技术资料

Hematopoiesis is tightly controlled by transcription regulatory networks,but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin(-)Sca-1(+)c-Kit(+)) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells,which express C/EBPα,we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP(+) cells represent a significant subset of multipotent hematopoietic progenitors,which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition,Cebpa/EYFP(+) cells compose a fraction of early thymic progenitors with robust myeloid potential. However,Cebpa/EYFP(+) multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages,respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors. View Publication

In mammals DNA methylation occurs at position 5 of cytosine in a CpG context and regulates gene expression. It plays an important role in diseases and inhibitors of DNA methyltransferases (DNMTs)--the enzymes responsible for DNA methylation--are used in clinics for cancer therapy. The most potent inhibitors are 5-azacytidine and 5-azadeoxycytidine. Zebularine (1-(beta-D-ribofuranosyl)-2(1H)- pyrimidinone) is another cytidine analog described as a potent inhibitor that acts by forming a covalent complex with DNMT when incorporated into DNA. Here we bring additional experiments to explain its mechanism of action. First,we observe an increase in the DNA binding when zebularine is incorporated into the DNA,compared to deoxycytidine and 5-fluorodeoxycytidine,together with a strong decrease in the dissociation rate. Second,we show by denaturing gel analysis that the intermediate covalent complex between the enzyme and the DNA is reversible,differing thus from 5-fluorodeoxycytidine. Third,no methylation reaction occurs when zebularine is present in the DNA. We confirm that zebularine exerts its demethylation activity by stabilizing the binding of DNMTs to DNA,hindering the methylation and decreasing the dissociation,thereby trapping the enzyme and preventing turnover even at other sites. View Publication

It has recently been shown that nucleosome distribution,histone modifications and RNA polymerase II (Pol II) occupancy show preferential association with exons (exon-intron marking")� View Publication

BACKGROUND The development of novel influenza vaccines inducing a broad immune response is an important objective. The aim of this study was to evaluate live vaccines which induce both strong humoral and cell-mediated immune responses against the novel human pandemic H1N1 influenza virus,and to show protection in a lethal animal challenge model. METHODOLOGY/PRINCIPAL FINDINGS For this purpose,the hemagglutinin (HA) and neuraminidase (NA) genes of the influenza A/California/07/2009 (H1N1) strain (CA/07) were inserted into the replication-deficient modified vaccinia Ankara (MVA) virus--a safe poxviral live vector--resulting in MVA-H1-Ca and MVA-N1-Ca vectors. These live vaccines,together with an inactivated whole virus vaccine,were assessed in a lung infection model using immune competent Balb/c mice,and in a lethal challenge model using severe combined immunodeficient (SCID) mice after passive serum transfer from immunized mice. Balb/c mice vaccinated with the MVA-H1-Ca virus or the inactivated vaccine were fully protected from lung infection after challenge with the influenza H1N1 wild-type strain,while the neuraminidase virus MVA-N1-Ca induced only partial protection. The live vaccines were already protective after a single dose and induced substantial amounts of neutralizing antibodies and of interferon-gamma-secreting (IFN-gamma) CD4- and CD8 T-cells in lungs and spleens. In the lungs,a rapid increase of HA-specific CD4- and CD8 T cells was observed in vaccinated mice shortly after challenge with influenza swine flu virus,which probably contributes to the strong inhibition of pulmonary viral replication observed. In addition,passive transfer of antisera raised in MVA-H1-Ca vaccinated immune-competent mice protected SCID mice from lethal challenge with the CA/07 wild-type virus. CONCLUSIONS/SIGNIFICANCE The non-replicating MVA-based H1N1 live vaccines induce a broad protective immune response and are promising vaccine candidates for pandemic influenza. View Publication

Human natural killer (NK) cells express an abundant level of 2B4 and CD2 on their surface. Their counter-receptors,CD48 and CD58,are also expressed on the NK cell surface,raising a question about the functional consequences of potential 2B4/CD48 and CD2/CD58 interactions. Using blocking antibodies specific to each receptor,we demonstrated that both 2B4/CD48 and CD2/CD58 interactions were essential for the development of NK effector functions: cytotoxicity and cytokine secretion. However,only 2B4/CD48,but not CD2/CD58,interactions were shown to be critical for the optimal NK cell proliferation in response to interleukin (IL)-2. IL-2-activated NK cells cultured in the absence of 2B4/CD48 or CD2/CD58 interactions were severely impaired for their ability to induce intracellular calcium mobilization and subsequent ERK activation upon tumor target exposure,suggesting that the early signaling pathway of NK receptors leading to impaired cytolysis and interferon (IFN)-γ secretion was inhibited. Nevertheless,these defects did not fully account for the reduced proliferation of NK cells in the absence of 2B4/CD48 interactions,because anti-CD2 or anti-CD58 monoclonal antibody (mAb)-treated NK cells,showing defective signaling and effector functions,displayed normal proliferation upon IL-2 stimulation. These results propose the signaling divergence between pathways leading to cell proliferation and cytotoxicity/cytokine release,which can be differentially regulated by 2B4 and CD2 during IL-2-driven NK cell activation. Collectively,these results reveal the importance of homotypic NK-to-NK cell cross-talk through 2B4/CD48 and CD2/CD58 pairs and further present their differential and overlapping roles in human NK cells. View Publication

The introduction of double stranded RNA (dsRNA) into the cytoplasm of mammalian cells usually leads to a potent antiviral response resulting in the rapid induction of interferon beta (IFNβ). This response can be mediated by a number of dsRNA sensors,including TLR3,MDA5,RIG-I and PKR. We show here that pluripotent human cells (human embryonic stem (hES) cells and induced pluripotent (iPS) cells) do not induce interferon in response to cytoplasmic dsRNA,and we have used a variety of approaches to learn the underlying basis for this phenomenon. Two major cytoplasmic dsRNA sensors,TLR3 and MDA5,are not expressed in hES cells and iPS cells. PKR is expressed in hES cells,but is not activated by transfected dsRNA. In addition,RIG-I is expressed,but fails to respond to dsRNA because its signaling adapter,MITA/STING,is not expressed. Finally,the interferon-inducible RNAse L and oligoadenylate synthetase enzymes are also expressed at very low levels. Upon differentiation of hES cells into trophoblasts,cells acquire the ability to respond to dsRNA and this correlates with a significant induction of expression of TLR3 and its adaptor protein TICAM-1/TRIF. Taken together,our results reveal that the lack of an interferon response may be a general characteristic of pluripotency and that this results from the systematic downregulation of a number of genes involved in cytoplasmic dsRNA signaling. View Publication

The ability of human embryonic stem cells (hESCs) to differentiate into essentially all somatic cell types has made them a valuable tool for studying human development and has positioned them for broad applications in toxicology,regenerative medicine,and drug discovery. This unit describes a protocol for the large-scale expansion and maintenance of hESCs in vitro. hESC cultures must maintain a balance between the cellular states of pluripotency and differentiation; thus,researchers must use care when growing these technically demanding cells. The culture system is based largely on the use of a proprietary serum-replacement product and basic fibroblast growth factor (bFGF),with mouse embryonic fibroblasts as a feeder layer. These conditions provide the basis for relatively inexpensive maintenance and expansion of hESCs,as well as their engineered counterparts,human induced pluripotent stem cells (hiPSCs). View Publication

Retinoids are important molecules involved in the development and homeostasis of the nervous system. As such,various retinoid derivatives are often found in culture media and supplement formulations to support the growth and maintenance of neural cells. However,all-trans-retinoic acid (ATRA) and its associated derivatives are light sensitive and are highly susceptible to isomerisation. This can lead to variability in retinoid concentrations and the nature of the retinoid species present in culture solutions which in turn can influence biological activity and introduce inconsistency. We have previously described the development of the synthetic retinoid derivative,EC23,as a chemically and light stable alternative that does not degrade and has biological activity similar to ATRA. In this study we demonstrate that the addition of exogenous retinoid can significantly enhance neuronal differentiation of both human neuroprogenitor and human embryonic stem cells. In the former,both ATRA and EC23 induced increased maturation and stabilisation of the axonal cytoskeleton. However,EC23 was particularly potent at lower nanomolar concentrations resulting in significantly greater neurogenesis than ATRA. In ES cells enhanced motor neuron marker expression was also detected in response to both retinoids when incorporated into an established protocol for neuronal differentiation. We propose that synthetic retinoid EC23 represents a valuable addition to the formulation of new and existing culture supplements to enhance neuronal differentiation whilst enabling improved consistency. View Publication

TOX is a DNA-binding factor required for development of CD4(+) T cells,natural killer T cells and regulatory T cells. Here we document that both natural killer (NK) cell development and lymphoid tissue organogenesis were also inhibited in the absence of TOX. We found that the development of lymphoid tissue-inducer cells,a rare subset of specialized cells that has an integral role in lymphoid tissue organogenesis,required TOX. Tox was upregulated considerably in immature NK cells in the bone marrow,consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus,many cell lineages of the immune system share a TOX-dependent step for development. View Publication

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition,FOXM1 has been reported to contribute to oncogenesis in various cancers. However,it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study,we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform,and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells,through induction of G(2)/M cell cycle arrest,a decrease in the protein expression of Aurora kinase B,Survivin,Cyclin B1,S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21,56,32 and 18 for M1,M2,M4 and M5 subtypes,respectively). Compared with normal ALDH(hi) cells,FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover,the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition,depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary,we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus,inhibition of FOXM1 expression represents an attractive target for AML therapy. View Publication

Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways,which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency,but the precise role and regulation of NANOG are not well defined. View Publication

The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However,Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression,we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src. We found that purvalanol A,developed as a CDK inhibitor,potently suppressed the anchorage-independent growth of c-Src-transformed cells,indicating that the activation of CDKs contributes to the c-Src transformation. We also found that purvalanol A suppressed the c-Src activity as effectively as the Src-selective inhibitor PP2,and that it reverted the transformed morphology to a nearly normal shape with less cytotoxicity than PP2. Purvalanol A induced a strong G2-M arrest,whereas PP2 weakly acted on the G1-S transition. Furthermore,when compared with PP2,purvalanol A more effectively suppressed the growth of human colon cancer HT29 and SW480 cells,in which Src family kinases and CDKs are activated. These findings demonstrate that the coordinated inhibition of cell cycle progression and tyrosine kinase signaling by the multi-selective purvalanol A is effective in suppressing cancer progression associated with c-Src up-regulation. View Publication