技术资料

In order to investigate the biologic processes underlying and resulting from the megakaryocytic hyperplasia that characterizes idiopathic myelofibrosis (IMF),peripheral blood CD34+ cells isolated from patients with IMF,polycythemia vera (PV),and G-CSF-mobilized healthy volunteers were cultured in the presence of stem cell factor and thrombopoietin. IMF CD34+ cells generated 24-fold greater numbers of megakaryocytes (MKs) than normal CD34+ cells. IMF MKs were also shown to have a delayed pattern of apoptosis and to overexpress the antiapoptotic protein bcl-xL. MK hyperplasia in IMF is,therefore,likely a consequence of both the increased ability of IMF progenitor cells to generate MKs and a decreased rate of MK apoptosis. Media conditioned (CM) by CD61+ cells generated in vitro from CD34+ cells were then assayed for the levels of growth factors and proteases. Higher levels of transforming growth factor-beta (TGF-beta) and active matrix metalloproteinase-9 (MMP9) were observed in media conditioned with IMF CD61+ cells than normal or PV CD61+ cells. Both normal and IMF CD61+ cells produced similar levels of VEGF. MK-derived TGF-B and MMP-9,therefore,likely contribute to the development of many pathological epiphenomena associated with IMF. View Publication

Infection with a recombinant murine-feline gammaretrovirus,MoFe2,or with the parent virus,Moloney murine leukemia virus,caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective,in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state. View Publication

Ewing sarcoma gene EWS encodes a putative RNA-binding protein with proposed roles in transcription and splicing,but its physiological role in vivo remains undefined. Here,we have generated Ews-deficient mice and demonstrated that EWS is required for the completion of B cell development and meiosis. Analysis of Ews(-/-) lymphocytes revealed a cell-autonomous defect in precursor B lymphocyte (pre-B lymphocyte) development. During meiosis,Ews-null spermatocytes were deficient in XY bivalent formation and showed reduced meiotic recombination,resulting in massive apoptosis and complete arrest in gamete maturation. Inactivation of Ews in mouse embryonic fibroblasts resulted in premature cellular senescence,and the mutant animals showed hypersensitivity to ionizing radiation. Finally,we showed that EWS interacts with lamin A/C and that loss of EWS results in a reduced lamin A/C expression. Our findings reveal essential functions for EWS in pre-B cell development and meiosis,with proposed roles in DNA pairing and recombination/repair mechanisms. Furthermore,we demonstrate a novel role of EWS in cellular senescence,possibly through its interaction and modulation of lamin A/C. View Publication

The T-cell protein tyrosine phosphatase (TC-PTP) is a negative regulator of the Jak/Stat cytokine signaling pathway. Our study shows that the absence of TC-PTP leads to an early bone marrow B-cell deficiency characterized by hindered transition from the pre-B cell to immature B-cell stage. This phenotype is intrinsic to the B cells but most importantly due to bone marrow stroma abnormalities. We found that bone marrow stromal cells from TC-PTP(-/-) mice have the unique property of secreting 232-890 pg/mL IFN-gamma. These high levels of IFN-gamma result in 2-fold reduction in mitotic index on IL-7 stimulation of TC-PTP(-/-) pre-B cells and lower responsiveness of IL-7 receptor downstream Jak/Stat signaling molecules. Moreover,we noted constitutive phosphorylation of Stat1 in those pre-B cells and demonstrated that this was due to soluble IFN-gamma secreted by TC-PTP(-/-) bone marrow stromal cells. Interestingly,culturing murine early pre-B leukemic cells within a TC-PTP-deficient bone marrow stroma environment leads to a 40% increase in apoptosis in these malignant cells. Our results unraveled a new role for TC-PTP in normal B lymphopoiesis and suggest that modulation of bone marrow microenvironment is a potential therapeutic approach for selected B-cell leukemia. View Publication

Chaetocin,a thiodioxopiperazine natural product previously unreported to have anticancer effects,was found to have potent antimyeloma activity in IL-6-dependent and -independent myeloma cell lines in freshly collected sorted and unsorted patient CD138(+) myeloma cells and in vivo. Chaetocin largely spares matched normal CD138(-) patient bone marrow leukocytes,normal B cells,and neoplastic B-CLL (chronic lymphocytic leukemia) cells,indicating a high degree of selectivity even in closely lineage-related B cells. Furthermore,chaetocin displays superior ex vivo antimyeloma activity and selectivity than doxorubicin and dexamethasone,and dexamethasone- or doxorubicin-resistant myeloma cell lines are largely non-cross-resistant to chaetocin. Mechanistically,chaetocin is dramatically accumulated in cancer cells via a process inhibited by glutathione and requiring intact/unreduced disulfides for uptake. Once inside the cell,its anticancer activity appears mediated primarily through the imposition of oxidative stress and consequent apoptosis induction. Moreover,the selective antimyeloma effects of chaetocin appear not to reflect differential intracellular accumulation of chaetocin but,instead,heightened sensitivity of myeloma cells to the cytotoxic effects of imposed oxidative stress. Considered collectively,chaetocin appears to represent a promising agent for further study as a potential antimyeloma therapeutic. View Publication

Genes that are strongly repressed after B-cell activation are candidates for being inactivated,mutated,or repressed in B-cell malignancies. Krüppel-like factor 4 (Klf4),a gene down-regulated in activated murine B cells,is expressed at low levels in several types of human B-cell lineage lymphomas and leukemias. The human KLF4 gene has been identified as a tumor suppressor gene in colon and gastric cancer; in concordance with this,overexpression of KLF4 can suppress proliferation in several epithelial cell types. Here we investigate the effects of KLF4 on pro/pre-B-cell transformation by v-Abl and BCR-ABL,oncogenes that cause leukemia in mice and humans. We show that overexpression of KLF4 induces arrest and apoptosis in the G1 phase of the cell cycle. KLF4-mediated death,but not cell-cycle arrest,can be rescued by Bcl-XL overexpression. Transformed pro/pre-B cells expressing KLF4 display increased expression of p21CIP and decreased expression of c-Myc and cyclin D2. Tetracycline-inducible expression of KLF4 in B-cell progenitors of transgenic mice blocks transformation by BCR-ABL and depletes leukemic pre-B cells in vivo. Collectively,our work identifies KLF4 as a putative tumor suppressor in B-cell malignancies. View Publication

Stem cell leukemia/T cell acute leukemia 1 (SCL/TAL1) plays a key role in the development of murine primitive hematopoiesis but its functions in adult definitive hematopoiesis are still unclear. Using lentiviral delivery of TAL1-directed shRNA in human hematopoietic cells,we show that decreased expression of TAL1 induced major disorders at different levels of adult hematopoietic cell development. Erythroid and myeloid cell production in cultures was dramatically decreased in TAL1-directed shRNA-expressing cells,whereas lymphoid B-cell development was normal. These results confirm the role of TAL1 in the erythroid compartment and show TLA1's implication in the function of myeloid committed progenitors. Moreover,long-term cultures and transplantation of TAL1-directed shRNA-expressing CD34+ cells into irradiated nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice led to dramatically low levels of human cells of all lineages including the B-lymphoid lineage,strongly suggesting that TAL1 has a role in the early commitment of hematopoietic stem cells (HSCs) in humans. Cultures and transplantation experiments performed with mouse Sca1+ cells gave identical results. Altogether,these observations definitively show that TAL1 participates in the regulation of hematopoiesis from HSCs to myeloid progenitors,and pinpoint TAL1 as a master protein of human and murine adult hematopoiesis. View Publication

Activating mutations of c-KIT lead to ligand-independent growth. Internal tandem duplications (ITDs) of exon 11,which encodes the juxtamembrane domain (JMD),are constitutively activating mutations found in 7% of gastrointestinal stromal tumors (GISTs) but have not been described in childhood acute myeloid leukemia (AML). DNA and cDNA from 60 children with AML were screened by polymerase chain reaction (PCR) for mutations of the JMD. A complex ITD (kit cITD) involving exon 11 and exon 12 was identified with a relative frequency of 7% (4/60). The human kit cITDs were inserted into the murine c-Kit backbone and expressed in Ba/F3 cells. KIT cITD induced factorindependent growth and apoptosis resistance,and exhibited constitutive autophosphorylation. KIT cITD constitutively activated the PI3K/AKT pathway and phosphorylated STAT1,STAT3,STAT5,and SHP-2. Imatinib (IM) or rapamycin (Rap) led to complete inhibition of growth,with IC50 values at nanomolar levels. IM and Rap synergistically inhibited growth and surmounted KIT cITD-induced apoptosis resistance. IM but not LY294002 inhibited phosphorylation of STAT3 and STAT5,suggesting aberrant cross talk between PI3K- and STAT-activating pathways. The findings presented may have immediate therapeutic impact for a subgroup of childhood AML-expressing c-KIT mutations. View Publication

Transcription factors are critical for instructing the development of B lymphocytes from multipotential progenitor cells in the bone marrow (BM). Here,we show that the absence of STAT3 impaired B-cell development. Mice selectively lacking STAT3 in BM progenitor cells displayed reduced numbers of mature B cells,both in the BM and in the periphery. The reduction in the B-cell compartment included reduced percentages and numbers of pro-B,pre-B,and immature B cells in the absence of STAT3,whereas the number of pre-pro-B cells was increased. We found that pro-B and pre-B-cell populations lacking STAT3 were hyporesponsive to IL-7 because of a decreased number of IL-7-responsive cells rather than decreased expression or signaling of IL-7Ralpha. Moreover,STAT3-deficient mice displayed enhanced apoptosis in the pro-B population when deprived of survival factors,suggesting that at least 2 mechanisms (impaired differentiation and enhanced apoptosis) are involved in the mutant phenotype. Last,BM transplantation confirmed that impaired B lymphopoiesis in the absence of STAT3 was caused by a cell autonomous defect. In sum,these studies defined a specific role for STAT3 in early B-cell development,probably acting at the pre-pro-B transition by contributing to the survival of IL-7-responsive progenitors. View Publication

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine,a potent inhibitor of PNP,was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK),we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization,phosphorylation of p53 at Ser15,and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation,changes in mitochondrial membrane potential,and PARP cleavage. Based on these data,a phase 2 clinical trial of forodesine has been initiated for CLL patients. View Publication

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 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro,bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-),PAX5(+),TdT(+),IgH rearranged)/myeloid (CD11b/Mac1(+),c-fms(+),lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast,young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model." View Publication