技术资料

After DNA replication,sister chromatids must be untangled,or decatenated,before mitosis so that chromatids do not tear during anaphase. Topoisomerase IIalpha (Topo IIalpha) is the major decatenating enzyme. Topo IIalpha inhibitors prevent decatenation,causing cells to arrest during mitosis. Here we report that acute myeloid leukemia cells fail to arrest at the mitotic decatenation checkpoint,and their progression through this checkpoint is regulated by the DNA repair component Metnase (also termed SETMAR). Metnase contains a SET histone methylase and transposase nuclease domain,and is a component of the nonhomologous end-joining DNA double-strand break repair pathway. Metnase interacts with Topo IIalpha and enhances its decatenation activity. Here we show that multiple types of acute leukemia cells have an attenuated mitotic arrest when decatenation is inhibited and that in an acute myeloid leukemia (AML) cell line this is mediated by Metnase. Of further importance,Metnase permits continued proliferation of these AML cells even in the presence of the clinical Topo IIalpha inhibitor VP-16. In vitro,purified Metnase prevents VP-16 inhibition of Topo IIalpha decatenation of tangled DNA. Thus,Metnase expression levels may predict AML resistance to Topo IIalpha inhibitors,and Metnase is a potential therapeutic target for small molecule interference. View Publication

BACKGROUND: Omega 3 fatty acids have been found to inhibit proliferation,induce apoptosis,and promote differentiation in various cell types. The processes of cell survival,expansion,and differentiation are of key importance in the regulation of hematopoiesis. We investigated the role of omega 3 fatty acids in controlling the frequency of various myeloid progenitor cells in the bone marrow of mice. Increased progenitor cell frequency and blocked differentiation are characteristics of hematopoietic disorders of the myeloid lineage,such as myeloproliferative diseases and myeloid leukemias. RESULTS: We found that increasing the proportion of omega 3 fatty acids relative to the proportion of omega 6 fatty acids in the diet caused increased differentiation and reduced the frequency of myeloid progenitor cells in the bone marrow of mice. Furthermore,this had no adverse effect on peripheral white blood cell counts. CONCLUSION: Our results indicate that omega 3 fatty acids impact hematopoietic differentiation by reducing myeloid progenitor cell frequency in the bone marrow and promoting progenitor cell differentiation. Further exploration of this discovery could lead to the use of omega 3 fatty acids as a therapeutic option for patients that have various disorders of hematopoiesis. View Publication

The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway commonly occurs in cancers and is a crucial event in tumorigenesis. Chronic myelogenous leukemia (CML) is characterized by a reciprocal chromosomal translocation (9;22) that generates the Bcr-Abl fusion gene. The PI3K/Akt pathway is activated by Bcr-Abl chimera protein and mediates the leukemogenesis in CML. However,the mechanism by which Bcr-Abl activates the PI3K/Akt pathway is not completely understood. In the present study,we found that pleckstrin homology domain leucine-rich repeat protein phosphatases 1 and 2 (PHLPP1 and PHLPP2) were depleted in CML cells. We investigated the interaction between PHLPPs and Bcr-Abl in CML cell lines and Bcr-Abl+ progenitor cells from CML patients. The Abl kinase inhibitors and depletion of Bcr-Abl induced the expression of PHLPP1 and PHLPP2,which dephosphorylated Ser-473 on Akt1,-2,and -3,resulting in inhibited proliferation of CML cells. The reduction of PHLPP1 and PHLPP2 expression by short interfering RNA in CML cells weakened the Abl kinase inhibitor-mediated inhibition of proliferation. In colony-forming unit-granulocyte,erythroid,macrophage,megakaryocyte; colony-forming unit-granulocyte,macrophage; and burst-forming unit-erythroid,treatment with the Abl kinase inhibitors and depletion of Bcr-Abl induced PHLPP1 and PHLPP2 expression and inhibited colony formation of Bcr-Abl+ progenitor cells,whereas depletion of PHLPP1 and PHLPP2 weakened the inhibition of colony formation activity by the Abl kinase inhibitors in Bcr-Abl+ progenitor cells. Thus,Bcr-Abl represses the expression of PHLPP1 and PHLPP2 and continuously activates Akt1,-2,and -3 via phosphorylation on Ser-473,resulting in the proliferation of CML cells. View Publication

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results,we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age,77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600,etoposide 100) and 8A (tipifarnib 400,etoposide 200). In vivo,tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as NCT00112853. View Publication

Earlier reports have suggested that the BCR/ABL oncogene,associated with chronic myeloid leukemia,induces a mutator phenotype; however,it is unclear whether this leads to long-term changes in chromosomes and whether the phenotype is found in primary chronic myelogeneous leukemia (CML) cells. We have addressed both these issues. BCR/ABL-expressing cell lines show an increase in DNA breaks after treatment with etoposide as compared to control cells. However,although BCR/ABL-expressing cell lines have an equivalent cell survival,they have an increase in chromosomal translocations after DNA repair as compared to control cells. This demonstrates that BCR/ABL expression decreases the fidelity of DNA repair. To see whether this is true in primary CML samples,normal CD34+ progenitor cells and CML progenitor cells were treated with etoposide. CML progenitor cells have equivalent survival but have an increase in DNA double-strand breaks (DSBs). Spectral karyotyping demonstrates new chromosomal translocations in CML cells,but not normal progenitor cells,consistent with error-prone DNA repair. Taken together,these data demonstrate that BCR/ABL enhances the accumulation of DSBs and alters the apoptotic threshold in CML leading to error-prone DNA repair. View Publication

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

Hematopoiesis during development is a dynamic process,with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis,the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1),best known for its proinflammatory properties,has radioprotective effects on adult bone marrow HSCs,induces HSC mobilization,and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1,IL-1 receptors (IL-1Rs),and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM,and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic,endothelial,and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation,concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development,acting to limit the differentiation of some HSCs along the myeloid lineage. View Publication

Mef2c is a MADS (MCM1-agamous-deficient serum response factor) transcription factor best known for its role in muscle and cardiovascular development. A causal role of up-regulated MEF2C expression in myelomonocytic acute myeloid leukemia (AML) has recently been demonstrated. Due to the pronounced monocytic component observed in Mef2c-induced AML,this study was designed to assess the importance of Mef2c in normal myeloid differentiation. Analysis of bone marrow (BM) cells manipulated to constitutively express Mef2c demonstrated increased monopoiesis at the expense of granulopoiesis,whereas BM isolated from Mef2c(Delta/-) mice showed reduced levels of monocytic differentiation in response to cytokines. Mechanistic studies showed that loss of Mef2c expression correlated with reduced levels of transcripts encoding c-Jun,but not PU.1,C/EBPalpha,or JunB transcription factors. Inhibiting Jun expression by short-interfering RNA impaired Mef2c-mediated inhibition of granulocyte development. Moreover,retroviral expression of c-Jun in BM cells promoted monocytic differentiation. The ability of Mef2c to modulate cell-fate decisions between monocyte and granulocyte differentiation,coupled with its functional sensitivity to extracellular stimuli,demonstrate an important role in immunity--and,consistent with findings of other myeloid transcription factors,a target of oncogenic lesions in AML. View Publication

The c-Myb gene encodes a transcription factor required for proliferation and survival of normal myeloid progenitors and leukemic blast cells. Targeting of c-Myb by antisense oligodeoxynucleotides has suggested that myeloid leukemia blasts (including chronic myelogenous leukemia [CML]-blast crisis cells) rely on c-Myb expression more than normal progenitors,but a genetic approach to assess the requirement of c-Myb by p210(BCR/ABL)-transformed hematopoietic progenitors has not been taken. We show here that loss of a c-Myb allele had modest effects (20%-28% decrease) on colony formation of nontransduced progenitors,while the effect on p210(BCR/ABL)-expressing Lin(-) Sca-1(+) and Lin(-) Sca-1(+)Kit(+) cells was more pronounced (50%-80% decrease). Using a model of CML-blast crisis,mice (n = 14) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/w) marrow cells developed leukemia rapidly and had a median survival of 26 days,while only 67% of mice (n = 12) injected with p210(BCR/ABL)-transduced p53(-/-)c-Myb(w/d) marrow cells died of leukemia with a median survival of 96 days. p210(BCR/ABL)-transduced c-Myb(w/w) and c-Myb(w/d) marrow progenitors expressed similar levels of the c-Myb-regulated genes c-Myc and cyclin B1,while those of Bcl-2 were reduced. However,ectopic Bcl-2 expression did not enhance colony formation of p210(BCR/ABL)-transduced c-Myb(w/d) Lin(-)Sca-1(+)Kit(+) cells. Together,these studies support the requirement of c-Myb for p210(BCR/ABL)-dependent leukemogenesis. View Publication

TRAIL induces apoptosis in a variety of tumor cells. Our laboratory found that human neutrophils contain an intracellular reservoir of prefabricated TRAIL that is released after stimulation with Mycobacterium bovis bacillus Calmette-Guérin. In this study,we examined the subcellular distribution of TRAIL in freshly isolated neutrophils. Neutrophil granules,secretory vesicles (SV),and plasma membrane vesicles were isolated by subcellular fractionation,followed by free-flow electrophoresis,and examined by ELISA and immunoblot. TRAIL was found in all membrane-bound fractions with the highest amounts in the fractions enriched in azurophilic granule (AG) and SV. Immunofluorescence confocal microscopy showed that TRAIL colocalized independently with myeloperoxidase (MPO),lactoferrin (LF),and albumin,respective markers of AG,specific granules,and SV. Furthermore,immunotransmission electron microscopy demonstrated that TRAIL colocalized intracellularly with MPO and albumin. We examined TRAIL expression in PLB-985 cells induced with dimethylformamide and in CD34-positive stem cells treated with G-CSF. Quantitative RT-PCR analysis showed that TRAIL was expressed in each stage of development,whereas MPO and LF were only expressed at distinct times during differentiation. Collectively,these findings suggest that TRAIL is expressed throughout neutrophil development,resulting in a broad distribution among different granule subtypes. View Publication

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms,active mutant WASp(I294T) was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell,decreased proliferation,and increased apoptosis. Cells became binucleated,suggesting a failure of cytokinesis,and micronuclei were formed,indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis,filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation,and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization. View Publication

In CD34(+) acute myeloid leukemia (AML),the malignant stem cells reside in the CD38(-) compartment. We have shown before that the frequency of such CD34(+)CD38(-) cells at diagnosis correlates with minimal residual disease (MRD) frequency after chemotherapy and with survival. Specific targeting of CD34(+)CD38(-) cells might thus offer therapeutic options. Previously,we found that C-type lectin-like molecule-1 (CLL-1) has high expression on the whole blast compartment in the majority of AML cases. We now show that CLL-1 expression is also present on the CD34(+)CD38(-) stem- cell compartment in AML (77/89 patients). The CD34(+)CLL-1(+) population,containing the CD34(+)CD38(-)CLL-1(+) cells,does engraft in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with outgrowth to CLL-1(+) blasts. CLL-1 expression was not different between diagnosis and relapse (n = 9). In remission,both CLL-1(-) normal and CLL-1(+) malignant CD34(+)CD38(-) cells were present. A high CLL-1(+) fraction was associated with quick relapse. CLL-1 expression is completely absent both on CD34(+)CD38(-) cells in normal (n = 11) and in regenerating bone marrow controls (n = 6). This AML stem-cell specificity of the anti-CLL-1 antibody under all conditions of disease and the leukemia-initiating properties of CD34(+)CLL-1(+) cells indicate that anti-CLL-1 antibody enables both AML-specific stem-cell detection and possibly antigen-targeting in future. View Publication