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 βthalassemia and sickle cell anemia.

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening

“…In order to discover novel fetal hemoglobin (HbF) inducers from the collected chemical library, we performed a first screening based on the K562.GR reporter system. The clone K562.GR contains a reporter construct with EGFP (enhanced green fluorescent protein) and RFP (red fluorescent protein) genes under the control of the human γ-globin or β -globin gene promoters, respectively, and it has been validated as a high throughput screening (HTS) system in previous publications [ 29 , 30 ]. With this system, a potential HbF inducer can be easily identified by fluorescence-activated cell sorting (FACS) detection of the specific fluorescent protein, in response to the activation of the corresponding promoter.…”

“…On the contrary, if the tested compound does not induce either the γ-globin or the β -globin promoter, no fluorescence is detected. Despite the fact that in this study we focused on HbF inducers, it should be underlined that the system would also allow the identification of transcription activators of the β -globin promoter, in the case RFP is increased instead of EGFP [ 29 , 30 ].…”

“…Interestingly, while most of the compounds were active only at the highest concentration (10 µM), showing no induction activity at the other concentrations, some of them, such as 56, 58, and 59, also showed a relevant activity at low concentrations (1 µM). The 1.35 value was identified as a minimum cut-off in previous articles, where the same K562.GR cellular biosensor was validated using well-known fetal hemoglobin inducers, such as 4,4′,6-Trimethylangelicin (TMA) and HU [ 29 , 30 , 34 ].…”

“…The human erythroid leukemia K562 cells [ 47 ] and the derived cellular biosensor K562.GR [ 29 , 30 ] were cultured in RPMI 1640 medium (Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS; Biowest, Nuaillé, France) with addition of 100 units/mL penicillin and 100 μg/mL streptomycin (Pen-Strep; Lonza Group, Basel, Switzerland).…”

“…In this work, we have screened a small chemical library constituted of 150 compounds, randomly selected, and exhibiting heterogeneous chemotypes, using our cellular biosensor K562.GR 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 [ 29 ]. This system is suitable for identifying the induction ability on the transcription of γ -globin and β -globin genes in erythroid cells in an efficient and reproducible way [ 29 , 30 ]. This investigation resulted in the identification of a subset of compounds that were ultimately analyzed as Hb inducers on primary cell cultures obtained from β -thalassemia patients.…”