技术资料

Although natural killer (NK) cells are well known for their ability to kill tumors,few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here,we show that human leukocyte antigen class I(low) tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1,ULBP2,and ULBP3. In line with the NK receptor ligand expression profiles,antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy. 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

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

During development of the neural tube,inhibition of the Notch response as well as the activation of the Sonic Hedgehog (Shh) response results in the formation of neuronal cell types. To determine whether Shh and Notch act independently,we tested the effects of the Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) on neuralized,embryonic stem (ES) cell-derived embryoid bodies (EBs),while varying the levels of Shh pathway activation. Shh-resistant EBs were derived from Smo null ES cells,while EBs with constitutive high level of Shh pathway activation were derived from Ptc1 null ES cells. Intermediate levels of Shh pathway activation was achieved by the addition of ShhN to the EB culture medium. It was found that DAPT-mediated inhibition of the Notch response resulted in enhanced neuronal differentiation. In the absence of Shh,more interneurons were detected,while the main effect of DAPT on EBs with an activated Shh response was the precocious loss of ventral neuronal precursor-specific markers. View Publication

AIM: The recently published REPAIR-AMI and ASTAMI trial showed differences in contractile recovery of left ventricular function after infusion of bone marrow-derived cells in acute myocardial infarction. Since the trials used different protocols for cell isolation and storage (REPAIR-AMI: Ficoll,storage in X-vivo 10 medium plus serum; ASTAMI: Lymphoprep,storage in NaCl plus plasma),we compared the functional activity of BMC isolated by the two different protocols. METHODS AND RESULTS: The recovery of total cell number,colony-forming units (CFU),and the number of mesenchymal stem cells were significantly reduced to 77 +/- 4%,83 +/- 16%,and 65 +/- 15%,respectively,when using the ASTAMI protocol compared with the REPAIR protocol. The capacity of the isolated BMC to migrate in response to stromal cell-derived factor 1 (SDF-1) was profoundly reduced when using the ASTAMI cell isolation procedure (42 +/- 8% and 78 +/- 3% reduction in healthy and CAD-patient cells,respectively). Finally,infusion of BMC into a hindlimb ischaemia model demonstrated a significantly blunted blood-flow-recovery by BMC isolated with the ASTAMI protocol (54 +/- 6% of the effect obtained by REPAIR cells). Comparison of the individual steps identified the use of NaCl and plasma for cell storage as major factors for functional impairment of the BMC. CONCLUSION: Cell isolation protocols have a major impact on the functional activity of bone marrow-derived progenitor cells. The assessment of cell number and viability may not entirely reflect the functional capacity of cells in vivo. Additional functional testing appears to be mandatory to assure proper cell function before embarking on clinical cell therapy trials. View Publication

Remarkably,apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice,but not other strains of mice,to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins,Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1,but not Bcl-2,Bcl-X(L),Bax,A1,or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand,Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis. View Publication

Neutropenia and neutrophil dysfunction are common in many diseases,although their etiology is often unclear. Previous views held that there was a single ER enzyme,glucose-6-phosphatase-alpha (G6Pase-alpha),whose activity--limited to the liver,kidney,and intestine--was solely responsible for the final stages of gluconeogenesis and glycogenolysis,in which glucose-6-phosphate (G6P) is hydrolyzed to glucose for release to the blood. Recently,we characterized a second G6Pase activity,that of G6Pase-beta (also known as G6PC),which is also capable of hydrolyzing G6P to glucose but is ubiquitously expressed and not implicated in interprandial blood glucose homeostasis. We now report that the absence of G6Pase-beta led to neutropenia; defects in neutrophil respiratory burst,chemotaxis,and calcium flux; and increased susceptibility to bacterial infection. Consistent with this,G6Pase-beta-deficient (G6pc3-/-) mice with experimental peritonitis exhibited increased expression of the glucose-regulated proteins upregulated during ER stress in their neutrophils and bone marrow,and the G6pc3-/- neutrophils exhibited an enhanced rate of apoptosis. Our results define a molecular pathway to neutropenia and neutrophil dysfunction of previously unknown etiology,providing a potential model for the treatment of these conditions. View Publication

Vascular remodeling,rather than vasoconstriction,is believed to account for high vascular resistance in severe pulmonary arterial hypertension (PAH). We have found previously that acute Rho kinase inhibition nearly normalizes PAH in chronically hypoxic rats that have no occlusive neointimal lesions. Here we examined whether Rho kinase-mediated vasoconstriction was also important in a rat model of severe occlusive PAH. Adult rats were exposed to chronic hypoxia ( approximately 10% O(2)) after subcutaneous injection of the vascular endothelial growth factor receptor inhibitor SUGEN 5416. Hemodynamic measurements were made in anesthetized rats after 2 weeks of hypoxia (early group) and 3 weeks of hypoxia plus 2 weeks of normoxia (late group). Both groups developed PAH,with greater severity in the late group. In the early group,intravenous fasudil was more effective than intravenous bradykinin,inhaled NO,or intravenous iloprost in reducing right ventricular systolic pressure. Despite more occlusive vascular lesions,fasudil also markedly reduced right ventricular systolic pressure in late-stage rats. Blood-perfused lungs from late-stage rats showed spontaneous vasoconstriction,which was reversed partially by the endothelin A receptor blocker BQ123 and completely by fasudil or Y-27632. Phosphorylation of MYPT1,a downstream target of Rho kinase,was increased in lungs from both groups of rats,and fasudil (intravenous) reversed the increased phosphorylation in the late group. Thus,in addition to structural occlusion,Rho kinase-mediated vasoconstriction is an important component of severe PAH in SUGEN 5416/hypoxia-exposed rats,and PAH can be significantly reduced in the setting of a severely remodeled lung circulation if an unconventional vasodilator is used. View Publication

Fasudil,a Rho-kinase inhibitor,may improve insulin signaling. However,its long-term effect on metabolic abnormalities and its preventive effect on diabetic nephropathy are still unknown. We assessed these effects of fasudil in insulin-resistant diabetic rats,comparing them with those of an angiotensin II receptor blocker,olmesartan. Male Otsuka Long-Evans Tokushima fatty (OLETF) and Long-Evans Tokushima Otsuka,non-diabetic control,rats at 15 weeks of age were used. OLETF rats were randomized to receive a low or a high dose of fasudil or olmesartan for 25 weeks. To examine the therapeutic effects after the development of diabetes,OLETF rats at 30 weeks of age were given fasudil for 10 weeks. Administration of high-dose fasudil completely suppressed the development of diabetes,obesity,and dyslipidemia and increased serum adiponectin levels in OLETF rats. High-dose olmesartan also decreased hemoglobin A1c and increased serum adiponectin. There was a significant correlation between hemoglobin A1c and serum adiponectin or free fatty acid levels. The treatment with high-dose fasudil ameliorated proteinuria,glomerulosclerosis,renal interstitial fibrosis,and macrophage infiltration in OLETF rats. Olmesartan,even at the low dose,suppressed renal complications. The treatment with fasudil after the development of diabetes improved the metabolic abnormalities in OLETF rats,but could not suppress the progression of nephropathy. We conclude that the long-term treatment with fasudil prevents the development of diabetes,at least in part,by improving adipocyte differentiation in insulin-resistant diabetic rats. Early use of fasudil may prevent diabetic nephropathy. View Publication

BACKGROUND AND OBJECTIVES: Chemokines are soluble mediators involved in angiogenesis,cellular growth control and immunomodulation. In the present study we investigated the effects of various chemokines on proliferation of acute myelogenous leukemia (AML) cells and constitutive chemokine release by primary AML cells. DESIGN AND METHODS: Native human AML cells derived from 68 consecutive patients were cultured in vitro. We investigated AML cell proliferation (3H-thymidine incorporation,colony formation),chemokine receptor expression,constitutive chemokine release and chemotaxis of normal peripheral blood mononuclear cells. RESULTS: Exogenous chemokines usually did not have any effect on AML blast proliferation in the absence of hematopoietic growth factors,but when investigating growth factor-dependent (interleukin 3 + granulocyte-macrophage colony-stimulating factor + stem cell factor) proliferation in suspension cultures the following patient subsets were identified: (i) patients whose cells showed chemokine-induced growth enhancement (8 patients); (ii) divergent effects on proliferation (15 patients); and (iii) no effect (most patients). These patient subsets did not differ in chemokine receptor expression,but,compared to CD34- AML cells,CD34+ cells showed higher expression of several receptors. Chemokines also increased the proliferation of clonogenic AML cells from the first subset of patients. Furthermore,a broad constitutive chemokine release profile was detected for most patients,and the following chemokine clusters could be identified: CCL2-4/CXCL1/8,CCL5/CXCL9-11 (possibly also CCL23) and CCL13/17/22/24/CXCL5 (possibly also CXCL6). Only the CCL2-4/CXCL1/8 cluster showed significant correlations between corresponding mRNA levels and NFkB levels/activation. The chemotaxis of normal immunocompetent cells for patients without constitutive chemokine release was observed to be decreased. INTERPRETATION AND CONCLUSIONS: Differences in chemokine responsiveness as well as chemokine release contribute to patient heterogeneity in AML. Patients with AML can be classified into distinct subsets according to their chemokine responsiveness and chemokine release profile. View Publication

We have found that a fungal strain,Talaromyces wortmannin KY12420,produces a potent inhibitor of smooth muscle myosin light chain kinase (MLCK). This active product,designated as MS-54,was isolated and purified from the culture broth of the fungus and identified as wortmannin. The inhibition of MLCK by wortmannin was prevented by a high concentration of ATP. The activity of the catalytic domain,which was disclosed by partial tryptic digestion,was also inhibited by wortmannin. These results suggest that wortmannin acts at or near to the catalytic site of the enzyme. It was shown clearly by kinetic analyses,preincubation studies,and dialysis experiments that the inhibitory action of wortmannin on MLCK was irreversible. Under the condition of preincubation for 3 min,0.3 microM wortmannin inhibited the activity of MLCK,while 10 microM wortmannin had no effect on the activities of cAMP-dependent protein kinase,cGMP-dependent protein kinase,and calmodulin-dependent protein kinase II,and had little effect on protein kinase C activity. These data expressed clearly the marked selectivity of the compound for MLCK. Furthermore,wortmannin also inhibited both the phosphorylation of myosin light chain and the contraction in rat thoracic aorta stimulated with KCl,which indicates the effectiveness of the compound in the cellular level as an MLCK inhibitor. View Publication

The hemopoietic microenvironment consists of a diverse repertoire of cells capable of providing signals that influence hemopoietic stem cell function. Although the role of osteoblasts and vascular endothelial cells has recently been characterized,the function of the most abundant cell type in the bone marrow,the adipocyte,is less defined. Given the emergence of a growing number of adipokines,it is possible that these factors may also play a role in regulating hematopoiesis. Here,we investigated the role of adiponectin,a secreted molecule derived from adipocytes,in hemopoietic stem cell (HSC) function. We show that adiponectin is expressed by components of the HSC niche and its receptors AdipoR1 and AdipoR2 are expressed by HSCs. At a functional level,adiponectin influences HSCs by increasing their proliferation,while retaining the cells in a functionally immature state as determined by in vitro and in vivo assays. We also demonstrate that adiponectin signaling is required for optimal HSC proliferation both in vitro and in long term hemopoietic reconstitution in vivo. Finally we show that adiponectin stimulation activates p38 MAPK,and that inhibition of this pathway abrogates adiponectin's proliferative effect on HSCs. These studies collectively identify adiponectin as a novel regulator of HSC function and suggest that it acts through a p38 dependent pathway. View Publication