技术资料

Protein tyrosine phosphatase 1B (PTP-1B) is a ubiquitously expressed cytosolic phosphatase with the ability to dephosphorylate JAK2 and TYK2,and thereby down-regulate cytokine receptor signaling. Furthermore,PTP-1B levels are up-regulated in certain chronic myelogenous leukemia patients,which points to a potential role for PTP-1B in myeloid development. The results presented here show that the absence of PTP-1B affects murine myelopoiesis by modifying the ratio of monocytes to granulocytes in vivo. This bias toward monocytic development is at least in part due to a decreased threshold of response to CSF-1,because the PTP-1B -/- bone marrow presents no abnormalities at the granulocyte-monocyte progenitor level but produces significantly more monocytic colonies in the presence of CSF-1. This phenomenon is not due to an increase in receptor levels but rather to enhanced phosphorylation of the activation loop tyrosine. PTP-1B -/- cells display increased inflammatory activity in vitro and in vivo through the constitutive up-regulation of activation markers as well as increased sensitivity to endotoxin. Collectively,our data indicate that PTP-1B is an important modulator of myeloid differentiation and macrophage activation in vivo and provide a demonstration of a physiological role for PTP-1B in immune regulation. View Publication

The control of tyrosine phosphorylation depends on the fine balance between kinase and phosphatase activities. Protein tyrosine phosphatase 1B (PTP-1B) and T cell protein tyrosine phosphatase (TC-PTP) are 2 closely related phosphatases known to control cytokine signaling. We studied the functional redundancy of PTP-1B and TC-PTP by deleting 1 or both copies of these genes by interbreeding TC-PTP and PTP-1B parental lines. Our results indicate that the double mutant (tcptp(-/-)ptp1b(-/-)) is lethal at day E9.5-10.5 of embryonic development with constitutive phosphorylation of Stat1. Mice heterozygous for TC-PTP on a PTP-1B-deficient background (tcptp(+/-)ptp1b(-/-)) developed signs of inflammation. Macrophages from these animals were highly sensitive to IFN-gamma,as demonstrated by increased Stat1 phosphorylation and nitric oxide production. In addition,splenic T cells demonstrated increased IFN-gamma secretion capacity. Mice with deletions of single copies of TC-PTP and PTP-1B (tcptp(+/-)ptp1b(+/-)) exhibited normal development,confirming that these genes are not interchangeable. Together,these data indicate a nonredundant role for PTP-1B and TC-PTP in the regulation of IFN signaling. View Publication

BACKGROUND Central to appropriate thrombin formation at sites of vascular injury is the concerted assembly of plasma- and/or platelet-derived factor (F) Va and FXa on the activated platelet surface. While the plasma-derived procofactor,FV,must be proteolytically activated by α-thrombin to FVa to function in prothrombinase,the platelet molecule is released from α-granules in a partially activated state,obviating the need for proteolytic activation. OBJECTIVES The current study was performed to test the hypothesis that subsequent to its endocytosis by megakaryocytes,plasma-derived FV is proteolytically processed to form the platelet-derived pool. METHODS & RESULTS Subsequent to FV endocytosis,a time-dependent increase in FV proteolytic products was observed in megakaryocyte lysates by SDS-PAGE followed by phosphorimaging or western blotting. This cleavage was specific and resulted in the formation of products similar in size to FV/Va present in a platelet lysate as well as to the α-thrombin-activated FVa heavy chain and light chain,and their respective precursors. Other proteolytic products were unique to endocytosed FV. The product/precursor relationships of these fragments were defined using anti-FV heavy and light chain antibodies with defined epitopes. Activity measurements indicated that megakaryocyte-derived FV fragments exhibited substantial FVa cofactor activity that was comparable to platelet-derived FV/Va. CONCLUSIONS Taken together,these observations suggest that prior to its packaging in α-granules endocytosed FV undergoes proteolysis by one or more specific megakaryocyte protease(s) to form the partially activated platelet-derived pool. View Publication

The mixed lineage leukemia (MLL) proto-oncogenic protein is a histone-lysine N-methyltransferase that is produced by proteolytic cleavage and self-association of the respective functionally distinct subunits (MLL(N) and MLL(C)) to form a holocomplex involved in epigenetic transcriptional regulation. On the basis of studies in Drosophila it has been suggested that the separated subunits might also have distinct functions. In this study,we used a genetically engineered mouse line that lacked MLL(C) to show that the MLL(N)-MLL(C) holocomplex is responsible for MLL functions in various developmental processes. The stability of MLL(N) is dependent on its intramolecular interaction with MLL(C),which is mediated through the first and fourth plant homeodomain (PHD) fingers (PHD1 and PHD4) and the phenylalanine/tyrosine-rich (FYRN) domain of MLL(N). Free MLL(N) is destroyed by a mechanism that targets the FYRN domain,whereas free MLL(C) is exported to the cytoplasm and degraded by the proteasome. PHD1 is encoded by an alternatively spliced exon that is occasionally deleted in T-cell leukemia,and its absence produces an MLL mutant protein that is deficient for holocomplex formation. Therefore,this should be a loss-of-function mutant allele,suggesting that the known tumor suppression role of MLL may also apply to the T-cell lineage. Our data demonstrate that the dissociated MLL subunits are subjected to distinct degradation pathways and thus not likely to have separate functions unless the degradation mechanisms are inhibited. View Publication

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However,it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias,we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition,overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover,PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia. View Publication

BACKGROUND: Persistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However,since in most of the studies reported in literature the engraftment state was observed in the nucleated cells,in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit - erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow. DESIGN AND METHODS: The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C,-c,-D,-E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit - erythroid colonies singly picked out after 14 days of incubation. RESULTS: The proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%,46%,15% and 25% at day 1364,1385,1314 and 932,respectively. Similar results were obtained for the erythroid precursors,analyzing the donor/recipient origin of the burst-forming unit - erythroid colonies. In contrast,on the same days of observation,the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%,100%,73% and 90%. Conclusions Our results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant,although the biological mechanisms underlying these observations need further investigation. View Publication

Definitive hematopoietic stem and progenitor cells (HSCs/Ps) originating from the yolk sac and/or para-aorta-splanchno-pleura/aorta-gonad-mesonephros are hypothesized to colonize the fetal liver,but mechanisms involved are poorly defined. The Rac subfamily of Rho GTPases has been shown to play essential roles in HSC/P localization to the bone marrow following transplantation. Here,we study the role of Rac1 in HSC/P migration during ontogeny and seeding of fetal liver. Using a triple-transgenic approach,we have deleted Rac1 in HSCs/Ps during very early embryonic development. Without Rac1,there was a decrease in circulating HSCs/Ps in the blood of embryonic day (E) 10.5 embryos,while yolk sac definitive hematopoiesis was quantitatively normal. Intraembryonic hematopoiesis was significantly impaired in Rac1-deficient embryos,culminating with absence of intra-aortic clusters and fetal liver hematopoiesis. At E10.5,Rac1-deficient HSCs/Ps displayed decreased transwell migration and impaired inter-action with the microenvironment in migration-dependent assays. These data suggest that Rac1 plays an important role in HSC/P migration during embryonic development and is essential for the emergence of intraembryonic hematopoiesis. View Publication

We evaluated the feasibility and efficacy of a reduced-intensity conditioning (RIC) regimen of fludarabine and melphalan to achieve rapid complete donor chimerism after allogeneic stem cell transplantation (SCT) in patients with metastatic solid tumors. Between January 1999 and January 2003,8 patients with metastatic breast cancer (BC) and 15 with metastatic renal cell carcinoma (RCC) underwent allogeneic SCT after an RIC regimen of 5 days of fludarabine and 2 days of melphalan. Filgrastim-mobilized stem cells from HLA-identical related or unrelated donors were infused. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus and methotrexate. All 22 evaluable patients had 100% donor chimerism at day 30 and at all measurement times thereafter. One patient died 19 days after SCT. Nine patients (39%) had grades II to IV acute GVHD and 10 patients (43%) had chronic GVHD. Five patients (22%) died of nonrelapse treatment-related complications. Treatment-related disease response was seen in 10 patients (45%),with 3 complete responses,2 partial responses,and 5 minor responses. Fludarabine-melphalan is a feasible and effective RIC regimen for allogeneic SCT in metastatic BC and RCC. It induces rapid complete donor chimerism without the need for donor lymphocyte infusion. Tumor regression associated with GVHD is consistent with graft-versus-tumor effect. View Publication

Casitas B-cell lymphoma (Cbl)-family E3 ubiquitin ligases are negative regulators of tyrosine kinase signaling. Recent work has revealed a critical role of Cbl in the maintenance of hematopoietic stem cell (HSC) homeostasis,and mutations in CBL have been identified in myeloid malignancies. Here we show that,in contrast to Cbl or Cbl-b single-deficient mice,concurrent loss of Cbl and Cbl-b in the HSC compartment leads to an early-onset lethal myeloproliferative disease in mice. Cbl,Cbl-b double-deficient bone marrow cells are hypersensitive to cytokines,and show altered biochemical response to thrombopoietin. Thus,Cbl and Cbl-b play redundant but essential roles in HSC regulation,whose breakdown leads to hematological abnormalities that phenocopy crucial aspects of mutant Cbl-driven human myeloid malignancies. View Publication

RARA (retinoic acid receptor alpha) haploinsufficiency is an invariable consequence of t(15;17)(q22;q21) translocations in acute promyelocytic leukemia (APL). Retinoids and RARA activity have been implicated in hematopoietic self-renewal and neutrophil maturation. We and others therefore predicted that RARA haploinsufficiency would contribute to APL pathogenesis. To test this hypothesis,we crossed Rara(+/-) mice with mice expressing PML (promyelocytic leukemia)-RARA from the cathepsin G locus (mCG-PR). We found that Rara haploinsufficiency cooperated with PML-RARA,but only modestly influenced the preleukemic and leukemic phenotype. Bone marrow from mCG-PR(+/-) × Rara(+/-) mice had decreased numbers of mature myeloid cells,increased ex vivo myeloid cell proliferation,and increased competitive advantage after transplantation. Rara haploinsufficiency did not alter mCG-PR-dependent leukemic latency or penetrance,but did influence the distribution of leukemic cells; leukemia in mCG-PR(+/-) × Rara(+/-) mice presented more commonly with low to normal white blood cell counts and with myeloid infiltration of lymph nodes. APL cells from these mice were responsive to all-trans retinoic acid and had virtually no differences in expression profiling compared with tumors arising in mCG-PR(+/-) × Rara(+/+) mice. These data show that Rara haploinsufficiency (like Pml haploinsufficiency and RARA-PML) can cooperate with PML-RARA to influence the pathogenesis of APL in mice,but that PML-RARA is the t(15;17) disease-initiating mutation. View Publication

The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the C" of the carboxyl-terminal CaaX motif). After farnesylation� View Publication

The β-hemoglobinopathies sickle cell disease and β-thalassemia are among the most common human genetic disorders worldwide. Hemoglobin A2 (HbA2,α₂δ₂) and fetal hemoglobin (HbF,α₂γ₂) both inhibit the polymerization of hemoglobin S,which results in erythrocyte sickling. Expression of erythroid Kruppel-like factor (EKLF) and GATA1 is critical for transitioning hemoglobin from HbF to hemoglobin A (HbA,α₂β₂) and HbA2. The lower levels of δ-globin expression compared with β-globin expression seen in adulthood are likely due to the absence of an EKLF-binding motif in the δ-globin proximal promoter. In an effort to up-regulate δ-globin to increase HbA2 expression,we created a series of EKLF-GATA1 fusion constructs composed of the transactivation domain of EKLF and the DNA-binding domain of GATA1,and then tested their effects on hemoglobin expression. EKLF-GATA1 fusion proteins activated δ-,γ-,and β-globin promoters in K562 cells,and significantly up-regulated δ- and γ-globin RNA transcript and protein expression in K562 and/or CD34(+) cells. The binding of EKLF-GATA1 fusion proteins at the GATA1 consensus site in the δ-globin promoter was confirmed by chromatin immunoprecipitation assay. Our studies demonstrate that EKLF-GATA1 fusion proteins can enhance δ-globin expression through interaction with the δ-globin promoter,and may represent a new genetic therapeutic approach to β-hemoglobinopathies. View Publication