技术资料

Four different classes of HDACs have been identified in humans so far. Classes I,II and IV are zinc-dependent amidohydrolases,while III is a family of phylogenetically conserved NAD-dependent protein deacetylases/ADP-ribosyltransferase with a well-defined role in modifying chromatin conformation and altering the accessibility of the damaged sites of DNA for repair enzymes. Sirtuins are histone deacetylases (HDACs) of class III that cleave off acetyl groups from acetyl-lysine residues in histones and non-histone proteins. As sirtuins are involved in many physiological and pathological processes,their activity has been associated with different human diseases,including cancer. Especially two sirtuin members,SIRT1 and SIRT2,have been found to antagonize p53-dependent transcriptional activation and apoptosis in response to DNA damage by catalyzing p53 deacetylation. The findings that SIRT1 levels are increased in a number of tumors highlight the oncogenic role of sirtuins,in particular,in the down-modulation of p53 oncosuppressor activity. Along this lane,cancers carrying wild-type (wt) p53 protein are known to deregulate its activity by other mechanisms. Therefore,inhibition of SIRT1 and SIRT2,aimed at restoring wt-p53 transcriptional activity in tumors that retain the ability to express normal p53,might represent a valid therapeutic cancer approach specially when combined with standard therapies. This review will be focused on sirtuin inhibitors,with a specific attention on inhibitors of SIRT1 and SIRT2. Among them,nicotinamide and its analogs,sirtinol,A3 and M15,splitomicin,HR73 and derivatives,cambinol and derivatives,EX 527,kinase inhibitors,suramin,4-dihydropyridine derivatives,tenovins,TRIPOS 360702,AC 93253,3-arylideneindolinones,CSC8 and CSC13 will also be described. View Publication

Neuron-specific in vitro screening strategies have the potential to accelerate the evaluation of chemicals for neurotoxicity. We examined neurite outgrowth as a measure of neuronal response with a commercially available rat cortex progenitor cell model,where cells were exposed to a chemical during a period of cell differentiation. In control cultures,the fraction of beta-III-tubulin positive neurons and their neurite length increased significantly with time,indicating differentiation of the progenitor cells. Expression of glial fibrillary acidic protein,an astrocyte marker,also increased significantly with time. By seeding progenitor cells at varying densities,we demonstrated that neurite length was influenced by cell-cell spacing. After ten days,cultures seeded at densities of 1000 cells/mm(2) or lower had significantly shorter neurites than cultures seeded at densities of 1250 cells/mm(2) or higher. Progenitor cells were exposed to lithium,a neuroactive chemical with diverse modes of action. Cultures exposed to 30 mmol/L or 10 mmol/L lithium chloride (LiCl) had significantly lower metabolic activity than control cultures,as reported by adenosine triphosphate content,and no neurons were observed after ten days of exposure. Cultures exposed to 3 mmol/L,1 mmol/L,or 0.3 mmol/L LiCl,which encompass lithium's therapeutic range,had metabolic activity similar to control cultures. These cultures exhibited concentration-dependent decreases in neurite outgrowth after ten days of LiCl exposure. Neurite outgrowth results were relatively robust,regardless of the evaluation methodology. This work demonstrates that measurement of neurite outgrowth in differentiating progenitor cell cultures can be a sensitive endpoint for neuronal response under non-cytotoxic exposure conditions. View Publication

Cytochrome b561 (Cyt-b561) proteins constitute a family of trans-membrane proteins that are present in a wide variety of organisms. Two of their characteristic properties are the reducibility by ascorbate (ASC) and the presence of two distinct b-type hemes localized on two opposite sides of the membrane. Here we show that the tonoplast-localized and the putative tumor suppressor Cyt-b561 proteins can be reduced by other reductants than ASC and dithionite. A detailed spectral analysis of the ASC-dependent and dihydrolipoic acid (DHLA)-dependent reduction of these two Cyt-b561 proteins is also presented. Our results are discussed in relation to the known antioxidant capability of DHLA as well as its role in the regeneration of other antioxidant compounds of cells. These results allow us to speculate on new biological functions for the trans-membrane Cyt-b561 proteins. View Publication

Osteoarthritis (OA) is a degenerative joint disease that involves the destruction of articular cartilage and eventually leads to disability. Molecules that promote the selective differentiation of multipotent mesenchymal stem cells (MSCs) into chondrocytes may stimulate the repair of damaged cartilage. Using an image-based high-throughput screen,we identified the small molecule kartogenin,which promotes chondrocyte differentiation (median effective concentration = 100 nM),shows chondroprotective effects in vitro,and is efficacious in two OA animal models. Kartogenin binds filamin A,disrupts its interaction with the transcription factor core-binding factor β subunit (CBFβ),and induces chondrogenesis by regulating the CBFβ-RUNX1 transcriptional program. This work provides new insights into the control of chondrogenesis that may ultimately lead to a stem cell-based therapy for osteoarthritis. View Publication

Recent studies have proposed that chronic treatment with antidepressants increases neurogenesis in the adult hippocampus. However,the effect of antidepressants on fetal neural stem cells (NSCs) has not been well defined. Our study shows the dose-dependent effects of fluoxetine on the proliferation and neural differentiation of NSCs. Fluoxetine,even at nanomolar concentrations,stimulated proliferation of NSCs and increased the number of βIII-tubulin (Tuj 1)- and neural nucleus marker (NeuN)-positive cells,but not glial fibrillary acidic protein (GFAP)-positive cells. These results suggest that fluoxetine can enhance neuronal differentiation. In addition,fluoxetine has protective effects against cell death induced by oligomeric amyloid beta (Aβ(42)) peptides. Taken together,these results clearly show that fluoxetine promotes both the proliferation and neuronal differentiation of NSCs and exerts protective effects against Aβ(42)-induced cytotoxicities in NSCs,which suggest that the use of fluoxetine is applicable for cell therapy for various neurodegenerative diseases,such as Alzheimer's and Parkinson's diseases by its actions in NSCs. View Publication

α-Lipoic acid (LA) is a thiol with antioxidant properties that protects against oxidative stress-induced apoptosis. LA is absorbed from the diet,taken up by cells and tissues,and subsequently reduced to dihydrolipoic acid (DHLA). In view of the recent application of DHLA as a hydrophilic nanomaterial preparation,determination of its biosafety profile is essential. In the current study,we examined the cytotoxic effects of DHLA on mouse embryos at the blastocyst stage,subsequent embryonic attachment and outgrowth in vitro,in vivo implantation by embryo transfer,and early embryonic development in an animal model. Blastocysts treated with 50 μM DHLA exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably,the implantation success rates of blastocysts pretreated with DHLA were lower than that of their control counterparts. Moreover,in vitro treatment with 50 μM DHLA was associated with increased resorption of post-implantation embryos and decreased fetal weight. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 100 μM DHLA led to decreased early embryo development,specifically,inhibition of development to the blastocyst stage. However,it appears that concentrations of DHLA lower than 50 μM do not exert a hazardous effect on embryonic development. Our results collectively indicate that in vitro and in vivo exposure to concentrations of DHLA higher than 50 μM DHLA induces apoptosis and retards early pre- and post-implantation development,and support the potential of DHLA to induce embryonic cytotoxicity. View Publication

Dental pulp is particularly interesting in regenerative medicine because of the accessibility and differentiation potential of the tissue. Dental pulp has an early developmental origin with multi-lineage differentiation potential as a result of its development during childhood and adolescence. However,no study has previously identified the presence of stem cell populations with embryonic-like phenotypes in human dental pulp from the third molar. In the present work,we describe a new population of dental pulp pluripotent-like stem cells (DPPSCs) that were isolated by culture in medium containing LIF,EGF and PDGF. These cells are SSEA4(+),OCT3/4(+),NANOG(+),SOX2(+),LIN28(+),CD13(+),CD105(+),CD34(-),CD45(-),CD90(+),CD29(+),CD73(+),STRO1(+) and CD146(-),and they show genetic stability in vitro based on genomic analysis with a newly described CGH technique. Interestingly,DPPSCs were able to form both embryoid-body-like structures (EBs) in vitro and teratoma-like structures that contained tissues derived from all three embryonic germ layers when injected in nude mice. We examined the capacity of DPPSCs to differentiate in vitro into tissues that have similar characteristics to mesoderm,endoderm and ectoderm layers in both 2D and 3D cultures. We performed a comparative RT-PCR analysis of GATA4,GATA6,MIXL1,NANOG,OCT3/4,SOX1 and SOX2 to determine the degree of similarity between DPPSCs,EBs and human induced pluripotent stem cells (hIPSCs). Our analysis revealed that DPPSCs,hIPSC and EBs have the same gene expression profile. Because DPPSCs can be derived from healthy human molars from patients of different sexes and ages,they represent an easily accessible source of stem cells,which opens a range of new possibilities for regenerative medicine. View Publication

Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors,nerve growth factor and brain-derived neurotrophic factor,share substantial amino acid sequence identity. We have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene,neurotrophin-3,that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain,suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development. View Publication

Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced,but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5),which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9),is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However,LSD1 is not essential for the maintenance of ESC identity. Instead,ESCs lacking LSD1 activity fail to differentiate fully,and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers,LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex,which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation,which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states. 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

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