技术资料

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore,overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria,committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU,TMZ,and MMS,which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy. View Publication

Flt3 ligand (Flt3L) is a nonredundant cytokine in type I interferon-producing cell (IPC) and dendritic cell (DC) development,and IPC and DC differentiation potential is confined to Flt3+ hematopoietic progenitor cells. Here,we show that overexpression of human Flt3 in Flt3- (Flt3(-)Lin(-)IL-7Ralpha(-)Thy1.1(-)c-Kit+) and Flt3+ (Flt3(+)Lin(-)IL-7Ralpha(-)Thy1.1(-)c-Kit+) hematopoietic progenitors rescues and enhances their IPC and DC differentiation potential,respectively. In defined hematopoietic cell populations,such as Flt3- megakaryocyte/erythrocyte-restricted progenitors (MEPs),enforced Flt3 signaling induces transcription of IPC,DC,and granulocyte/macrophage (GM) development-affiliated genes,including STAT3,PU.1,and G-/M-/GM-CSFR,and activates differentiation capacities to these lineages. Moreover,ectopic expression of Flt3 downstream transcription factors STAT3 or PU.1 in Flt3- MEPs evokes Flt3 receptor expression and instructs differentiation into IPCs,DCs,and myelomonocytic cells,whereas GATA-1 expression and consecutive megakaryocyte/erythrocyte development is suppressed. Based on these data,we propose a demand-regulated,cytokine-driven DC and IPC regeneration model,in which high Flt3L levels initiate a self-sustaining,Flt3-STAT3- and Flt3-PU.1-mediated IPC and DC differentiation program in Flt3+ hematopoietic progenitor cells. View Publication

We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However,a small number of regenerated myofibers were detected,suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally,we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore,we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells. View Publication

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal-recessive motor neuron disease caused by mutations in the immunoglobulin micro-binding protein 2. We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase (ALDH) activity to modify disease progression of nmd mice,an animal model of SMARD1. ALDH(hi)SSC(lo) stem cells are self-renewing and multipotent and when intrathecally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns. Transplanted nmd animals presented delayed disease progression,sparing of motor neurons and ventral root axons and increased lifespan. To further investigate the molecular events responsible for these differences,microarray and real-time reverse transcription-polymerase chain reaction analyses of wild-type,mutated and transplanted nmd spinal cord were undertaken. We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling,as well as an up-regulation of genes related to the chromatin organization in nmd compared with wild-type mice,suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd-transplanted mice expressed high transcript levels for genes related to neurogenesis such as doublecortin (DCX),LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogeneses may contribute to the observed nmd phenotype amelioration. View Publication

Stem cells are undifferentiated cells defined by their ability to self-renew and differentiate to progenitors and terminally differentiated cells. Stem cells have been isolated from almost all tissues,and an emerging idea is that they share common characteristics such as the presence of ATP-binding cassette transporter G2 and high telomerase and aldehyde dehydrogenase (ALDH) activity,raising the hypothesis of a set of universal stem cell markers. In the present study,we describe the isolation of primitive neural stem cells (NSCs) from adult and embryonic murine neurospheres and dissociated tissue,based on the expression of high levels of ALDH activity. Single-cell suspension was stained with a fluorescent ALDH substrate termed Aldefluor and then analyzed by flow cytometry. A population of cells with low side scatter (SSC(lo)) and bright ALDH (ALDH(br)) activity was isolated. SSC(lo)ALDH(br) cells are capable of self-renewal and are able to generate new neurospheres and neuroepithelial stem-like cells. Furthermore,these cells are multipotent,differentiating both in neurons and macroglia,as determined by immunocytochemistry and real-time reverse transcription-polymerase chain reaction analysis. To evaluate the engraftment potential of SSC(lo)ALDH(br) cells in vivo,we transplanted them into mouse brain. Donor-derived neurons with mature morphology were detected in the cortex and subcortical areas,demonstrating the capacity of this cell population to differentiate appropriately in vivo. The ALDH expression assay is an effective method for direct identification of NSCs,and improvement of the stem cell isolation protocol may be useful in the development of a cell-mediated therapeutic strategy for neurodegenerative diseases. View Publication

Thrombopoietin (TPO) is a potent regulator of megakaryopoiesis and stimulates megakaryocyte (MK) progenitor expansion and MK differentiation. In this study,we show that TPO induces activation of the mammalian target of rapamycin (mTOR) signaling pathway,which plays a central role in translational regulation and is required for proliferation of MO7e cells and primary human MK progenitors. Treatment of MO7e cells,human CD34+,and primary MK cells with the mTOR inhibitor rapamycin inhibits TPO-induced cell cycling by reducing cells in S phase and blocking cells in G0/G1. Rapamycin markedly inhibits the clonogenic growth of MK progenitors with high proliferative capacity but does not reduce the formation of small MK colonies. Addition of rapamycin to MK suspension cultures reduces the number of MK cells,but inhibition of mTOR does not significantly affect expression of glycoproteins IIb/IIIa (CD41) and glycoprotein Ib (CD42),nuclear polyploidization levels,cell size,or cell survival. The downstream effectors of mTOR,p70 S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1),are phosphorylated by TPO in a rapamycin- and LY294002-sensitive manner. Part of the effect of the phosphatidyl inositol 3-kinase pathway in regulating megakaryopoiesis may be mediated by the mTOR/S6K/4E-BP1 pathway. In conclusion,these data demonstrate that the mTOR pathway is activated by TPO and plays a critical role in regulating proliferation of MK progenitors,without affecting differentiation or cell survival. View Publication

Whether cytokines can modulate the fate of primitive hematopoietic progenitor cells (HPCs) through successive in vitro cell divisions has not been established. Single human marrow CD34+CD38-/lo cells in the G0 phase of cell cycle were cultured under 7 different cytokine combinations,monitored for proliferation on days 3,5,and 7,then assayed for long-term culture-initiating cell (LTC-IC) function on day 7. LTC-IC function was then retrospectively correlated with prior number of in vitro cell divisions to determine whether maintenance of LTC-IC function after in vitro cell division is dependent on cytokine exposure. In the presence of proliferation progression signals,initial cell division was independent of cytokine stimulation,suggesting that entry of primitive HPCs into the cell cycle is a stochastic property. However,kinetics of proliferation beyond day 3 and maintenance of LTC-IC function were sensitive to cytokine stimulation,such that LTC-IC underwent an initial long cell cycle,followed by more synchronized shorter cycles varying in length depending on the cytokine combination. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) transplantation studies revealed analogous results to those obtained with LTC-ICs. These data suggest that although exit from quiescence and commitment to proliferation might be stochastic,kinetics of proliferation,and possibly fate of primitive HPCs,might be modulated by extrinsic factors. View Publication

GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs),whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac,fetal liver,and adult bone marrow. However,HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also,cytotoxic drug-induced regeneration studies show a clear GATA-2 dose-related proliferation defect in adult bone marrow. Thus,GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow. View Publication

The HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors,and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis,we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner,pre-B-cell leukemia transcription factor-1 (PBX1). Additionally,we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo,HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4,a region frequently deleted in HOXA9-induced AMLs. In vitro,HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1. View Publication

We show here that the process of megakaryocytic differentiation requires the presence of the recently discovered protein tescalcin. Tescalcin is dramatically upregulated during the differentiation and maturation of primary megakaryocytes or upon PMA-induced differentiation of K562 cells. This upregulation requires sustained signaling through the ERK pathway. Overexpression of tescalcin in K562 cells initiates events of spontaneous megakaryocytic differentiation,such as expression of specific cell surface antigens,inhibition of cell proliferation,and polyploidization. Conversely,knockdown of this protein in primary CD34+ hematopoietic progenitors and cell lines by RNA interference suppresses megakaryocytic differentiation. In cells lacking tescalcin,the expression of Fli-1,Ets-1,and Ets-2 transcription factors,but not GATA-1 or MafB,is blocked. Thus,tescalcin is essential for the coupling of ERK cascade activation with the expression of Ets family genes in megakaryocytic differentiation. View Publication

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA,encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast,no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines,mice,dogs,nonhuman primates,and human subjects. Here,we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose,and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells,such hits were overrepresented in leukemic clones,pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases,indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings,we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery. View Publication

Taking advantage of fluorescent substrates for their metabolic marker aldehyde dehydrogenase (ALDH),hematopoietic stem cells (HSC) were defined as SSC(lo)ALDH(br) - reflecting their low orthogonal light scattering and bright fluorescence intensity in flow cytometry. Based thereon,we investigated the usefulness of ALDH activity for characterizing HSC graft quality,particularly under stress conditions. We first compared the expression of ALDH vs CD34 in bone marrow and peripheral blood stem cell (PBSC) samples over 7 days. We noted that (i) only ALDH activity but not CD34 expression strongly reflected colony-forming ability over time,and that (ii) PBSC grafts stored at room temperature lost most of their progenitor cells within just 48 h. We then retrospectively related ALDH and CD34 expression as well as granulocyte-macrophage colony-forming units (CFU-GM) potential for 19 cryopreserved allogeneic PBSC grafts to engraftment data. Strikingly,in all six patients who received markedly decreased numbers of SSC(lo)ALDH(br) cells,this was associated not only with almost complete loss of CFU-GM potential but also with delayed establishment/permanent absence of full hematopoietic donor cell chimerism,whereas all other patients showed early complete donor chimerism. In conclusion,we suggest to measure ALDH activity as a surrogate marker for HSC activity,and to transport and store PBSC under controlled cooling conditions. View Publication