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Development and characterization of cellular biosensors for HTS of erythroid differentiation inducers targeting the transcriptional activity of γ-globin and β-globin gene promoters

  • Giulia Breveglieri
  • Francesca Salvatori
  • Alessia Finotti
  • Lucia Carmela Cosenza
  • Cristina Zuccato
  • Nicoletta Bianchi
  • Laura Breda
  • Stefano Rivella
  • Alberto Bresciani
  • Monica Bisbocci
  • Monica Borgatti
  • Roberto GambariEmail author
Research Paper
  • 30 Downloads
Part of the following topical collections:
  1. New Developments in Biosensors

Abstract

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of β-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and β-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and β-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the β-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from β-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of β-thalassemia and sickle cell anemia.

Graphical abstract

Keywords

HbF inducers Thalassemia Reporter systems HTS Erythroid differentiation Sickle cell anemia 

Abbreviations

HbF

Fetal hemoglobin

SCA

Sickle cell anemia

ErPC

Erythroid precursor cell

GFP

Green fluorescence protein

EGFP

Enhanced green fluorescence protein

RFP

Red fluorescence protein

LCR

Locus control region

FACS

Fluorescence activated cell sorting

PCR

Polymerase chain reaction

HTS

High-throughput screening

HU

Hydroxyurea

Notes

Acknowledgments

Roberto Gambari is funded by Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo), CIB (Consorzio Interuniversitario per le Biotecnologie), UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Β-Thalassemia; n. 306201-FP7-HEALTH-2012-INNOVATION-1), by Wellcome Trust (Innovator Award 208872/Z/17/Z) and by AIFA (AIFA-2016-02364887). This research activity was also supported by Associazione Veneta per la Lotta alla Talassemia (AVLT), Rovigo.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

The β-thalassemia patients have been recruited at Thalassemic Day Hospital (DHT) of the University Hospital “Sant’Anna” (Ferrara, Italy). The approval of the collection and processing of the human biological samples for this research was carried out by the Ethics Committee of Ferrara District, number 06/2013 (approved on June 20, 2013). The study complies with the Declaration of Helsinki, the principles of Good Clinical Practice, and all further applicable regulations in accordance with the ethical standards. All samples of peripheral blood have been obtained after written documentation of informed consent from patient or legal representative. Copies of the consents have been collected for archiving by Thalassemic Day Hospital (DHT) of the University Hospital “Sant’Anna” (Ferrara, Italy).

Supplementary material

216_2019_1959_MOESM1_ESM.pdf (4.4 mb)
ESM 1 (PDF 4.42 MB)

References

  1. 1.
    Testa U. Fetal hemoglobin chemical inducers for treatment of hemoglobinopathies. Ann Hematol. 2009;88:505–28.CrossRefGoogle Scholar
  2. 2.
    Gambari R. Foetal hemoglobin inducers and thalassemia: novel achievements. Blood Transfus. 2009;8:5–7.Google Scholar
  3. 3.
    Ataga KI. Novel therapies in sickle cell disease. Hematology. 2009;2009:54–61.CrossRefGoogle Scholar
  4. 4.
    Fard AD, Hosseini SA, Shahjahani M, Salari F, Jaseb K. Evaluation of novel fetal hemoglobin inducer drugs in treatment of β-hemoglobinopathy disorders. Int J Hematol Oncol Stem Cell Res. 2013;7:47–54.Google Scholar
  5. 5.
    Fibach E, Burke LP, Schechter AN, Noguchi CT, Rodgers GP. Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with cell anemia or β-thalassemia. Blood. 1993;81:1630–5.Google Scholar
  6. 6.
    Cao H, Stamatoyannopoulos G, Jung M. Induction of human gamma globin gene expression by histone deacetylase inhibitors. Blood. 2004;103:701–9.CrossRefGoogle Scholar
  7. 7.
    Cortesi R, Gui V, Osti F, Nastruzzi C, Gambari R. Human leukemic K562 cells treated with cytosine arabinoside: enhancement of erythroid differentiation by retinoic acid and retinol. Eur J Haematol. 1998;61:295–301.CrossRefGoogle Scholar
  8. 8.
    Bianchi N, Osti F, Rutigliano C, Corradini FG, Borsetti E, Tomassetti M, et al. The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells. Br J Haematol. 1999;104:258–65.CrossRefGoogle Scholar
  9. 9.
    Mischiati C, Sereni A, Lampronti I, Bianchi N, Borgatti M, Prus E, et al. Rapamycin-mediated induction of gamma-globin mRNA accumulation in human erythroid cells. Br J Haematol. 2004;126:612–21.CrossRefGoogle Scholar
  10. 10.
    Bianchi N, Chiarabelli C, Borgatti M, Mischiati C, Fibach E, Gambari R. Accumulation of gamma-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine. Br J Haematol. 2001;113:951–61.CrossRefGoogle Scholar
  11. 11.
    Bianchi N, Ongaro F, Chiarabelli C, Gualandi L, Mischiati C, Bergamini P, et al. Induction of erythroid differentiation of human K562 cells by cisplatin analogs. Biochem Pharmacol. 2000;60:31–40.CrossRefGoogle Scholar
  12. 12.
    Lampronti I, Bianchi N, Borgatti M, Fibach E, Prus E, Gambari R. Accumulation of gamma-globin mRNA in human erythroid cells treated with angelicin. Eur J Haematol. 2003;71:189–95.CrossRefGoogle Scholar
  13. 13.
    Lozzio CB, Lozzio BB. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood. 1975;45:321–34.Google Scholar
  14. 14.
    Voss JW, Caron C. Inducers of gamma globin gene expression and screening assays therefor. United States Patent 5700640, 1997.Google Scholar
  15. 15.
    Asano H, Stamatoyannopoulos G. Activation of beta-globin promoter by erythroid Krüppel-like factor. Mol Cell Biol. 1998;18:102–9.CrossRefGoogle Scholar
  16. 16.
    Skarpidi E, Vassilopoulos G, Li Q, Stamatoyannopoulos G. Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin. Blood. 2000;96:321–6.Google Scholar
  17. 17.
    Orford M, Nefedov M, Vadolas J, Zaibak F, Williamson R, Ioannou PA. Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET recombination. Nucleic Acids Res. 2000;28:E84.CrossRefGoogle Scholar
  18. 18.
    Vadolas J, Wardan H, Orford M, Voullaire L, Zaibak F, Williamson R, et al. Development of sensitive fluorescent assays for embryonic and fetal haemoglobin inducers using the human beta-globin locus in erythropoietic cells. Blood. 2002;100:4209–16.CrossRefGoogle Scholar
  19. 19.
    Haley JD, Smith DE, Schwedes J, Brennan R, Pearce C, Moore C, et al. Identification and characterization of mechanistically distinct inducers of gamma-globin transcription. Biochem Pharmacol. 2003;66:1755–68.CrossRefGoogle Scholar
  20. 20.
    Jia CP, Huang SZ, Yan JB, Xiao YP, Ren ZR, Zeng YT. Effects of human locus control region elements HS2 and HS3 on human beta-globin gene expression in transgenic mouse. Blood Cells Mol Dis. 2003;31:360–9.CrossRefGoogle Scholar
  21. 21.
    Dietzel S, Zolghadr K, Hepperger C, Belmont AS. Differential large-scale chromatin compaction and intranuclear positioning of transcribed versus non-transcribed transgene arrays containing beta-globin regulatory sequences. J Cell Sci. 2004;117:4603–14.CrossRefGoogle Scholar
  22. 22.
    Vadolas J, Wardan H, Orford M, Williamson R, Ioannou PA. Cellular genomic reporter assays for screening and evaluation of inducers of fetal hemoglobin. Hum Mol Genet. 2004;13:223–33.CrossRefGoogle Scholar
  23. 23.
    Zoueva OP, Rodgers GP. Inhibition of beta protein 1 expression enhances beta-globin promoter activity and beta-globin mRNA levels in the human erythroleukemia (K562) cell line. Exp Hematol. 2004;32:700–8.CrossRefGoogle Scholar
  24. 24.
    Migliaccio G, Di Baldassarre A, Di Rico C, Di Noia A, Nakamoto B, Cao H, et al. Spontaneous switch from Agamma- to beta-globin promoter activity in a stable transfected dual reporter vector. Blood Cells Mol Dis. 2005;34:174–80.CrossRefGoogle Scholar
  25. 25.
    Di Baldassarre A, Di Rico M, Di Noia A, Bonfini T, Iacone A, Marchisio M, et al. Protein kinase Calpha is differentially activated during neonatal and adult erythropoiesis and favors expression of a reporter gene under the control of the (A)gamma globin-promoter in cellular models of hemoglobin switching. J Cell Biochem. 2007;101:411–24.CrossRefGoogle Scholar
  26. 26.
    Breveglieri G, Salvatori F, Finotti A, Bertuzzi I, Destro F, Falzoni S, et al. Cellular biosensors for the identification of fetal hemoglobin inducers. Minerva Biotec. 2007;19:123–32.Google Scholar
  27. 27.
    Yan J, Xiao Y, Wang S, Gong Z, Huang S, Zeng Y. Expression of green fluorescent protein under the regulation of human locus control region elements HS2 and HS3 in transgenic mice. Int J Hematol. 2008;88:36–42.CrossRefGoogle Scholar
  28. 28.
    Xu X, von Löhneysen K, Soldau K, Noack D, Vu A, Friedman JS. A novel approach for in vivo measurement of mouse red cell redox status. Blood. 2011;118:3694–7.CrossRefGoogle Scholar
  29. 29.
    Chan KS, Xu J, Wardan H, McColl B, Orkin S, Vadolas J. Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation. FASEB J. 2012;26:1736–44.CrossRefGoogle Scholar
  30. 30.
    Kao BR, McColl B, Vadolas J. Generation of BAC reporter cell lines for drug discovery. Methods Mol Biol. 2015;1227:323–43.CrossRefGoogle Scholar
  31. 31.
    Papadopoulos P, Gutiérrez L, van der Linden R, Kong-A-San J, Maas A, Drabek D, et al. A dual reporter mouse model of the human β-globin locus: applications and limitations. PLoS One. 2012;7:e51272.CrossRefGoogle Scholar
  32. 32.
    Peterson KR, Costa FC, Fedosyuk H, Neades RY, Chazelle AM, Zelenchuk L, et al. A cell-based high-throughput screen for novel chemical inducers of fetal hemoglobin for treatment of hemoglobinopathies. PLoS One. 2014;9:e107006.CrossRefGoogle Scholar
  33. 33.
    McColl B, Kao BR, Lourthai P, Chan K, Wardan H, Roosjen M, et al. An in vivo model for analysis of developmental erythropoiesis and globin gene regulation. FASEB J. 2014;28:2306–17.CrossRefGoogle Scholar
  34. 34.
    Boosalis MS, Sangerman JI, White GL, Wolf RF, Shen L, Dai Y, et al. Novel inducers of fetal globin identified through high throughput screening (HTS) are active in vivo in anemic baboons and transgenic mice. PLoS One. 2015;10:e0144660.CrossRefGoogle Scholar
  35. 35.
    Breda L, Kleinert DA, Casu C, Casula L, Cartegni L, Fibach E, et al. A preclinical approach for gene therapy of beta-thalassemia. Ann N Y Acad Sci. 2010;1202:134–40.CrossRefGoogle Scholar
  36. 36.
    Fibach E, Bianchi N, Borgatti M, Prus E, Gambari R. Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells. Blood. 2003;102:1276–81.CrossRefGoogle Scholar
  37. 37.
    May C, Rivella S, Callegari J, Heller G, Gaensler KM, Luzzatto L, et al. Therapeutic haemoglobin synthesis in β-thalassemic mice expressing lentivirus-encoded human β-globin. Nature. 2000;406:82–6.CrossRefGoogle Scholar
  38. 38.
    Perrine SP, Miller BA, Faller DV, Cohen RA, Vichinsky EP, Hurst D, et al. Sodium butyrate enhances fetal globin gene expression in erythroid progenitors of patients with Hb SS and beta thalassemia. Blood. 1989;74:454–9.Google Scholar
  39. 39.
    Brognara E, Lampronti I, Breveglieri G, Accetta A, Corradini R, Manicardi A, et al. C(5) modified uracil derivatives showing antiproliferative and erythroid differentiation inducing activities on human chronic myelogenous leukemia K562 cells. Eur J Pharmacol. 2011;672:30–7.CrossRefGoogle Scholar
  40. 40.
    Bianchi N, Zuccato C, Lampronti I, Borgatti M, Gambari R. Fetal hemoglobin inducers from the natural world: a novel approach for identification of drugs for the treatment of {beta}-thalassemia and sickle-cell anemia. Evid Based Complement Alternat Med. 2009;6:141–51.CrossRefGoogle Scholar
  41. 41.
    Salvatori F, Breveglieri G, Zuccato C, Finotti A, Bianchi N, Borgatti M, et al. Production of beta-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous beta(0)39 thalassemia patients. Am J Hematol. 2009;84:720–8.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Giulia Breveglieri
    • 1
    • 2
  • Francesca Salvatori
    • 3
  • Alessia Finotti
    • 1
  • Lucia Carmela Cosenza
    • 1
    • 2
  • Cristina Zuccato
    • 1
  • Nicoletta Bianchi
    • 4
  • Laura Breda
    • 5
  • Stefano Rivella
    • 5
  • Alberto Bresciani
    • 6
  • Monica Bisbocci
    • 6
  • Monica Borgatti
    • 1
    • 2
  • Roberto Gambari
    • 1
    Email author
  1. 1.Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular BiologyUniversity of FerraraFerraraItaly
  2. 2.Biotechnology CenterUniversity of FerraraFerraraItaly
  3. 3.Department of Chemical and Pharmaceutical SciencesUniversity of FerraraFerraraItaly
  4. 4.Department of Biomedical Sciences and Specialist Surgery, Section of Biochemistry, Molecular Biology and Medical GeneticsUniversity of FerraraFerraraItaly
  5. 5.Hematology DivisionChildren’s Hospital of PhiladelphiaPhiladelphiaUSA
  6. 6.IRBM Science Park SpAPomezia (Rome)Italy

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