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

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule,the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals,testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed,there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover,the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor,AG490. View Publication

The cellular reservoir for Kaposi sarcoma-associated herpesvirus (KSHV) infection in the hematopoietic compartment and mechanisms governing latent infection and reactivation remain undefined. To determine susceptibility of human CD34+ hematopoietic progenitor cells (HPCs) to infection with KSHV,purified HPCs were exposed to KSHV,and cells were differentiated in vitro and in vivo. Clonogenic colony-forming activity was significantly suppressed in KSHV-infected CD34+ cells,and viral DNA was predominantly localized to granulocyte-macrophage colonies differentiated in vitro. rKSHV.219 is a recombinant KSHV construct that expresses green fluorescent protein from a cellular promoter active during latency and red fluorescent protein from a viral lytic promoter. Infection of CD34+ HPCs with rKSHV.219 showed similar patterns of infection,persistence,and hematopoietic suppression in vitro in comparison with KSHV. rKSHV.219 infection was detected in human CD14+ and CD19+ cells recovered from NOD/SCID mouse bone marrow and spleen following reconstitution with rKSHV.219-infected CD34+ HPCs. These results suggest that rKSHV.219 establishes persistent infection in NOD/SCID mice and that virus may be disseminated following differentiation of infected HPCs into the B-cell and monocyte lineages. CD34+ HPCs may be a reservoir for KSHV infection and may provide a continuous source of virally infected cells in vivo. View Publication

The novel immunosuppressant FTY720 activates sphingosine 1-phosphate receptors (S1PRs) that affect responsiveness of lymphocytes to chemokines such as stromal cell-derived factor 1 (SDF-1),resulting in increased lymphocyte homing to secondary lymphoid organs. Since SDF-1 and its receptor CXCR4 are also involved in bone marrow (BM) homing of hematopoietic stem and progenitor cells (HPCs),we analyzed expression of S1PRs and the influence of FTY720 on SDF-1/CXCR4-mediated effects in human HPCs. By reverse transcriptase-polymerase chain reaction (RT-PCR),S1PRs were expressed in mobilized CD34+ HPCs,particularly in primitive CD34+/CD38- cells. Incubation of HPCs with FTY720 resulted in prolonged SDF-1-induced calcium mobilization and actin polymerization,and substantially increased SDF-1-dependent in vitro transendothelial migration,without affecting VLA-4,VLA-5,and CXCR4 expression. In nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice,the number of CD34+/CD38- cells that homed to the BM after 18 hours was significantly raised by pretreatment of animals and cells with FTY720,tending to result in improved engraftment. In addition,in vitro growth of HPCs (week-5 cobblestone area-forming cells [CAFCs]) was 2.4-fold increased. We conclude that activation of S1PRs by FTY720 increases CXCR4 function in HPCs both in vitro and in vivo,supporting homing and proliferation of HPCs. In the hematopoietic microenvironment,S1PRs are involved in migration and maintenance of HPCs by modulating the effects of SDF-1. View Publication

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications,and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here,we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF),thrombopoietin (TPO),insulin-like growth factor 2 (IGF-2),and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses,there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM side population" (SP) cells� View Publication

Bacterial magnetic particles (BacMPs) are efficient platforms of proteins for surface display systems. In this study,mononuclear cells from peripheral blood were separated using BacMPs expressing protein A on the BacMP membrane surface (protein A-BacMPs),which were complexed with the Fc fragment of anti-mouse IgG antibody. The procedure of positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8,CD14,CD19,CD20) prior to treatment with protein A-BacMP binding with rabbit anti-mouse IgG secondary antibodies. Flow cytometric analysis showed that approximately 97.5 +/- 1.7% of CD19(+) and CD20(+) cells were involved in the positive fraction after magnetic separation. The ratio of the negative cells in the negative fraction was approximately 97.6 +/-1.4%. This indicates that CD19(+) and CD20(+) cells can be efficiently separated from mononuclear cells. Stem cell marker (CD34) positive cells were also separated using protein A-BacMP binding with antibody. May-Grunwald Giemsa stain showed a high nuclear/cytoplasm ratio,which indicates a typical staining pattern of stem cells. The separated cells had the capability of colony formation as hematopoietic stem cells. Furthermore,the inhibitory effect of magnetic cell separation on CD14(+) cells was evaluated by measurement of cytokine in the culture supernatant by ELISA when the cells were cultured with or without lipopolysaccharide (LPS). The induction of IL1-beta,TNFalpha,and IL6 was observed in the presence of 1 ng/mL LPS in all fractions. On the other hand,in the absence of LPS,BacMPs had little immunopotentiation to CD14(+) cells as well as that of artificial magnetic particles,although TNFalpha and IL6 were slightly induced in the absence of LPS in the positive fraction. View Publication

Tetraspanins are thought to facilitate the formation of multiprotein complexes at cell surfaces,but evidence illuminating the biologic importance of this role is sparse. Tetraspanin CD151 forms very stable laminin-binding complexes with integrins alpha3beta1 and alpha6beta1 in kidney and alpha3beta1 and alpha6beta4 in skin. It is encoded by a gene at the same position on chromosome 11p15.5 as the MER2 blood group gene. We show that CD151 expresses the MER2 blood group antigen and is located on erythrocytes. We examined CD151 in 3 MER2-negative patients (2 are sibs) of Indian Jewish origin with end-stage kidney disease. In addition to hereditary nephritis the sibs have sensorineural deafness,pretibial epidermolysis bullosa,and beta-thalassemia minor. The 3 patients are homozygous for a single nucleotide insertion (G383) in exon 5 of CD151,causing a frameshift and premature stop signal at codon 140. The resultant truncated protein would lack its integrin-binding domain. We conclude that CD151 is essential for the proper assembly of the glomerular and tubular basement membrane in kidney,has functional significance in the skin,is probably a component of the inner ear,and could play a role in erythropoiesis. View Publication

Gene therapy for a wide variety of disorders would be greatly enhanced by the development of vectors that could be targeted for gene delivery to specific populations of cells. We describe here high-efficiency targeted transduction based on a novel targeting strategy that exploits the ability of retroviruses to incorporate host cell proteins into the surface of the viral particle as they bud through the plasma membrane. Ecotropic retroviral particles produced in cells engineered to express the membrane-bound form of stem cell factor (mbSCF) transduce both human cell lines and primary cells with high efficiency in a strictly c-kit (SCF receptor)-dependent fashion. The availability of efficient targeted vectors provides a platform for the development of a new generation of therapies using in vivo gene delivery. View Publication

Internal tandem duplications (ITDs) of the FMS-like tyrosine kinase 3 (FLT3) receptor tyrosine kinase are found in approximately 30% of patients with acute myelogenous leukemia (AML) and are associated with a poor prognosis. FLT3 ITD mutations result in constitutive kinase activation and are thought to be pathogenetically relevant,implicating FLT3 as a plausible therapeutic target. MLN518 (formerly CT53518) is a small molecule inhibitor of the FLT3,KIT,and platelet-derived growth-factor receptor (PDGFR) tyrosine kinases with significant activity in murine models of FLT3 ITD-positive leukemia. Given the importance of FLT3 and KIT for normal hematopoietic progenitor cells,we analyzed the effect of MLN518 on murine hematopoiesis under steady-state conditions,after chemotherapy-induced myelosuppression,and during bone marrow transplantation. In these assays,we show that MLN518 has mild toxicity toward normal hematopoiesis at concentrations that are effective in treating FLT3 ITD-positive leukemia in mice. We also demonstrate that MLN518 preferentially inhibits the growth of blast colonies from FLT3 ITD-positive compared with ITD-negative patients with AML,at concentrations that do not significantly affect colony formation by normal human progenitor cells. In analogy to imatinib mesylate in BCR-ABL-positive acute leukemia,MLN518-induced remissions may not be durable. Our studies provide the basis for integrating this compound into chemotherapy and transplantation protocols. View Publication

Erythropoietin (EPO) is the hormone necessary for development of erythrocytes from immature erythroid cells. EPO activates Jun N-terminal kinase (JNK),a member of the mitogen-activated protein kinase (MAPK) family in the EPO-dependent murine erythroid HCD57 cells. Therefore,we tested if JNK activity supported proliferation and/or survival of these cells. Treatment with the JNK inhibitor SP600125 inhibited JNK activity and EPO-dependent proliferation of HCD57 cells and the human EPO-dependent cell lines TF-1 and UT7-EPO. SP600125 also increased the fraction of cells in G2/M. Introduction of a dominant-negative form of JNK1 inhibited EPO-dependent proliferation in HCD57 cells but did not increase the fraction of cells in G2/M. Constitutive JNK activity was observed in primary murine erythroid progenitors. Treatment of primary mouse bone marrow cells with the SP600125 inhibitor reduced the number of erythroid burst-forming units (BFU-e's) but not the more differentiated erythroid colony-forming units (CFU-e's),and SP600125 protected the BFU-e's from apoptosis induced by cytosine arabinoside,demonstrating that the SP600125 inhibited proliferation of the BFU-e's. Therefore,JNK activity appears to be an important regulator of proliferation in immature,primary erythroid cells and 3 erythroid cell lines but may not be required for the survival or proliferation of CFU-e's or proerythroblasts. View Publication

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Exposure of murine bone marrow (BM) cells to ionizing radiation (IR; 4 Gy) resulted in textgreater95% inhibition of the frequency of various day types of cobblestone area-forming cells in association with the induction of apoptosis in hematopoietic stem cell alike cells (Lin(-) ScaI(+) c-kit(+) cells; IR: 64.8 +/- 0.4% versus control: 20.4 +/- 0.5%; P textless 0.001) and progenitors (Lin(-) ScaI(-) c-kit(+) cells; IR: 46.2 +/- 1.4% versus control: 7.8 +/- 0.5%; P textless 0.001). Incubation of murine BM cells with busulfan (BU; 30 micro M) for 6 h also inhibited the cobblestone area-forming cell frequency but failed to cause a significant increase in apoptosis in these two types of hematopoietic cells. After 5 weeks of long-term BM cell culture,33% and 72% of hematopoietic cells survived IR- and BU-induced damage,respectively,as compared with control cells,but they could not form colony forming units-granulocyte macrophages. Moreover,these surviving cells expressed an increased level of senescence-associated beta-galactosidase,p16(Ink4a),and p19(Arf). These findings suggest that IR inhibits the function of hematopoietic stem cell alike cells and progenitors primarily by inducing apoptosis,whereas BU does so mainly by inducing premature senescence. In addition,induction of premature senescence in BM hematopoietic cells also contributes to IR-induced inhibition of their hematopoietic function. Interestingly,the induction of hematopoietic cell senescence by IR,but not by BU,was associated with an elevation in p53 and p21(Cip1/Waf1) expression. This suggests that IR induces hematopoietic cell senescence in a p53-p21(Cip1/Waf1)-dependent manner,whereas the induction of senescence by BU bypasses the p53-p21(Cip1/Waf1) pathway. View Publication