A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level γ-Globin Inducing Compounds.
The identification of drugs capable of reactivating γ-globin to ameliorate β-thalassemia and Sickle Cell anemia is still a challenge, as available γ-globin inducers still have limited clinical indications. High-throughput screenings (HTS) aimed to identify new potentially therapeutic drugs require suitable first-step-screening methods combining the possibility to detect variation in the γ/β globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing β-globin (β-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of γ- and β-globin content at single-cell level. The assay was validated by using the known globin inducers hemin, hydroxyurea and butyric acid and further tested in a pilot screening that confirmed HDACs as targets for γ-globin induction (as proved by siRNA-mediated HDAC3 knockdown and by treatment with HDACs inhibitors entinostat and dacinostat) and identified Heme-oxygenases as novel candidate targets for γ-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased γ-globin expression. This result is particularly interesting as several metalloporphyrins have already been developed for clinical uses and could be tested (alone or in combination with other drugs) to improve pharmacological γ-globin reactivation for the treatment of β-hemoglobinopathies.
Department of Biology, Nerviano Medical Sciences S.r.l., Nerviano, Milano, Italy.
Marta
M
Durlak
Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milano, Italy.
Cristina
C
Fugazza
Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milano, Italy.
Sudharshan
S
Elangovan
Istituto di Ricerca Genetica e Biomedica del Consiglio Nazionale delle Ricerche, Cagliari, Italy.
Maria Giuseppina
MG
Marini
Istituto di Ricerca Genetica e Biomedica del Consiglio Nazionale delle Ricerche, Cagliari, Italy.
Maria Franca
MF
Marongiu
Dipartimento di Sanità Pubblica, Medicina Clinica e Molecolare, Università degli Studi di Cagliari, Cagliari, Italy.
Paolo
P
Moi
Department of Biology, Nerviano Medical Sciences S.r.l., Nerviano, Milano, Italy.
Ivan
I
Fraietta
Department of Biology, Nerviano Medical Sciences S.r.l., Nerviano, Milano, Italy.
Paolo
P
Cappella
Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milano, Italy.
Gloria
G
Barbarani
Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milano, Italy.
Isaura
I
Font-Monclus
Dipartimento di Scienze della Salute, Università degli studi di Milano-Bicocca, Monza, Italy.
Mario
M
Mauri
Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milano, Italy.
Sergio
S
Ottolenghi
Department of Biology, Nerviano Medical Sciences S.r.l., Nerviano, Milano, Italy.
Fabio
F
Gasparri
Dipartimento di Biotecnologie e Bioscienze, Università degli studi di Milano-Bicocca, Milano, Italy.
Antonella
A
Ronchi
D000755:Anemia, Sickle Cell / Q000378:metabolism; D020148:Butyric Acid / Q000378:metabolism; D005319:Fetal Hemoglobin / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000378:metabolism; D020014:K562 Cells; D055544:beta-Globins / Q000032:analysis* / Q000378:metabolism; D017086:beta-Thalassemia / Q000378:metabolism; D055545:gamma-Globins / Q000032:analysis* / Q000378:metabolism
D055544:beta-Globins; D055545:gamma-Globins; D020148:Butyric Acid; D005319:Fetal Hemoglobin; D006918:Hydroxyurea