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Although thalidomide has been shown to improve anemia in some patients with myelodysplastic syndromes and stimulates erythropoietin in patients with multiple myeloma, thalidomide's specific effects on ␥-globin gene expression during erythroid differentiation have not been studied. Here, we investigated the effects of thalidomide on ␥-globin gene expression and the involved signaling pathway using an ex vivo culture system of primary human CD34 ؉ cells. We found that thalidomide induced ␥-globin mRNA expression in a dose-dependent manner, but had no effect on ␤-globin expression. We also demonstrated that intracellular reactive oxygen species (ROS) levels were increased by treatment with thalidomide for 48 hours (from day 3 to day 5). Western blot analysis demonstrated that thalidomide activated the p38 mitogenactivated protein kinase ( IntroductionThalidomide is a synthetic glutamic acid derivative that was originally prescribed as a sedative and antinausea medicine, but later withdrawn from the market due to its teratogenic effects. 1 However, thalidomide has made a remarkable comeback since the discovery of its immunomodulatory and anti-inflammatory effects, which have led to its use as a treatment for various proinflammatory and autoimmune conditions. 2,3 It is currently being used to treat a number of diseases, including dermatologic, infectious, autoimmune, and hematologic disorders, especially multiple myeloma. 4 Thalidomide has shown promising results in the treatment of multiple myeloma due to its antiangiogenic activity. 5 The precise mechanisms whereby thalidomide exerts its therapeutic effect are still unknown.Thalidomide's effects may be related to its ability to repress cytokine-induced nuclear factor-B (NF-B), tumor necrosis factor-␣ (TNF-␣), vascular endothelial growth factor (VEGF), and prostaglandin E2 (PGE 2 ) synthesis, as well as its ability to increase production of reactive oxygen species (ROS) that play a role in vitro. 3,[6][7][8][9] ROS can induce or suppress the expression of several genes and can affect cell-signaling pathways by altering the activities of certain protein kinases and transcription factors. The pathway by which thalidomide exerts its teratogenicity is also under debate. However, it has been observed that the teratogenic properties of thalidomide may be due to a species-specific conversion to free-radical intermediates that result in DNA damage. 10,11 In addition to these biologically based effects, recent clinical trials have confirmed that thalidomide may improve anemia and, less frequently, other cytopenias in a proportion of younger patients with low-risk myelodysplastic syndromes, and stimulates erythropoiesis in patients with multiple myeloma. 12,13 Moreover, thalidomide and its derivatives have been reported to reduce or even eliminate the need for red blood cell transfusions in some anemic patients with myelodysplasia. 14 Hence, it is of special interest to evaluate the effect of thalidomide on fetal hemoglobin synthesis in adult erythropoiesis using an in vitro...

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We report here a lentiviral vector system for regulated transgene expression. We used the tetracycline repressor fused with a transcriptional suppression domain (tTS) to specifically suppress transgene expression. Human cells were first transduced with a tTS-expressing vector and subsequently transduced with a second lentiviral vectorcontaining transgene controlled by a regular promoter adjacent to a high-affinity tTS-binding site (tetO). After optimizing the location of the tetO site in the latter vector, we achieved a better inducible transgene expression than the previous lentiviral vectors using the tetracycline repressor systems. In this new system, the transgene transcription from a cellular promoter such as EF1␣ or ubiquitin-C promoter is suppressed by the tTS bound to the nearby tetO site. In the presence of the tetracycline analog doxycycline (Dox), however, the tTS binding is released from the transgene vector and transcription from the promoter is restored. Thus, this system simply adds an extra level of regulation, suitable for any types of promoters (ubiquitous or cell-specific). We tested this tTSsuppressive, Dox-inducible system in 293T cells, human multipotent hematopoietic progenitor cells, and three human embryonic stem cell lines, using a dual-gene vector containing the green fluorescent protein reporter or a cellular gene. We observed a tight suppression in the uninduced state. However, the suppression is reversible, and transgene expression was restored at 5 ng/ml Dox. The lentiviral vectors containing the tTS-suppressive, Dox-inducible system offer a universal, inducible, and reversible transgene expression system in essentially any mammalian cell types, including human embryonic stem cells. STEM CELLS 2007;25:779 -789