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Adaptive transcriptional responses to oxygen deprivation (hypoxia) are mediated by the hypoxia-inducible factors (HIFs),heterodimeric transcription factors composed of two basic helix-loop-helix-PAS family proteins. The transcriptional activity of HIF is determined by the hypoxic stabilization of the HIF-alpha proteins. HIF-1alpha and HIF-2alpha exhibit high sequence homology but have different mRNA expression patterns; HIF-1alpha is expressed ubiquitously whereas HIF-2alpha expression is more restricted to certain tissues,e.g.,the endothelium,lung,brain,and neural crest derivatives. Germ-line deletion of either HIF subunit is embryonic lethal with unique features suggesting important roles for both HIF-alpha isoforms. Global deletion of Hif-2alpha results in distinct phenotypes depending on the mouse strain used for the mutation,clearly demonstrating an important role for HIF-2alpha in mouse development. The function of HIF-2alpha in adult life,however,remains incompletely understood. In this study,we describe the generation of a conditional murine Hif-2alpha allele and the effect of its acute postnatal ablation. Under very stringent conditions,we ablate Hif-2alpha after birth and compare the effect of acute global deletion of Hif-2alpha and Hif-1alpha. Our results demonstrate that HIF-2alpha plays a critical role in adult erythropoiesis,with acute deletion leading to anemia. Furthermore,although HIF-1alpha was first purified and cloned based on its affinity for the human erythropoietin (EPO) 3' enhancer hypoxia response element (HRE) and regulates Epo expression during mouse embryogenesis,HIF-2alpha is the critical alpha isoform regulating Epo under physiologic and stress conditions in adults. View Publication

The erythroid response to acute anemia relies on the rapid expansion in the spleen of a specialized population of erythroid progenitors termed stress BFU-E. This expansion requires BMP4/Madh5-dependent signaling in vivo; however,in vitro,BMP4 alone cannot recapitulate the expansion of stress BFU-E observed in vivo,which suggests that other signals are required. In this report we show that mutation of the Kit receptor results in a severe defect in the expansion of stress BFU-E,indicating a role for the Kit/SCF signaling pathway in stress erythropoiesis. In vitro analysis showed that BMP4 and SCF are necessary for the expansion of stress BFU-E,but only when spleen cells were cultured in BMP4 + SCF at low-oxygen concentrations did we recapitulate the expansion of stress BFU-E observed in vivo. Culturing spleen cells in BMP4,SCF under hypoxic conditions resulted in the preferential expansion of erythroid progenitors characterized by the expression of Kit,CD71,and TER119. This expression pattern is also seen in stress erythroid progenitors isolated from patients with sickle cell anemia and patients with beta-thalassemia. Taken together these data demonstrate that SCF and hypoxia synergize with BMP4 to promote the expansion and differentiation of stress BFU-E during the recovery from acute anemia. View Publication

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However,clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody,we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here,we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells,lymphocytes,or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors,suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely,2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs,melphalan,or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore,administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma. View Publication

Hematopoietic stem cells are resistant to HIV-1 infection. Here,we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21(Waf1/Cip1/Sdi1) (p21),a known regulator of stem cell pool size,restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs,p27(Kip1) and p18(INK4C),had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase,SIVmac-251,and the other cell-intrinsic inhibitors of HIV-1,Trim5alpha,PML,Murr1,and IFN-alpha,were unaffected by p21. Therefore,p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected sanctuary" in HIV disease." View Publication

OBJECTIVE: Our laboratory has established two unique methods to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of both protocols is the separation of whole bone marrow into small-sized cells by counter-flow elutriation. We sought to provide the scientific community with an alternate approach to acquire our stem cells by replacing elutriation with the use of density-gradient centrifugation. METHODS: The elutriated fraction 25 population was characterized based on density using a discontinuous gradient. The long-term reconstituting potential of whole bone marrow cells collected at each density interface was determined by subjecting the fractions to the two-day homing protocol,transplanting them into lethally irradiated recipient mice,and assessing peripheral blood chimerism. We also investigated the ability of high-density bone marrow cells isolated in conjunction with the ALDH protocol to repopulate the hematopoietic system of myeloablated recipients. RESULTS: Bone marrow cells collected at the high-density interface of 1.081/1.087 g/mL (fraction 3) had the capacity for homing to marrow and the ability to provide long-term hematopoietic reconstitution. Fraction three lineage-depleted ALDH-bright cells could also engraft and provide long-term hematopoiesis at limiting dilutions. CONCLUSIONS: Density-gradient centrifugation can be used in conjunction with either of our stem cell isolation protocols to obtain cells with long-term reconstitution ability. We anticipate that this strategy will encourage and enable investigators to study the biology of HSCs isolated using functional characteristics. View Publication

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells,addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC),we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population,which,despite its clonality,comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC,capable of repopulating secondary liquid cultures of CRA assays,confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle,in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However,quiescent LSC were also detected,capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC,believed to sustain minimal residual disease in vivo,were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines,which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Antineutrophil cytoplasmic antibodies (ANCAs) with specificity for proteinase 3 (PR3) are central to a form of ANCA-associated vasculitis. Membrane PR3 (mPR3) is expressed only on a subset of neutrophils. The aim of this study was to determine the mechanism of PR3 surface expression on human neutrophils. Neutrophils were isolated from patients and healthy controls,and hematopoietic stem cells from cord blood served as a model of neutrophil differentiation. Surface expression was analyzed by flow cytometry and confocal microscopy,and proteins were analyzed by Western blot experiments. Neutrophil subsets were separated by magnetic cell sorting. Transfection experiments were carried out in HEK293 and HL60 cell lines. Using neutrophils from healthy donors,patients with vasculitis,and neutrophilic differentiated stem cells we found that mPR3 display was restricted to cells expressing neutrophil glycoprotein NB1,a glycosylphosphatidylinositol (GPI)-linked surface receptor. mPR3 expression was decreased by enzymatic removal of GPI anchors from cell membranes and was absent in a patient with paroxysmal nocturnal hemoglobinuria. PR3 and NB1 coimmunoprecipitated from and colocalized on the neutrophil plasma membrane. Transfection with NB1 resulted in specific PR3 surface binding in different cell types. We conclude that PR3 membrane expression on neutrophils is mediated by the NB1 receptor. View Publication

Mutations in the RUNX1 gene are found at high frequencies in minimally differentiated acute myelogenous leukemia. In addition to null mutations,many of the mutations generate Runx1 DNA-binding (RDB) mutants. To determine if these mutants antagonize wild-type protein activity,cDNAs were transduced into murine bone marrow or human cord blood cells using retroviral vectors. Significantly,the RDB mutants did not act in a transdominant fashion in vivo to disrupt Runx1 activity in either T-cell or platelet development,which are highly sensitive to Runx1 dosage. However,RDB mutant expression impaired expansion and differentiation of the erythroid compartment in which Runx1 expression is normally down-regulated,showing that a RDB-independent function is incompatible with erythroid differentiation. Significantly,both bone marrow progenitors expressing RDB mutants or deficient for Runx1 showed increased replating efficiencies in vitro,accompanied by the accumulation of myeloblasts and dysplastic progenitors,but the effect was more pronounced in RDB cultures. Disruption of the interface that binds CBFbeta,an important cofactor of Runx1,did not impair RDB mutant replating activity,arguing against inactivation of Runx1 function by CBFbeta sequestration. We propose that RDB mutants antagonize Runx1 function in early progenitors by disrupting a critical balance between DNA-binding-independent and DNA-binding-dependent signaling. View Publication

The T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of the Jak/Stat cytokine signaling pathway. Our study shows that the absence of TC-PTP leads to an early bone marrow B-cell deficiency characterized by hindered transition from the pre-B cell to immature B-cell stage. This phenotype is intrinsic to the B cells but most importantly due to bone marrow stroma abnormalities. We found that bone marrow stromal cells from TC-PTP(-/-) mice have the unique property of secreting 232-890 pg/mL IFN-gamma. These high levels of IFN-gamma result in 2-fold reduction in mitotic index on IL-7 stimulation of TC-PTP(-/-) pre-B cells and lower responsiveness of IL-7 receptor downstream Jak/Stat signaling molecules. Moreover,we noted constitutive phosphorylation of Stat1 in those pre-B cells and demonstrated that this was due to soluble IFN-gamma secreted by TC-PTP(-/-) bone marrow stromal cells. Interestingly,culturing murine early pre-B leukemic cells within a TC-PTP-deficient bone marrow stroma environment leads to a 40% increase in apoptosis in these malignant cells. Our results unraveled a new role for TC-PTP in normal B lymphopoiesis and suggest that modulation of bone marrow microenvironment is a potential therapeutic approach for selected B-cell leukemia. View Publication

The transcription factor PAX5 is a critical regulator of B-cell commitment and development. Although normally not expressed in myeloid progenitors,PAX5 has recently been shown to be frequently expressed in myeloid malignancies and to suppress expression of myeloid differentiation genes,compatible with an effect on the differentiation or maintenance of myeloid progenitors. However,previous studies in which PAX5 was ectopically expressed in normal myeloid progenitors in vivo and in vitro provided conflicting results as to the effect of PAX5 on myeloid development. Herein,we demonstrate that on ectopic expression of PAX5 in bone marrow multipotent stem/progenitor cells,cells with a biphenotypic B220(+)GR-1/MAC-1(+) phenotype are produced. These remain cytokine-dependent,but unlike control-transduced cells they sustain long-term generation of myeloid progenitors in vitro and remain capable of myeloid differentiation. Notably,PAX5(+)B220(+)GR-1/MAC-1(+) myeloid progenitors coexpress,at the single-cell level,myeloid genes and otherwise B-cell-specific PAX5 target genes. These findings establish that ectopic expression of PAX5 introduces extensive self-renewal properties in otherwise short-lived myeloid progenitors. Along with the established ectopic expression of PAX5 in acute myeloid leukemia,this motivates a careful investigation of the potential involvement of ectopic PAX5 expression in myeloid and biphenotypic leukemias. View Publication

Expression of the constitutively activated TEL/PDGFbetaR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia. TEL/PDGFbetaR activates multiple signal transduction pathways in cell-culture systems,and expression of the TEL-PDGFRB fusion gene induces myeloproliferative disease (MPD) in mice. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB-mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays. Fetal liver hematopoietic stem and progenitor cells harboring targeted deletion of both Stat5a and Stat5b (Stat5ab(null/null)) genes were refractory to transformation by TEL-PDGFRB in methylcellulose colony assays. Notably,these cell populations were maintained in Stat5ab(null/null) fetal livers and succumbed to transformation by c-Myc. Surprisingly,targeted disruption of either Stat5a or Stat5b alone also impaired TEL-PDGFRB-mediated transformation. Survival of TPiGFP--textgreaterStat5a(-/-) and TPiGFP--textgreaterStat5a(+/-) mice was significantly prolonged,demonstrating significant sensitivity of TEL-PDGFRB-induced MPD to the dosage of Stat5a. TEL-PDGFRB-mediated MPD was incompletely penetrant in TPiGFP--textgreaterStat5b(-/-) mice. In contrast,Src family kinases Lyn,Hck,and Fgr and the Stat family member Stat1 were dispensable for TEL-PDGFRB disease. Together,these data demonstrate that Stat5a and Stat5b are dose-limiting mediators of TEL-PDGFRB-induced myeloproliferation. View Publication

T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study,we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed,soluble Notch ligands that allows the titration of T cell lineage commitment,we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast,their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically,this is not due to differences in receptor expression,because early T lineage precursors,bone marrow lineage marker-negative,Sca-1-positive,c-Kit-positive and common lymphoid progenitor cells,express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment. View Publication