Fetal hemoglobin induction by histone deacetylase inhibitors involves generation of reactive oxygen species.
OBJECTIVE
Several compounds, including butyrate and trichostatin A, have been shown to activate gamma-gene expression via p38 mitogen-activated protein kinase (MAPK) signaling. In eukaryotic cells, reactive oxygen species (ROS) act as signaling molecules to mediate phosphorylation of tyrosine kinases such as p38 MAPK to regulate gene expression. Therefore, we determined the role of the reactive oxygen species hydrogen peroxide (H(2)O(2)) in drug-mediated fetal hemoglobin (HbF) induction.


METHODS
H(2)O(2) levels were measured using 2',7'-dichlorofluorescein-diacetate in K562 cells after drug treatments. To confirm a role for H(2)O(2) in HbF induction, studies were completed with the mitochondrial respiratory chain inhibitor myxothiazole, which prevents ROS generation. The ability of myxothiazole to block gamma-globin mRNA accumulation and HbF induction was measured in K562 cells and burst-forming unit-erythroid colonies respectively using quantitative real-time PCR and alkaline denaturation.


RESULTS
Butyrate and trichostastin A stimulated p38 MAPK phosphorylation via a H(2)O(2)-dependent mechanism. Pretreatment with myxothiazole to inhibit ROS formation or SB203580 to impede p38 MAPK signaling attenuated gamma-gene activation in K562 cells and HbF induction in erythroid progenitors. However, myxothiazole had no effect on the ability of hydroxyurea to induce HbF.


CONCLUSIONS
The findings presented herein support a ROS-p38 MAPK cell signaling mechanism for HbF induction by butyrate and trichostatin A.
University of Texas at Dallas, Department of Molecular and Cell Biology, Richardson, TX 75083, USA.
Cheng-Hui
CH
Hsiao
Wei
W
Li
Tzu-Fang
TF
Lou
B Surendra
BS
Baliga
Betty S
BS
Pace
D015153:Blotting, Western; D002087:Butyrates / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005319:Fetal Hemoglobin / Q000096:biosynthesis*; D005914:Globins / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology; D020014:K562 Cells; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction; D017382:Reactive Oxygen Species; D015398:Signal Transduction; D024483:Vitamin K 3 / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D002087:Butyrates; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D017382:Reactive Oxygen Species; C012589:trichostatin A; D024483:Vitamin K 3; D005914:Globins; D005319:Fetal Hemoglobin; D006861:Hydrogen Peroxide; D048051:p38 Mitogen-Activated Protein Kinases