A Novel High-Content Immunofluorescence Assay as a Tool to Identify at the Single Cell Level gamma-Globin Inducing Compounds

作者：Durlak Marta; Fugazza Cristina; Elangovan Sudharshan; Marini Maria Giuseppina; Marongiu Maria Franca; Moi Paolo; Fraietta Ivan; Cappella Paolo; Barbarani Gloria; Font Monclus Isaura; Mauri Mario; Ottolenghi Sergio; Gasparri Fabio; Ronchi Antonella^*

来源：PLos One, 2015, 10(10): e0141083.

DOI：10.1371/journal.pone.0141083

The identification of drugs capable of reactivating gamma-globin to ameliorate beta-thalassemia and Sickle Cell anemia is still a challenge, as available gamma-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 gamma/beta globin ratio with the robustness of a cell line. We took advantage of a K562 cell line variant expressing beta-globin (beta-K562) to set up a new multiplexed high-content immunofluorescence assay for the quantification of gamma- and beta-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 gamma-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 gamma-globin induction. Indeed, Heme-oxygenase2 siRNA knockdown as well as its inhibition by Tin protoporphyrin-IX (TinPPIX) greatly increased gamma-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 gamma-globin reactivation for the treatment of beta-hemoglobinopathies.

• 出版日期2015-10-28