技术资料

Histone lysine methylation has important roles in the organization of chromatin domains and the regulation of gene expression. To analyze its function and modulate its activity,we screened for specific inhibitors against histone lysine methyltransferases (HMTases) using recombinant G9a as the target enzyme. From a chemical library comprising 125,000 preselected compounds,seven hits were identified. Of those,one inhibitor,BIX-01294 (diazepin-quinazolin-amine derivative),does not compete with the cofactor S-adenosyl-methionine,and selectively impairs the G9a HMTase and the generation of H3K9me2 in vitro. In cellular assays,transient incubation of several cell lines with BIX-01294 lowers bulk H3K9me2 levels that are restored upon removal of the inhibitor. Importantly,chromatin immunoprecipitation at several G9a target genes demonstrates reversible reduction of promoter-proximal H3K9me2 in inhibitor-treated mouse ES cells and fibroblasts. Our data identify a biologically active HMTase inhibitor that allows for the transient modulation of H3K9me2 marks in mammalian chromatin. View Publication

MEK/ERK signaling plays a crucial role in a diverse set of cellular functions including cell proliferation,differentiation and survival,and recently has been reported to negatively regulate mouse embryonic stem cell (mESC) self-renewal by antagonizing STAT3 activity. However,its role in human ESCs (hESCs) remains unclear. Here we investigated the functions of MEK/ERK in controlling hESC activity. We demonstrated that MEK/ERK kinases were targets of fibroblast growth factor (FGF) pathway in hESCs. Surprisingly,we found that,in contrast to mESCs,high basal MEK/ERK activity was required for maintaining hESCs in an undifferentiated state. Inhibition of MEK/ERK activity by specific MEK inhibitors PD98059 and U0126,or by RNA interference,rapidly caused the loss of self-renewal capacity. We also showed that MEK/ERK signaling cooperated with phosphoinositide 3-kinase (PI3K)/AKT signaling in maintaining hESC pluripotency. However,MEK/ERK signaling had little or no effect on regulating hESC proliferation and survival,in contrast to PI3K/AKT signaling. Taken together,these findings reveal the unique and crucial role of MEK/ERK signaling in the determination of hESC cell fate and expand our understanding of the molecular mechanisms behind the FGF pathway maintenance of hESC pluripotency. Importantly,these data make evident the striking differences in the control of self-renewal between hESCs and mESCs. View Publication

Adaptive transcriptional responses to oxygen deprivation (hypoxia) are mediated by the hypoxia-inducible factors (HIFs),heterodimeric transcription factors composed of two basic helix-loop-helix-PAS family proteins. The transcriptional activity of HIF is determined by the hypoxic stabilization of the HIF-alpha proteins. HIF-1alpha and HIF-2alpha exhibit high sequence homology but have different mRNA expression patterns; HIF-1alpha is expressed ubiquitously whereas HIF-2alpha expression is more restricted to certain tissues,e.g.,the endothelium,lung,brain,and neural crest derivatives. Germ-line deletion of either HIF subunit is embryonic lethal with unique features suggesting important roles for both HIF-alpha isoforms. Global deletion of Hif-2alpha results in distinct phenotypes depending on the mouse strain used for the mutation,clearly demonstrating an important role for HIF-2alpha in mouse development. The function of HIF-2alpha in adult life,however,remains incompletely understood. In this study,we describe the generation of a conditional murine Hif-2alpha allele and the effect of its acute postnatal ablation. Under very stringent conditions,we ablate Hif-2alpha after birth and compare the effect of acute global deletion of Hif-2alpha and Hif-1alpha. Our results demonstrate that HIF-2alpha plays a critical role in adult erythropoiesis,with acute deletion leading to anemia. Furthermore,although HIF-1alpha was first purified and cloned based on its affinity for the human erythropoietin (EPO) 3' enhancer hypoxia response element (HRE) and regulates Epo expression during mouse embryogenesis,HIF-2alpha is the critical alpha isoform regulating Epo under physiologic and stress conditions in adults. View Publication

The erythroid response to acute anemia relies on the rapid expansion in the spleen of a specialized population of erythroid progenitors termed stress BFU-E. This expansion requires BMP4/Madh5-dependent signaling in vivo; however,in vitro,BMP4 alone cannot recapitulate the expansion of stress BFU-E observed in vivo,which suggests that other signals are required. In this report we show that mutation of the Kit receptor results in a severe defect in the expansion of stress BFU-E,indicating a role for the Kit/SCF signaling pathway in stress erythropoiesis. In vitro analysis showed that BMP4 and SCF are necessary for the expansion of stress BFU-E,but only when spleen cells were cultured in BMP4 + SCF at low-oxygen concentrations did we recapitulate the expansion of stress BFU-E observed in vivo. Culturing spleen cells in BMP4,SCF under hypoxic conditions resulted in the preferential expansion of erythroid progenitors characterized by the expression of Kit,CD71,and TER119. This expression pattern is also seen in stress erythroid progenitors isolated from patients with sickle cell anemia and patients with beta-thalassemia. Taken together these data demonstrate that SCF and hypoxia synergize with BMP4 to promote the expansion and differentiation of stress BFU-E during the recovery from acute anemia. View Publication

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However,clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody,we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the alpha2 domain of HLA-A. Here,we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells,lymphocytes,or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors,suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely,2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs,melphalan,or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore,administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma. View Publication

Ras activation is crucial for lymphocyte development and effector function. Both T and B lymphocytes contain two types of Ras activators: ubiquitously expressed SOS and specifically expressed Ras guanyl nucleotide-releasing protein (RasGRP). The need for two activators is enigmatic since both are activated following antigen receptor stimulation. In addition,RasGRP1 appears to be dominant over SOS in an unknown manner. The crystal structure of SOS provides a clue: an unusual allosteric Ras-GTP binding pocket. Here,we demonstrate that RasGRP orchestrates Ras signaling in two ways: (i) by activating Ras directly and (ii) by facilitating priming of SOS with RasGTP that binds the allosteric pocket. Priming enhances SOS' in vivo activity and creates a positive RasGTP-SOS feedback loop that functions as a rheostat for Ras activity. Without RasGRP1,initiation of this loop is impaired because SOS' catalyst is its own product (RasGTP)-hence the dominance of RasGRP1. Introduction of an active Ras-like molecule (RasV12C40) in T- and B-cell lines can substitute for RasGRP function and enhance SOS' activity via its allosteric pocket. The unusual RasGRP-SOS interplay results in sensitive and robust Ras activation that cannot be achieved with either activator alone. We hypothesize that this mechanism enables lymphocytes to maximally respond to physiologically low levels of stimulation. View Publication

In this study,we analyzed IL-2-activated polyclonal natural killer (NK) cells derived from 2 patients affected by leukocyte adhesion deficiency type I (LAD1),an immunodeficiency characterized by mutations of the gene coding for CD18,the beta subunit shared by major leukocyte integrins. We show that LAD1 NK cells express normal levels of various triggering NK receptors (and coreceptors) and that mAb-mediated engagement of these receptors results in the enhancement of both NK cytolytic activity and cytokine production. Moreover,these activating NK receptors were capable of recognizing their specific ligands on target cells. Thus,LAD1 NK cells,similarly to normal NK cells,were capable of killing most human tumor cells analyzed and produced high amounts of IFN-gamma when cocultured in presence of target cells. Murine target cells represented a common exception,as they were poorly susceptible to LAD1 NK cells. Finally,LAD1 NK cells could efficiently kill or induce maturation of monocyte-derived immature dendritic cells (DCs). Altogether our present study indicates that in LAD1 patients,3 important functions of NK cells (eg,cytotoxicity,IFN-gamma production,and DC editing) are only marginally affected and provides new insight on the cooperation between activating receptors and LFA-1 in the induction of NK cell activation and function. View Publication

Toll-like receptors (TLR) recognize pathogen-associated molecular patterns and play important roles in the innate immune system. While single-stranded viral RNA is the natural ligand of TLR7/TLR8,the imidazoquinoline resiquimod (R-848) is recognized as a potent synthetic agonist of TLR7/TLR8. We investigated the effects of TLR7/8 activation on lipid mediator production in polymorphonuclear leukocytes exposed to R-848. Although R-848 had minimal effects by itself,it strongly enhanced leukotriene B4 formation on subsequent stimulation by fMLP,platelet-activating factor,and the ionophore A23187. R-848 acted via TLR8 but not TLR7 as shown by the lack of effect of the TLR7-specific ligand imiquimod. Priming with R-848 also resulted in enhanced arachidonic acid release and platelet-activating factor formation following fMLP stimulation,as well as enhanced prostaglandin E2 synthesis following the addition of arachidonic acid. Western blot analysis demonstrated that R-848 induced the phosphorylation of the cytosolic phospholipase A2alpha,promoted 5-lipoxygenase translocation and potently stimulated the expression of the type 2 cyclooxygenase. Bafilomycin A1,an inhibitor of endosomal acidification,efficiently inhibited all R-848-induced effects. These studies demonstrate that TLR8 signaling strongly promotes inflammatory lipid mediator biosynthesis and provide novel insights on innate immune response to viral infections. View Publication

We have investigated the mechanism of inhibition of enoyl-acyl carrier protein reductase of Plasmodium falciparum (PfENR) by triclosan in the presence of a few important catechins and related plant polyphenols. The examined flavonoids inhibited PfENR reversibly with Ki values in the nanomolar range,EGCG being the best with 79 +/- 2.67 nM. The steady-state kinetics revealed time dependent inhibition of PfENR by triclosan,demonstrating that triclosan exhibited slow tight-binding kinetics with PfENR in the presence of these compounds. Additionally,all of them potentiated the binding of triclosan with PfENR by a two-step mechanism resulting in an overall inhibition constant of triclosan in the low picomolar concentration range. The high affinities of tea catechins and the potentiation of binding of triclosan in their presence are readily explained by molecular modeling studies. The enhancement in the potency of triclosan induced by these compounds holds great promise for the development of effective antimalarial therapy. View Publication

OBJECTIVE: Our laboratory has established two unique methods to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of both protocols is the separation of whole bone marrow into small-sized cells by counter-flow elutriation. We sought to provide the scientific community with an alternate approach to acquire our stem cells by replacing elutriation with the use of density-gradient centrifugation. METHODS: The elutriated fraction 25 population was characterized based on density using a discontinuous gradient. The long-term reconstituting potential of whole bone marrow cells collected at each density interface was determined by subjecting the fractions to the two-day homing protocol,transplanting them into lethally irradiated recipient mice,and assessing peripheral blood chimerism. We also investigated the ability of high-density bone marrow cells isolated in conjunction with the ALDH protocol to repopulate the hematopoietic system of myeloablated recipients. RESULTS: Bone marrow cells collected at the high-density interface of 1.081/1.087 g/mL (fraction 3) had the capacity for homing to marrow and the ability to provide long-term hematopoietic reconstitution. Fraction three lineage-depleted ALDH-bright cells could also engraft and provide long-term hematopoiesis at limiting dilutions. CONCLUSIONS: Density-gradient centrifugation can be used in conjunction with either of our stem cell isolation protocols to obtain cells with long-term reconstitution ability. We anticipate that this strategy will encourage and enable investigators to study the biology of HSCs isolated using functional characteristics. View Publication

OBJECTIVE The p15INK4B tumor suppressor is frequently silenced by promoter hypermethylation in myelodysplastic syndrome and acute myeloid leukemia (AML). Clinically approved DNA methylation inhibitors,such as 5-aza-2'-deoxycytidine,can reverse p15INK4B promoter methylation,but widespread clinical use of these inhibitors is limited by their toxicity and instability in aqueous solution. The cytidine analog zebularine is a stable DNA methylation inhibitor that has minimal toxicity in vitro and in vivo. We evaluated zebularine effects on p15INK4B reactivation and cell growth in vitro to investigate a potential role for zebularine in treating myeloid malignancies. METHODS We examined the specific effects of zebularine on reexpression of transcriptionally silenced p15INK4B and its global effects on cell cycle and apoptosis in AML cell lines and primary patient samples. RESULTS Zebularine treatment of AML193,which has a densely methylated p15INK4B promoter,results in a dose-dependent increase in p15INK4B expression that correlates with CpG island promoter demethylation and enrichment of local histone acetylation. We observed enhanced p15INK4B induction following co-treatment with zebularine and the histone deacetylase inhibitor Trichostatin A. Zebularine inhibits cell proliferation,arrests cells at G(2)/M,and induces apoptosis at dosages that effectively demethylate the p15INK4B promoter. Zebularine treatment of KG-1 cells and AML patient blasts with hypermethylated p15INK4B promoters also reactivates p15INK4B reexpression and induces apoptosis. CONCLUSION Zebularine is an effective inhibitor of p15INK4B methylation and cell growth in human AML in vitro. Our results extend the spectrum of zebularine effects to nonepithelial malignancies and provide a strong rationale for evaluating its clinical utility in the treatment of myeloid malignancies. View Publication

We showed that resistance to severe hypoxia defines hierarchical levels within normal hematopoietic populations and that hypoxia modulates the balance between generation of progenitors and maintenance of hematopoietic stem cells (HSC) in favor of the latter. This study deals with the effects of hypoxia (0.1% oxygen) in vitro on Friend's murine erythroleukemia (MEL) cells,addressing the question of whether a clonal leukemia cell population comprise functionally different cell subsets characterized by different hypoxia resistance. To identify leukemia stem cells (LSC),we used the Culture Repopulating Ability (CRA) assay we developed to quantify in vitro stem cells capable of short-term reconstitution (STR). Hypoxia strongly inhibited the overall growth of MEL cell population,which,despite its clonality,comprised progenitors characterized by markedly different hypoxia-resistance. These included hypoxia-sensitive colony-forming cells and hypoxia-resistant STR-type LSC,capable of repopulating secondary liquid cultures of CRA assays,confirming what was previously shown for normal hematopoiesis. STR-type LSC were found capable not only of surviving in hypoxia but also of being mostly in cycle,in contrast with the fact that almost all hypoxia-surviving cells were growth-arrested and with what we previously found for HSC. However,quiescent LSC were also detected,capable of delayed culture repopulation with the same efficiency as STR-like LSC. The fact that even quiescent LSC,believed to sustain minimal residual disease in vivo,were found within the MEL cells indicates that all main components of leukemia cell populations may be present within clonal cell lines,which are therefore suitable to study the sensitivity of individual components to treatments. Disclosure of potential conflicts of interest is found at the end of this article. View Publication