搜索结果: 'gmp t cell activator'

Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK,its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491,whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly,their activities in cell lines were highly divergent. Unlike PHA-767491,XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines,this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors,we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491,including Chk1 and PKR kinase inhibitors,but had divergent chemical scaffolds from known DDK inhibitors. Taken together,these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific,biologically active DDK inhibitors for use as chemotherapeutic agents. View Publication

The HOX family of homeobox genes plays an important role in normal and malignant hematopoiesis. Dysregulated HOX gene expression profoundly effects the proliferation and differentiation of hematopoietic stem cells (HSCs) and committed progenitors,and aberrant activation of HOX genes is a common event in human myeloid leukemia. HOXB6 is frequently overexpressed in human acute myeloid leukemia (AML). To gain further insight into the role of HOXB6 in hematopoiesis,we overexpressed HOXB6 in murine bone marrow using retrovirus-mediated gene transfer. We also explored structure-function relationships using mutant HOXB6 proteins unable to bind to DNA or a key HOX-binding partner,pre-B-cell leukemia transcription factor-1 (PBX1). Additionally,we investigated the potential cooperative interaction with myeloid ecotropic viral integration site 1 homolog (MEIS1). In vivo,HOXB6 expanded HSCs and myeloid precursors while inhibiting erythropoiesis and lymphopoiesis. Overexpression of HOXB6 resulted in AML with a median latency of 223 days. Coexpression of MEIS1 dramatically shortened the onset of AML. Cytogenetic analysis of a subset of HOXB6-induced AMLs revealed recurrent deletions of chromosome bands 2D-E4,a region frequently deleted in HOXA9-induced AMLs. In vitro,HOXB6 immortalized a factor-dependent myelomonocytic precursor capable of granulocytic and monocytic differentiation. These biologic effects of HOXB6 were largely dependent on DNA binding but independent of direct interaction with PBX1. View Publication

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined,reliable,and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC),as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein,vitronectin (VN),or laminin (LN) have been shown to support hPSC expansion in a static environment. However,they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology,consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein,cell attachment efficiency and cell spreading are improved,thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates,which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 $\$ during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 $\$ indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation,whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines,thus confirming the robustness of this scalable expansion process in a defined environment. View Publication

Although modulation of protein levels is an important tool for study of protein function,it is difficult or impossible to knockdown or knockout genes that are critical for cell growth or viability. For such genes,a conditional knockdown approach would be valuable. The FKBP protein-based destabilization domain (DD)-tagging approach,which confers instability to the tagged protein in the absence of the compound Shield-1,has been shown to provide rapid control of protein levels determined by Shield-1 concentration. Although a strategy to knock-in DD-tagged protein at the endogenous loci has been employed in certain parasite studies,partly due to the relative ease of knock-in as a result of their mostly haploid lifecycles,this strategy has not been demonstrated in diploid or hyperploid mammalian cells due to the relative difficulty of achieving complete knock-in in all alleles. The recent advent of CRISPR/Cas9 homing endonuclease-mediated targeted genome cleavage has been shown to allow highly efficient homologous recombination at the targeted locus. We therefore assessed the feasibility of using CRISPR/Cas9 to achieve complete knock-in to DD-tag the essential gene Treacher Collins-Franceschetti syndrome 1 (TCOF1) in human 293T cells. Using a double antibiotic selection strategy to select clones with at least two knock-in alleles,we obtained numerous complete knock-in clones within three weeks of initial transfection. DD-TCOF1 expression in the knock-in cells was Shield-1 concentration-dependent,and removal of Shield-1 resulted in destabilization of DD-TCOF1 over the course of hours. We further confirmed that the tagged TCOF1 retained the nucleolar localization of the wild-type untagged protein,and that destabilization of DD-TCOF1 resulted in impaired cell growth,as expected for a gene implicated in ribosome biogenesis. CRISPR/Cas9-mediated homologous recombination to completely knock-in a DD tag likely represents a generalizable and efficient strategy to achieve rapid modulation of protein levels in mammalian cells. View Publication

BACKGROUND: Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients,eliminate virus infections,then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs),they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs,but there was little apparent expansion of these cells in patients. In that study,VSTs were gene-modified on day 19 of culture and we hypothesized that by this time,sufficient T-cell differentiation may have occurred to limit the subsequent proliferative capacity of the transduced T-cells. To facilitate the clinical testing of this hypothesis in a project supported by the NHLBI-PACT mechanism,we developed and optimized a good manufacturing practices (GMP) compliant method for the early transduction of VSTs directed to Epstein-Barr virus (EBV),Adenovirus (AdV) and cytomegalovirus (CMV) using a CAR directed to the tumor-associated antigen disialoganglioside (GD2). RESULTS: Ad-CMVpp65-transduced EBV-LCLs effectively stimulated VSTs directed to all three viruses (triVSTs). Transduction efficiency on day three was increased in the presence of cytokines and high-speed centrifugation of retroviral supernatant onto retronectin-coated plates,so that under optimal conditions up to 88% of tetramer-positive VSTs expressed the GD2.CAR. The average transduction efficiency of early-and late transduced VSTs was 55 ± 4% and 22 ± 5% respectively,and early-transduced VSTs maintained higher frequencies of T cells with central memory or intermediate memory phenotypes. Early-transduced VSTs also had higher proliferative capacity and produced higher levels of TH1 cytokines IL-2,TNF-α,IFN-γ,MIP-1α,MIP-1β and other cytokines in vitro. CONCLUSIONS: We developed a rapid and GMP compliant method for the early transduction of multivirus-specific T-cells that allowed stable expression of high levels of a tumor directed CAR. Since a proportion of early-transduced CAR-VSTs had a central memory phenotype,they should expand and persist in vivo,simultaneously protecting against infection and targeting residual malignancy. This manufacturing strategy is currently under clinical investigation in patients receiving allogeneic HSCT for relapsed neuroblastoma and B-cell malignancies (NCT01460901 using a GD2.CAR and NCT00840853 using a CD19.CAR). View Publication

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation,and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI,and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I),we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI,we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation,and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together,our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation,and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. View Publication

All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When exposed to the pluripotent human embryonal carcinoma (EC) stem cell line,TERA2.cl.SP12,ATRA induces ectoderm differentiation and the formation of neuronal cell types. We have previously generated synthetic analogues of retinoic acid (EC23 and EC19) which also induce the differentiation of EC cells. Even though EC23 and EC19 have similar chemical structures,they have differing biochemical effects in terms of EC cell differentiation. EC23 induces neuronal differentiation in a manner similar to ATRA,whereas EC19 directs the cells to form epithelial-like derivatives. Previous MALDI-TOF MS analysis examined the response of TERA2.cl.SP12 cells after exposure to ATRA,EC23 and EC19 and further demonstrated the similarly in the effect of ATRA and EC23 activity whilst responses to EC19 were very different. In this study,we show that Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS) coupled with appropriate scatter correction and multivariate analysis can be used as an effective tool to further investigate the differentiation of human pluripotent stem cells and monitor the alternative affects different retinoid compounds have on the induction of differentiation. FT-IRMS detected differences between cell populations as early as 3 days of compound treatment. Populations of cells treated with different retinoid compounds could easily be distinguished from one another during the early stages of cell differentiation. These data demonstrate that FT-IRMS technology can be used as a sensitive screening technique to monitor the status of the stem cell phenotype and progression of differentiation along alternative pathways in response to different compounds. View Publication

A variety of factors produced by stromal fibroblasts,including Flt3-ligand (FL),interleukin-11 (IL-11),Steel factor (SF),and IL-7,have been implicated in stimulating the production of pre-B cells and myeloid cells from primitive hematopoietic precursors. To investigate their relative roles in this process,either as single-acting or synergistic agents,we compared the yield and types of cells produced after 2 weeks from small numbers of Sca-1+ Lin- (i.e.,B220-,Ly-1-,Gr-1-,and Ter-119-) day 14.5 murine fetal liver cells placed in stromal cell-free cultures containing all possible combinations of FL,SF,IL-7,and IL-11. None of these factors alone supported the production (or survival) of any cells beyond 1 week: only pairs of factors consisting of either FL or SF plus either IL-11 or IL-7 were effective in this regard,with FL plus IL-11 being the most potent pair (approximately 7 x 10(4) cells obtained per 100 Sca-1+ Lin- input cells). The maximum numbers of cells were produced in the presence of FL,IL-11,and IL-7: these included both B220+ and Mac-1+/Gr-1+ cells (approximately 10(6) and approximately 2 x 10(5),respectively,per 100 Sca-1+ Lin- input cells). Both of these lineages were also obtained with each of the other possible three-factor combinations,albeit with variable effectiveness. Omission of either FL or IL-7 caused the greatest reduction in the yield of B220+ cells (approximately 130-fold and approximately 80-fold,respectively). Omission of IL-11 and,to a lesser extent,FL caused the greatest reduction in the yield of Mac-1+/Gr-1+ cells (approximately 90-fold and approximately 3-fold,respectively). When fetal calf serum was replaced with a defined serum substitute,the out put of B220+ cells remained the same but myelopoiesis was consistently enhanced (approximately 5- to 20-fold). These findings support a model involving factor redundancy in the extracellular signals required to stimulate the production and amplification of both lymphoid and myeloid cells from early Sca-1+ Lin- cells. They also reveal quantitative differences in the abilities of different competent factor combinations to promote this process,which may be further modulated by the presence of undefined serum components. View Publication

Human embryonic stem cells (hESCs) exhibit unique cell cycle structure,self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells,but its role in hESCs remains unclear. Here,we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly,knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly,FOXM1 depletion sensitized hESCs to oxidative stress. Moreover,genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1,which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed a substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together,our findings provide the first evidence to support FOXM1 as an important regulator of cell cycle progression and defense against oxidative stress in hESCs. View Publication

Dpep is a cell-penetrating peptide that targets transcription factors ATF5,CEBPB and CEBPD to selectively suppress growth and survival of diverse tumor cell types in vitro and in vivo. Due to these actions and its apparent safety,the peptide has potential as a cancer therapeutic. How Dpep might be combined with other anti-cancer agents to achieve synergistic efficacy and to overcome possible peptide resistance has not been assessed in depth. Based on prior work indicating that Dpep promotes apoptotic cancer cell death and up-regulates multiple pro-apoptotic and tumor suppressor genes,we studied combinations of Dpep with ABT-263,a pro-apoptotic BCL2 family inhibitor,and decitabine,a hypomethylating drug. Combining Dpep with each agent alone or together synergistically suppressed the growth of a range of solid and liquid tumor cell types. Moreover,the combinations synergistically inhibited the growth of cells lines that were selected either in vivo or in vitro for Dpep resistance. Finally,we tested the combination of Dpep with ABT-263 in a mouse melanoma xenograft model. The combination more effectively inhibited tumor growth than either agent alone and,in contrast to vehicle or ABT-263,produced a 40% durable survival rate. Taken together,these observations highlight potential drug partners for the therapeutic development of Dpep. View Publication

It is anticipated that gamma-secretase inhibitors (gamma-Sec-I) that modulate Notch processing will alter differentiation in tissues whose architecture is governed by Notch signaling. To explore this hypothesis,Han Wistar rats were dosed for up to 5 days with 10-100 micromol/kg b.i.d. gamma-Sec-I from three chemical series that inhibit Notch processing in vitro at various potencies (Notch IC(50)). These included an arylsulfonamide (AS) (142 nM),a dibenzazepine (DBZ) (1.7 nM),and a benzodiazepine (BZ) (2.2 nM). The DBZ and BZ caused dose-dependent intestinal goblet cell metaplasia. In contrast,the AS produced no detectable in vivo toxicity,despite higher exposure to free drug. In a time course using BZ,small intestinal crypt cell and large intestinal glandular cell epithelial apoptosis was observed on days 1-5,followed by goblet cell metaplasia on days 2-5 and crypt epithelial and glandular epithelial regenerative hyperplasia on days 4-5. Gene expression profiling of duodenal samples from BZ-dosed animals revealed significant time-dependent deregulation of mRNAs for various panendocrine,hormonal,and transcription factor genes. Somatostatin,secretin,mucin,CCK,and gastrin mRNAs were elevated twofold or more by day 2,and a number of candidate early-predictive" genes were altered on days 1-2� View Publication

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15,a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients,significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues,and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors,we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore,STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM,53BP1,and MDC1. Furthermore,protein levels of these DNA damage response molecules are reduced by IL-15,as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally,pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients. View Publication