参考文献

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain,two consensus sequences that are targets for phosphotyrosine binding domains,a proline-rich region,and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties,we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase. View Publication

A major goal of experimental and clinical hematology is the identification of mechanisms and conditions that support the expansion of transplantable hematopoietic stem cells. In normal marrow,such cells appear to be identical to (or represent a subset of) a population referred to as long-term-culture-initiating cells (LTC-ICs) so-named because of their ability to produce colony-forming cell (CFC) progeny for textgreater or = 5 weeks when cocultured with stromal fibroblasts. Some expansion of LTC-ICs in vitro has recently been described,but identification of the factors required and whether LTC-IC self-renewal divisions are involved have remained unresolved issues. To address these issues,we examined the maintenance and/or generation of LTC-ICs from single CD34+ CD38- cells cultured for variable periods under different culture conditions. Analysis of the progeny obtained from cultures containing a feeder layer of murine fibroblasts engineered to produce steel factor,interleukin (IL)-3,and granulocyte colony-stimulating factor showed that approximately 20% of the input LTC-ICs (representing approximately 2% of the original CD34+ CD38- cells) executed self-renewal divisions within a 6-week period. Incubation of the same CD34+ CD38- starting populations as single cells in a defined (serum free) liquid medium supplemented with Flt-3 ligand,steel factor,IL-3,IL-6,granulocyte colony-stimulating factor,and nerve growth factor resulted in the proliferation of initial cells to produce clones of from 4 to 1000 cells within 10 days,approximately 40% of which included textgreater or = 1 LTC-IC. In contrast,in similar cultures containing methylcellulose,input LTC-ICs appeared to persist but not divide. Overall the LTC-IC expansion in the liquid cultures was 30-fold in the first 10 days and 50-fold by the end of another 1-3 weeks. Documentation of human LTC-IC self-renewal in vitro and identification of defined conditions that permit their extensive and rapid amplification should facilitate analysis of the molecular mechanisms underlying these processes and their exploitation for a variety of therapeutic applications. View Publication

Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment,20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage. Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines,suggesting paracrine and/or autocrine growth regulation. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells,and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias,attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth. Here we show that leukaemic cells from patients in relapse have constitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker,AG-490,selectively blocks leukaemic cell growth in vitro and in vivo by inducing programmed cell death,with no deleterious effect on normal haematopoiesis. View Publication

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The available techniques for directed gene manipulation in the mouse are unprecedented in any multicellular organism and make the mouse an invaluable tool for unraveling all aspects of mammalian biology. To realize fully the potential of these genetic tools requires that phenotypic analysis be efficient,rapid,and complete. Genetic chimeras and mosaics,in which mutant cells are mixed with wild-type cells,can be used to augment standard analysis of intact mutant animals and alleviate the time required and the expense involved in generating and maintaining multiple strains of mutant mice. View Publication

The bcr-abl oncogene,present in 95% of patients with chronic myelogenous leukemia (CML),has been implicated as the cause of this disease. A compound,designed to inhibit the Abl protein tyrosine kinase,was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML,there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias. View Publication

In cultures of embryonic striatum,we previously reported that EGF induces the proliferation of single precursor cells,which give rise to spheres of undifferentiated cells that can generate neurons and glia. We report here that,in vitro,these embryonic precursor cells exhibit properties and satisfy criteria representative of stem cells. The EGF-responsive cell was able to generate the three major phenotypes of the mammalian CNS--neurons,astrocytes,and oligodendrocytes. Approximately 90% of both primary spheres and secondary expanded clones,derived from the primary spheres,contained all three cell types. The increase in frequency of EGF-generated spheres,from 1% in primary culture to close to 20% in secondary culture,and the large number of clonally derived secondary spheres that could be generated from a single primary sphere indicate that EGF induces both renewal and expansion of the precursor cell itself. In population studies,the EGF-responsive cells were carried through 10 passages,resulting in a 10(7)-fold increase in cell number,without losing their proliferative and multilineage potential. Thus,this study describes the first demonstration,through clonal and population analyses in vitro,of a mammalian CNS stem cell that proliferates in response to an identified growth factor (EGF) and produces the three principal cell types of the CNS. View Publication

All-trans retinoic acid (ATRA) is successfully used in the cyto-differentiating treatment of acute promyelocytic leukemia (APL). Paradoxically,APL cells express PML-RAR,an aberrant form of the retinoic acid receptor type alpha (RAR alpha) derived from the leukemia-specific t(15;17) chromosomal translocation. We show here that AM580,a stable retinobenzoic derivative originally synthesized as a RAR alpha agonist,is a powerful inducer of granulocytic maturation in NB4,an APL-derived cell line,and in freshly isolated APL blasts. After treatment of APL cells with AM580 either alone or in combination with granulocyte colony-stimulating factor (G-CSF),the compound induces granulocytic maturation,as assessed by determination of the levels of leukocyte alkaline phosphatase,CD11b,CD33,and G-CSF receptor mRNA,at concentrations that are 10- to 100-fold lower than those of ATRA necessary to produce similar effects. By contrast,AM580 is not effective as ATRA in modulating the expression of these differentiation markers in the HL-60 cell line and in freshly isolated granulocytes obtained from the peripheral blood of chronic myelogenous leukemia patients during the stable phase of the disease. In NB4 cells,two other synthetic nonselective RAR ligands are capable of inducing LAP as much as AM580,whereas RAR beta- or RAR gamma-specific ligands are totally ineffective. These results show that AM580 is more powerful than ATRA in modulating the expression of differentiation antigens only in cells in which PML-RAR is present. Binding experiments,using COS-7 cells transiently transfected with PML-RAR and the normal RAR alpha,show that AM580 has a lower affinity than ATRA for both receptors. However,in the presence of PML-RAR,the synthetic retinoid is a much better transactivator of retinoic acid-responsive element-containing promoters than the natural retinoid,whereas,in the presence of RAR alpha,AM580 and ATRA have similar activity. This may explain the strong cyto-differentiating potential of AM580 in PML-RAR-containing leukemic cells. View Publication

Under appropriate conditions in culture,embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic,endothelial,muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo,indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development. View Publication

The site-directed recombinase Cre can be employed to delete or express genes in cell lines or animals. Clearly,the ability to control remotely the activity of this enzyme would be highly desirable. To this end we have constructed expression vectors for fusion proteins consisting of the Cre recombinase and a mutated hormone-binding domain of the murine oestrogen receptor. The latter still binds the anti-oestrogen drug tamoxifen but no longer 17 beta-oestradiol. We show here that in embryonic stem cells expressing such fusion proteins,tamoxifen can efficiently induce Cre-mediated recombination,thereby activating a stably integrated LacZ reporter gene. In the presence of either 10 microM tamoxifen or 800 nM 4-hydroxy-tamoxifen,recombination of the LacZ gene is complete within 3-4 days. By placing a tamoxifen-binding domain on both ends of the Cre protein,the enzymatic activity of Cre can be even more tightly controlled. Transgenic mice expressing such an tamoxifen-inducible Cre enzyme may thus provide a new and useful genetic tool to mutate or delete genes at specific times during development or in adult animals. View Publication

Fumonisin B1 (FB1),a mycotoxin produced by the fungus Fusarium moniliforme,which is a common contaminant of corn,is suspected to be a cause of human esophageal cancer. FB1 is hepatotoxic and hepatocarcinogenic in rats,and although the mechanisms involved have not been clarified,the latter is associated with a weak initiating activity. The effects of FB1 on the activity of protein serine/threonine phosphatases (PPs) (PP1,PP2A,PP2B,PP2C and PP5/T/K/H) were investigated in the present study. Inhibition of dephosphorylation was noted for all five PPs with IC50 values of 80 microM-3000 microM. Among the five PPs examined,PP5 was most sensitive with an IC50 of 80 microM. This concentration is comparable to that estimated to be reached in the rat body by feeding FB1 to obtain hepatic tumors. Inhibition of PP5 could thus play important roles in the toxicity and carcinogenic action of FB1. View Publication

Neutral endopeptidase (NEP) may regulate peptide-induced inflammation in the respiratory tract. It is of interest to determine which respiratory resident cells express NEP. Trachea and bronchi from seven nonsmoking,nonasthmatic subjects were examined. NEP messenger ribonucleic acid (mRNA) was characterized by Northern blot hybridization of cultured human tracheobronchial epithelial and smooth muscle cells,and reverse transcriptase-polymerase chain reaction (RT-PCR) in trachea and bronchi. In situ hybridization with biotin- and 35S-labelled antisense complementary ribonucleic acid (cRNA) probes was used to determine the distribution of NEP mRNA in human bronchial mucosa. NEP-immunoreactive material was detected using MEK10 murine monoclonal antibodies and the immunogold method with silver enhancement. NEP mRNA was 4.5 kb in size in the cultured human smooth muscle and epithelial cells by Northern blot analysis. No evidence was found by RT-PCR for truncated,alternatively spliced NEP mRNAs,such as del exon 16 or del exons 5-18 in human bronchus. NEP mRNA was detected by in situ hybridization in epithelial cells,submucosal glands,bronchial smooth muscle and endothelium. NEP-immunoreactive material was identified in the epithelium,submucosal glands,bronchial smooth muscle,and endothelium,demonstrating an excellent correlation between the distribution of NEP mRNA and the cell surface protein. NEP mRNA and immunoreactive material were excluded from epithelial goblet cell and submucosal gland mucous cell vacuoles. We conclude that the various sites of NEP protein and mRNA expression correlate with the locations of peptide receptors and NEP enzyme function,and are consistent with the hypothesis that NEP may regulate peptide-induced inflammation in human bronchi. View Publication