技术资料

Long non-coding RNAs (lncRNAs) are a numerous class of newly discovered genes in the human genome,which have been proposed to be key regulators of biological processes,including stem cell pluripotency and neurogenesis. However,at present very little functional characterization of lncRNAs in human differentiation has been carried out. In the present study,we address this using human embryonic stem cells (hESCs) as a paradigm for pluripotency and neuronal differentiation. With a newly developed method,hESCs were robustly and efficiently differentiated into neurons,and we profiled the expression of thousands of lncRNAs using a custom-designed microarray. Some hESC-specific lncRNAs involved in pluripotency maintenance were identified,and shown to physically interact with SOX2,and PRC2 complex component,SUZ12. Using a similar approach,we identified lncRNAs required for neurogenesis. Knockdown studies indicated that loss of any of these lncRNAs blocked neurogenesis,and immunoprecipitation studies revealed physical association with REST and SUZ12. This study indicates that lncRNAs are important regulators of pluripotency and neurogenesis,and represents important evidence for an indispensable role of lncRNAs in human brain development. View Publication

The RSK (90 kDa ribosomal S6 kinase) family comprises a group of highly related serine/threonine kinases that regulate diverse cellular processes,including cell growth,proliferation,survival and motility. This family includes four vertebrate isoforms (RSK1,RSK2,RSK3 and RSK4),and single family member orthologues are also present in Drosophila and Caenorhabditis elegans. The RSK isoforms are downstream effectors of the Ras/ERK (extracellular-signal-regulated kinase) signalling pathway. Significant advances in the field of RSK signalling have occurred in the past few years,including several new functions ascribed to the RSK isoforms,the discovery of novel protein substrates and the implication of different RSK isoforms in cancer. Collectively,these new findings increase the diversity of biological functions regulated by RSK,and highlight potential new directions of research. In the present paper,we review the structure,expression and activation mechanisms of the RSK isoforms,and discuss their physiological roles on the basis of established substrates and recent discoveries. View Publication

Signaling mediates cellular responses to extracellular stimuli. The c-Jun NH(2)-terminal kinase (JNK) pathway exemplifies one subgroup of the mitogen-activated protein (MAP) kinases,which,besides having established functions in stress response,also contribute to development by an unknown mechanism. We show by genome-wide location analysis that JNK binds to a large set of active promoters during the differentiation of stem cells into neurons. JNK-bound promoters are enriched with binding motifs for the transcription factor NF-Y but not for AP-1. NF-Y occupies these predicted sites,and overexpression of dominant-negative NF-YA reduces the JNK presence on chromatin. We find that histone H3 Ser10 (H3S10) is a substrate for JNK,and JNK-bound promoters are enriched for H3S10 phosphorylation. Inhibition of JNK signaling in post-mitotic neurons reduces phosphorylation at H3S10 and the expression of target genes. These results establish MAP kinase binding and function on chromatin at a novel class of target genes during stem cell differentiation. View Publication

Symptomatic dengue virus infection ranges in disease severity from an influenza-like illness to life-threatening shock. One model of the mechanism underlying severe disease proposes that weakly neutralizing,dengue serotype cross-reactive antibodies induced during a primary infection facilitate virus entry into Fc receptor-bearing cells during a subsequent secondary infection,increasing viral replication and the release of cytokines and vasoactive mediators,culminating in shock. This process has been termed antibody-dependent enhancement of infection and has significantly hindered vaccine development. Much of our understanding of this process has come from studies using mouse monoclonal antibodies (MAbs); however,antibody responses in mice typically exhibit less complexity than those in humans. A better understanding of the humoral immune response to natural dengue virus infection in humans is sorely needed. Using a high-efficiency human hybridoma technology,we isolated 37 hybridomas secreting human MAbs to dengue viruses from 12 subjects years or even decades following primary or secondary infection. The majority of the human antibodies recovered were broadly cross-reactive,directed against either envelope or premembrane proteins,and capable of enhancement of infection in vitro; few exhibited serotype-specific binding or potent neutralizing activity. Memory B cells encoding enhancing antibodies predominated in the circulation,even two or more decades following infection. Mapping the epitopes and activity of naturally occurring dengue antibodies should prove valuable in determining whether the enhancing and neutralizing activity of antibodies can be separated. Such principles could be used in the rational design of vaccines that enhance the induction of neutralizing antibodies,while lowering the risk of dengue shock syndrome. View Publication

The MEK1 and MEK2 inhibitor GSK1120212 is currently in phase II/III clinical development. To identify predictive biomarkers,sensitivity to GSK1120212 was profiled for 218 solid tumor cell lines and 81 hematologic malignancy cell lines. For solid tumors,RAF/RAS mutation was a strong predictor of sensitivity. Among RAF/RAS mutant lines,co-occurring PIK3CA/PTEN mutations conferred a cytostatic response instead of a cytotoxic response for colon cancer cells that have the biggest representation of the comutations. Among KRAS mutant cell lines,transcriptomics analysis showed that cell lines with an expression pattern suggestive of epithelial-to-mesenchymal transition were less sensitive to GSK1120212. In addition,a proportion of cell lines from certain tissue types not known to carry frequent RAF/RAS mutations also seemed to be sensitive to GSK1120212. Among these were breast cancer cell lines,with triple negative breast cancer cell lines being more sensitive than cell lines from other breast cancer subtypes. We identified a single gene DUSP6,whose expression was associated with sensitivity to GSK1120212 and lack of expression associated with resistance irrelevant of RAF/RAS status. Among hematologic cell lines,acute myeloid leukemia and chronic myeloid leukemia cell lines were particularly sensitive. Overall,this comprehensive predictive biomarker analysis identified additional efficacy biomarkers for GSK1120212 in RAF/RAS mutant solid tumors and expanded the indication for GSK1120212 to patients who could benefit from this therapy despite the RAF/RAS wild-type status of their tumors. View Publication

A method for the generation of human induced pluripotent stem (iPS) cells was established. This method employs adenovirus carrying the ecotropic retrovirus receptor mCAT1 and Moloney murine leukemia virus (MMLV)-based retroviral vectors carrying the four transcription factors POU5F1 (OCT3/4),KLF4,SOX2,and MYC (c-Myc) (Masaki H & Ishikawa T Stem Cell Res 1:105-15,2007). The differentiation of human iPS cells into hepatic cells was performed by a stepwise protocol (Song Z et al. Cell Res 19:1233-42,2009). These cells have potential as patient-specific in vitro models for studying disease etiology and could be used in drug discovery programs tailored to deal with genetic variations in drug efficacy and toxicity. View Publication

Human pluripotent stem cells (hPSCs),including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs),share the properties of unlimited self-renewal and the capacity to become any cell type in the body,making them well suited for regenerative medicine and cell therapy. So far,almost all hPSC lines have been directly or indirectly exposed to animal-derived products,which would hinder their use for clinical purposes. One of the biggest challenges in this area is to remove animal components from the derivation,propagation,and cryopreservation of hPSCs. Moreover,the presence of undefined components of animal or human origin in culture system may interfere with the interpretation of the effect of exogenous agents on the growth and differentiation of hPSCs and are prone to significant variability. To explore hPSC expansion in defined,xeno-free conditions,2 different groups of culture systems were used to culture different hESC and hiPSC lines. Our results suggested that (1) medium,matrix,and exogenous factors have synergistic effects on the adhesion and growth of hPSCs; (2) cooperation of exogenous factors including basic fibroblast growth factor,Rho-associated kinase inhibitor (ROCK),and other growth factors is critical for hPSC adhesion and proliferation; (3) basal media have different effects on hPSC attachment to the culture surface; and (4) a medium or matrix component can work synergistically in one culture system,and not at all in another. In this study,we found that Vitronectin/TeSR2 and PDL/HEScGRO (Y-27632) systems were optimal for maintaining the long-term culture of 3 hESC lines and 2 hiPSC lines under defined,xeno-free conditions. View Publication

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation,and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and,with quantitative cell division tracking and fate monitoring,identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion,during directed differentiation,to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system. View Publication

There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB),used in the Aldeflour assay,has been considered a specific inhibitor for ALDH1A1 isoform. In this study,we explore the effects of human ALDH isoenzymes,ALDH1A2 and ALDH2,on drug resistance and proliferation,and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay,Western blot,RT-PCR,and Aldefluor assay. Both cell lines,with either ALDH1A2 or ALDH2,exhibited higher cell proliferation rates,higher clonal efficiency,and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors,DEAB and disulfiram,we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore,our TLDA results revealed that ALDH1,ALDH7,ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition,other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells. View Publication

Bone marrow (BM) hematopoietic cells are selectively sensitive to polycyclic aromatic hydrocarbons (PAH) in vivo. 7,12-Dimethylbenz(a)anthracene (DMBA),but not benzo(a)pyrene (BP),depletes BM hematopoietic cells in C57BL/6 mice. This difference is due to a BP-selective aryl hydrocarbon receptor (AhR)-mediated recovery. Colony-forming unit assays show suppression of lymphoid progenitors by each PAH within 6 h but a subsequent recovery,exclusively after BP treatment. Suppression of myeloid progenitors (6 h) occurs only for DMBA. Each progenitor responded equally to DMBA and BP in congenic mice expressing the PAH-resistant AhR (AhR(d)). AhR,therefore,mediates this BP recovery in each progenitor type. These PAH suppressions depend on Cyp1b1-mediated metabolism. Paradoxically,few genes responded to DMBA,whereas 12 times more responded to BP. Progenitor suppression by DMBA,therefore,occurs with minimal effects on the general BM population. Standard AhR-mediated stimulations (Cyp1a1,Cyp1b1,Ahrr) were similar for each PAH and for the specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin but were absent in AhR(d) mice. A group of 12 such AhR responses was sustained from 6 to 24 h. A second,larger set of BP responses (chemokines,cytokines,cyclooxygenase 2) differed in two respects; DMBA responses were low and BP responses declined extensively from 6 to 24 h. A third cluster exhibited BP-induced increases in protective genes (Nqo1,GST-mu) that appeared only after 12 h. Conversion of BP to quinones contributes oxidative signaling not seen with DMBA. We propose that genes in this second cluster,which share oxidative signaling and AhR activation,provide the AhR-dependent protection of hematopoietic progenitors seen for BP. View Publication

Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and,accordingly,ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes,however,inhibit ARF/p53 and only infrequently select for ARF or p53 mutations,suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK,the initiating oncogene of anaplastic large cell lymphomas (ALCLs),induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly,human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a,which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence,and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors. View Publication

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC),an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L,containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39,was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs,alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,triciribine,GDC-0941,and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice,replicated well in GSCs,and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,whereas their combination with MG18L synergized in killing GSCs and glioma cell lines,but not human astrocytes,through enhanced induction of apoptosis. Importantly,synergy was independent of inhibitor sensitivity. In vivo,the combination of MG18L and LY294002 significantly prolonged survival of mice,as compared with either agent alone,achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM,being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication