技术资料

In mice there are two families of MHC class I-specific receptors,namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1(b) and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently,how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study,we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers,but not Ly49 molecules,and their cytotoxicity is inhibited by Qa-1(b) on target cells. Using RT-PCR analysis,we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order,with their order of acquisition being first CD94; subsequently NKG2D,NKG2A,and NKG2E; and finally,NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells,and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells,suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic,orderly,and cumulative. View Publication

BACKGROUND AND OBJECTIVES: Leptin receptors can be expressed by acute myelogenous leukemia (AML) cells,but the functional effects of leptin on native AML blasts have not been characterized in detail. We investigated systemic leptin levels in AML patients and in vitro effects of leptin on cultured AML blasts. DESIGN AND METHODS: Serum leptin levels were compared for patients with untreated AML and healthy controls. Native AML blasts were derived from a large group of consecutive patients,and effects of leptin on proliferation (suspension cultures and colony formation),constitutive cytokine secretion,differentiation and apoptosis regulation were assayed in vitro. RESULTS: Systemic leptin levels were decreased in patients with untreated AML,and leptin levels in acute leukemia patients were not altered during severe chemotherapy-induced cytopenia and complicating febrile neutropenia. In vitro studies demonstrated that leptin increased AML blast release of interleukin (IL) 1beta,IL6,tumor necrosis factor (TNF) alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF). This enhancing effect showed no correlation with CD34 expression and was not dependent on the presence of serum,induction of differentiation or alteration of caspase 3 activity with decreased in vitro apoptosis. Leptin also increased spontaneous AML blast proliferation,whereas divergent effects on blast proliferation were observed in the presence of exogenous cytokines. The in vitro effects were usually observed at concentrations exceeding the systemic levels. INTERPRETATION AND CONCLUSIONS: Our results suggest that systemic leptin levels alone do not have a major influence on native AML blasts,but the systemic levels in combination with local leptin release in the bone marrow may affect the functional characteristics of these cells. View Publication

The lysophospholipids,lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P),are now recognized as important extracellular signaling molecules. These lipid mediators are pleiotropic; among the most common cellular responses are mitogenesis,cell survival (anti-apoptosis),inhibition of adenylyl cyclase and calcium mobilization. Physiologic events associated with these mediators include platelet aggregation,vasopressor activity,wound healing,immune modulation,and angiogenesis. Many of the actions of LPA and S1P are mediated through a set of eight G protein-coupled receptors. Five of these are S1P-prefering while the remaining three are LPA receptors. These receptors are expressed widely and in aggregate signal through a variety of heterotrimeric G proteins. The lysophospholipid receptor family is referred to commonly as the Edg" group (e.g.� View Publication

The disease diabetes mellitus arises as a consequence of a failure of the beta-cells in the islets of Langerhans of the pancreas to produce insulin in the amounts required to meet the needs of the body. Whole pancreas or islet transplants in patients with severe diabetes effectively restore insulin production. A lack of availability of donor pancreata requires the development of alternative sources of islets such as the ex vivo culture and differentiation of stem/progenitor cells. Earlier we discovered multipotential progenitor cells in islets isolated from adult human pancreata that express the neural stem cell marker nestin: nestin-positive islet-derived progenitor cells (NIPs). Recently it was shown that the exclusion of the Hoechst 33342 dye,which defines the pluripotential side population (SP) of hematopoietic stem cells,is mediated by the ATP-binding cassette transporter,ABCG2. Here we report that the human islet-derived NIPs contain a substantial subpopulation of SP cells that co-express ABCG2,MDR1,and nestin. Thus NIPs may be a potential source of adult pluripotential stem/progenitor cells useful for the production of islet tissue for transplantation into diabetic subjects. View Publication

Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor beta (TGF-beta) is a member of a large family of pleiotropic cytokines that are involved in many biological processes,including growth control,differentiation,migration,cell survival,adhesion,and specification of developmental fate,in both normal and diseased states. TGF-beta superfamily members signal through a receptor complex comprising a type II and type I receptor,both serine/threonine kinases. Here,we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-beta type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7,which are very highly related to ALK5 in their kinase domains. It has no effect on the other,more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this,we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-beta signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542,we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK,JNK,or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum. View Publication

Reactive oxygen (ROS) and nitrogen oxide (RNOS) species are produced as by-products of oxidative metabolism. A major function for ROS and RNOS is immunological host defense. Recent evidence indicate that ROS and RNOS may also function as signaling molecules. However,high levels of ROS and RNOS have been considered to potentially damage cellular macromolecules and have been implicated in the pathogenesis and progression of various chronic diseases. alpha-Lipoic acid and dihydrolipoic acid exhibit direct free radical scavenging properties and as a redox couple,with a low redox potential of -0.32 V,is a strong reductant. Several studies provided evidence that alpha-lipoic acid supplementation decreases oxidative stress and restores reduced levels of other antioxidants in vivo. However,there is also evidence indicating that alpha-lipoic acid and dihydrolipoic acid may exert prooxidant properties in vitro. alpha-Lipoic acid and dihydrolipoic acid were shown to promote the mitochondrial permeability transition in permeabilized hepatocytes and isolated rat liver mitochondria. Dihydrolipoic acid also stimulated superoxide anion production in rat liver mitochondria and submitochondrial particles. alpha-Lipoic acid was recently shown to stimulate glucose uptake into 3T3-L1 adipocytes by increasing intracellular oxidant levels and/or facilitating insulin receptor autophosphorylation presumably by oxidation of critical thiol groups present in the insulin receptor beta-subunit. Whether alpha-lipoic acid and/or dihydrolipoic acid-induced oxidative protein modifications contribute to their versatile effects observed in vivo warrants further investigation. View Publication

OBJECTIVE: The aim of this study was the preclinical evaluation of imatinib mesylate (Gleevec,formerly STI571) in conjunction with arsenic trioxide (As2O3,Trisenox) for the treatment of chronic myelogenous leukemia (CML). MATERIALS AND METHODS: Tetrazolium-based cell line proliferation assays (MTT assays) were performed to determine the cytotoxicity of As2O3 alone and in combination with imatinib. Cell lines tested in this study were Bcr-Abl-expressing cells (K562,MO7p210,32Dp210) and parental cells (MO7e,32D). Isobologram analysis was performed manually and using the median effect method. In vitro cytotoxicity also was determined in colony-forming assays using CML patient cells. Western blot analysis was performed to detect Bcr-Abl protein levels in K562 cells exposed to As2O3 at graded concentrations. Bcr-Abl protein level kinetics were correlated with cell viability (trypan blue count) and activated caspase-3 detected by flow cytometry. RESULTS: We show additive to synergistic cytotoxicity in Bcr-Abl+ cell lines depending on inhibitory concentrations and cell type. Results obtained by colony-forming assays confirmed the findings in cell line proliferation assays. Flow cytometric detection of activated caspase-3 revealed synergistic activity in K562 cells. Treatment of K562 cells with As2O3 alone led to down-regulation of Bcr-Abl protein within 24 hours,even at low doses. The decline of Bcr-Abl preceded activation of caspase-3 and the loss of viable cells. CONCLUSIONS: Favorable cytotoxicity and proapoptotic activity of imatinib in conjunction with As2O3 and specific down-regulation of Bcr-Abl protein levels by As2O3 in K562 cells indicate that As2O3 in combination with imatinib might be useful for circumventing resistance to imatinib monotherapy. View Publication

5-Azacytidine was first synthesized almost 40 years ago. It was demonstrated to have a wide range of anti-metabolic activities when tested against cultured cancer cells and to be an effective chemotherapeutic agent for acute myelogenous leukemia. However,because of 5-azacytidine's general toxicity,other nucleoside analogs were favored as therapeutics. The finding that 5-azacytidine was incorporated into DNA and that,when present in DNA,it inhibited DNA methylation,led to widespread use of 5-azacytidine and 5-aza-2'-deoxycytidine (Decitabine) to demonstrate the correlation between loss of methylation in specific gene regions and activation of the associated genes. There is now a revived interest in the use of Decitabine as a therapeutic agent for cancers in which epigenetic silencing of critical regulatory genes has occurred. Here,the current status of our understanding of the mechanism(s) by which 5-azacytosine residues in DNA inhibit DNA methylation is reviewed with an emphasis on the interactions of these residues with bacterial and mammalian DNA (cytosine-C5) methyltransferases. The implications of these mechanistic studies for development of less toxic inhibitors of DNA methylation are discussed. View Publication

Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies,ES cells can differentiate into derivatives of all 3 primary germ layers,including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart,such as atrial-like,ventricular-like,sinus nodal-like,and Purkinje-like cells,have been established. During differentiation,cardiac-specific genes as well as proteins,receptors,and ion channels are expressed in a developmental continuum,which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro gain-of-function" or "loss-of-function" genetic studies. Recently� View Publication

ZD6474 [N-(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine]is a potent,p.o. active,low molecular weight inhibitor of kinase insert domain-containing receptor [KDR/vascular endothelial growth factor receptor (VEGFR) 2] tyrosine kinase activity (IC(50) = 40 nM). This compound has some additional activity versus the tyrosine kinase activity of fms-like tyrosine kinase 4 (VEGFR3;IC(50) = 110 nM) and epidermal growth factor receptor (EGFR/HER1; IC(50) = 500 nM) and yet demonstrates selectivity against a range of other tyrosine and serine-threonine kinases. The activity of ZD6474 versus KDR tyrosine kinase translates into potent inhibition of vascular endothelial growth factor-A (VEGF)-stimulated endothelial cell (human umbilical vein endothelial cell) proliferation in vitro (IC(50) = 60 nM). Selective inhibition of VEGF signaling has been demonstrated in vivo in a growth factor-induced hypotension model in anesthetized rat: administration of ZD6474 (2.5 mg/kg,i.v.) reversed a hypotensive change induced by VEGF (by 63%) but did not significantly affect that induced by basic fibroblast growth factor. Once-daily oral administration of ZD6474 to growing rats for 14 days produced a dose-dependent increase in the femoro-tibial epiphyseal growth plate zone of hypertrophy,which is consistent with inhibition of VEGF signaling and angiogenesis in vivo. Administration of 50 mg/kg/day ZD6474 (once-daily,p.o.) to athymic mice with intradermally implanted A549 tumor cells also inhibited tumor-induced neovascularization significantly (63% inhibition after 5 days; P textless 0.001). Oral administration of ZD6474 to athymic mice bearing established (0.15-0.47 cm(3)),histologically distinct (lung,prostate,breast,ovarian,colon,or vulval) human tumor xenografts or after implantation of aggressive syngeneic rodent tumors (lung,melanoma) in immunocompetent mice,produced a dose-dependent inhibition of tumor growth in all cases. Statistically significant antitumor activity was evident in each model with at least 25 mg/kg ZD6474 once daily (P textless 0.05,one-tailed t test). Histological analysis of Calu-6 tumors treated with 50 mg/kg/day ZD6474 for 24 days showed a significant reduction (textgreater70%) in CD31 (endothelial cell) staining in nonnecrotic regions. ZD6474 also restrained growth of much larger (0.9 cm(3) volume) Calu-6 lung tumor xenografts and induced profound regression in established PC-3 prostate tumors of 1.4 cm(3) volume. ZD6474 is currently in Phase I clinical development as a once-daily oral therapy in patients with advanced cancer. View Publication

Regulatory T cells (T(reg)) that prevent autoimmune diseases by suppression of self-reactive T cells may also suppress the immune response against cancer. In mice,depletion of T(reg) by Ab therapy leads to more efficient tumor rejection. T(reg)-mediated suppression of antitumor immune responses may partly explain the poor clinical response to vaccine-based immunotherapy for human cancer. In this study,we measured the prevalence of T(reg) that coexpress CD4 and CD25 in the PBLs,tumor-infiltrating lymphocytes,and regional lymph node lymphocytes from 65 patients with either pancreas or breast cancer. In breast cancer patients (n = 35),pancreas cancer patients (n = 30),and normal donors (n = 35),the prevalence of T(reg) were 16.6% (SE 1.22),13.2% (SE 1.13),and 8.6% (SE 0.71) of the total CD4(+) cells,respectively. The prevalence of T(reg) were significantly higher in breast cancer patients (p textless 0.01) and pancreas cancer patients (p textless 0.01) when compared with normal donors. In tumor-infiltrating lymphocytes and lymph node lymphocytes,the T(reg) prevalence were 20.2% (SE 3.93) and 20.1% (SE 4.3),respectively. T(reg) constitutively coexpressed CTLA-4 and CD45RO markers,and secreted TGF-beta and IL-10 but did not secrete IFN-gamma. When cocultured with activated CD8(+) cells or CD4(+)25(-) cells,T(reg) potently suppressed their proliferation and secretion of IFN-gamma. We conclude that the prevalence of T(reg) is increased in the peripheral blood as well as in the tumor microenvironment of patients with invasive breast or pancreas cancers. These T(reg) may mitigate the immune response against cancer,and may partly explain the poor immune response against tumor Ags. View Publication

Mechanism-based inhibitors of enzymes,which mimic reactive intermediates in the reaction pathway,have been deployed extensively in the analysis of metabolic pathways and as candidate drugs. The inhibition of cytosine-[C5]-specific DNA methyltransferases (C5 MTases) by oligodeoxynucleotides containing 5-azadeoxycytidine (AzadC) and 5-fluorodeoxycytidine (FdC) provides a well-documented example of mechanism-based inhibition of enzymes central to nucleic acid metabolism. Here,we describe the interaction between the C5 MTase from Haemophilus haemolyticus (M.HhaI) and an oligodeoxynucleotide duplex containing 2-H pyrimidinone,an analogue often referred to as zebularine and known to give rise to high-affinity complexes with MTases. X-ray crystallography has demonstrated the formation of a covalent bond between M.HhaI and the 2-H pyrimidinone-containing oligodeoxynucleotide. This observation enables a comparison between the mechanisms of action of 2-H pyrimidinone with other mechanism-based inhibitors such as FdC. This novel complex provides a molecular explanation for the mechanism of action of the anti-cancer drug zebularine. View Publication