技术资料

Histone deacetylase (HDAC) inhibitors (HDI) induce endoplasmic reticulum (ER) stress and apoptosis,while promoting autophagy,which promotes cancer cell survival when apoptosis is compromised. Here,we determined the in vitro and in vivo activity of the combination of the pan-HDI panobinostat and the autophagy inhibitor chloroquine against human estrogen/progesterone receptor and HER2 (triple)-negative breast cancer (TNBC) cells. Treatment of MB-231 and SUM159PT cells with panobinostat disrupted the hsp90/histone deacetylase 6/HSF1/p97 complex,resulting in the upregulation of hsp. This was accompanied by the induction of enhanced autophagic flux as evidenced by increased expression of LC3B-II and the degradation of the autophagic substrate p62. Treatment with panobinostat also induced the accumulation and colocalization of p62 with LC3B-II in cytosolic foci as evidenced by immunofluorescent confocal microscopy. Inhibition of panobinostat-induced autophagic flux by chloroquine markedly induced the accumulation of polyubiquitylated proteins and p62,caused synergistic cell death of MB-231 and SUM159PT cells,and inhibited mammosphere formation in MB-231 cells,compared with treatment with each agent alone. Finally,in mouse mammary fat pad xenografts of MB-231 cells,a tumor size-dependent induction of heat shock response,ER stress and autophagy were observed. Cotreatment with panobinostat and chloroquine resulted in reduced tumor burden and increased the survival of MB-231 breast cancer xenografts. Collectively,our findings show that cotreatment with an autophagy inhibitor and pan-HDI,for example,chloroquine and panobinostat results in accumulation of toxic polyubiquitylated proteins,exerts superior inhibitory effects on TNBC cell growth,and increases the survival of TNBC xenografts. View Publication

Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here,we show that three members of the Ccr4-Not complex,Cnot1,Cnot2,and Cnot3,play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts,these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs,Cnot1,Cnot2,and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead,they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly,these Cnot genes are also necessary for the maintenance of human ESCs,and silencing them mainly lead to TE and primitive endoderm differentiation. Together,our results indicate that Cnot1,Cnot2,and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs. View Publication

In vitro exposure of neural progenitor cell (NPC) populations to reduced O(2) (e.g. 3% versus 20%) can increase their proliferation,survival and neuronal differentiation. Our objective was to determine if an acute (textless1hr),in vivo exposure to intermittent hypoxia (AIH) alters expansion and/or differentiation of subsequent in vitro cultures of NPC from the subventricular zone (SVZ). Neonatal C57BL/6 mice (postnatal day 4) were exposed to an AIH paradigm (20×1 minute; alternating 21% and 10% O(2)). Immediately after AIH,SVZ tissue was isolated and NPC populations were cultured and assayed either as neurospheres (NS) or as adherent monolayer cells (MASC). AIH markedly increased the capacity for expansion of cultured NS and MASC,and this was accompanied by increases in a proliferation maker (Ki67),MTT activity and hypoxia-inducible factor-1α (HIF-1α) signaling in NS cultures. Peptide blockade experiments confirmed that proteins downstream of HIF-1α are important for both proliferation and morphological changes associated with terminal differentiation in NS cultures. Finally,immunocytochemistry and Western blotting experiments demonstrated that AIH increased expression of the neuronal fate determination transcription factor Pax6 in SVZ tissue,and this was associated with increased neuronal differentiation in cultured NS and MASC. We conclude that in vivo AIH exposure can enhance the viability of subsequent in vitro SVZ-derived NPC cultures. AIH protocols may therefore provide a means to prime" NPC prior to transplantation into the injured central nervous system." View Publication

R848,also known as resiquimod,acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study,we examined the effects of R848 on differentiation,survival,and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition,it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However,R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6,IL-12,interferon (IFN)-γ,and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-β was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-β to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7. View Publication

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice,while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover,gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice,resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells,and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline. View Publication

The anti-diabetic drug metformin is rapidly emerging as a potential anti-cancer agent. Metformin,effective in treating type 2 diabetes and the insulin resistance syndromes,improves insulin resistance by reducing hepatic gluconeogenesis and by enhancing glucose uptake by skeletal muscle. Epidemiological studies have consistently associated metformin use with decreased cancer incidence and cancer-related mortality. Furthermore,numerous preclinical and clinical studies have demonstrated anti-cancer effects of metformin,leading to an explosion of interest in evaluating this agent in human cancer. The effects of metformin on circulating insulin levels indicate a potential efficacy towards cancers associated with hyperinsulinaemia; however,metformin may also directly inhibit tumour growth. In this review,we describe the mechanism of action of metformin and summarise the epidemiological,clinical and preclinical evidence supporting a role for metformin in the treatment of cancer. In addition,the challenges associated with translating preclinical results into therapeutic benefit in the clinical setting will be discussed. View Publication

MicroRNAs (miRNA) comprise a group of short ribonucleic acid molecules implicated in regulation of key biological processes and functions at the post-transcriptional level. Ionizing radiation (IR) causes DNA damage and generally triggers cellular stress response. However,the role of miRNAs in IR-induced response in human embryonic stem cells (hESC) has not been defined yet. Here,by using system biology approaches,we show for the first time,that miRNAome undergoes global alterations in hESC (H1 and H9 lines) after IR. Interrogation of expression levels of 1,090 miRNA species in irradiated hESC showed statistically significant changes in 54 genes following 1 Gy of X-ray exposures; global miRNAome alterations were found to be highly temporally and cell line--dependent in hESC. Time-course studies showed that the 16 hr miRNAome radiation response of hESC is much more robust compared to 2 hr-response signature (only eight genes),and may be involved in regulating the cell cycle. Quantitative real-time PCR performed on some miRNA species confirms the robustness of our miRNA microarray platform. Positive regulation of differentiation-,cell cycle-,ion transport- and endomembrane system-related processes were predicted to be negatively affected by miRNAome changes in irradiated hESC. Our findings reveal a fundamental role of miRNAome in modulating the radiation response,and identify novel molecular targets of radiation in hESC. View Publication

The identification of succinate dehydrogenase (SDH),fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate,respectively,which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes,including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2,which catalyse the interconversion of isocitrate and 2-OG,are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG,but not (S)-2HG,stimulates EGLN activity,leading to diminished HIF levels,which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy. View Publication

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However,HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C,in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29,CD73,CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2),OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also,the cells showed high expression of MSC markers CD29,CD73,CD44 and CD105,but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion,HUCB is a good source of MSC using this new technique. View Publication

γ-Secretase-mediated cleavage of amyloid precursor protein (APP) results in the production of Alzheimer disease-related amyloid-β (Aβ) peptides. The Aβ42 peptide in particular plays a pivotal role in Alzheimer disease pathogenesis and represents a major drug target. Several γ-secretase modulators (GSMs),such as the nonsteroidal anti-inflammatory drugs (R)-flurbiprofen and sulindac sulfide,have been suggested to modulate the Alzheimer-related Aβ production by targeting the APP. Here,we describe novel GSMs that are selective for Aβ modulation and do not impair processing of Notch,EphB2,or EphA4. The GSMs modulate Aβ both in cell and cell-free systems as well as lower amyloidogenic Aβ42 levels in the mouse brain. Both radioligand binding and cellular cross-competition experiments reveal a competitive relationship between the AstraZeneca (AZ) GSMs and the established second generation GSM,E2012,but a noncompetitive interaction between AZ GSMs and the first generation GSMs (R)-flurbiprofen and sulindac sulfide. The binding of a (3)H-labeled AZ GSM analog does not co-localize with APP but overlaps anatomically with a γ-secretase targeting inhibitor in rodent brains. Combined,these data provide compelling evidence of a growing class of in vivo active GSMs,which are selective for Aβ modulation and have a different mechanism of action compared with the original class of GSMs described. View Publication

The ability of somatic cells to reprogram their ATP-generating machinery into a Warburg-like glycolytic metabotype while overexpressing stemness genes facilitates their conversion into either induced pluripotent stem cells (iPSCs) or tumor-propagating cells. AMP-activated protein kinase (AMPK) is a metabolic master switch that senses and decodes intracellular changes in energy status; thus,we have evaluated the impact of AMPK activation in regulating the generation of iPSCs from nonstem cells of somatic origin. The indirect and direct activation of AMPK with the antidiabetic biguanide metformin and the thienopyridone A-769662,respectively,impeded the reprogramming of mouse embryonic and human diploid fibroblasts into iPSCs. The AMPK activators established a metabolic barrier to reprogramming that could not be bypassed,even through p53 deficiency,a fundamental mechanism to greatly improve the efficiency of stem-cell production. Treatment with metformin or A-769662 before the generation of iPSC colonies was sufficient to drastically decrease iPSC generation,suggesting that AMPK activation impedes early stem cell genetic reprogramming. Monitoring the transcriptional activation status of each individual reprogramming factor (i.e.,Oct4,Sox2,Klf4 and c-Myc) revealed that AMPK activation notably prevented the transcriptional activation of Oct4,the master regulator of the pluripotent state. AMPK activation appears to impose a normalized metabolic flow away from the required pro-immortalizing glycolysis that fuels the induction of stemness and pluripotency,endowing somatic cells with an energetic infrastructure that is protected against reprogramming. AMPK-activating anti-reprogramming strategies may provide a roadmap for the generation of novel cancer therapies that metabolically target tumor-propagating cells. View Publication

Targeted FISH analysis is an essential component of the management of plasma cell myeloma for identification of cytogenetic abnormalities. The purpose of this study was to evaluate the column-free method,RoboSep® (RS),for sorting CD138-expressing cells in bone marrow aspirates. Comparative analysis of column-based and RS methodologies was carried out on 54 paired bone marrow aspirate validation samples from patients undergoing work-up for plasma cell dyscrasia. Abnormalities detected by FISH analysis using an IGH@/CCND1 probe set were seen in 54% with RS,and 44% with column-based. We found a statistically significant difference between the yield of abnormalities detected in paired positive cases (p = 0.0001). An additional 183 consecutive post-validation samples sorted by RS showed recurrent genetic abnormalities in 85/120 (71%) of successfully sorted samples with ≥ 1% plasma cells but in none of 63 samples in which FISH analysis was completed on samples that could not be sorted due to insufficient plasma cells upon cell sorting. The column-free method successfully sorted PC,when present in ≥ 1% of cells,for detection of abnormalities by FISH. Furthermore,our data suggest that FISH analysis should not be performed on samples with an inadequate yield at the cell selection step. View Publication