技术资料

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However,transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT),driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs,which can be administered to kill residual untransduced,diseased HSCs,whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells,transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin,leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia. View Publication

Developing novel therapies that suppress self-renewal of leukemia stem cells may reduce the likelihood of relapses and extend long-term survival of patients with acute myelogenous leukemia (AML). AML1-ETO (AE) is an oncogene that plays an important role in inducing self-renewal of hematopoietic stem/progenitor cells (HSPCs),leading to the development of leukemia stem cells. Previously,using a zebrafish model of AE and a whole-organism chemical suppressor screen,we have discovered that AE induces specific hematopoietic phenotypes in embryonic zebrafish through a cyclooxygenase (COX)-2 and β-catenin-dependent pathway. Here,we show that AE also induces expression of the Cox-2 gene and activates β-catenin in mouse bone marrow cells. Inhibition of COX suppresses β-catenin activation and serial replating of AE(+) mouse HSPCs. Genetic knockdown of β-catenin also abrogates the clonogenic growth of AE(+) mouse HSPCs and human leukemia cells. In addition,treatment with nimesulide,a COX-2 selective inhibitor,dramatically suppresses xenograft tumor formation and inhibits in vivo progression of human leukemia cells. In summary,our data indicate an important role of a COX/β-catenin-dependent signaling pathway in tumor initiation,growth,and self-renewal,and in providing the rationale for testing potential benefits from common COX inhibitors as a part of AML treatments. View Publication

The most frequent translocation t(8;21) in acute myeloid leukemia (AML) generates the chimeric AML1/ETO protein,which blocks differentiation and induces self-renewal in hematopoietic progenitor cells. The underlying mechanisms mediating AML1/ETO-induced self-renewal are largely unknown. Using expression microarray analysis,we identified the Groucho-related amino-terminal enhancer of split (AES) as a consistently up-regulated AML1/ETO target. Elevated levels of AES mRNA and protein were confirmed in AML1/ETO-expressing leukemia cells,as well as in other AML specimens. High expression of AES mRNA or protein was associated with improved survival of AML patients,even in the absence of t(8;21). On a functional level,knockdown of AES by RNAi in AML1/ETO-expressing cell lines inhibited colony formation. Similarly,self-renewal induced by AML1/ETO in primary murine progenitors was inhibited when AES was decreased or absent. High levels of AES expression enhanced formation of immature colonies,serial replating capacity of primary cells,and colony formation in colony-forming unit-spleen assays. These findings establish AES as a novel AML1/ETO-induced target gene that plays an important role in the self-renewal phenotype of t(8;21)-positive AML. View Publication

The Notch1-RBP-Jkappa and the transcription factor Runx1 pathways have been independently shown to be indispensable for the establishment of definitive hematopoiesis. Importantly,expression of Runx1 is down-regulated in the para-aortic splanchnopleural (P-Sp) region of Notch1- and Rbpsuh-null mice. Here we demonstrate that Notch1 up-regulates Runx1 expression and that the defective hematopoietic potential of Notch1-null P-Sp cells is successfully rescued in the OP9 culture system by retroviral transfer of Runx1. We also show that Hes1,a known effector of Notch signaling,potentiates Runx1-mediated transactivation. Together with the recent findings in zebrafish,Runx1 is postulated to be a cardinal down-stream mediator of Notch signaling in hematopoietic development throughout vertebrates. Our findings also suggest that Notch signaling may modulate both expression and transcriptional activity of Runx1. View Publication

Establishment of angiogenic circuits that orchestrate blood vessel development and remodeling requires an exquisite balance between the activities of pro- and antiangiogenic factors. However,the logic that permits complex signal integration by vascular endothelium is poorly understood. We demonstrate that a neuropeptide View Publication

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells,we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma,particularly by monocytes and stromal cells,but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration,HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines,through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether,these data identify ErbB receptors as putative therapeutic targets in multiple myeloma. View Publication

Mouse embryonic fibroblasts (MEFs) deficient for pocket proteins (i.e.,pRB/p107-,pRB/p130-,or pRB/p107/p130-deficient MEFs) have lost proper G(1) control and are refractory to Ras(V12)-induced senescence. However,pocket protein-deficient MEFs expressing Ras(V12) were unable to exhibit anchorage-independent growth or to form tumors in nude mice. We show that depending on the level of pocket proteins,loss of adhesion induces G(1) and G(2) arrest,which could be alleviated by overexpression of the TBX2 oncogene. TBX2-induced transformation occurred only in the absence of pocket proteins and could be attributed to downregulation of the p53/p21(CIP1) pathway. Our results show that a balance between the pocket protein and p53 pathways determines the level of transformation of MEFs by regulating cyclin-dependent kinase activities. Since transformation of human fibroblasts also requires ablation of both pathways,our results imply that the mechanisms underlying transformation of human and mouse cells are not as different as previously claimed. View Publication

The myeloproliferative disorder of mice lacking the Src homology 2 (SH2)-containing 5' phosphoinositol phosphatase,SHIP,underscores the need for closely regulating phosphatidylinositol 3-kinase (PI3K) pathway activity,and hence levels of phosphatidylinositol species during hematopoiesis. The role of the 3' phosphoinositol phosphatase Pten in this process is less clear,as its absence leads to embryonic lethality. Despite Pten heterozygosity being associated with a lymphoproliferative disorder,we found no evidence of a hematopoietic defect in Pten(+/-) mice. Since SHIP shares the same substrate (PIP(3)) with Pten,we hypothesized that the former might compensate for Pten haploinsufficiency in the marrow. Thus,we examined the effect of Pten heterozygosity in SHIP(-/-) mice,predicting that further dysregulation of PIP(3) metabolism would exacerbate the pheno-type of the latter. Indeed,compared with SHIP(-/-) mice,Pten(+/-)SHIP(-/-) animals developed a myelodysplastic phenotype characterized by increased hepatosplenomegaly,extramedullary hematopoiesis,anemia,and thrombocytopenia. Consistent with a marrow defect,clonogenic assays demonstrated reductions in committed myeloid and megakaryocytic progenitors in these animals. Providing further evidence of a Pten(+/-)SHIP(-/-) progenitor abnormality,reconstitution of irradiated mice with marrows from these mice led to a marked defect in short-term repopulation of peripheral blood by donor cells. These studies suggest that the regulation of the levels and/or ratios of PI3K-derived phosphoinositol species by these 2 phosphatases is critical to normal hematopoiesis. View Publication

Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy,but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular,the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that,together with other factors,can expand mouse bone marrow HSCs in culture. Here,we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF,TPO,and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers,as assayed by NOD/SCID transplantation. A serum-free culture containing SCF,TPO,FGF-1,angiopoietin-like 5,and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs,a number potentially applicable to several clinical processes including HSC transplantation. View Publication

The physiologic roles of angiopoietin-like proteins (Angptls) in the hematopoietic system remain unknown. Here we show that hematopoietic stem cells (HSCs) in Angptl3-null mice are decreased in number and quiescence. HSCs transplanted into Angptl3-null recipient mice exhibited impaired repopulation. Bone marrow sinusoidal endothelial cells express high levels of Angptl3 and are adjacent to HSCs. Importantly,bone marrow stromal cells or endothelium deficient in Angptl3 have a significantly decreased ability to support the expansion of repopulating HSCs. Angptl3 represses the expression of the transcription factor Ikaros,whose unregulated overexpression diminishes the repopulation activity of HSCs. Angptl3,as an extrinsic factor,thus supports the stemness of HSCs in the bone marrow niche. View Publication