技术资料

Chromosomal rearrangements of the 11p15 locus have been identified in hematopoietic malignancies,resulting in translocations involving the N-terminal portion of the nucleoporin gene NUP98. Fifteen different fusion partner genes have been identified for NUP98,and more than one half of these are homeobox transcription factors. By contrast,the NUP98 fusion partner in t(11;20) is Topoisomerase I (TOP1),a catalytic enzyme recognized for its key role in relaxing supercoiled DNA. We now show that retrovirally engineered expression of NUP98-TOP1 in murine bone marrow confers a potent in vitro growth advantage and a block in differentiation in hematopoietic precursors,evidenced by a competitive growth advantage in liquid culture,increased replating efficient of colony-forming cells (CFCs),and a marked increase in spleen colony-forming cell output. Moreover,in a murine bone marrow transplantation model,NUP98-TOP1 expression led to a lethal,transplantable leukemia characterized by extremely high white cell counts,splenomegaly,and mild anemia. Strikingly,a mutation to a TOP1 site to inactivate the isomerase activity essentially left unaltered the growth-promoting and leukemogenic effects of NUP98-TOP1. These findings,together with similar biologic effects reported for NUP98-HOX fusions,suggest unexpected,overlapping functions of NUP98 fusion genes,perhaps related to common DNA binding properties. View Publication

Generating lentiviral vectors pseudotyped with different viral glycoproteins (GPs) may modulate the physicochemical properties of the vectors,their interaction with the host immune system,and their host range. We have investigated the capacity of a panel of GPs of both retroviral (amphotropic murine leukemia virus [MLV-A]; gibbon ape leukemia virus [GALV]; RD114,feline endogenous virus) and nonretroviral (fowl plague virus [FPV]; Ebola virus [EboV]; vesicular stomatitis virus [VSV]; lymphocytic choriomeningitis virus [LCMV]) origins to pseudotype lentiviral vectors derived from simian immunodeficiency virus (SIVmac251). SIV vectors were efficiently pseudotyped with the FPV hemagglutinin,VSV-G,LCMV,and MLV-A GPs. In contrast,the GALV and RD114 GPs conferred much lower infectivity to the vectors. Capitalizing on the conservation of some structural features in the transmembrane domains and cytoplasmic tails of the incorporation-competent MLV-A GP and in RD114 and GALV GPs,we generated chimeric GPs encoding the extracellular and transmembrane domains of GALV or RD114 GPs fused to the cytoplasmic tail (designated TR) of MLV-A GP. Importantly,SIV-derived vectors pseudotyped with these GALV/TR and RD114/TR GP chimeras had significantly higher titers than vectors coated with the parental GPs. Additionally,RD114/TR-pseudotyped vectors were efficiently concentrated and were resistant to inactivation induced by the complement of both human and macaque sera,indicating that modified RD114 GP-pseudotyped lentiviral vectors may be of particular interest for in vivo gene transfer applications. Furthermore,as compared to vectors pseudotyped with other retroviral GPs or with VSV-G,RD114/TR-pseudotyped vectors showed augmented transduction of human and macaque primary blood lymphocytes and CD34+ cells. View Publication

The steps to leukemia following an in utero fusion of MLL (HRX,ALL-1) to a partner gene in humans are not known. Introduction of the Mll-AF9 fusion gene into embryonic stem cells results in leukemia in mice with cell-type specificity similar to humans. In this study we used myeloid colony assays,immunophenotyping,and transplantation to evaluate myelopoiesis in Mll-AF9 mice. Colony assays demonstrated that both prenatal and postnatal Mll-AF9 tissues have significantly increased numbers of CD11b(+)/CD117(+)/Gr-1(+/-) myeloid cells,often in compact clusters. The self-renewal capacity of prenatal myeloid progenitors was found to decrease following serial replating of colony-forming cells. In contrast,early postnatal myeloid progenitors increased following replating; however,the enhanced self-renewal of early postnatal myeloid progenitor cells was limited and did not result in long-term cell lines or leukemia in vivo. Unlimited replating,long-term CD11b/Gr-1(+) myeloid cell lines,and the ability to produce early leukemia in vivo in transplantation experiments,were found only in mice with overt leukemia. Prenatal Mll-AF9 tissues had reduced total (mature and progenitor) CD11b/Gr-1(+) cells compared with wild-type tissues. Colony replating,immunophenotyping,and cytochemistry suggest that any perturbation of cellular differentiation from the prenatal stage onward is partial and largely reversible. We describe a novel informative in vitro and in vivo model system that permits study of the stages in the pathogenesis of Mll fusion gene leukemia,beginning in prenatal myeloid cells,progressing to a second stage in the postnatal period and,finally,resulting in overt leukemia in adult animals. View Publication

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors,namely insulin receptor substrate-2 (IRS2),Src homology 2 domain-containing inositol 5'-phosphatase (SHIP),Grb2-associated binder-1 (Gab1),and the Epo receptor (EpoR). Using different in vitro systems,we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors,Akt,FKHRL1,and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR),through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR,or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors,but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors,the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally,our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2),which,in turn,down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation. View Publication

Although it has been shown that unfractionated bone marrow,hematopoietic stem cells,common myeloid progenitors,and bipotent megakaryocyteerythrocyte progenitors can give rise to megakaryocyte colonies in culture,monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here,we use a monoclonal antibody to the megakaryocyte-associated surface protein,CD9,to purify MKPs from the c-kit(+)Sca-1(-)IL7Ralpha(-)Thy1.1(-)Lin(-) fraction of adult C57BLKa-Thy1.1 bone marrow. The CD9(+) fraction contained a subset of CD41(+)FcgammaR(lo)CD34(+)CD38(+) cells that represent approximately 0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit-megakaryocytes and occasionally burst-forming unit-megakaryocytes,with a plating efficiency textgreater60% at the single-cell level. In vivo,MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for approximately 3 weeks. Common myeloid progenitors and megakaryocyteerythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures,indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns. View Publication

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation,which is only partially successful in the absence of an HLA genoidentical donor,thus justifying research to find an alternative therapeutic approach. To this end,RAG-2-deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2-/-Sca-1(+) selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)-based MND (myeloproliferative sarcoma virus enhancer,negative control region deleted,dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2-/- sublethally irradiated mice (3Gy). Two months later,T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens,allogeneic cells,and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy,a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether,this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2-/- mice,constituting a significant step toward clinical application. View Publication

The role of thrombopoietin (Tpo) in promoting hematopoiesis has been extensively studied in late fetal,neonatal,and adult mice. However,the effects of Tpo on early yolk sac hematopoiesis have been largely unexplored. We examined whole embryos or the cells isolated from embryo proper and yolk sacs and identified both Tpo and c-mpl (Tpo receptor) mRNA transcripts in tissues as early as embryonic day 6.5 (E6.5). Presomite whole embryos and somite-staged yolk sac and embryo proper cells were plated in methylcellulose cultures and treated with selected hematopoietic growth factors in the presence or absence of Tpo. Tpo alone failed to promote colony-forming unit (CFU) formation. However,in the presence of other growth factors,Tpo caused a substantial dose-dependent reduction in primitive and definitive erythroid CFU growth in cultures containing E7.5 and E8.0 whole embryos and E8.25 to 9.5 yolk sac-derived cells. Meanwhile,Tpo treatment resulted in a substantial dose-dependent increase in CFU-mixed lineage (CFU-Mix) and CFU-megakaryocyte (CFU-Meg) formation in cultures containing cells from similar staged tissues. Addition of Tpo to cultures of sorted E9.5 yolk sac c-Kit(+)CD34(+) hematopoietic progenitors also inhibited erythroid CFU growth but augmented CFU-Mix and CFU-Meg activity. Effects of Tpo on CFU growth were blocked in the presence of a monoclonal antibody with Tpo-neutralizing activity but not with control antibody. Thus,under certain growth factor conditions,Tpo directly inhibits early yolk sac erythroid CFU growth but facilitates megakaryocyte and mixed lineage colony formation. View Publication

The chimeric transcription factor E2a-Hlf is an oncoprotein associated with a subset of acute lymphoblastic leukemias of early B-lineage derivation. We employed a retroviral transduction-transplantation approach to evaluate the oncogenic effects of E2a-Hlf on murine B-cell progenitors harvested from adult bone marrow. Expression of E2a-Hlf induced short-lived clusters of primary hematopoietic cells but no long-term growth on preformed bone marrow stromal cell layers comprised of the AC6.21 cell line. Coexpression with Bcl-2,however,resulted in the sustained self-renewal of early preB-I cells that required stromal and interleukin-7 (IL-7) support for growth in vitro. Immortalized cells were unable to induce leukemias after transplantation into nonirradiated syngeneic hosts,unlike the leukemic properties and cytokine independence of preB-I cells transformed by p190(Bcr-Abl) under identical in vitro conditions. However,bone marrow cells expressing E2a-Hlf in combination with Bcl-2,but not E2a-Hlf alone,induced leukemias in irradiated recipients with long latencies,demonstrating both a requirement for suppression of apoptosis and the need for further secondary mutations in leukemia pathogenesis. Coexpression of IL-7 substituted for Bcl-2 to induce the in vitro growth of pre-B cells expressing E2a-Hlf,but leukemic conversion required additional abrogation of undefined stromal requirements and was associated with alterations in the Arf/Mdm2/p53 pathway. Thus,E2a-Hlf enhances the self-renewal of bone marrow B-cell progenitors without inciting a p53 tumor surveillance response or abrogating stromal and cytokine requirements for growth,which are nevertheless abrogated during progression to a leukemogenic phenotype. View Publication

Transfer of therapeutic genes to human hematopoietic stem cells (HSCs) using complex vectors at clinically relevant efficiencies remains a major challenge. Recently we described a stable retroviral vector that sustains long-term expression of green fluorescent protein (GFP) and a human beta-globin gene in the erythroid progeny of transduced murine HSCs. We now report the efficient transduction of primitive human CD34(+) fetal liver or cord blood cells with this vector and expression of the beta-globin transgene in the erythroid progeny of these human cells for at least 2 months. After growth factor prestimulation and then a 2- to 3-day exposure to the virus,35% to 55% GFP(+) progeny were seen in assays of transduced colony-forming cells,primitive erythroid precursors that generate large numbers of glycophorin A(+) cells in 3-week suspension cultures,and 6-week long-term culture-initiating cells. In immunodeficient mice injected with unselected infected cells,5% to 15% of the human cells regenerated in the marrow (including the erythroid cells) were GFP(+) 3 and 6 weeks after transplantation. Importantly,the numbers of GFP(+) human lymphoid and either granulopoietic or erythroid cells in individual mice 6 weeks after transplantation were significantly correlated,indicative of the initial transduction of human multipotent cells with in vivo repopulating activity. Expression of the transduced beta-globin gene in human cells obtained directly from the mice or after their differentiation into erythroid cells in vitro was demonstrated by reverse transcriptase-polymerase chain reaction using specific primers. These experiments represent a significant step toward the realization of a gene therapy approach for human beta-globin gene disorders. View Publication

Patients with heart failure are predisposed to infections and anemia,possibly due to reduced hematopoiesis. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is increased in heart failure,and it inhibits normal hematopoiesis,partly due to apoptosis through the effector molecule Fas. We examined bone marrow progenitor cells of mice with heart failure induced by acute myocardial infarction. The fraction of progenitor cells in mice with heart failure was only approximately 40% of control. Measured with in vitro clonal assays,the proliferative capacity of the progenitor cells in mice with heart failure was reduced to approximately 50% of control. Flow cytometry with specific markers revealed a threefold increase in apoptosis among progenitor cells from mice with heart failure. In these mice,TNF-alpha/Fas expression was increased in bone marrow natural killer (NK) and T cells,and these lymphocytes showed increased cytolytic activity in vitro against progenitor cells. We conclude that the TNF-alpha/Fas pathway in lymphocytes is activated in the bone marrow during heart failure,which may play a pathogenic role in the observed decrease in hematopoiesis. View Publication

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular,primitive leukemia cells,often termed leukemia stem cells,are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins,presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation,CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise,we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly,these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism,which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1),as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism,an intrinsic property of primary human AML cells. View Publication

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4,which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast,at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia. View Publication