技术资料

The Sox6 transcription factor plays critical roles in various cell types,including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells,we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar,evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo,and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied,in erythroid cells,by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation,mediated by the double Sox6 binding site within its own promoter,may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation. View Publication

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups,making these studies difficult. Here,we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion,the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs,especially for their homing potential. View Publication

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro,bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-),PAX5(+),TdT(+),IgH rearranged)/myeloid (CD11b/Mac1(+),c-fms(+),lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast,young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model." View Publication

Neutrophils are the most abundant WBCs and have an essential role in the clearance of pathogens. Tight regulation of neutrophil numbers and their recruitment to sites of inflammation is critical in maintaining a balanced immune response. In various inflammatory conditions,such as rheumatoid arthritis,vasculitis,cystic fibrosis,and inflammatory bowel disease,increased serum G-CSF correlates with neutrophilia and enhanced neutrophil infiltration into inflamed tissues. We describe a fully human therapeutic anti-G-CSFR antibody (CSL324) that is safe and well tolerated when administered via i.v. infusion to cynomolgus macaques. CSL324 was effective in controlling G-CSF-mediated neutrophilia when administered either before or after G-CSF. A single ascending-dose study showed CSL324 did not alter steady-state neutrophil numbers,even at doses sufficient to completely prevent G-CSF-mediated neutrophilia. Weekly infusions of CSL324 (%10 mg/kg) for 3 wk completely neutralized G-CSF-mediated pSTAT3 phosphorylation without neutropenia. Moreover,repeat dosing up to 100 mg/kg for 12 wk did not result in neutropenia at any point,including the 12-wk follow-up after the last infusion. In addition,CSL324 had no observable effect on basic neutrophil functions,such as phagocytosis and oxidative burst. These data suggest that targeting G-CSFR may provide a safe and effective means of controlling G-CSF-mediated neutrophilia as observed in various inflammatory diseases. View Publication

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here,we identify a newly discovered human repopulating cell,distinct from previously identified repopulating cells,that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells,or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR,Thy-1 and CD34). In contrast to CD34+ subfractions,Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations,providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation. View Publication

Here we report the discovery of ON044580,an α-benzoyl styryl benzyl sulfide that possesses potent inhibitory activity against two unrelated kinases,JAK2 and BCR-ABL,and exhibits cytotoxicity to human tumor cells derived from chronic myelogenous leukemia (CML) and myelodysplasia (MDS) patients or cells harboring a mutant JAK2 kinase. This novel spectrum of activity is explained by the non-ATP-competitive inhibition of JAK2 and BCR-ABL kinases. ON044580 inhibits mutant JAK2 kinase and the proliferation of JAK2(V617F)-positive leukemic cells and blocks the IL-3-mediated phosphorylation of JAK2 and STAT5. Interestingly,this compound also directly inhibits the kinase activity of both wild-type and imatinib-resistant (T315I) forms of the BCR-ABL kinase. Finally,ON044580 effectively induces apoptosis of imatinib-resistant CML patient cells. The apparently unrelated JAK2 and BCR-ABL kinases share a common substrate,STAT5,and such substrate competitive inhibitors represent an alternative therapeutic strategy for development of new inhibitors. The novel mechanism of kinase inhibition exhibited by ON044580 renders it effective against mutant forms of kinases such as the BCR-ABL(T315I) and JAK2(V617F). Importantly,ON044580 selectively reduces the number of aneuploid cells in primary bone marrow samples from monosomy 7 MDS patients,suggesting another regulatory cascade amenable to this agent in these aberrant cells. Data presented suggest that this compound could have multiple therapeutic applications including monosomy 7 MDS,imatinib-resistant CML,and myeloproliferative neoplasms that develop resistance to ATP-competitive agents. View Publication

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons,astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker,the aldehyde dehydrogenase activity,we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency,BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly,we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting. View Publication

Activated tyrosine kinases have been frequently implicated in the pathogenesis of cancer,including acute myeloid leukemia (AML),and are validated targets for therapeutic intervention with small-molecule kinase inhibitors. To identify novel activated tyrosine kinases in AML,we used a discovery platform consisting of immunoaffinity profiling coupled to mass spectrometry that identifies large numbers of tyrosine-phosphorylated proteins,including active kinases. This method revealed the presence of an activated colony-stimulating factor 1 receptor (CSF1R) kinase in the acute megakaryoblastic leukemia (AMKL) cell line MKPL-1. Further studies using siRNA and a small-molecule inhibitor showed that CSF1R is essential for the growth and survival of MKPL-1 cells. DNA sequence analysis of cDNA generated by 5'RACE from CSF1R coding sequences identified a novel fusion of the RNA binding motif 6 (RBM6) gene to CSF1R gene generated presumably by a t(3;5)(p21;q33) translocation. Expression of the RBM6-CSF1R fusion protein conferred interleukin-3 (IL-3)-independent growth in BaF3 cells,and induces a myeloid proliferative disease (MPD) with features of megakaryoblastic leukemia in a murine transplant model. These findings identify a novel potential therapeutic target in leukemogenesis,and demonstrate the utility of phosphoproteomic strategies for discovery of tyrosine kinase alleles. 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

Natural killer (NK) cell differentiation from pluripotent CD34(+) human hematopoietic stem cells or oligopotent lymphoid progenitors has already been reported. In the present study,long-term cultures of the CD56(-)/CD34(-) myeloid-like adherent cell fraction (ACF) from umbilical cord blood (UCB),characterized by the expression of CD14(+) as well as other myeloid markers,were set up with flt3 ligand (FL) and interleukin-15 (IL-15). The UCB/ACF gradually expressed the CD56 marker,which reached fairly high levels (approximately 90% of the cells were CD56(+)) by day 15. FL plus IL-15-driven ACF/CD56(+) cells progressively expressed a mature NK functional program lysing both NK- and lymphokine-activate killer (LAK)-sensitive tumor targets and producing high levels of interferon-gamma (IFN-gamma),granulocyte-macrophage colony-stimulating factor,tumor necrosis factor alpha,and IL-10 upon stimulation with IL-12 and IL-18. Similar results were obtained when highly purified CD14(+) cells from UCB were cultured with FL and IL-15. In contrast,UCB/CD34(+) cells cultured under the same conditions showed a delayed expression of CD56 and behaved functionally differently in that they exhibited NK but not LAK cytotoxicity and produced significantly fewer cytokines. Kinetic studies on the phenotype of UCB/ACF or UCB/CD14(+) cells cultured in the presence of FL and IL-15 showed a rapid decrease in CD14 expression after day 5,which reached levels of zero by day 20. Approximately 60% of the CD56(+) derived from the UCB/ACF or the UCB/CD14(+) cells coexpressed CD14 by day 5. Taken together,our data support the role of CD14(+) myeloid-like cells within UCB as a novel progenitor for lymphoid NK cells. View Publication