技术资料

The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines,suggesting that WT1 participates in regulating the proliferation of leukemic cells. However,the role of WT1 in normal hematopoiesis is not well understood. To investigate this question,we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1,although contributing efficiently to other organ systems,make only a minimal contribution to the hematopoietic system in chimeras,indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition,fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E),erythroid colony-forming unit (CFU-E),and colony-forming unit-granulocyte macrophage-erythroid-megakaryocyte (CFU-GEMM). However,transplantation of fetal liver hematopoietic cells lacking WT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1. View Publication

The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures,respectively. In erythrocytes,the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However,the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme,designated IGnT3. The genes for IGnT1 (reported in 1993),IGnT2 (also presented in this study),and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells,IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover,reticulocytes expressed the IGnT3 transcript,but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree,individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006GtextgreaterA; Gly336Arg) and exon 3 (1049GtextgreaterA; Gly350Glu),respectively,of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3,not by IGnT1 or IGnT2. View Publication

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling,quiescent progenitors are present in most samples. In the current study,(3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0),G(1),and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results,with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly,after 72 hours in serum-free culture with or without Steel factor (SF),Flt-3 ligand (FL),and interleukin-3 (IL-3),most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours,1.2% to 37%,and 0% to 7.6% in cultures without and with growth factors [GFs],respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF,FL,IL-3,and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition,3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary,quiescent leukemic cells,including NOD/SL-ICs,are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs. View Publication

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM,respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck),A-431 (vulva),BT474 (breast),CaLu-3 (lung),and N87 (gastric). Normal human foreskin fibroblasts,nontumorigenic epithelial cells (HB4a),and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure,average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were textless 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator,AKT,was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest,the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016,and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines,concentrations of GW2016 were reached where outgrowth did not occur. Furthermore,growth arrest and cell death were observed in parallel experiments,as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally,twice daily,with complete inhibition of tumor growth at the higher dose. Together,these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2. View Publication

Purmorphamine,which is a 2,6,9-trisubstituted purine compound,was discovered through cell-based high-throughput screening from a heterocycle combinatorial library. It differentiates multipotent mesenchymal progenitor cells into an osteoblast lineage. It will serve as a unique chemical tool to study the molecular mechanisms of osteogenesis of stem cells and bone development. View Publication

Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription,nuclear import,and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell-based gene therapy trials performed to date,cells have been activated via their cognate antigen receptor. To couple activation with gene transfer,we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However,transduction of highly purified resting T cells with these anti-CD3-displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here,we describe interleukin-7 (IL-7)-displaying HIV-1-derived vectors. Like recombinant IL-7,these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore,a single exposure to the IL-7-displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells,preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether,these results suggest that IL-7-displaying vectors could constitute interesting tools for T-cell-targeted gene therapy. View Publication

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines,but its role in proliferation,differentiation,and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells,we generated green fluorescent protein (GFP)-tagged versions of wild type CIS,a mutant harboring an inactivated SH2 domain (CIS R107K),and a mutant with a deletion of the SOCS Box (CISDeltaBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence,both are essential for the growth inhibitory effect of CIS,whereas the CIS SOCS box is dispensable. Accordingly,the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly,apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus,CIS negatively regulates signaling at two levels,apoptosis and proliferation,and thereby sets a threshold for signal transduction. View Publication

BACKGROUND: The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults,Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover,stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure,however: little is known about the communication between the pathway suppressor Patched (Ptc),a multipass transmembrane protein that directly binds Hh,and the pathway activator Smoothened (Smo),a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc. RESULTS: We have identified and characterized a synthetic non-peptidyl small molecule,Hh-Ag,that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro,while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null,but not Smo-null,mouse embryos. Biochemical studies with Hh-Ag,the Hh-signaling antagonist cyclopamine,and a novel Hh-signaling inhibitor Cur61414,reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc,as each binds directly to Smo. CONCLUSIONS: Smo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions. View Publication

The CD45 antigen is present on all cells of the hematopoietic lineage. Using a murine model,we have determined whether a lytic CD45 monoclonal antibody can produce persistent aplasia and whether it could facilitate syngeneic or allogeneic stem cell engraftment. After its systemic administration,we found saturating quantities of the antibody on all cells expressing the CD45 antigen,both in marrow and in lymphoid organs. All leukocyte subsets in peripheral blood were markedly diminished during or soon after anti-CD45 treatment,but only the effect on the lymphoid compartment was sustained. In contrast to the prolonged depletion of T and B lymphocytes from the thymus and spleen,peripheral blood neutrophils began to recover within 24 hours after the first anti-CD45 injection and marrow progenitor cells were spared from destruction,despite being coated with saturating quantities of anti-CD45. Given the transient effects of the monoclonal antibody on myelopoiesis and the more persistent effects on lymphopoiesis,we asked whether this agent could contribute to donor hematopoietic engraftment following nonmyeloablative transplantation. Treatment with anti-CD45 alone did not enhance syngeneic engraftment,consistent with its inability to destroy progenitor cells and permit competitive repopulation with syngeneic donor stem cells. By contrast,the combination of anti-CD45 and an otherwise inactive dose of total-body irradiation allowed engraftment of H2 fully allogeneic donor stem cells. We attribute this result to the recipient immunosuppression produced by depletion of CD45(+) lymphocytes. Monoclonal antibodies of this type may therefore have an adjunctive role in nonmyeloablative conditioning regimens for allogeneic stem cell transplantation. View Publication

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Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However,HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs,we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein,and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells,HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo,which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However,high HOXB4 expression substantially impaired the myeloerythroid differentiation program,and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P textless.03) and in vivo (P =.01). Furthermore,HOXB4 overexpression also significantly reduced B-cell output (P textless.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials. 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