技术资料

Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins,chromosomal instability,or DNA damage is associated with p53/p21 activation,culminating in either senescence or apoptosis,depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH,in contrast to circulating pituitary-derived endocrine GH,has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway,with subsequent marked induction of intracellular pituitary GH in vitro. In contrast,GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo,treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland,as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence,likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary,in nonpituitary cells GH exerts antiapoptotic properties. Thus,the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence. View Publication

Replacing defective retinal pigment epithelial (RPE) cells with those derived from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) is a potential strategy for treating retinal degenerative diseases. Early clinical trials have demonstrated that hESC-derived or hiPSC-derived RPE cells can be delivered safely as a suspension to the human eye. The next step is transplantation of hESC/hiPSC-derived RPE cells as cell sheets that are more physiological. We have developed a tissue-engineered product consisting of hESC-derived RPE cells grown as sheets on human amniotic membrane as a biocompatible substrate. We established a surgical approach to engraft this tissue-engineered product into the subretinal space of the eyes of rats with photoreceptor cell loss. We show that transplantation of the hESC-RPE cell sheets grown on a human amniotic membrane scaffold resulted in rescue of photoreceptor cell death and improved visual acuity in rats with retinal degeneration compared to hESC-RPE cells injected as a cell suspension. These results suggest that tissue-engineered hESC-RPE cell sheets produced under good manufacturing practice conditions may be a useful approach for treating diseases of retinal degeneration. View Publication

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However,the cell types,their gene expression dynamics,and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types,including five subtypes of radial glia-like cells and four progenitors. In the mouse,two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species,but with clear differences in cell proliferation,developmental timing,and dopaminergic neuron development. Additionally,we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells,at a single-cell level. Thus,our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies. View Publication

Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous neoplasm in which JAK2 inhibition has demonstrated reductions in inflammatory cytokines and promising clinical activity. We hypothesize that annotation of inflammatory cytokines may uncover mutation-independent cytokine subsets associated with novel CMML prognostic features. A Luminex cytokine profiling assay was utilized to profile cryopreserved peripheral blood plasma from 215 CMML cases from three academic centers,along with center-specific,age-matched plasma controls. Significant differences were observed between CMML patients and healthy controls in 23 out of 45 cytokines including increased cytokine levels in IL-8,IP-10,IL-1RA,TNF-alpha$,IL-6,MCP-1/CCL2,hepatocyte growth factor (HGF),M-CSF,VEGF,IL-4,and IL-2RA. Cytokine associations were identified with clinical and genetic features,and Euclidian cluster analysis identified three distinct cluster groups associated with important clinical and genetic features in CMML. CMML patients with decreased IL-10 expression had a poor overall survival when compared to CMML patients with elevated expression of IL-10 (P = 0.017),even when adjusted for ASXL1 mutation and other prognostic features. Incorporating IL-10 with the Mayo Molecular Model statistically improved the prognostic ability of the model. These established cytokines,such as IL-10,as prognostically relevant and represent the first comprehensive study exploring the clinical implications of the CMML inflammatory state. View Publication

CD8 T cells can play both a protective and pathogenic role in inflammation and autoimmune development. Recent studies have highlighted the ability of CD8 T cells to function as T follicular helper (Tfh) cells in the germinal center in the context of infection. However,whether this phenomenon occurs in autoimmunity and contributes to autoimmune pathogenesis is largely unexplored. In this study,we show that CD8 T cells acquire a CD4 Tfh profile in the absence of functional regulatory T cells in both the IL-2-deficient and scurfy mouse models. Depletion of CD8 T cells mitigates autoimmune pathogenesis in IL-2-deficient mice. CD8 T cells express the B cell follicle-localizing chemokine receptor CXCR5,a principal Tfh transcription factor Bcl6,and the Tfh effector cytokine IL-21. CD8 T cells localize to the B cell follicle,express B cell costimulatory proteins,and promote B cell differentiation and Ab isotype class switching. These data reveal a novel contribution of autoreactive CD8 T cells to autoimmune disease,in part,through CD4 follicular-like differentiation and functionality. View Publication

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K,which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 $\mu$mol/L (MCF7 cells) and 0.17 $\mu$mol/L (SW620 cells),and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 $\mu$mol/L (MCF7 cells) and more than 10 $\mu$mol/L (SW620 cells). Five-day exposure to 1 $\mu$mol/L KU-0060648 inhibited cell proliferation by more than 95{\%} in MCF7 cells but only by 55{\%} in SW620 cells. In clonogenic survival assays,KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells,but not in DNA-PKcs-deficient cells,thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts,concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold,without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors. View Publication

Yaffe and colleagues discuss the issues surrounding the authentication and quality of induced pluripotent stem cells. View Publication

Economic development has become a prominent issue for state governments. Nevertheless,states vary in the economic policies they choose. Two general approaches to the issue are discussed: the maintenance/attraction strategy and the creation strategy. Factor analysis allows us to gauge state effort on these two criteria. Regression analysis shows that political culture is an important factor in predicting which approach a state chooses,with traditionalistic states favoring the maintenance/attraction strategy,and moralistic states favoring the creation alternative. Other predictors of state policy choices include the condition of the economy and the diffusion of innovations. Also discussed is the interaction of political culture with other relevant variables in shaping state policies. View Publication

Here,we report that genome editing by CRISPR-Cas9 induces a p53-mediated DNA damage response and cell cycle arrest in immortalized human retinal pigment epithelial cells,leading to a selection against cells with a functional p53 pathway. Inhibition of p53 prevents the damage response and increases the rate of homologous recombination from a donor template. These results suggest that p53 inhibition may improve the efficiency of genome editing of untransformed cells and that p53 function should be monitored when developing cell-based therapies utilizing CRISPR-Cas9. View Publication

Both targeted therapy and immunotherapy have been used successfully to treat melanoma,but the development of resistance and poor response rates to the individual therapies has limited their success. Designing rational combinations of targeted therapy and immunotherapy may overcome these obstacles,but requires assessment in preclinical models with the capacity to respond to both therapeutic classes. Herein,we describe the development and characterization of a novel,immunogenic variant of the BrafV600ECdkn2a-/-Pten-/- YUMM1.1 tumor model that expresses the immunogen,ovalbumin (YOVAL1.1). We demonstrate that,unlike parental tumors,YOVAL1.1 tumors are immunogenic in vivo and can be controlled by immunotherapy. Importantly,YOVAL1.1 tumors are sensitive to targeted inhibitors of BRAFV600E and MEK,responding in a manner consistent with human BRAFV600E melanoma. The YOVAL1.1 melanoma model is transplantable,immunogenic and sensitive to clinical therapies,making it a valuable platform to guide strategic development of combined targeted therapy and immunotherapy approaches in BRAFV600E melanoma. View Publication

Laminin and fibronectin are glycoproteins that influence cell behavior and mediate cell/substratum adhesion. We have examined the interaction of these macromolecules with the serine protease plasminogen activator (PA) in two types of extracellular matrices; one produced by the murine Engelbreth-Holm-Swarm (EHS) tumor (Matrigel),and another by normal kidney epithelial cells in culture. Matrigel was found to contain significant quantities of tissue-type PA (tPA). Two of the major components of Matrigel,laminin and type IV collagen,were also examined. Tissue-type PA was associated with purified preparations of laminin; however,it was not found associated with type IV collagen. Normal kidney epithelial cells in culture secrete large amounts of urokinase (UK) and deposit a subepithelial matrix containing both laminin and fibronectin. These matrix macromolecules were isolated from the deposited matrix by immunoprecipitation,examined by zymography,and found to contain UK. The potential role of this interaction in the mechanisms of cell migration and matrix remodeling is discussed. View Publication

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study,we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly,increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD),independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated,and inhibited or enhanced by KD of CRBN or IKZF3,respectively,independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent,as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity. View Publication