title: Cellular genomic reporter assays for screening and evaluation of inducers of fetal hemoglobin.
abstract: Reactivation of fetal hemoglobin (HbF) expression using pharmacological agents represents a potential strategy for the therapy of beta-thalassemia, sickle cell disease, HbE and other beta-hemoglobinopathies. However, the drugs currently available have low efficacy and specificity and are associated with high toxicity. We describe the development of stable cellular genomic reporter assays (GRAs) based on the green fluorescence protein (EGFP) gene under the Ggamma-globin promoter in the intact human beta-globin locus. We show that human erythroleukemic cell lines stably transfected with a Ggamma-EGFP beta-globin locus construct can maintain a uniform basal level of EGFP expression over long periods of continuous culture and that induction of EGFP expression parallels the induction of the endogenous globin genes. We compared the EGFP-induction potency of a number of chemotherapeutic agents, including histone deacetylase inhibitors and DNA-binding agents. We show that hydroxyurea and butyrate result in moderate levels of induction (70-80%) but with an additive inductive effect. Among the DNA-binding agents tested, cisplatin was the most potent inducer of HbF expression, (442+/-32%), a level which is comparable to hemin (764+/-145%). These results indicate that cellular GRAs containing Ggamma-EGFP-modified beta-globin locus constructs can be used to develop novel inducers of HbF synthesis for the therapy of beta-hemoglobinopathies.
authors.affiliation: The Murdoch Childrens Research Institute, The University of Melbourne, Royal Children's Hospital, Melbourne, Australia.
authors.forename: Jim
authors.initials: J
authors.lastname: Vadolas
authors.forename: Hady
authors.initials: H
authors.lastname: Wardan
authors.forename: Michael
authors.initials: M
authors.lastname: Orford
authors.forename: Robert
authors.initials: R
authors.lastname: Williamson
authors.forename: Panayiotis A
authors.initials: PA
authors.lastname: Ioannou
mesh_terms: D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005319:Fetal Hemoglobin / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D015894:Genome, Human; D005914:Globins / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins; D006427:Hemin / Q000494:pharmacology; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D020014:K562 Cells; D008164:Luminescent Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D002087:Butyrates; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D006427:Hemin; D005914:Globins; D005319:Fetal Hemoglobin; D002945:Cisplatin; D006918:Hydroxyurea