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

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.

• Authors: Giulia Breveglieri, Francesca Salvatori, Alessia Finotti, Lucia Carmela Cosenza, Cristina Zuccato, Nicoletta Bianchi, Laura Breda, Stefano Rivella, Alberto Bresciani, Monica Bisbocci, Monica Borgatti, Roberto Gambari
• Journal: Analytical and bioanalytical chemistry (Anal Bioanal Chem) Vol. 411 Issue 29 Pg. 7669-7680 (Nov 2019) ISSN: 1618-2650 [Electronic] Germany
• PMID: 31273412 (Publication Type: Journal Article)

Chemical References

• beta-Globins
• enhanced green fluorescent protein
• gamma-Globins
• Green Fluorescent Proteins
• Fetal Hemoglobin

Topics

• Biosensing Techniques
• Cell Differentiation (drug effects)
• Erythrocytes (cytology, drug effects)
• Fetal Hemoglobin (genetics)
• Green Fluorescent Proteins (genetics)
• High-Throughput Screening Assays (methods)
• Humans
• K562 Cells
• Promoter Regions, Genetic
• Reproducibility of Results
• Transcription, Genetic
• beta-Globins (genetics)
• gamma-Globins (genetics)