ch lenalidomide pretreatment both augmented and extended the duration of phosphorylation of the transcription factor. We next evaluated whether lenalidomide increased binding PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19683258 of STAT5 to DNA using electrophoretic mobility shift assay to influence transcriptional response. We found that lenalidomide increased and sustained the binding of the transcription factor to DNA in UT7 cells when given prior to rhEpo. These data indicate lenalidomide potentiates EpoR signaling in UT7 erythroid progenitors through increased STAT5 DNA binding, which may account for the promotion of erythroid gene specific transcriptional response described by Ebert et al. We next investigated the effects of lenalidomide on EpoR signaling following rhEpo stimulation in primary MDS erythroid progenitors by flow cytometry. Primitive erythroid precursors characterized by CD45dim, CD71high and GlyAlow, were analyzed for changes in STAT5 phosphorylation. Lenalidomide pretreatment markedly increased STAT5 phosphorylation in response to rhEpo stimulation in MDS erythroid progenitors compared to rhEpo treatment alone. 7 / 18 Lenalidomide Induces Lipid Rafts in MDS Erythroid Precursors pretreatment. To determine if potentiation of EpoR signaling by lenalidomide influenced colony-forming capacity, we assessed colony recovery with and without lenalidomide treatment in UT7 cells and primary MDS bone marrow mononuclear cells. UT7 cells were pretreated for 2 h with lenalidomide, then plated in cytokine containing methylcellulose with rhEpo. Combination treatment with lenalidomide and rhEpo yielded a significant increase in colony number compared to rhEpo alone . To further verify this, we performed colony formation assays using primary MDS BM-MNCs, with 2 to 6 patients evaluated at each indicated concentration of lenalidomide. We found more than a two-fold increase in erythroid burst forming units following lenalidomide pretreatment, whereas there was minimal change in the number of mixed colony forming units-granulocyte, erythrocyte, monocyte, and megakaryocyte 8 / 18 Lenalidomide Induces Lipid Rafts in MDS Erythroid Precursors or, suggesting an erythroid specific effect. Although we did not find a dose dependent increase in erythroid colonies, these data are consistent with previous get 2883-98-9 findings by Ebert et al. where maximal augmentation of BFU-E recovery with lenalidomide was observed at concentrations 1 mM. ROCK inhibition prevents F-actin polymerization and raft assembly induced by lenalidomide We previously reported that rhEpo induction of lipid raft assembly was Rac GTPase dependent. Inhibition of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19681941 Rho-associated protein kinase, ROCK, and 9 / 18 Lenalidomide Induces Lipid Rafts in MDS Erythroid Precursors Rac GTPase inhibited recruitment of EpoR into the raft fractions after Epo stimulation. To determine whether ROCK was similarly involved in the induction of lipid rafts by lenalidomide, UT7 cells were treated with lenalidomide either with or without pretreatment with 100 mM of the ROCK inhibitor, Y27632, for 30 min. Pretreatment with 
Y-27632 inhibited the induction of lipid rafts by lenalidomide as shown by GM-1 dot blot analysis and by western blot of Lyn kinase from isolated cellular fractions. These findings were confirmed by confocal microscopy imaging of lipid rafts. Moreover, lenalidomide induction of actin filament polymerization was also inhibited by the ROCK inhibitor, as analyzed by confocal microscopy of phalloidin stained cells. These findings implicate ROCK in