参考文献

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055,an ATP-competitive mTOR inhibitor,by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors,including RMS. Therefore,in the present study,we searched for AZD8055-based combination therapies. Here,we identify a new synergistic lethality of AZD8055 together with ABT-737,a BH3 mimetic that antagonizes Bcl-2,Bcl-xL,and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index textless 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level,as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential,activation of caspases,and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055,the PI3K/mTOR inhibitor NVP-BEZ235,the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis,whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly,molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly,knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737,our findings have important implications for the development of molecular targeted therapies for RMS. View Publication

Gene silencing agents such as small interfering RNA (siRNA) and microRNA offer the promise to modulate expression of almost every gene for the treatment of human diseases including cancer. However,lack of vehicles for effective systemic delivery to the disease organs has greatly limited their in vivo applications. In this study,we developed a high capacity polycation-functionalized nanoporous silicon (PCPS) platform comprised of nanoporous silicon microparticles functionalized with arginine-polyethyleneimine inside the nanopores for effective delivery of gene silencing agents. Incubation of MDA-MB-231 human breast cancer cells with PCPS loaded with STAT3 siRNA (PCPS/STAT3) or GRP78 siRNA (PCPS/GRP78) resulted in 91 and 83% reduction of STAT3 and GRP78 gene expression in vitro. Treatment of cells with a microRNA-18a mimic in PCPS (PCPS/miR-18) knocked down 90% expression of the microRNA-18a target gene ATM. Systemic delivery of PCPS/STAT3 siRNA in murine model of MDA-MB-231 breast cancer enriched particles in tumor tissues and reduced STAT3 expression in cancer cells,causing significant reduction of cancer stem cells in the residual tumor tissue. At the therapeutic dosage,PCPS/STAT3 siRNA did not trigger acute immune response in FVB mice,including changes in serum cytokines,chemokines,and colony-stimulating factors. In addition,weekly dosing of PCPS/STAT3 siRNA for four weeks did not cause signs of subacute toxicity based on changes in body weight,hematology,blood chemistry,and major organ histology. Collectively,the results suggest that we have developed a safe vehicle for effective delivery of gene silencing agents. View Publication

The optical isomers of the 1,4-dihydropyridine BAY K 8644 were studied in isolated rabbit aorta and heart preparations. The (-)-enantiomer has the known vasoconstricting and positive inotropic properties of the Ca agonistic compound. In contrast,its antipode shows at about 10-50 times higher concentrations the vasodilating and negative inotropic effects of Ca antagonistic drugs. It is concluded that neither simple chemical nor physical actions can be responsible for the opposite effects of Ca antagonistic and Ca agonistic dihydropyridines. View Publication

Neutrophils are recruited from the blood to sites of inflammation,where they contribute to immune defense but may also cause tissue damage. During inflammation,neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function-associated antigen 1 (LFA-1),which can be induced by selectin engagement. Here,we demonstrate that a subset of P-selectin glycoprotein ligand-1 (PSGL-1) molecules is constitutively associated with L-selectin. Although this association does not require the known lectin-like interaction between L-selectin and PSGL-1,the signaling output is dependent on this interaction and the cytoplasmic tail of L-selectin. The PSGL-1-L-selectin complex signals through Src family kinases,ITAM domain-containing adaptor proteins,and other kinases to ultimately result in LFA-1 activation. The PSGL-1-L-selectin complex-induced signaling effects on neutrophil slow rolling and recruitment in vivo demonstrate the functional importance of this pathway. We conclude that this is a signaling complex specialized for sensing adhesion under flow. View Publication

Asymmetry of cell fate is one fundamental property of stem cells,in which one daughter cell self-renews,whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms,such as plants,fungi,and mammals,has already been shown. However,before this current work,asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs),and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing,differentiating human and mouse ESCs. Moreover,we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3),indicating a molecular mechanism that regulates this phenomenon. Furthermore,our data support the hypothesis that retention of chromatids with the old" template DNA preserves the epigenetic memory of cell fate� View Publication

A metabolic biomarker-based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery-phase detection of potential human developmental toxicants. In this study,metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure-based biomarker assay using these metabolites,along with a cytotoxicity endpoint,was then developed using a 9-point dose–response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity,an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy,but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity,100% specificity). The assay had a high concordance (≥75%) with existing in vivo models,demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests. View Publication

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies,disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT),an inherited form of fatal arrhythmia. Here,we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs,both in resting conditions and after $\$-adrenergic stimulation,resembling the cardiac phenotype of the patients. Furthermore,treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine),an antiarrhythmic drug that inhibits Ca(2+)/calmodulin-dependent serine-threonine protein kinase II (CaMKII),drastically reduced the presence of DADs in CVPT-CMs,rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition,intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients,whereas in the wild-type clusters,only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice,the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells,supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies. View Publication

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors,such as SOX2,may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis,i.e.,the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state,adds new layers of complexity to cancer biology,because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis,we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor $$ (ER$$)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state,such as strong aldehyde dehydrogenase activity,as detected by ALDEFLUOR,and overexpression of the SSEA-4 and CD44 breast CSC markers,the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor $$ (ER$$),which is a pivotal integrator of the genomic and nongenomic E 2/ER$$ signaling pathways,drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover,SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells,and the highest levels of phospho-Ser118-ER$$ occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E 2/ER$$ signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression,i.e.,SOX2 can promote non-genomic E 2 signaling that leads to nuclear phospho-Ser118-ER$$,which ultimately exacerbates genomic ER signaling in response to E 2. Because E 2 stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140,which targets SOX2,the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator,SOX2,and the local and systemic ability of E 2 to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy. View Publication

Background The AAVS1 locus is viewed as a ‘safe harbor' for transgene insertion into human genome. In the present study,we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected,stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting,25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette,we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Conclusions Given high targeting efficiency,stability in expression of inserted transgene and flexibility in transgene exchange,the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research,drug development,regenerative medicine and gene therapy. Copyright textcopyright 2013 John Wiley & Sons,Ltd. View Publication

Maternal-effect mutations in NLRP7 cause rare biparentally inherited hydatidiform moles (BiHMs),abnormal pregnancies containing hypertrophic vesicular trophoblast but no embryo. BiHM trophoblasts display abnormal DNA methylation patterns affecting maternally methylated germline differentially methylated regions (gDMRs),suggesting that NLRP7 plays an important role in reprogramming imprinted gDMRs. How NLRP7—a component of the CATERPILLAR family of proteins involved in innate immunity and apoptosis—causes these specific DNA methylation and trophoblast defects is unknown. Because rodents lack NLRP7,we used human embryonic stem cells to study its function and demonstrate that NLRP7 interacts with YY1,an important chromatin-binding factor. Reduced NLRP7 levels alter DNA methylation and accelerate trophoblast lineage differentiation. NLRP7 thus appears to function in chromatin reprogramming and DNA methylation in the germline or early embryonic development,functions not previously associated with members of the NLRP family. View Publication

AIMS: Mesenchymal stem cells (MSCs) are widely used for cell therapy,particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity,critical elements for their survival and differentiation,have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and,more generally,to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. METHODS AND RESULTS: MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes,a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand,A-769662,a well-characterized AMPK activator,was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice,providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation,we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved,whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. CONCLUSION: MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation,p27 being involved in this regulation. View Publication

Aldehyde dehydrogenase (ALDH) has been identified in stem cells from both normal and cancerous tissues. This study aimed to evaluate the potential of ALDH as a universal brain tumour initiating cell (BTIC) marker applicable to primary brain tumours and their biological role in maintaining stem cell status. Cells from various primary brain tumours (24paediatric and 6 adult brain tumours) were stained with Aldefluor and sorted by flow cytometry. We investigated the impact of ALDH expression on BTIC characteristics in vitro and on tumourigenic potential in vivo. Primary brain tumours showed universal expression of ALDH,with 0.3-28.9% of the cells in various tumours identified as ALDH(+). The proportion of CD133(+) cells within ALDH(+) is higher than ALDH cells. ALDH(+) cells generate neurospheres with high proliferative potential,express neural stem cell markers and differentiate into multiple nervous system lineages. ALDH(+) cells tend to show high expression of induced pluripotent stem cell-related genes. Notably,targeted knockdown of ALDH1 by shRNA interference in BTICs potently disturbed their self-renewing ability. After 3months,ALDH(+) cells gave rise to tumours in 93% of mice whereas ALDH cells did not. The characteristic pathology of mice brain tumours from ALDH(+) cells was similar to that of human brain tumours,and these cells are highly proliferative in vivo. Our data suggest that primary brain tumours contain distinct subpopulations of cells that have high expression levels of ALDH and BTIC characteristics. ALDH might be a potential therapeutic target applicable to primary brain tumours. View Publication