技术资料

The aryl hydrocarbon receptor (AhR) mediates the carcinogenicity of a family of environmental contaminants,the most potent being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Increased incidence of lymphoma and leukemia in humans is associated with TCDD exposure. Although AhR activation by TCDD has profound effects on the immune system,precise cellular and molecular mechanisms have yet to be determined. These studies tested the hypothesis that alteration of marrow populations following treatment of mice with TCDD is due to an effect on hematopoietic stem cells (HSCs). Treatment with TCDD resulted in an increased number and proliferation of bone marrow (BM) populations enriched for HSCs. There was a time-dependent decrease in B-lineage cells with a concomitant increase in myeloid populations. The decrease in the B-cell lineage colony-forming unit-preB progenitors along with a transient increase in myeloid progenitors were consistent with a skewing of lineage development from lymphoid to myeloid populations. However,HSCs from TCDD-treated mice exhibited diminished capacity to reconstitute and home to marrow of irradiated recipients. AhR messenger RNA was expressed in progenitor subsets but is downregulated during HSC proliferation. This result was consistent with the lack of response following the exposure of 5-fluorouracil-treated mice to TCDD. The direct exposure of cultured BM cells to TCDD inhibited the growth of immature hematopoietic progenitor cells,but not more mature lineage-restricted progenitors. Overall,these data are consistent with the hypothesis that TCDD,through AhR activation,alters the ability of HSCs to respond appropriately to signals within the marrow microenvironment. View Publication

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment,proliferation,and differentiation. Given its critical functions,c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay,we found that miR-15a directly binds the 3'-UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic,functionality of binding was shown. The mimic decreased c-Myb expression,and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3'-UTR partially rescued the miR-15a induced cell-cycle block. Of interest,the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally,in studies using normal human CD34(+) cells,we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation,and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate,these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis. View Publication

Fanconi anemia (FA) is an inherited recessive DNA repair disorder mainly characterized by bone marrow failure and cancer predisposition. Studies in mosaic FA patients have shown that reversion of one inherited germ-line mutation resulting in a functional allele in one or a few hematopoietic stem cells (HSCs) can lead to the proliferation advantage of corrected cells,thus over time normalizing the hematologic status of the patient. In contrast to these observations,it is still unclear whether ex vivo genetic correction of FA HSCs also provides a similar proliferation advantage to FA HSCs. Using an FA mouse model with a marked hematopoietic phenotype,the FA-D1 (Brca2(Delta27/Delta27)) mice,we demonstrate that the lentivirus-mediated gene therapy of FA HSCs results in the progressive expansion of genetically corrected clones in mild-conditioned FA-D1 recipients. Consistent with these data,hematopoietic progenitors from FA recipients progressively became mitomycin C resistant and their chromosomal instability was reverted. No evidence of myelodysplasia,leukemias,or abnormal clonal repopulation was observed at multiple time points in primary or secondary recipients. Our results demonstrate that ectopic expression of BRCA2 confers a beneficial in vivo proliferation advantage to FA-D1 HSCs that enables the full hematopoietic repopulation of FA recipients with genetically corrected cells. View Publication

Hematopoiesis during development is a dynamic process,with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis,the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1),best known for its proinflammatory properties,has radioprotective effects on adult bone marrow HSCs,induces HSC mobilization,and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1,IL-1 receptors (IL-1Rs),and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM,and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic,endothelial,and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation,concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development,acting to limit the differentiation of some HSCs along the myeloid lineage. View Publication

Endoglin is an accessory receptor for TGF-beta signaling and is required for normal hemangioblast,early hematopoietic,and vascular development. We have previously shown that an upstream enhancer,Eng -8,together with the promoter region,mediates robust endothelial expression yet is inactive in blood. To identify hematopoietic regulatory elements,we used array-based methods to determine chromatin accessibility across the entire locus. Subsequent transgenic analysis of candidate elements showed that an endothelial enhancer at Eng +9 when combined with an element at Eng +7 functions as a strong hemato-endothelial enhancer. Chromatin immunoprecipitation (ChIP)-chip analysis demonstrated specific binding of Ets factors to the promoter as well as to the -8,+7+9 enhancers in both blood and endothelial cells. By contrast Pu.1,an Ets factor specific to the blood lineage,and Gata2 binding was only detected in blood. Gata2 was bound only at +7 and GATA motifs were required for hematopoietic activity. This modular assembly of regulators gives blood and endothelial cells the regulatory freedom to independently fine-tune gene expression and emphasizes the role of regulatory divergence in driving functional divergence. View Publication

The growth factors that drive the division and differentiation of stem cells during early human embryogenesis are unknown. The secretion of endorphins,progesterone (P(4)),human chorionic gonadotropin,17beta-estradiol,and gonadotropin-releasing hormone by trophoblasts that lie adjacent to the embryoblast in the blastocyst suggests that these pregnancy-associated factors may directly signal the growth and development of the embryoblast. To test this hypothesis,we treated embryoblast-derived human embryonic stem cells (hESCs) with ICI 174,864,a delta-opioid receptor antagonist,and RU-486 (mifepristone),a P(4) receptor competitive antagonist. Both antagonists potently inhibited the differentiation of hESC into embryoid bodies,an in vitro structure akin to the blastocyst containing all three germ layers. Furthermore,these agents prevented the differentiation of hESC aggregates into columnar neuroectodermal cells and their organization into neural tube-like rosettes as determined morphologically. Immunoblot analyses confirmed the obligatory role of these hormones; both antagonists inhibited nestin expression,an early marker of neural precursor cells normally detected during rosette formation. Conversely,addition of P(4) to hESC aggregates induced nestin expression and the formation of neuroectodermal rosettes. These results demonstrate that trophoblast-associated hormones induce blastulation and neurulation during early human embryogenesis. View Publication

The role of epitope-specific TCR repertoire diversity in the control of HIV-1 viremia is unknown. Further analysis at the clonotype level is important for understanding the structural aspects of the HIV-1 specific repertoire that directly relate to CTL function and ability to suppress viral replication. In this study,we performed in-depth analysis of T cell clonotypes directed against a dominantly recognized HLA B57-restricted epitope (KAFSPEVIPMF; KF11) and identified common usage of the TCR beta-chain TRBV7 in eight of nine HLA B57 subjects examined,regardless of HLA B57 subtype. Despite this convergent TCR gene usage,structural and functional assays demonstrated no substantial difference in functional or structural avidity between TRBV7 and non-TRBV7 clonotypes and this epitopic peptide. In a subject where TRBV7-usage did not confer cross-reactivity against the dominant autologous sequence variant,another circulating TCR clonotype was able to preferentially recognize the variant peptide. These data demonstrate that despite selective recruitment of TCR for a conserved epitope over the course of chronic HIV-1 infection,TCR repertoire diversity may benefit the host through the ability to recognize circulating epitope variants. View Publication

Human embryonic stem (ES) cells can be maintained in an undifferentiated state if the culture medium is first conditioned on a layer of mouse embryonic fibroblast (MEF) feeder cells. Here we show that human ES cell proliferation is coordinated by MEF-secreted heparan sulfate proteoglycans (HSPG) in conditioned medium (CM). These HSPG and other heparinoids can stabilize basic fibroblast growth factor (FGF2) in unconditioned medium at levels comparable to those observed in CM. They also directly mediate binding of FGF2 to the human ES cell surface,and their removal from CM impairs proliferation. Finally,we have developed a purification scheme for MEF-secreted HSPG in CM. Using column chromatography,immunoblotting,and mass spectrometry-based proteomic analysis,we have identified multiple HSPG species in CM. The results demonstrate that HSPG are key signaling cofactors in CM-based human ES cell culture. View Publication

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors,where it initiates critical cell survival and proliferation signals after stimulation by its ligand,thrombopoietin (TPO). As a result,a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore,they show very low-level expression of Mpl on platelets and megakaryocytes,and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However,impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels,which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis. View Publication

Epithelial-mesenchymal transition (EMT) plays important roles in various physiological and pathological processes,and is regulated by signaling pathways mediated by cytokines,including transforming growth factor beta (TGFbeta). Embryonic endothelial cells also undergo differentiation into mesenchymal cells during heart valve formation and aortic maturation. However,the molecular mechanisms that regulate such endothelial-mesenchymal transition (EndMT) remain to be elucidated. Here we show that TGFbeta plays important roles during mural differentiation of mouse embryonic stem cell-derived endothelial cells (MESECs). TGFbeta2 induced the differentiation of MESECs into mural cells,with a decrease in the expression of the endothelial marker claudin 5,and an increase in expression of the mural markers smooth muscle alpha-actin,SM22alpha and calponin,whereas a TGFbeta type I receptor kinase inhibitor inhibited EndMT. Among the transcription factors involved in EMT,Snail was induced by TGFbeta2 in MESECs. Tetracycline-regulated expression of Snail induced the differentiation of MESECs into mural cells,whereas knockdown of Snail expression abrogated TGFbeta2-induced mural differentiation of MESECs. These results indicate that Snail mediates the actions of endogenous TGFbeta signals that induce EndMT. View Publication

With their unique ability to differentiate into all cell types,embryonic stem (ES) cells hold great therapeutic promise. To improve the efficiency of embryoid body (EB)-mediated ES cell differentiation,we studied murine EBs on the basis of their size and found that EBs with an intermediate size (diameter 100-300 microm) are the most proliferative,hold the greatest differentiation potential,and have the lowest rate of cell death. In an attempt to promote the formation of this subpopulation,we surveyed several biocompatible substrates with different surface chemical parameters and identified a strong correlation between hydrophobicity and EB development. Using self-assembled monolayers of various lengths of alkanethiolates on gold substrates,we directly tested this correlation and found that surfaces that exhibit increasing hydrophobicity enrich for the intermediate-size EBs. When this approach was applied to the human ES cell system,similar phenomena were observed. Our data demonstrate that hydrophobic surfaces serve as a platform to deliver uniform EB populations and may significantly improve the efficiency of ES cell differentiation. View Publication