Thalidomide Induces gamma-Globin Gene Expression in Adult Primary Erythroid Cells through Increased Intracellular Reactive Oxygen Species Mediated Activation of p38 MAPK Signal Pathway.

Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), including increases in both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomide’s therapeutic effect are still being defined. We hypothesize that thalidomide induce the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin gene expression in a dose-dependent manner. The averaged gamma/gamma+beta percentage ratio was 12.89% ± 0.11% in cultures treatment with the highest concentration of 100uM thalidomide compared with 3.21% ± 0.07% in Epo alone (P<0.01). Interestingly, we found that intracellular ROS level was significantly increased by treated with 100uM thalidomide for 48 hours, from day 3 to day 5. We can not rule-out an effect of thalidomide on ROS generation beyond day 6, as concomitant hemoglobin formation at this time also induce the H2DCF-DA dye generate the fluroscence. We also found by Western blot analysis that thalidomide activated the p38 MAPK signaling pathway in a time- and dose-dependent manner. Thalidomide also increased the histone H3 phospho-acetylation and histone H4 acetylation. In contrast, treatment with the anti-oxidant N-acetylcycteine (NAC) inhibited the thalidomide induced p38 activity and histone H3, H4 acetylation. These data indicate that thalidomide induced the gamma-globin gene increase via activate p38 MAPK signaling pathway as well as histone modification associated with the generation of ROS.