技术资料

Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus,inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study,we investigated the effects of imetelstat (GRN163L),a potent telomerase inhibitor,on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro,telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally,imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat,but not control oligonucleotides,also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after textless4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice,concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat,suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. View Publication

Microglial cells play a dynamic role in the brain beyond their established function of immune surveillance. Activated microglia play key roles in neural development,neuroinflammation,neural repair and neurotoxicity. They are particularly important in several neurodegenerative diseases in which sustained microglial activation contributes to the progression of neurodegenerative processes. Consequently,understanding microglial function in CNS health and disease has become an area of active research in recent years. However,a significant obstacle to progress in this field has been the inherent difficulties in obtaining large amounts of primary microglial cells to routinely perform mechanistic studies and characterize signaling pathways regulating the dynamics of microglial activation. Herein,we describe a novel column-free magnetic separation protocol for high-yield isolation of primary microglia from mouse postnatal mixed glial cultures. The procedure is based on optimized culture conditions that enable high microglial cell densities in confluent mixed glial cultures followed by highly efficient recovery of pure microglia by magnetic separation. The novel column-free magnetic separation system utilizes tetrameric antibody complexes (TAC) with dual specificity for CD11b-PE labeled microglia and dextran magnetic nanoparticles. An FcR blocker (anti-CD16/32) is added to enhance the purity of the microglial separation by preventing non-specific labeling of other cell types. This procedure yields on average textgreater3×10�?� microglial cells per mouse pup,with a remarkable purity of 97% and recovery of around 87% of microglia from the mixed glial population. Importantly,the microglia obtained by this method are fully functional and respond like cells obtained by conventional isolation techniques. View Publication

To exploit the full potential of human pluripotent stem cells for regenerative medicine,developmental biology and drug discovery,defined culture conditions are needed. Media of known composition that maintain human embryonic stem (hES) cells have been developed,but finding chemically defined,robust substrata has proven difficult. We used an array of self-assembled monolayers to identify peptide surfaces that sustain pluripotent stem cell self-renewal. The effective substrates displayed heparin-binding peptides,which can interact with cell-surface glycosaminoglycans and could be used with a defined medium to culture hES cells for more than 3 months. The resulting cells maintained a normal karyotype and had high levels of pluripotency markers. The peptides supported growth of eight pluripotent cell lines on a variety of scaffolds. Our results indicate that synthetic substrates that recognize cell-surface glycans can facilitate the long-term culture of pluripotent stem cells. View Publication

The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines,such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics,consistent with the requirement for conformational change. Improving interactions in this allosteric pocket,as well as establishing binding interactions in the ATP pocket,enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series,BIRB 796,has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture. View Publication

Telomere length must be tightly regulated in highly proliferative tissues,such as the lymphohematopoietic system. Under steady-state conditions,the levels and functionality of hematopoietic-committed or multipotent progenitors were not affected in late-generation telomerase-deficient mice (mTerc(-/-)) with critically short telomeres. Evaluation of self-renewal potential of mTerc(-/-) day-12 spleen colony-forming units demonstrated no alteration as compared with wildtype progenitors. However,the replating ability of mTerc(-/-) granulocyte-macrophage CFUs (CFU-GMs) was greatly reduced as compared with wildtype CFU-GMs,indicating a diminished capacity of late-generation mTerc(-/-) committed progenitors when forced to proliferate. Long-term bone marrow cultures of mTerc(-/-) bone marrow (BM) cells show a reduction in proliferative capacity; this defect can be mainly attributed to the hematopoietic,not to the stromal,mTerc(-/-) cells. In serial and competitive transplantations,mTerc(-/-) BM stem cells show reduced long-term repopulating capacity,concomitant with an increase in genetic instability compared with wildtype cells. Nevertheless,in competitive transplantations late-generation mTerc(-/-) precursors can occasionally overcome this proliferative impairment and reconstitute irradiated recipients. In summary,our results demonstrate that late-generation mTerc(-/-) BM cells with short telomeres,although exhibiting reduced proliferation ability and reduced long-term repopulating capacity,can still reconstitute myeloablated animals maintaining stem cell function. View Publication

Overexpression of the breast cancer resistance protein (BCRP) efflux pump in human cancer cell lines results in resistance to a variety of cytostatic agents. The aim of this study was to analyze BCRP protein expression and activity in acute myeloid leukemia (AML) samples and to determine whether it is up-regulated due to clonal selection at relapse/refractory disease. BCRP protein expression was measured flow cytometrically with the monoclonal antibodies BXP-34 and BXP-21 in 20 paired samples of de novo and relapsed/refractory AML. BXP-34/immunoglobulin G1 ratios were observed of 1.6 +/- 0.5 (mean +/- SD,range 0.8-2.7) and BXP-21/immunoglobulin G2a ratios of 4.9 +/- 3.0 (range 1.1-14.5) in the patient samples versus 9.8 +/- 6.8 and 6.5 +/- 2.4,respectively,in the MCF-7 cell line. BCRP activity was determined flow cytometrically by measuring mitoxantrone accumulation in absence and presence of the inhibitor fumitremorgin C. Mitoxantrone accumulation,expressed as mean fluorescence intensity (MFI),varied between 44 and 761 MFI (227 +/- 146 MFI) and correlated inversely with BCRP expression (r = -0.58,P textless.001). Addition of fumitremorgin C showed a small increase in mitoxantrone accumulation (11 +/- 29 MFI,n = 40) apart from the effect of PSC833 and MK-571. No consistent up-regulation of BCRP expression or activity was observed at relapse/refractory disease; some cases showed an increase and other cases a decrease at relapse. Relatively high BCRP expression correlated with immature immunophenotype,as determined by expression of the surface marker CD34 (r = 0.54,P =.001). In conclusion,this study shows that BCRP protein is expressed at low but variable levels in AML,especially in immature CD34(+) cells. BCRP was not consistently up-regulated in relapsed/refractory AML. 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

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

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

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