MATERIALS AND METHODS HUVEC. HUVECs were obtained from umbilical cords from cesarean sections. The cells were cultured by standard methods in the presence of heparin and endothelial-cell growth supplement (7). They were characterized by their homogeneous and typical cobblestone morphology, factor VIII antigen positivity, and the presence of Weibel-Palade bodies on electron microscopy. HUVECs were used for these studies after three to five passages.For clonal culture to exclude contamination with hematopoietic stem cells, HUVECs (1-5 x 105 cells per ml) were plated in methylcellulose cultures as described (8), with the modification that half of the fetal bovine serum was replaced with human umbilical cord blood serum. Hematopoietic growth factors were added as follows: rhEpo, 2 units/ml; stem-cell factor, 10 pg/ml; granulocyte/macrophage-colonystimulating factor, 200 units/ml; interleukin 3, 200 units/ml; endothelial-cell growth factor, 20 ktg/ml.Preparation of mRNA. After 1-5 x 107 cells were harvested and washed twice with phosphate-buffered saline, RNA was extracted with guanidinium thiocyanate and lauryl sarcosinate (9). mRNA was adsorbed onto oligo(dT)-cellulose columns (Pharmacia) and, after the columns were washed with high-and low-salt solutions, was eluted with 10 mM Tris'HCl buffer containing 1 mM EDTA (pH 7.4) at 650C. Total amount and concentration of mRNA were determined spectrophotometrically and confirmed by agarose gel electrophoresis.

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We have studied the patterns of expression of various genes during maturation of normal human adult erythroid precursors cultured in a two-phase liquid culture method. In the first phase, peripheral blood mononuclear cells are cultured for one week in the presence of a combination of growth factors, but not erythropoietin (Epo). In Phase II, Epo is included in the medium. Cell samples were taken throughout phase II, and expression of globins, transcription factors, and cytokine receptors was assayed by RT-PCR and quantified by phosphor imaging. We have divided phase II into stages: early (days 0-5), intermediate (days 6-10) and late (days 11-15) and measured maximum expression of each gene. During early phase II, gamma-globin, Spl, and GATA-2 mRNAs were expressed at their highest levels. As the cells matured during the intermediate period, GATA-2 levels remained high, and then declined, while the transcription factors GATA-1, EKLF, NF-E2, and the Epo receptor (EpoR) reached maximum expression. In late phase II, beta-globin increased and reached its maximum level of expression. This erythroid culture system appears to recapitulate normal adult erythropoiesis at the molecular level, and thus may be a suitable model to examine the molecular basis of severe congenital or acquired disorders of erythropoiesis.

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...