Although erythroid cells and megakaryocytes arise from a common progenitor, their

Although erythroid cells and megakaryocytes arise from a common progenitor, their terminal maturation follows very different paths; erythroid cells undergo cell-cycle exit and enucleation, whereas megakaryocytes continue to progress through the cell cycle but skip late stages of mitosis to become polyploid cells. survival of proliferating cells, survivin-deficient hematopoietic progenitors failed to give rise to either erythroid or megakaryocytic colonies. Together, these studies show that whereas survivin expression is essential for megakaryocyte and erythroid progenitors, its down-regulation is required for terminal differentiation of megakaryocytes. and = 0.004 for difference in CD41 expression and 0.008 for difference in Ter119 expression (Student’s test). ( 0.05 (MannCWhitney test). Cell Staining, Antibodies, and Flow Cytometry. Surface staining for human CD41, human Rabbit polyclonal to Caspase 3.This gene encodes a protein which is a member of the cysteine-aspartic acid protease (caspase) family.Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis.Caspases exist as inactive proenzymes which undergo pro CD42, and mouse Ter119 (BD Pharmingen) was performed by using phycoerythrin (PE)-conjugated antibodies in Ca2+-free, Mg2+-free PBS with 2% serum. Surface staining for mouse CD41 or CD61 was analyzed by using a purified anti-mouse CD41 or CD61 antibody (BD Pharmingen), followed by staining with PE or PE-Cy5-conjugated secondary antibody (Jackson ImmunoResearch). Cytoplasmic staining for Lenalidomide distributor survivin expression was performed as described (14), with a polyclonal anti-survivin antibody (AF886; R & D Systems) after fixing the cells in 2% paraformaldehyde, followed by permeabilization with Perm/Wash buffer (BD Biosciences). All flow cytometry was performed on a FACScan (BD Biosciences), and data were analyzed by using flojo software. The anti-survivin mouse mAb (6E4; Cell Signaling Technology, Beverly, MA) was used for immunofluorescence staining. Western blot analyses to detect survivin in K562 cells were performed by using the polyclonal and monoclonal anti-survivin antibodies, and analyses to detect survivin Lenalidomide distributor in primary cells were performed with the polyclonal anti-survivin antibody. Results To determine whether megakaryopoiesis is associated with a decrease in survivin expression, we induced megakaryocytic differentiation in the bipotential human cell line K562 with the phorbol ester PMA. At 96 h after PMA treatment, the cells exhibited basophilic cytoplasm, indented nuclei, and staining for both CD41 and CD42, consistent with megakaryocytic differentiation (Fig. 1and data not shown). As expected, there was a marked reduction in survivin expression in this population of cells (Fig. 1differentiation from CD34+ cells through the orthochromatic stage of maturation (Fig. 1as compared with the other panels. The mouse monoclonal anti-survivin antibody used in this experiment gave results that were consistent with the known localization of survivin in mitotic cells (8). (= 0.004), down-regulation of survivin is likely to be an important step in megakaryopoiesis. Conversely, Lenalidomide distributor overexpression of survivin led to an increase in the number of Ter119+ cells (Fig. 2= 0.008). Of note, analysis of survivin expression in purified Ter119+ and CD41+ cells by qRT-PCR verified that survivin was overexpressed in the terminally differentiated cells by 4- to 7-fold (data not shown). Together, these results suggest that elevated levels of survivin favor the expansion of erythroid cells over megakaryocytes. Because survivin overexpression might be predicted to interfere with polyploidization, we next compared the DNA content of CD41+ cells generated in the presence or absence of ectopically expressed survivin. We found that there was an accumulation of CD41+ cells with a 4DNA content and a concomitant diminution in the fraction of cells reaching a ploidy of 4in the survivin-overexpressing population in comparison with the control MIGR1-infected cells (Fig. 2= 0.01; Fig. 4and 0.01. Our RNAi studies suggested that megakaryocytes and erythroid cells exhibit differential requirements for survivin. To determine unambiguously whether survivin is required for development of only one or Lenalidomide distributor both of these lineages, we performed hematopoietic colony assays with bone marrow progenitors from survivin conditionally targeted mice (15). First, we harvested bone marrow from 8- to 10-week-old survivin+/+, survivinfl/+, and survivinfl/fl mice; infected these cells with retroviruses that expressed either Cre and GFP or GFP alone; sorted for GFP+ cells; and then performed colony-forming assays. Heterozygous loss of survivin resulted in 50% Lenalidomide distributor reduction in survivin mRNA expression (Fig. 5and 0.02; **, 0.01; ***, 0.0002. Note that the difference between CFU-Mk+/+ to +/fl was not significant (Student’s test). Data from the colony assays revealed that erythroid progenitors are more sensitive to the levels of survivin than those of the megakaryocyte lineage. Heterozygous survivinfl/+ progenitors expressing Cre gave rise to 50% BFU-Es in comparison with those expressing GFP alone (Fig. 5as compared.