    Article  CAS  PubMed  Google Scholar 

  20. Ahmed MM (2004) Regulation of radiation-induced apoptosis by early growth response-1 gene in solid tumors. Curr Cancer Drug Targets 4:43–52

    Article  CAS  PubMed  Google Scholar 

  21. Datta R, Taneja N, Sukhatme VP et al (1993) 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 U S A 90:2419–2422

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Joki T, Nakamura M, Ohno T (1995) Activation of the radiosensitive EGR-1 promoter induces expression of the herpes simplex virus thymidine kinase gene and sensitivity of human glioma cells to ganciclovir. Hum Gene Ther 6:1507–1513

    Article  CAS  PubMed  Google Scholar 

  23. Scott SD, Marples B, Hendry JH et al (2000) A radiation-controlled molecular switch for use in gene therapy of cancer. Gene Ther 7:1121–1125

    Article  CAS  PubMed  Google Scholar 

  24. Scott SD, Joiner MC, Marples B (2002) Optimizing radiation-responsive gene promoters for radiogenetic cancer therapy. Gene Ther 9:1396–1402

    Article  CAS  PubMed  Google Scholar 

  25. Coulter JA, McCarthy HO, Worthington J et al (2008) The radiation-inducible pE9 promoter driving inducible nitric oxide synthase radiosensitizes hypoxic tumour cells to radiation. Gene Ther 15:495–503

    Article  CAS  PubMed  Google Scholar 

  26. Sambucetti LC, Cherrington JM, Wilkinson GW, Mocarski ES (1989) NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J 8:4251–4258

    PubMed Central  CAS  PubMed  Google Scholar 

  27. Chen J, Stinski MF (2002) Role of regulatory elements and the MAPK/ERK or p38 MAPK pathways for activation of human cytomegalovirus gene expression. J Virol 76:4873–4885

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Iyer M, Wu L, Carey M et al (2001) Two-step transcriptional amplification as a method for imaging reporter gene expression using weak promoters. Proc Natl Acad Sci U S A 98:14595–14600

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Karpati G, Lochmüller H (1997) The scope of gene therapy in humans: scientific, safety and ethical considerations. Neuromuscul Disord 7:273–276

    Article  CAS  PubMed  Google Scholar 

  30. Wang Z, Sun Y (2010) Targeting p53 for novel anticancer therapy. Transl Oncol 3:1–12

    Article  PubMed Central  PubMed  Google Scholar 

  31. Vichalkovski A, Gresko E, Hess D et al (2010) PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p53 activity in response to DNA damage. Oncogene 29:3554–3565

    Article  CAS  PubMed  Google Scholar 

  32. Valsesia-Wittmann S, Magdeleine M, Dupasquier S et al (2004) Oncogenic cooperation between H-Twist and N-Myc overrides failsafe programs in cancer cells. Cancer Cell 6:625–630

    Article  CAS  PubMed  Google Scholar 

  33. Giaccia AJ, Kastan MB (1998) The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev 12:2973–2983

    Article  CAS  PubMed  Google Scholar 

  34. Ko LJ, Prives C (1996) p53: puzzle and paradigm. Genes Dev 10:1054–1072

    Article  CAS  PubMed  Google Scholar 

  35. McCarthy HO, Worthington J, Barrett E et al (2007) p21((WAF1))-mediated transcriptional targeting of inducible nitric oxide synthase gene therapy sensitizes tumours to fractionated radiotherapy. Gene Ther 14:246–255

    Article  CAS  PubMed  Google Scholar 

  36. Chastel C, Jiricny J, Jaussi R (2004) Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy. DNA Repair (Amst) 3:201–215

    Article  CAS  Google Scholar 

  37. Sherman ML, Datta R, Hallahan DE et al (1990) Ionizing radiation regulates expression of the c-jun protooncogene. Proc Natl Acad Sci U S A 87:5663–5666

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Das A, Chendil D, Dey S et al (2001) Ionizing radiation down-regulates p53 protein in primary Egr-1−/− mouse embryonic fibroblast cells causing enhanced resistance to apoptosis. J Biol Chem 276:3279–3286

    Article  CAS  PubMed  Google Scholar 

  39. Hsieh YJ, Liu RS, Hwu L et al (2007) Cre/loxP system controlled by specific promoter for radiation-mediated gene therapy of hepatoma. Anticancer Res 27:1571–1579

    CAS  PubMed  Google Scholar 

  40. Caposio P, Luganini A, Bronzini M et al (2010) The Elk-1 and serum response factor binding sites in the major immediate-early promoter of human cytomegalovirus are required for efficient viral replication in quiescent cells and compensate for inactivation of the NF-kappaB sites in proliferating cells. J Virol 84:4481–4493

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Silver DP, Livingston DM (2001) Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol Cell 8:233–243

    Article  CAS  PubMed  Google Scholar 

  42. Semprini S, Troup TJ, Kotelevtseva N et al (2007) Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques. Nucleic Acids Res 35:1402–1410

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  43. Thyagarajan B, Guimarães MJ, Groth AC, Calos MP (2000) Mammalian genomes contain active recombinase recognition sites. Gene 244:47–54

    Article  CAS  PubMed  Google Scholar 

  44. Loonstra A, Vooijs M, Beverloo HB et al (2001) Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci U S A 98:9209–9214

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  45. Jeannotte L, Aubin J, Bourque S et al (2011) Unsuspected effects of a lung-specific Cre deleter mouse line. Genesis 49:152–159

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by the grants: NSC 102-2314-B-010-038-MY3, NSC 102-2627-M-010-003 (National Science Council, Taiwan), MOST 103-2314-B-037-007 (Ministry of Science and Technology), MOHW104-TDU-B-211-124-001 (Department of Health), and V103C-132 (Taipei Veterans General Hospital). The authors thank the technical support from Molecular and Genetic Imaging Core, Taiwan Mouse Clinic (MOST 103-2325-B-001-015), which is funded by the National Research Program for Biopharmaceuticals (NRPB) at the Ministry of Science and Technology (MOST) of Taiwan, and Ms. Tsuey-Ling Jan for the assistance of preparing the manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Medical Imaging and Radiological Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan

    Ya-Ju Hsieh & Sharon Chia-Ju Chen

  2. Molecular and Genetic Imaging Core/Taiwan Mouse Clinic, National Comprehensive Mouse Phenotyping and Drug Testing Center, Taipei, Taiwan

    Luen Hwu & Ren-Shyan Liu

  3. Biomedical Imaging Research Center, National Yang-Ming University, Taipei, Taiwan

    Luen Hwu, Chien-Chih Ke, Fu-Du Chen & Ren-Shyan Liu

  4. Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, Taiwan

    Ai-Lin Huang & Ren-Shyan Liu

  5. Hwa Hsia University of Technology, Taipei, Taiwan

    Fu-Du Chen

  6. Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan

    Shyh-Jong Wu

  7. Faculty of Chinese Medicine, Macau University of Science and Technology, Macau, China

    Yong-Hua Zhao

  8. Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan

    Ren-Shyan Liu

  9. Biophotonic and Molecular Imaging Research Center, National Yang-Ming University, Taipei, Taiwan

    Ren-Shyan Liu

  10. National PET/Cyclotron Center and Department of Nuclear Medicine, Taipei Veterans General Hospital, Taipei, Taiwan

    Ren-Shyan Liu

  11. Division of Nuclear Medicine, National Yang-Ming University Medical School, 155, Sec. 2, Linong Street, Taipei, 11221, Taiwan

    Ren-Shyan Liu

Authors
  1. Ya-Ju Hsieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luen Hwu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chien-Chih Ke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ai-Lin Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fu-Du Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shyh-Jong Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sharon Chia-Ju Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yong-Hua Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ren-Shyan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ren-Shyan Liu.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hsieh, YJ., Hwu, L., Ke, CC. et al. Demonstration of Tightly Radiation-Controlled Molecular Switch Based on CArG Repeats by In Vivo Molecular Imaging. Mol Imaging Biol 17, 802–810 (2015). https://doi.org/10.1007/s11307-015-0843-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11307-015-0843-7

Key words

Search

Navigation