技术资料

The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosa-associated lymphoid tissue (MALT)-type lymphomas,and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence,constitutive nuclear factor-kappaB (NF-kappaB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis,Emu-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless,API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IkappaB kinase gamma (IKKgamma),indicative for enhanced NF-kappaB activation,was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition,we show that the API2-MALT1 fusion resided in the cholesterol- and sphingolipid-enriched membrane microdomains,termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts,which is mediated by the API2 portion,is sufficient to trigger NF-kappaB activation via enhanced polyubiquitination of IKKgamma. Taken together,these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-kappaB activation. View Publication

The Wiskott-Aldrich syndrome protein (WASP) is a product of the gene defective in an Xid disorder,Wiskott-Aldrich syndrome. WASP expression is limited to hemopoietic cells,and WASP regulates the actin cytoskeleton. It has been reported that monocytes/macrophages from WASP-deficient Wiskott-Aldrich syndrome patients are severely defective in chemotaxis,resulting in recurrent infection. However,the molecular basis of such chemotactic defects is not understood. Recently,the WASP N-terminal region was found to bind to the three mammalian verprolin homologs: WASP interacting protein (WIP); WIP and CR16 homologous protein (WICH)/WIP-related protein (WIRE); and CR16. Verprolin was originally found to play an important role in the regulation of actin cytoskeleton in yeast. We have shown that WASP,WIP,and WICH/WIRE are expressed predominantly in the human monocyte cell line THP-1 and that WIP and WICH/WIRE are involved in monocyte chemotaxis. When WASP binding to verprolins was blocked,chemotactic migration of monocytes was impaired in both THP-1 cells and primary human monocytes. Increased expression of WASP and WIP enhanced monocyte chemotaxis. Blocking WASP binding to verprolins impaired cell polarization but not actin polymerization. These results indicate that a complex of WASP with mammalian verprolins plays an important role in chemotaxis of monocytes. Our results suggest that WASP and mammalian verprolins function as a unit in monocyte chemotaxis and that the activity of this unit is critical to establish cell polarization. In addition,our results also indicate that the WASP-verprolin complex is involved in other functions such as podosome formation and phagocytosis. View Publication

IL-7 plays a major role in T lymphocyte homeostasis and has been proposed as an immune adjuvant for lymphopenic patients. This prospect is based,at least in part,on the short-term expansion of peripheral T cells in rIL7-treated mice and primates. Nevertheless,in vivo,following initial increases in T cell proliferation and numbers,lymphocytes return to a quiescent state. As the bases for this cell cycle exit have not yet been elucidated,it is important to assess the long-term biological effects of IL-7 on quiescent human T lymphocyte subsets. In this study,we find that IL-7-stimulated CD4+ naive lymphocytes enter into cell cycle with significantly delayed kinetics as compared with the memory population. Importantly though,these lymphocytes exit from the cell cycle despite the continuous replenishment of rIL-7. This response is distinct in memory and naive CD4+ lymphocytes with memory cells starting to exit from cycle by day 10 vs day 18 for naive cells. Return to quiescence is associated with a cessation in IL-7R signaling as demonstrated by an abrogation of STAT-5 phosphorylation,despite an up-regulation of surface IL-7Ralpha. Indeed,an initial 10-fold decrease in IL-7Ralpha mRNA levels is followed by increased IL-7Ralpha expression in naive as well as memory T cells,with kinetics paralleling cell cycle exit. Altogether,our data demonstrate that IL-7 promotes the extended survival of both naive and memory CD4+ T cells,whereas cycling of these two subsets is distinct and transient. Thus,IL-7 therapy should be designed to allow optimal responsiveness of naive and memory T cell subsets. View Publication

Somatic activation of a conditional targeted Kras(G12D) allele induces a fatal myeloproliferative disease in mice that closely models juvenile and chronic myelomonocytic leukemia. These mice consistently develop severe and progressive anemia despite adequate numbers of clonogenic erythroid progenitors in the bone marrow and expanded splenic hematopoiesis. Ineffective erythropoiesis is characterized by impaired differentiation. These results demonstrate that endogenous levels of oncogenic Ras have cell lineage-specific effects and support efforts to modulate Ras signaling for therapy of anemia in patients with myelodysplastic syndromes and myeloproliferative disorders. View Publication

Stem cells hold a great potential for the regeneration of damaged tissues in cardiovascular or musculoskeletal diseases. Unfortunately,problems such as limited availability,control of cell fate,and allograft rejection need to be addressed before therapeutic applications may become feasible. Generation of multipotent progenitors from adult differentiated cells could be a very attractive alternative to the limited in vitro self-renewal of several types of stem cells. In this direction,a recently synthesized unnatural purine,named reversine,has been proposed to induce reversion of adult cells to a multipotent state,which could be then converted into other cell types under appropriate stimuli. Our study suggests that reversine treatment transforms primary murine and human dermal fibroblasts into myogenic-competent cells both in vitro and in vivo. Moreover,this is the first study to demonstrate that plasticity changes arise in primary mouse and human cells following reversine exposure. View Publication

B cell-activating factor of the tumor necrosis factor (TNF) family,or BAFF,is mainly produced in monocytes and dendritic cells,and indispensable for proliferation,differentiation and survival of B cells. BAFF is a type II membrane-bound protein and the extracellular C-terminal fragment is released from the cells as soluble BAFF (sBAFF),which binds to specific receptors on B cells. Accumulating evidence suggests that BAFF plays an important role in the pathogenesis of autoimmune diseases,such as systemic lupus erythematosus (SLE). In this study,we developed a sensitive sandwich ELISA system to quantify the amount of sBAFF using our own mAb. Treatment of peripheral T cells of SLE patients with an anti-CD3 antibody triggered robust expression of BAFF and subsequent release of sBAFF from the cells. On the other hand,the stimulus induced only marginal elevation of sBAFF from normal T cells. These data indicate that BAFF is expressed in T cells upon stimulation at least under pathological conditions. Expression of BAFF was also largely induced in a human T cell line,Loucy (American Type Tissue Collection CRL-2629),in response to several stimuli,while other T cell lines so far examined produced the cytokine almost constitutively. These data suggest that Loucy recapitulates some of the characteristics of SLE T cells. Investigation of molecular and cellular mechanisms of production of BAFF in Loucy demonstrated that expression of BAFF was regulated through a signal transduction pathway which involves c-jun NH2-terminal kinase and p38,and that shedding of BAFF was catalyzed by a membrane-bound protease,furin. View Publication

Chronic myeloid leukemia (CML) is caused by the constitutively activated tyrosine kinase breakpoint cluster (BCR)-ABL. Current frontline therapy for CML is imatinib,an inhibitor of BCR-ABL. Although imatinib has a high rate of clinical success in early phase CML,treatment resistance is problematic,particularly in later stages of the disease,and is frequently mediated by mutations in BCR-ABL. Dasatinib (BMS-354825) is a multitargeted tyrosine kinase inhibitor that targets oncogenic pathways and is a more potent inhibitor than imatinib against wild-type BCR-ABL. It has also shown preclinical activity against all but one of the imatinib-resistant BCR-ABL mutants tested to date. Analysis of the crystal structure of dasatinib-bound ABL kinase suggests that the increased binding affinity of dasatinib over imatinib is at least partially due to its ability to recognize multiple states of BCR-ABL. The structure also provides an explanation for the activity of dasatinib against imatinib-resistant BCR-ABL mutants. View Publication

Diamond-Blackfan anemia (DBA) is a broad developmental disease characterized by anemia,bone marrow (BM) erythroblastopenia,and an increased incidence of malignancy. Mutations in ribosomal protein gene S19 (RPS19) are found in approximately 25% of DBA patients; however,the role of RPS19 in the pathogenesis of DBA remains unknown. Using global gene expression analysis,we compared highly purified multipotential,erythroid,and myeloid BM progenitors from RPS19 mutated and control individuals. We found several ribosomal protein genes downregulated in all DBA progenitors. Apoptosis genes,such as TNFRSF10B and FAS,transcriptional control genes,including the erythropoietic transcription factor MYB (encoding c-myb),and translational genes were greatly dysregulated,mostly in diseased erythroid cells. Cancer-related genes,including RAS family oncogenes and tumor suppressor genes,were significantly dysregulated in all diseased progenitors. In addition,our results provide evidence that RPS19 mutations lead to codownregulation of multiple ribosomal protein genes,as well as downregulation of genes involved in translation in DBA cells. In conclusion,the altered expression of cancer-related genes suggests a molecular basis for malignancy in DBA. Downregulation of c-myb expression,which causes complete failure of fetal liver erythropoiesis in knockout mice,suggests a link between RPS19 mutations and reduced erythropoiesis in DBA. View Publication

During hematopoietic differentiation of human embryonic stem cells (hESCs),early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays,their phenotype has not been defined. Here,using hESC differentiation in coculture with OP9 stromal cells,we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45,persisted on differentiating hematopoietic cells,and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore,we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)),expressed KDR,VE-cadherin,and CD105 endothelial proteins,and expressed GATA-2,GATA-3,RUNX1,C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile. View Publication

We have previously described a novel series of low molecular weight protein tyrosine kinase inhibitors which we named tyrphostins. The characteristic active pharmacophore of these compounds was the hydroxy-cis-benzylidenemalononitrile moiety. In this article we describe three novel groups of tyrphostins: (i) one group has the phenolic moiety of the cis-benzylidenemalononitrile replaced either with other substituted benzenes or with heteroaromatic rings,(ii) another is a series of conformationally constrained derivatives of hydroxy-cis-benzylidenemalononitriles in which the malononitrile moiety is fixed relative to the aromatic ring,and (iii) two groups of compounds in which the position trans to the benzenemalononitrile has been substituted by ketones and amides. Among the novel tyrphostins examined we found inhibitors which discriminate between the highly homologous EGF receptor kinase (HER1) and ErbB2/neu kinase (HER2). These findings may lead to selective tyrosine kinase blockers for the treatment of diseases in which ErbB2/neu is involved. View Publication