SpringerLink
Log in

Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application

  • Original article
  • Published:
European Journal of Nuclear Medicine and Molecular Imaging Aims and scope Submit manuscript

Abstract

Purpose

The aim of this study was to develop a cellular model for the concurrent imaging of reporter genes expression by using positron emission tomography (PET) and bioluminescence imaging (BLI) for the assessment of estrogen receptor activity in vivo in a breast cancer model.

Methods

Two reporters were chosen: a mutated form of the dopaminergic D2 receptor (D2R80A) for PET imaging, and the Firefly Luciferase for BLI. The presence of an IRES sequence between the two reporters ensured the coordinated expression driven by the same regulatory sequence containing an estrogen responsive element (ERE). To prevent chromatin effects on reporter expression, the construct was flanked by insulator sequences (Matrix Attachment Region, MAR).

Results

In vitro studies showed that the vector was efficient in coordinating the expression of the two genes. Moreover, stably transfected cells implanted in recipient animals maintained their capacity to express the reporters and react to systemic treatments permitting the in vivo study of ERs activity by PET and BLI imaging. In vitro expression analysis after long-term treatments showed different behaviour of the two reporter proteins in monitoring estrogen-dependent transcription outlining the importance of multi-reporter systems. With this model, PET and BLI can be applied to the concurrent evaluation of gene expression induced by estrogen and its analogues by using a bicistronic construct.

Conclusion

The combined features of rapid, sensitive, sequential BLI and tomographic and quantitative PET imaging will allow the use of this strategy for the in vivo evaluation of molecular processes also for pharmacodynamic studies.

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

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Ottobrini L, Ciana P, Biserni A, Lucignani G, Maggi A. Molecular imaging: a new way to study molecular processes in vivo. Mol Cell Endocrinol 2006;246:69–75.

    Article  PubMed  CAS  Google Scholar 

  2. Lucignani G, Ottobrini L, Martelli C, Rescigno M, Clerici M. Molecular imaging of cell-mediated cancer immunotherapy. Trends Biotechnol 2006;24:410–8.

    Article  PubMed  CAS  Google Scholar 

  3. Maggi A, Ciana P. Reporter mice and drug discovery and development. Nat Rev Drug Discov 2005;4:249–55.

    Article  PubMed  CAS  Google Scholar 

  4. Ciana P, Di Luccio G, Belcredito S, Pollio G, Vegeto E, Tatangelo L, et al. Engineering of a mouse for the in vivo profiling of estrogen receptor activity. Mol Endocrinol 2001;15:1104–13.

    Article  PubMed  CAS  Google Scholar 

  5. Thompson JF, Hayes LS, Lloyd DB. Modulation of firefly luciferase stability and impact on studies of gene regulation. Gene 1991;103:171–7.

    Article  PubMed  CAS  Google Scholar 

  6. Ciana P, Ghisletti S, Mussi P, Eberini I, Vegeto E, Maggi A. Estrogen receptor alpha, a molecular switch converting transforming growth factor-alpha-mediated proliferation into differentiation in neuroblastoma cells. J Biol Chem 2003;278:31737–44.

    Article  PubMed  CAS  Google Scholar 

  7. Hoffman GE, Merchenthaler I, Zup SL. Neuroprotection by ovarian hormones in animal models of neurological disease. Endocrine 2006;29:217–31.

    Article  PubMed  CAS  Google Scholar 

  8. Vegeto E, Belcredito S, Etteri S, Ghisletti S, Brusadelli A, Meda C, et al. Estrogen receptor-alpha mediates the brain antiinflammatory activity of estradiol. Proc Natl Acad Sci 2003;100:9614–9.

    Article  PubMed  CAS  Google Scholar 

  9. Paulmurugan R, Gambhir SS. An intramolecular folding sensor for imaging estrogen receptor-ligand interactions. Proc Natl Acad Sci 2006;103:15883–8.

    Article  PubMed  CAS  Google Scholar 

  10. Liang Q, Gotts J, Satyamurthy N, Barrio J, Phelps ME, Gambhir SS, et al. Noninvasive, repetitive, quantitative measurement of gene expression from a bicistronic message by positron emission tomography, following gene transfer with adenovirus. Mol Ther 2002;6:73–82.

    Article  PubMed  CAS  Google Scholar 

  11. Ray P, De A, Min JJ, Tsien RY, Gambhir SS. Imaging tri-fusion multimodality reporter gene expression in living subjects. Cancer Res 2004;64:1323–30.

    Article  PubMed  CAS  Google Scholar 

  12. Ponomarev V, Doubrovin M, Serganova I, Vider J, Shavrin A, Beresten T, et al. A novel triple-modality reporter gene for whole-body fluorescent, bioluminescent, and nuclear noninvasive imaging. Eur J Nucl Med Mol Imaging 2004;31:740–51.

    Article  PubMed  CAS  Google Scholar 

  13. Sammarco MC, Grabczyk E. A series of bidirectional tetracycline-inducible promoters provides coordinated protein expression. Anal Biochem 2005;346:210–6.

    Article  PubMed  CAS  Google Scholar 

  14. Liang Q, Satyamurthy N, Barrio JR, Toyokuni T, Phelps MP, Gambhir SS, et al. Noninvasive, quantitative imaging in living animals of a mutant dopamine D2 receptor reporter gene in which ligand binding is uncoupled from signal transduction. Gene Ther 2001;8:1490–8.

    Article  PubMed  CAS  Google Scholar 

  15. Kim DG, Kang HM, Jang SK, Shin HS. Construction of a bifunctional mrna in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus. Mol Cell Biol 1992;12:3636–43.

    PubMed  CAS  Google Scholar 

  16. Ma ZQ, Spreafico E, Pollio G, Santagati S, Conti E, Cattaneo E, et al. Activated estrogen receptor mediates growth arrest and differentiation of a neuroblastoma cell line. Proc Natl Acad Sci (USA) 1993;90:3740–44.

    Article  CAS  Google Scholar 

  17. Patrone C, Gianazza E, Santagati S, Agrati P, Maggi A. Divergent pathways regulate ligand-independent activation of ERα in SK-N-BE neuroblastoma and COS-1 renal carcinoma cells. Mol Endocrinol 1998;12:835–41.

    Article  PubMed  CAS  Google Scholar 

  18. Hsieh CH, Liu RS, Wang HE, Hwang JJ, Deng WP, Chen JC, et al. In vitro evaluation of herpes simplex virus type 1 thymidine kinase reporter system in dynamic studies of transcriptional gene regulation. Nucl Med Biol 2006;33:653–60.

    PubMed  CAS  Google Scholar 

  19. Seemann MD. Human PET/CT scanners: feasibility for oncological in vivo imaging in mice. Eur J Med Res 2004;9:468–72.

    PubMed  Google Scholar 

  20. Tatsumi M, Nakamoto Y, Traughber B, Marshall LT, Geschwind JF, Wahl RL. Initial experience in small animal tumor imaging with a clinical positron emission tomography/computed tomography scanner using 2-[F-18]fluoro-2-deoxy-D-glucose. Cancer Res 2003;63:6252–7.

    PubMed  CAS  Google Scholar 

  21. DeGrado TR, Turkington TG, Williams JJ, Stearns CW, Hoffman JM, Coleman RE. Performance characteristics of a whole-body PET scanner. J Nucl Med 1994;35:1398–406.

    PubMed  CAS  Google Scholar 

  22. Stief A, Winter DM, Stratling WH, Sippel AE. A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 1989;341:343–5.

    Article  PubMed  CAS  Google Scholar 

  23. Ciana P, Scarlatti F, Biserni A, Ottobrini L, Brena A, Lana A, et al. The dynamics of estrogen receptor activity. Maturitas 2006;54:315–20.

    Article  PubMed  CAS  Google Scholar 

  24. Zhang W, Purchio AF, Chen K, Wu J, Lu L, Coffee R, et al. A transgenic mouse model with a luciferase reporter for studying in vivo transcriptional regulation of the human CYP3A4 gene. Drug Metab Dispos 2003;31:1054–64.

    Article  PubMed  CAS  Google Scholar 

  25. Robertson GR, Field J, Goodwin B, Bierach S, Tran M, Lehnert A, et al. Transgenic mouse models of human CYP3A4 gene regulation. Mol Pharmacol 2003;64:42–50.

    Article  PubMed  CAS  Google Scholar 

  26. Gossen JA, de Leeuw WJ, Tan CH, Zwarthoff EC, Berends F, Lohman PH, et al. Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc Natl Acad Sci (USA) 1989;86:7971–5.

    Article  CAS  Google Scholar 

  27. Sacco MG, Zecca L, Bagnasco L, Chiesa G, Parolini C, Bromley P, et al. A transgenic mouse model for the detection of cellular stress induced by toxic inorganic compounds. Nat Biotechnol 1997;15:1392–7.

    Article  PubMed  CAS  Google Scholar 

  28. Wirth D, Christians E, Munaut C, Dessy C, Foidart JM, Gustin P. Differential heat shock gene hsp70-1 response to toxicants revealed by in vivo study of lungs in transgenic mice. Cell Stress Chaperones 2002;7:387–95.

    Article  PubMed  CAS  Google Scholar 

  29. Ciana P, Biserni A, Tatangelo L, Tiveron C, Sciarroni AF, Ottobrini L, et al. A novel peroxisome proliferator-activated receptor responsive element-luciferase reporter mouse reveals gender specificity of peroxisome proliferator-activated receptor activity in liver. Mol Endocrinol 2007;21:388–400.

    Article  PubMed  CAS  Google Scholar 

  30. Wetterwald A, van der Pluijm G, Que I, Sijmons B, Buijs J, Karperien M, et al. Optical Imaging of Cancer Metastasis to Bone Marrow. A Mouse Model of Minimal Residual Disease. Am J Pathol 2002;160:1143–53.

    PubMed  Google Scholar 

  31. MacLaren DC, Gambhir SS, Satyamurthy N, Barrio JR, Sharfstein S, Toyokuni T, et al. Repetitive, non-invasive imaging of the dopamine D2 receptor as a reporter gene in living animals. Gene Ther 1999;6:785–91.

    Article  PubMed  CAS  Google Scholar 

  32. Aung W, Okauchi T, Sato M, Saito T, Nakagawa H, Ishihara H, et al. In-vivo PET imaging of inducible D2R reporter transgene expression using [11C]FLB 457 as reporter probe in living rats. Nucl Med Commun 2005;26:259–68.

    Article  PubMed  CAS  Google Scholar 

  33. Sun FL, Elgin SC. Putting boundaries on silence. Cell 1999;99:459–62.

    Article  PubMed  CAS  Google Scholar 

  34. Burgess-Beusse B, Farrell C, Gaszner M, Litt M, Recillas-Targa F, Simpson M, et al. The insulation of genes from external enhancers and silencing chromatin. Proc Natl Acad Sci 2002;99 Suppl 4:16433–7.

    Article  PubMed  CAS  Google Scholar 

  35. Stief A, Winter DM, Stratling WH, Sippel AE. A nuclear DNA attachment element mediates elevated and position-independent gene activity. Nature 1989;341:343–5.

    Article  PubMed  CAS  Google Scholar 

  36. Ciana P, Raviscioni M, Mussi P, Vegeto E, Que I, Parker MG, et al. In vivo imaging of transcriptionally active estrogen receptors. Nat Med 2003;9:82–6.

    Article  PubMed  CAS  Google Scholar 

  37. Kim YJ, Dubey P, Ray P, Gambhir SS, Witte ON. Multimodality imaging of lymphocytic migration using lentiviral-based transduction of a tri-fusion reporter gene. Mol Imaging Biol 2004;6:331–40.

    Article  PubMed  Google Scholar 

  38. Blasberg RG. In vivo molecular-genetic imaging: multi-modality nuclear and optical combinations. Nucl Med Biol 2003;30:879–88.

    Article  PubMed  CAS  Google Scholar 

  39. Ray P, Bauer E, Iyer M, Barrio JR, Satyamurthy N, Phelps ME, et al. Monitoring gene therapy with reporter gene imaging. Seminars in Nuclear Medicine 2001;31:312–20.

    Article  PubMed  CAS  Google Scholar 

  40. Barrio JR, Satyamurthy N, Huang SC, Keen RE, Nissenson CH, Hoffman JM, et al. 3-(2′-[18F]Fluoroethyl)spiperone: in vivo biochemical and kinetic characterization in rodents, nonhuman primates, and humans. J Cereb Blood Flow Metab 1989;9:830–9.

    PubMed  CAS  Google Scholar 

  41. Knight AR, Misra A, Quirk K, Benwell K, Revell D, Kennett G, et al. Pharmacological characterisation of the agonist radioligand binding site of 5-HT2A, 5-HT2B and 5-HT2C receptors. Naunyn-Schmiedebergs Arch Pharmacol 2004;370:114–23.

    PubMed  CAS  Google Scholar 

  42. Choi DS, Maroteaux L. Immunohistochemical localisation of the serotonin 5-HT2B receptor in mouse gut, cardiovascular system, and brain. FEBS Lett 1996;391:45–51.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgement

We thank Pasquale Simonelli for the technical support and Daniel Seifert for radiopharmaceutical preparations. The study was supported by PRIN (2001068337), FIRB (RBAU017YPX) and AIRC and was part of the scientific programme of the European Network of Excellence EMIL (European Molecular Imaging Laboratories).

Author information

Authors and Affiliations

  1. Institute of Radiological Sciences, University of Milan, Unit of Nuclear Medicine, Hospital San Paolo, Via Di Rudinì, 8, 20142, Milan, Italy

    Luisa Ottobrini, Michela Lecchi, Cristina Martelli & Giovanni Lucignani

  2. Department of Pharmacological Sciences, University of Milan, Milan, Italy

    Paolo Ciana & Adriana Maggi

  3. CNR-IBFM, University of Milan-Bicocca, Scientific Institute H. S. Raffaele, Milan, Italy

    Rosamaria Moresco, Sara Belloli, Sergio Todde & Ferruccio Fazio

  4. Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Bio-X Program, Stanford University, Stanford, CA, USA

    Sanjiv Sam Gambhir

Authors
  1. Luisa Ottobrini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paolo Ciana
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rosamaria Moresco
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michela Lecchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sara Belloli
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Cristina Martelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sergio Todde
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ferruccio Fazio
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sanjiv Sam Gambhir
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Adriana Maggi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Giovanni Lucignani
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Giovanni Lucignani.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ottobrini, L., Ciana, P., Moresco, R. et al. Development of a bicistronic vector for multimodality imaging of estrogen receptor activity in a breast cancer model: preliminary application. Eur J Nucl Med Mol Imaging 35, 365–378 (2008). https://doi.org/10.1007/s00259-007-0578-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00259-007-0578-z

Keywords

Search

Navigation