Hydroxamide derivatives of short-chain fatty acids are potent inducers of human fetal globin gene expression

Objective. To examine whether hydroxamic acids are inducers of fetal hemoglobin expression, we tested the effects on gamma gene expression of butyric and propionic hydroxamic acids and of two other hydroxamic acids (SBHA and SAHA), which are potent inhibitors of histone deacetylase (HDAC). We also investigated whether there is a correlation between HDAC inhibitory activity of the compounds and their ability to induce gamma-globin gene expression. Materials and Methods. Effects on gamma-globin expression were assessed by two methods: 1) a screening assay in which specific gamma-globin gene inducers are recognized by their ability to increase gamma firefly luciferase activity significantly more than beta-renilla luciferase activity; and 2) measurements of beta-globin mRNA and the frequency of fetal hemoglobin-positive erythroblasts in cultures of burst-forming unit erythroid (BFU-E) from normal individuals. HDAC in vitro activity was measured with a partially purified rat liver HDAC and a fluorogenic substrate. Results. All compounds tested increased gamma firefly luciferase activity, gamma/gamma + beta mRNA ratios, and percentage of fetal hemoglobin-containing erythroblasts in BFU-E cultures, in a dose-dependent fashion. Butyryl-hydroxamic acid 100 muM increased the gamma/gamma + beta mRNA ratios by 5.8-fold and the frequency of fetal hemoglobin-containing erythroblasts by 4.1-fold. Propionyl-hydroxamic acid 150 muM increased the gamma/gamma + beta ratios by 6.3-fold and the fetal hemoglobin-containing erythroblasts by 3.9-fold. SBHA induced gamma-globin gene expression at very low concentrations, 5 to 20 muM in the luciferase system and 2 to 8 muM in BFU-E cultures; SAHA at 1 to 7.5 muM in the luciferase system and 1 to 2.5 muM in the BFU-E cultures. HDAC in vitro inhibition was observed in the millimolar range for propionate and butyrate. IC50 determinations led to values of 384 muM for propionyl-hydroxamate, 47 muM for butyryl-hydroxamate, 0.93 muM for SBHA, and 0.26 muM for SAHA. Conclusion. Our data indicate that hydroxamic acid-based HDAC inhibitors are potent gamma-globin gene inducers and that the concentration range of their effects on gamma gene expression can be correlated roughly with their HDAC inhibitory potencies. (C) 2003 International Society for Experimental Hematology. Published by Elsevier Science Inc.