技术资料

The microbial product wortmannin has previously been shown to be a potent inhibitor of phosphatidylinositol-3-kinase. In view of the potential role of this enzyme in transduction of mitogenic signals,we determined the cytotoxic activity of wortmannin against several human tumor cell lines in vitro. The most sensitive lines included GC3 colon carcinoma,IGROV1 ovarian carcinoma,and CCRF-CEM leukemia (IC-50s ranging from 0.7-2.1 microM). The cytotoxicity of wortmannin was decreased approximately 10-fold by serum-free conditions. Wortmannin was generally less active in low passage human breast cancer cell lines that overexpress either epidermal growth factor receptor or Her2/neu. Wortmannin was also tested for in vivo antitumor activity against seven murine tumor and ten human tumor xenograft models. Activity (textgreater 60% inhibition of tumor growth) was observed in only the C3H mammary carcinoma and the human BxPC-3 pancreatic carcinoma xenograft. In vivo antitumor activity did not correlate with in vitro sensitivity to wortmannin cytotoxicity. View Publication

DNA-dependent protein kinase (DNA-PK),which is involved in DNA double-stranded break repair and V(D)J recombination,comprises a DNA-targeting component called Ku and an approximately 460 kDa catalytic subunit,DNA-PKcs. Here,we describe the cloning of the DNA-PKcs cDNA and show that DNA-PKcs falls into the phosphatidylinositol (PI) 3-kinase family. Biochemical assays,however,indicate that DNA-PK phosphorylates proteins but has no detectable activity toward lipids. Strikingly,DNA-PKcs is most similar to PI kinase family members involved in cell cycle control,DNA repair,and DNA damage responses. These include the FKBP12-rapamycin-binding proteins Tor1p,Tor2p,and FRAP,S. pombe rad3,and the product of the ataxia telangiectasia gene,mutations in which lead to genomic instability and predisposition to cancer. The relationship of these proteins to DNA-PKcs provides important clues to their mechanisms of action. View Publication

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors,we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage-/CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer (stroma-free") or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane ("stroma-noncontact"). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a "stroma-free" culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3]� View Publication

The epithelial mucin produced by the MUC1 gene is present in the apical cell membrane of normal breast epithelial cells and is highly expressed in many breast cancers. Several studies have provided conflicting evidence regarding the relationship between MUC1 expression and survival in breast cancer patients. In this study a detailed immunohistological analysis of MUC1 expression was performed using monoclonal antibody BC2 and was related to other tumor characteristics and patient survival. Patients whose tumors showed MUC1 expression in greater than 75% of tumor cells had significantly poorer disease-free and overall survival (P textless .05). The proportion of cells showing cytoplasmic MUC1 expression was prognostically significant,but the proportion of cells that lined gland spaces showing apical membrane staining was of no prognostic significance. A high level of MUC1 expression was significantly associated with the presence of axillary node metastases and estrogen receptors but not with other tumor characteristics. View Publication

A class of pyridinyl imidazoles inhibit the MAP kinase homologue,termed here reactivating kinase (RK) [Lee et al. (1994) Nature 372,739-746]. We now show that one of these compounds (SB 203580) inhibits RK in vitro (IC50 = 0.6 microM),suppresses the activation of MAPKAP kinase-2 and prevents the phosphorylation of heat shock protein (HSP) 27 in response to interleukin-1,cellular stresses and bacterial endotoxin in vivo. These results establish that MAPKAP kinase-2 is a physiological RK substrate,and that HSP27 is phosphorylated by MAPKAP kinase-2 in vivo. The specificity of SB 203580 was indicated by its failure to inhibit 12 other protein kinases in vitro,and by its lack of effect on the activation of RK kinase and other MAP kinase cascades in vivo. We suggest that SB 203580 will be useful for identifying other physiological roles and targets of RK and MAPKAP kinase-2. View Publication

Thiazolidinedione derivatives are antidiabetic agents that increase the insulin sensitivity of target tissues in animal models of non-insulin-dependent diabetes mellitus. In vitro,thiazolidinediones promote adipocyte differentiation of preadipocyte and mesenchymal stem cell lines; however,the molecular basis for this adipogenic effect has remained unclear. Here,we report that thiazolidinediones are potent and selective activators of peroxisome proliferator-activated receptor gamma (PPAR gamma),a member of the nuclear receptor superfamily recently shown to function in adipogenesis. The most potent of these agents,BRL49653,binds to PPAR gamma with a Kd of approximately 40 nM. Treatment of pluripotent C3H10T1/2 stem cells with BRL49653 results in efficient differentiation to adipocytes. These data are the first demonstration of a high affinity PPAR ligand and provide strong evidence that PPAR gamma is a molecular target for the adipogenic effects of thiazolidinediones. Furthermore,these data raise the intriguing possibility that PPAR gamma is a target for the therapeutic actions of this class of compounds. View Publication

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We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded,leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus,the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides,but also in the regulated processing of precursors into active proteins. View Publication

The FLT3/FLK2 receptor tyrosine kinase is closely related to two receptors,c-Kit and c-Fms,which function with their respective ligands,Kit ligand and macrophage colony-stimulating factor to control differentiation of haematopoietic and non-haematopoietic cells. FLT3/FLK2 is thought to be present on haematopoietic stem cells and found in brain,placenta and testis. We have purified to homogeneity and partially sequenced a soluble form of the FLT3/FLK2 ligand produced by mouse thymic stromal cells. We isolated several mouse and human complementary DNAs that encode polypeptides with identical N termini and different C termini. Some variants contain hydrophobic transmembrane segments,suggesting that processing may be required to release soluble ligand. The purified ligand enhances the response of mouse stem cells and a primitive human progenitor cell population to other growth factors such as interleukins IL-3 and IL-6 and to granulocyte-macrophage colony-stimulating factor,and also stimulates fetal thymocytes. View Publication

Pluripotent embryonic stem cells (ESC,ES cells) of line D3 were differentiated in vitro and via embryo-like aggregates (embryoid bodies) of defined cell number into spontaneously beating cardiomyocytes. By using RT-PCR technique,alpha- and beta-cardiac myosin heavy chain (MHC) genes were found to be expressed in embryoid bodies of early to terminal differentiation stages. The exclusive expression of the beta-cardiac MHC gene detected in very early differentiated embryoid bodies proved to be dependent on the number of ES cells developing in the embryoid body. Cardiomyocytes enzymatically isolated from embryoid body outgrowths at different stages of development were further characterized by immunocytological and electrophysiological techniques. All cardiomyocytes appeared to be positive in immunofluorescence assays with monoclonal antibodies against cardiac-specific alpha-cardiac MHC,as well as muscle-specific sarcomeric myosin heavy chain and desmin. The patch-clamp technique allowed a more detailed characterization of the in vitro differentiated cardiomyocytes which were found to represent phenotypes corresponding to sinusnode,atrium or ventricle of the heart. The cardiac cells of early differentiated stage expressed pacemaker-like action potentials similar to those described for embryonic cardiomyocytes. The action potentials of terminally differentiated cells revealed shapes,pharmacological characteristics and hormonal regulation inherent to adult sinusnodal,atrial or ventricular cells. In cardiomyocytes of intermediate differentiation state,action potentials of very long duration (0.3-1 s) were found,which may represent developmentally controlled transitions between different types of action potentials. Therefore,the presented ES cell differentiation system permits the investigation of commitment and differentiation of embryonic cells into the cardiomyogenic lineage in vitro. View Publication

Most primitive hematopoietic cells appear to be normally quiescent in vivo,whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system,where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of primitive" (high proliferative potential)� View Publication

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060,and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor,with PGF2 alpha approximately fluprostenol textgreater PGD2 textgreater PGE2 textgreater U46619 textgreater iloprost. In summary,we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway. View Publication