Role of p21(Cip1/Waf1) in cell-cycle exit of endomitotic megakaryocytes.

The cyclin-dependent kinase inhibitor p21(Waf-1/Cip-1) is expressed at high level during megakaryocyte differentiation, but its precise function remains unknown. In this study, it is confirmed that p21 was expressed at a high level in hypoploid (2N and 4N) and polyploid (at least 8N) human megakaryocytes derived from CD34(+) cells. A high expression of p27(Kip1), p16, cyclin E, and cyclin D3 was also found in both populations associated with a hypophosphorylated form of retinoblastoma protein, suggesting that the majority of hypoploid and polyploid megakaryocytes are G(1)-arrested cells. As human megakaryocytes grown in vitro present a defect in their polyploidization, the study switched to the murine model. The modal ploidy of megakaryocytes derived from lineage-negative cells was 32N, and an elevated expression of p21 was found in high-ploidy megakaryocytes. In addition, p21 and p27 were coexpressed in the majority of mature polyploid megakaryocytes. The p21 was detected by immunofluorescence in megakaryocytes derived from p53(-/-) mice, demonstrating a p53-independent regulation during megakaryocyte differentiation. Megakaryocytopoiesis of p21(-/-) mice was subsequently studied. No marked abnormality in the ploidy of primary or cultured megakaryocytes was detected. Overexpression of p21 in p21(-/-) or normal murine megakaryocytes and in human megakaryocytes showed in all these cases a marked inhibition in megakaryocyte polyploidization. In conclusion, while a reciprocal relation is observed between p21 levels in megakaryocytes and the cycling state of the cells, p21 is not essential for the determination of the ploidy profile in normal megakaryocytes in vivo. However, high levels of its expression in cultured megakaryocytes arrest the endomitotic cell cycle.     

A combination of megakaryocyte growth and development factor and interleukin-1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes

Chemotherapy-induced thrombocytopenia is a major risk factor in cancer treatment. The transfusion of autologous ex vivo expanded megakaryocytes could be a new therapy to shorten the period of thrombocytopenia. Therefore we investigated, in a liquid culture system, the effect of various cytokine combinations composed of pegylated megakaryocyte growth and development factor (PEG-rHuMGDF), interleukin-1 (IL-1), IL-3, IL-6, IL-11 and stem cell factor (SCF) on the proliferation and differentiation of CD34+ cells, in order to define the most optimal and minimum levels of cytokine combinations for megakaryocyte expansion. Besides PEG-rHuMGDF, IL-1 was found to be important for optimal megakaryocyte expansion. Depletion of either SCF, IL-6 or IL-11 did not exert a large effect, but the absence of IL-1 strongly diminished the number of megakaryocytic cells. Addition of IL-3 to the combination PEG-rHuMGDF, IL-1, IL-6, IL-11 and SCF significantly reduced the number of megakaryocyte progenitors (CD34+CD41+ cells) and the number of CFU-Meg. Furthermore, we found a strong correlation between the number of CD34+CD41+ cells and the number of CFU-Meg obtained after 8 d culture. Our study shows that optimal ex vivo expansion of megakaryocytes is achieved by the combination of PEG-rHuMGDF and IL-1. The numbers of megakaryocytes and megakaryocyte progenitors (CD34+CD41+) obtained in our liquid culture system with the growth factor combination PEG-rHuMGDF and IL-1 are suitable for transfusion purposes.     

Cyclin D3 is essential for megakaryocytopoiesis

A normal cell cycle in most eukaryotic cells consists of a tightly regulated sequence of phases including DNA synthesis (S) followed by a gap (G2), mitosis (M), and a gap (G1). In the megakaryocytic lineage, the cells undergo endomitosis, which involves DNA synthesis in the absence of mitosis, thus giving rise to polyploid cells. We aimed at defining whether the megakaryocytic cell cycle consists of a continuous S phase or of G1/S phases and at determining which cyclins are involved in this process. Studies were performed in primary cultures of mouse bone marrow cells. DNA synthesis in megakaryocytes was followed by determining incorporation of a DNA precursor, bromodeoxyuridine (BrdU), into the cells by in situ staining for BrdU. These experiments showed that no more than 15% of the recognizable megakaryocytes in normal bone marrow are in the process of endomitosis, including S phases interrupted by short gaps. Using immunohistochemistry, we showed that mature megakaryocytes express the G1 phase cyclin and cyclin D3, but not the mitotic cyclin, cyclin B1. Under culture conditions that selectively promote megakaryocytopoiesis, antisense oligonucleotides designed to suppress cyclin D3 expression, but not sense oligonucleotides or antisense oligonucleotides to cyclin B1, dramatically suppress endomitosis and abrogate megakaryocyte development. Our results indicate that endoreduplication in megakaryocytes is associated with low levels of or the absence of cyclin B1, whereas progression through this process depends on the G1 phase for which cyclin D3 is crucial.     

Requirement of thrombopoietin-induced activation of ERK for megakaryocyte differentiation and of p38 for erythroid differentiation

Thrombopoietin (TPO) plays a critical role not only in proliferation and differentiation of megakaryocytes but also in erythroid differentiation. We have investigated whether the different pathway of mitogen-activated protein kinase (MAPK) after TPO stimulation may discriminate megakaryocyte and erythroid differentiation. In this study, we have used human CD34+ hematopoietic progenitor cells (HPCs) from cord blood (CB) in serum-free liquid culture supplemented with TPO, to compare the respective effects of specific inhibitors of MAPK kinase (MEK) (PD98059) and p38 MAP kinase (p38) (SB203580) on megakaryocyte and erythroid development. PD98059, but not SB203580, significantly suppressed TPO-induced megakaryocyte differentiation when examined by the expression of CD41 and polyploidy assay. In the presence of SB203580, CD34+/CD36+ erythroid progenitors clearly decreased, whereas they increased when cultured with PD98059. These results indicate that activation of extracellular-signal-regulated kinase (ERK) is required for TPO-induced megakaryocyte differentiation and that p38 is required for TPO-induced erythroid differentiation.     

CD34+ cells derived from fetal liver contained a high proportion of immature megakaryocytic progenitor cells

Endoreplication and maturation of the megakaryocyte (MK) may be retarded or delayed during ontogenesis. In this study, CD34+ cells were isolated from both human fetal liver and adult bone marrow and incubated with thrombopoietin (TPO). The cell number, morphological characteristics, platelet-associated antigen phenotype, maturation stage and DNA ploidy of CD41+ cells were examined from day 0 to day 12 in culture. 1) TPO stimulated the proliferation of fetal liver (FL)-derived CD34+ cells with a mean 73.14-fold increase of CD41+ cells after 12 d in culture. Adult BM-derived CD34+ cells increased only slightly, with a mean 8.18-fold increase of CD41+ cells. 2) Although the membrane phenotype of both FL CD34+-derived MKs and BM CD34+ -derived MKs analyzed with CD41a, CD42a, CD61 and CD34 were similar, all FL CD34+-derived MKs were in maturation stage I and II and in low ploidy (<4N) class. By comparison, BM CD34+ MKs possessed 15% MKs in maturation stage III and IV and with 23% MKs in high ploidy class ( > 4N). 3) Most of cultured FL-derived CD34+ cells did not have a well developed demarcation system (DM) and numerous alpha-granules after 12 d incubation. von Willebrand factor (vWF) appeared earlier on the cultured BM-derived CD34+ cells than on FL-derived CD34+ cells. 4) The expression of both cyclin E and cyclin B1 progressively increased in FL CD34+ cells induced by TPO during 12 d in culture. 5) The expression of cyclin D1 gradually decreased in FL CD34+ cells induced by TPO over 12 d incubation. 6) Immunocytochemical analysis showed that cyclin D3 was detected only in cytoplasm of cultured FL-derived CD34+ cells, whereas in both cytoplasm and nuclei of cultured BM-derived CD34+ cells. These data suggest that FL-derived CD34+ cells contain a high proportion of immature megakaryocytic progenitor cells. It further suggests that TPO can push these progenitor cells into proliferation by upregulating the expression of cyclins B1 and E, and drive a high proportion of cells into megakaryocytic lineage.     

XIAP通过调节CDK4／CDK6／cyclinD1复合物表达促进肝癌细胞增殖

目的研究肝癌细胞中XIAP与cDK4／cDK6／cyclinD1复合物之间的调控关系及在肝癌细胞增殖中的作用。方法前期研究用PathwayArray进行肝癌组织异常表达蛋白筛选,后期XIAP特异性抑制剂embelin处理Huh7细胞进行增殖、周期分析。Westernblot验证XIAP与cDK4／cDK6／cyclinD1复合物之间的调控关系。结果PathwayArray提示XIAP与CDK4、CDK6、cyclinD1之间存在联合表达。embelin引起Huh7细胞G1期阻滞、S期分布减少,抑制细胞增殖。XIAP可以调节Huh7细胞G1期蛋白CDK4、CDK6、cyclinD1表达。结论XIAP通过调节肝癌细胞G1期蛋白CDK4／CDK6／eyelinD1复合物的表达,促使细胞进入G1周期,从而促进其增殖。     

Development of human megakaryocytes: I. Hematopoietic progenitors (CD34+ bone marrow cells) are enriched with megakaryocytes expressing CD4

CD34 is expressed by essentially all human hematopoietic progenitors including cells of the megakaryocyte (MK) lineage. We have previously reported CD4 expression by some human MK (Blood 81:2,664, 1993). To study the role of maturation on CD4 expression by MK, we examined CD34+ bone marrow cells for their expression of CD41 (GPIIb-GPIIIa) and CD4 with specific monoclonal antibody (MoAb)-fluorochrome conjugates and for DNA polyploidization with propidium iodide or 7-aminoactinomycin D (7-AAD). Surprisingly, MK were at least 20-fold more common in the CD34+ progenitor pool (approximately 10%) than in the more mature CD34+ population (approximately 0.5%) of low density bone marrow cells. CD4 expression correlated with markers of immaturity in that CD4 was enriched among CD34+ cells, and the proportion of CD4+ MK declined with increasing ploidy. Almost all CD34+ polyploid ( > or = 8N) cells were CD4+. Despite these correlations with immaturity, CD34+CD4+ MK precursors were unable to produce MK colony-forming units (CFU-MK) when cultured under conditions that supported the growth of CFU-MK from CD34+CD4- MK lineage cells. MK became polyploid before the loss of either CD34 or CD4 expression. The presence of CD4 on these cells correlates with the onset of endomitotic reduplication and is associated with the loss of the ability of these cells to undergo normal mitotic division. The role of CD4 on immature MK as a differentiation antigen and/or receptor for the human immunodeficiency virus (HIV)-1 virus remains to be determined.     

Comparative analyses of megakaryocytes derived from cord blood and bone marrow

Thrombocytopenia is typically observed in patients undergoing cord blood transplantation. We hypothesized that delayed recovery of the platelet count might be caused by defects in the megakaryocytic differentiation pathway of cord blood progenitors. To test this hypothesis, we compared the features of in vitro megakaryocytopoiesis between cord blood progenitors and those in bone marrow cells after isolation of CD34+ cells as progenitors. The proliferative responses of the progenitors in cord blood are higher than those in bone marrow cells in the presence of interleukin (IL)-3, stem cell factor (SCF) and thrombopoietin (TPO). However, the ability to generate mature megakaryocytes was higher in bone marrow progenitors than in cord blood in the same in vitro culture system, when examined by the expression of CD41, polyploidy and proplatelet formation. Furthermore, an earlier induction of c-mpl protein, a receptor for TPO, was observed in the progenitors from bone marrow than in those from cord blood in the presence of SCF and IL-3. Therefore, the ability to generate mature megakaryocytes in bone marrow progenitors is superior to that in cord blood, and the delayed engraftment of platelets after cord blood transplantation might be attributed to the features of cord blood megakaryocyte progenitors.     

The in vitro effects of cytokines on expansion and migration of megakaryocyte progenitors

Increasing the number of megakaryocyte progenitors in stem cell transplants by ex vivo expansion culture may be an approach to accelerate platelet recovery in patients undergoing high-dose chemotherapy. We evaluated the effect of three different cytokine combinations on expansion, with special emphasis on the type of colony formation and migration of megakaryocytic cells. The number of clonogenic megakaryocyte progenitors (colony-forming units-megakaryocyte; CFU-Mk) with high- (> 20 cells/colony) and low-proliferative capacity (5-20 cells/colony) and the number of megakaryocytic (CD61+) cells were significantly increased by including interleukin 3 (IL-3) or IL-3 + IL-6 + IL-11 + Flt3-ligand to cultures containing megakaryocyte growth and development factor (MGDF) plus stem cell factor (SCF). No difference in the maturation of megakaryocytes from all three cytokine combinations to platelets were observed, as demonstrated by electron microscopy. In chemotaxis experiments, the migration towards stromal cell-derived factor 1 (SDF-1) was shown to be reduced for CD61+ cells and megakaryocyte progenitors cultured in other cytokines besides MGDF + SCF. The reduced migration was related to a lower expression of CXCR4, the receptor for SDF-1, on megakaryocytes from the proliferating cultures. These in vitro results demonstrate that expansion in IL-3 and other cytokines besides MGDF + SCF significantly impair the capacity of megakaryocytic cells to migrate.     

Differential requirements for survivin in hematopoietic cell development

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. In our efforts to identify genes that participate in this process, we discovered that survivin, a member of the inhibitor of apoptosis family that also has an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express survivin throughout their maturation, whereas megakaryocytes express approximately 4-fold lower levels of survivin mRNA and no detectable protein. To investigate the role of survivin in these lineages, we overexpressed or knocked down survivin from mouse bone marrow cells and then examined erythroid and megakaryocyte development. These studies revealed that overexpression of survivin antagonized megakaryocyte growth, maturation, and polyploidization but had no effect on erythroid development. This block in polyploidization was accompanied by increased expression of p21 and decreased expression of megakaryocyte genes such as von Willebrand factor and beta(1)-tubulin. In contrast, a reduction in survivin expression interfered with the formation of erythroid cells but not megakaryocytes. Last, consistent with the requirement for survivin in the 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.     

Expression of CD41 and c-mpl does not indicate commitment to the megakaryocyte lineage during haemopoietic development

Haemopoietic progenitors with the phenotype expected of early megakaryocyte precursors (CD34+ CD41+) were isolated from normal human bone marrow or induced in culture from CD34+ CD41- bone marrow cells by treatment with thrombopoietin (TPO) or IL-3. We found that although this population included the majority of cells that can form CFU-MK in culture, it also contained both erythroid and myeloid progenitors. The clonogenic potential of the CD34+ CD41+-induced cells was greater than that of isolated CD34+ CD41+ cells in that the isolated cells only formed CFU-MK and BFU-e, whereas the induced cells formed myeloid colonies as well. Glycophorin was found on isolated CD34+ CD41+ cells, not on induced cells. Its presence distinguished between MK and erythroid progenitors. Separation of a CD34+ CD41+ glycophorin A+ population resulted in the isolation of a highly purified population of BFU-e. A major portion of the cells that expressed CD34+ CD41+, in either cohort, were of the erythroid lineage. True MK progenitors were present in the CD34+ population in greater proportion than in whole marrow and were further enriched amongst CD34+ populations that expressed CD41. The presence of the thrombopoietin (TPO) receptor, c-mpl, did not correlate with inducibility of the gpIIbIIIa complex since essentially all CD34+ progenitors, including the earliest identifiable human haemopoietic progenitors (CD34+ CD38- cells), expressed c-mpl mRNA detectable by PCR regardless of their ultimate fate. Thus neither the expression of CD41 nor the expression of c-mpl was predictive of commitment to the MK lineage.     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

蛙皮素对人胃黏膜上皮永生细胞系cyclin D1/CDK4的影响

目的 研究蛙皮素对正常胃黏膜上皮细胞的促生长作用及其与细胞调控因子之间的关系。方法 应用人胃黏膜上皮永生细胞株GES－1,经蛙皮素及受体拮抗剂不同处理后,以MTT为指标研究对细胞增殖的影响;流式细胞仪观察对细胞cyclin D1表达的影响;利用Western印迹和增强化学发光法观察CDK4蛋白表达的变化;利用Sepharose微珠免疫沉淀方法检测对CDK4活性的影响。结果 蛙皮素在浓度为10^-7mol/L时对GES－1细胞有明显的促生长作用,对cyclin D1表达有促进作用;蛙皮素受体拮抗剂均抑制这些作用;同时还发现蛙皮素可使人胃黏膜上皮永生细胞系细胞的CDK4蛋白表达及其激酶活性增加。结论 蛙皮素促进人胃黏膜上皮细胞生长是通过使细胞周期调控因子cyclin D1和CDK4蛋白表达升高,CDK4激酶活性增加,促进细胞周期的进展而实现的。     

Overexpression of cyclin D1 moderately increases ploidy in megakaryocytes

BACKGROUND AND OBJECTIVES: Megakaryocytes undergo a unique cell cycle by which they replicate their complete genome many times in the absence of cytokinesis. In the search for regulators of the endomitotic cell cycle, we previously produced mice transgenic for cyclin D3 to identify this cyclin as able to enhance ploidy and to increase the number of differentiated cells in the megakaryocytic lineage. Of the D-type cyclins, cyclin D3 and to a much lesser extent cyclin D1, are present in megakaryocytes undergoing endomitosis and these cyclins are, respectively, markedly and moderately upregulated following exposure to the ploidy-promoting factor, Mpl-ligand. Our objective was to explore whether cyclin D1 can mimic the effect of cyclin D3 on ploidy in megakaryocytes. DESIGN AND METHODS: We generated transgenic mice overexpressing cyclin D1 in megakaryocytes and analyzed megakaryocyte ploidy, number and platelet levels in these mice and control mice. RESULTS: We show that transgenic mice in which cyclin D1 is overexpressed in megakaryocytes display higher ploidy level than the control mice, with no change in the number of differentiated cells of the megakaryocytic series, or in platelet level. INTERPRETATION AND CONCLUSIONS: Our models support a key role for D-type cyclins in the endomitotic cell cycle, and also indicate that although cyclin D3, from among the D cyclins, is unique in its high levels of expression in megakaryocytes, it is not unique in its ability to promote polyploidization.     

Expression of CD34 and platelet glycoproteins during human megakaryocytic differentiation.

Megakaryocyte (MK) progenitors express the CD34 antigen, but the precise stage along the MK differentiation at which the CD34 is turned off is not known. Purified marrow CD34+ cells give rise within 4 days in culture to rare mature MK, suggesting that some MK precursors bear the CD34 antigen. By multiparameter flow cytometry, CD34+ cells bearing platelet glycoproteins (GP) could be detected, but at a low frequency (less than 2% of the marrow CD34+ cells). We used an in vitro liquid suspension culture to selectively amplify MK differentiation. CD34+ cells were isolated after 6 days before a wave of mature MK. These cells gave rise within another 4 days in culture to numerous MK (up to 50%), showing that these CD34+ cells were greatly enriched in MK precursors. This was confirmed by ultrastructural studies that showed the presence of typical promegakaryoblasts. By flow cytometry, three populations of small cell size could be defined: CD34+ GPIIIa-, CD34+ GPIIIa+, and CD34- GPIIIa+ cells. The two GPIIIa+ populations were almost pure immature blastic MK. alpha-Granules were rare in the CD34+ GPIIIa+ cells, whereas they were more developed in the CD34- GPIIIa+ cells, which also exhibited demarcation membranes. Approximately 45% of the two GPIIIa+ cell populations were capable of undergoing at least one cell division and of giving rise to a polyploid progeny. However, proliferation and polyploidization capacities were higher in the CD34+ GPIIIa+ than in the CD34- GPIIIa+ cells. A small fraction of GPIIIa+ cells (about 10%) were able to give rise to MK colonies containing a maximum of 16 cells for the double-positive cells. GPIb was expressed on about sixfold less cells than GPIIIa, but was detected on a few CD34+ cells. Most double-stained (CD34+ GPIb+) cells were polyploid. CD34- GP+ cells (more mature) contained less polyploid MK than the CD34+ GP+ fraction. Altogether, these findings show that CD34 is still expressed on a polyploid transitional immature MK and that GPIIIa is present on some MK progenitors with low proliferative capacities. They also suggest that the expression of CD34 is related to the ability of the MK precursors to accomplish DNA synthesis (either cell division or endomitosis). Such a characterization will facilitate the investigation of the role of the different cytokines on MK differentiation.     

Overexpression of cyclin D1 in the Dami megakaryocytic cell line causes growth arrest

The maturation of megakaryocytes in vivo requires polyploidization or repeated duplication of DNA without cytokinesis. As DNA replication and cytokinesis are tightly regulated in somatic cells by cyclins and cyclin-dependent kinases, we sought to determine the pattern of cyclin gene expression in cells that undergo megakaryocytic differentiation and polyploidization. The Dami megakaryocytic cell line differentiates and increases ploidy in response to phorbol 12-myristate 13-acetate (PMA) stimulation in vitro. We used Northern blotting to analyze mRNA levels of cyclins A, B, C, D1, and E in PMA-induced Dami cells and found that cyclin D1 mRNA levels increased dramatically (18-fold). Similar increases in cyclin D1 mRNA were obtained for other cell lines (HEL and K562) with megakaryocytic properties, but not in HeLa cells. The increase in cyclin D1 was confirmed by Western immunoblotting of PMA-treated Dami cells. This finding suggested that cyclin D1 might participate in megakaryocyte differentiation by promoting endomitosis and/or inhibiting cell division. To address these possibilities, we constructed two stable Zn+2-inducible, cyclin D1-overexpressing Dami cell lines. Cyclin D1 expression alone was not sufficient to induce polyploidy, but in conjunction with PMA-induced differentiation, polyploidization was slightly enhanced. However, unlike other cell systems, cyclin D1 overexpression caused cessation of cell growth. Although the mechanism by which cyclin D1 may affect megakaryocyte differentiation is not clear, these data demonstrate that cyclin D1 is upregulated in differentiating megakaryocytic cells and may contribute to differentiation by arresting cell proliferation.     

PI3K/Akt-dependent Epo-induced signalling and target genes in human early erythroid progenitor cells

Erythropoietin (Epo) is the major regulator of differentiation, proliferation and survival of erythroid progenitors, but the Epo-induced changes in gene expression that lead to these effects are not fully understood. The aim of this study was to examine how Epo, via activation of phosphatidylinositol 3-kinase (PI3K)/Akt, exerts its role in the development of erythroid progenitors from CD34+ cells, and to identify early Epo target genes in human erythroid progenitors. In CD34+ progenitor cells, Epo alone was able to induce cell cycle progression as demonstrated by upregulation of cyclin D3, E and A leading to hyperphosphorylation of the retinoblastoma protein (RB). These effects were completely counteracted by the PI3K inhibitor LY294002. Furthermore, enforced expression of an activated form of Akt kinase highly augmented Epo-induced erythropoiesis. Fluorescent-activated cell sorting (FACS)-sorted CD34+CD71+CD45RA-GPA- erythroid progenitors stimulated with Epo in the presence or absence of LY294002 were subjected to gene expression profiling. Several novel target genes of Epo were identified, and the majority were regulated in a PI3K-dependent manner, including KIT (CD117) and CDH1 (E-cadherin). FACS analysis of Epo-stimulated erythroid progenitors showed that the increased mRNA expression of KIT and CDH1 was accompanied by an induction of the corresponding proteins CD117 and E-cadherin.