技术资料

The induction of senescence-like growth arrest has emerged as a putative contributor to the anticancer effects of chemotherapeutic agents. Clinical trials are underway to evaluate the efficacy of inhibitors for class I and II histone deacetylases to treat malignancies. However,a potential antiproliferative effect of inhibitor for Sirt1,which is an NAD(+)-dependent deacetylase and belongs to class III histone deacetylases,has not yet been explored. Here,we show that Sirt1 inhibitor,Sirtinol,induced senescence-like growth arrest characterized by induction of senescence-associated beta-galactosidase activity and increased expression of plasminogen activator inhibitor 1 in human breast cancer MCF-7 cells and lung cancer H1299 cells. Sirtinol-induced senescence-like growth arrest was accompanied by impaired activation of mitogen-activated protein kinase (MAPK) pathways,namely,extracellular-regulated protein kinase,c-jun N-terminal kinase and p38 MAPK,in response to epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I). Active Ras was reduced in Sirtinol-treated senescent cells compared with untreated cells. However,tyrosine phosphorylation of the receptors for EGF and IGF-I and Akt/PKB activation were unaltered by Sirtinol treatment. These results suggest that inhibitors for Sirt1 may have anticancer potential,and that impaired activation of Ras-MAPK pathway might take part in a senescence-like growth arrest program induced by Sirtinol. View Publication

Severe combined immunodeficiency (SCID) caused by mutations in RAG1 or RAG2 genes is characterized by a complete block in T- and B-cell development. The only curative treatment is allogeneic hematopoietic stem cell transplantation,which gives a high survival rate (90%) when an HLA-genoidentical donor exists but unsatisfactory results when only partially compatible donors are available. We have thus been interested in the development of a potential alternative treatment by using retroviral gene transfer of a normal copy of RAG1 cDNA. We show here that this approach applied to RAG-1-deficient mice restores normal B- and T-cell function even in the presence of a reduced number of mature B cells. The reconstitution is stable over time,attesting to a selective advantage of transduced progenitors. Notably,a high transgene copy number was detected in all lymphoid organs,and this was associated with a risk of lymphoproliferation as observed in one mouse. Altogether,these results demonstrate that correction of RAG-1 deficiency can be achieved by gene therapy in immunodeficient mice but that human application would require the use of self-inactivated vector to decrease the risk of lymphoproliferative diseases. View Publication

UNLABELLED: AC133 cells,a subpopulation of CD34+ hematopoietic stem cells,can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected,magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. MATERIALS AND METHODS: EPCs labeled with ferumoxide-protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma,and MRI was obtained at different stages of tumor development and size. RESULTS: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However,low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron-positive cells at the sites corresponding to low signal intensity on MRI. Confocal microscopy showed incorporation into the neovasculature,and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. CONCLUSION: MRI demonstrated the incorporation of FePro-labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors. View Publication

The beta-globin locus control region (LCR) is a large DNA element that is required for high-level expression of beta-like globin genes from the endogenous mouse locus or in transgenic mice carrying the human beta-globin locus. The LCR encompasses 6 DNaseI hypersensitive sites (HSs) that bind transcription factors. These HSs each contain a core of a few hundred base pairs (bp) that has most of the functional activity and exhibits high interspecies sequence homology. Adjoining the cores are 500- to 1000-bp flanks" with weaker functional activity and lower interspecies homology. Studies of human beta-globin transgenes and of the endogenous murine locus show that deletion of an entire HS (core plus flanks) moderately suppresses expression. However� View Publication

OBJECTIVES: The goal of this study was to modify mesenchymal stem cells (MSCs) cells with a hypoxia-regulated heme oxygenase-1 (HO-1) plasmid to enhance the survival of MSCs in acute myocardial infarction (MI) heart. BACKGROUND: Although stem cells are being tested clinically for cardiac repair,graft cells die in the ischemic heart because of the effects of hypoxia/reoxygenation,inflammatory cytokines,and proapoptotic factors. Heme oxygenase-1 is a key component in inhibiting most of these factors. METHODS: Mesenchymal stem cells from bone marrow were transfected with either HO-1 or LacZ plasmids. Cell apoptosis was assayed in vitro after hypoxia-reoxygen treatment. In vivo,1 x 10(6) of male MSC(HO-1),MSC(LacZ),MSCs,or medium was injected into mouse hearts 1 h after MI (n = 16/group). Cell survival was assessed in a gender-mismatched transplantation model. Apoptosis,left ventricular remodeling,and cardiac function were tested in a gender-matched model. RESULTS: In the ischemic myocardium,the MSC(HO-1) group had greater expression of HO-1 and a 2-fold reduction in the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells compared with the MSC(LacZ) group. At seven days after implantation,the survival MSC(HO-1) was five-fold greater than the MSC(LacZ) group; MSC(HO-1) also attenuated left ventricular remodeling and enhanced the functional recovery of infarcted hearts two weeks after MI. CONCLUSIONS: A hypoxia-regulated HO-1 vector modification of MSCs enhances the tolerance of engrafted MSCs to hypoxia-reoxygen injury in vitro and improves their viability in ischemic hearts. This demonstration is the first showing that a physiologically inducible vector expressing of HO-1 genes improves the survival of stem cells in myocardial ischemia. View Publication

CD4(+) T cells producing interleukin 17 (IL-17) are associated with autoimmunity,although the precise mechanisms that control their development are undefined. Here we present data that challenge the idea of a shared developmental pathway with T helper type 1 (T(H)1) or T(H)2 lineages and instead favor the idea of a distinct effector lineage we call 'T(H)-17'. The development of T(H)-17 cells from naive precursor cells was potently inhibited by interferon-gamma (IFN-gamma) and IL-4,whereas committed T(H)-17 cells were resistant to suppression by T(H)1 or T(H)2 cytokines. In the absence of IFN-gamma and IL-4,IL-23 induced naive precursor cells to differentiate into T(H)-17 cells independently of the transcription factors STAT1,T-bet,STAT4 and STAT6. These findings provide a basis for understanding how inhibition of IFN-gamma signaling enhances development of pathogenic T(H)-17 effector cells that can exacerbate autoimmunity. View Publication

Erythropoietin (Epo) stimulation of its receptor's downstream signaling pathways and optimum function of GATA-1 transcription factor are both essential for normal erythroid cell development. Epo-receptor (EpoR) signaling and GATA-1 regulate proliferation,survival,differentiation,and maturation of erythroid cells. Whether any signal that is generated by EpoR targets GATA-1 or affects GATA-1 transcriptional activity is not known. Here,we demonstrate that stimulation of EpoR results in phosphorylation of GATA-1 at serine 310 (S310) in primary fetal liver erythroid progenitors and in cultured erythroid cells. We show that phosphorylation of GATA-1 is important for Epo-induced maturation of fetal liver erythroid progenitor cells. The PI3-kinase/AKT signaling pathway is identified as a mediator of Epo-induced phosphorylation of GATA-1. AKT serine threonine kinase phosphorylates GATA-1S310 in vitro and in erythroid cells and enhances GATA-1 transcriptional activity. These data demonstrate that EpoR signaling phosphorylates GATA-1 and modulates its activity via the PI3-kinase/AKT signaling pathway. View Publication

Hematopoietic stem cells (HSCs) can be isolated from murine bone marrow by their ability to efflux the Hoechst 33342 dye. This method defines an extremely small and hematopoietically potent subset of cells known as the side population (SP). Recent studies suggest that transplanted single SP cells are capable of lymphohematopoietic repopulation at near absolute efficiencies. Here,we carefully reevaluate the hematopoietic potential of individual SP cells and find substantially lower rates of reconstitution. Our strategy involved the cotransplantation of single SP cells along with different populations of competitor cells that varied in their self-renewal capacity. Even with minimized HSC competition,SP cells were only able to reconstitute up to 35% of recipient mice. Furthermore,through immunophenotyping and clonal in vitro assays we find that SP cells are virtually homogeneous. Isolation of HSCs on the basis of Hoechst exclusion and a single cell-surface marker allows enrichment levels similar to that obtained with complex multicolor strategies. Altogether,our results indicate that even an extremely homogeneous HSC population,based on phenotype and dye efflux,cannot reconstitute mice at absolute efficiencies. View Publication

In the central nervous system,neural stem cells proliferate in the ventricular zone (VZ) and sequentially give rise to both neurons and glial cells in a temporally and spatially regulated manner,suggesting that stem cells may differ from one another in different brain regions and at different developmental stages. For the purpose of marking and purifying neural stem cells to ascertain whether such differences exist,we generated transgenic mice using promoters from Hes genes (pHes1 or pHes5) to drive expression of destabilized enhanced green fluorescent protein. In the developing brains of these transgenic mice,GFP expression was restricted to undifferentiated cells in the VZ,which could asymmetrically produce a Numb-positive neuronal daughter and a GFP-positive progenitor cell in clonal culture,indicating that they retain the capacity to self-renew. Our results suggest that pHes-EGFP transgenic mice can be used to explore similarities and differences among neural stem cells during development. View Publication

Adult retinal stem cells represent a possible cell source for the treatment of retinal degeneration. However,only a small number of stem cells reside in the ciliary margin. The present study aimed to promote the proliferation of adult retinal stem cells via the Wnt signaling pathway. Ciliary margin cells from 8-week-old mice were dissociated and cultured to allow sphere colony formation. Wnt3a,a glycogen synthase kinase (GSK) 3 inhibitor,fibroblast growth factor (FGF) 2,and a FGF receptor inhibitor were then applied in the culture media. The primary spheres were dissociated to prepare either monolayer or secondary sphere cultures. Wnt3a increased the size of the primary spheres and the number of Ki-67-positive proliferating cells in monolayer culture. The Wnt3a-treated primary sphere cells were capable of self-renewal and gave rise to fourfold the number of secondary spheres compared with nontreated sphere cells. These cells also retained their multilineage potential to express several retinal markers under differentiating culture conditions. The Wnt3a-treated cells showed nuclear accumulation of beta-catenin,and a GSK3 inhibitor,SB216763,mimicked the mitogenic activity of Wnt3a. The proliferative effect of SB216763 was attenuated by an FGF receptor inhibitor but was enhanced by FGF2,with Ki-67-positive cells reaching over 70% of the total cells. Wnt3a and SB216763 promoted the proliferation of retinal stem cells,and this was partly dependent on FGF2 signaling. A combination of Wnt and FGF signaling may provide a therapeutic strategy for in vitro expansion or in vivo activation of adult retinal stem cells. View Publication