Freezing response was measured without CS or US

Rafts in MDS Erythroid Precursors have been implicated in MDS pathogenesis, and may underlie the observed deficiency in raft assembly. Recent investigations have shown that expression of the unconventional Rac activating guanine nucleotide exchange factor, DOCK4, is decreased in MDS patients compared to age-matched controls. DOCK4 is a member of the CDM family of proteins, which are regulators of adherens junctions and cell migration. The DOCK4 promoter is hypermethylated in MDS, with consequent silencing of gene expression. A recent report showed that DOCK4 silencing in MDS was associated with diminished F-actin polymerization. Moreover, decreased DOCK4 expression was associated with increased erythrocyte fragility which was confirmed by gene knockdown experiments in primary erythroid progenitors. These findings provide a plausible pathobiological rationale for the ineffective erythropoiesis in MDS in which intrinsic cytoskeletal abnormalities arising from decreased PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1968231 DOCK4 initiated polymerization of actin impairs lipid raft assembly and growth factor receptor signaling. Rac GTPase dependent raft assembly, which in our investigations is 12 / 18 Lenalidomide Induces Lipid Rafts in MDS Erythroid Precursors partially rescued by lenalidomide, further supports this notion. The OPC 8212 effects of lenalidomide on DOCK4 expression and activity warrant further investigation. Although lenalidomide has been previously reported to activate GTPases, the mechanism by which this occurs is unknown. Recent findings have shown that IMiDs bind to the cereblon E3 ubiquitin ligase complex to inhibit ligase function, which accounts in part for the teratogenicity of thalidomide, as well as the antiproliferative effects in multiple myeloma. However, more recent reports suggest that lenalidomide instead binds to CRBN to induce its E3 ligase activity, causing degradation of the Ikaros transcription factors and accounting for the anti-tumor activity in myeloma. These conflicting data suggest that cereblon may be ambiguous in its function with respect to not only cellular pathway, but also to cell lineage. The role of cereblon, if any, in lenalidomideinduced raft formation remains unclear. Transfection approaches to alter cereblon gene expression also disrupt raft integrity, thus perhaps only the creation of a stable knockdown could properly address the role of cereblon in lenalidomide induction of raft assembly. In addition to modifying the E3 ligase activity of cereblon, we recently reported that lenalidomide inhibits the activity of the human homolog of the murine double minute-2 E3 ubiquitin ligase, MDM2. Inhibition of MDM2 autoubiquitination stabilizes the protein, permitting binding to and degradation of p53 in del clones. These findings suggest that lenalidomide may have broader E3 ligase inhibitory effects. It is possible that lenalidomide may activate GTPases through inhibition of E3 ligases involved in their degradation. Several ligases are known to ubiquitinate RhoA, including SMAD ubiquitination regulatory factor 1, SMURF1 and the CRL3 complex. Additionally, the HECT ligase, HACE1, has recently been implicated in the ubiquitination and degradation of Rac1. Investigation of the effects of lenalidomide on these E3 ligases and consequent effects on GTPase activation and actin cytoskeletal reorganization is currently underway. Methods Reagents and cells UT7 cells, acquired PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19682429 from American Type Culture Collection, were maintained in alpha-MEM supplemented with 20% F