搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor,nilotinib (AMN107),is significantly more potent against BCR-ABL than imatinib,and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib,indicating that sequential therapy with these 2 agents has clinical value. However,simultaneous,rather than sequential,administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons,including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here,we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity,including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further,using a highly quantifiable bioluminescent in vivo model,drug combinations were at least additive in antileukemic activity,compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase,the combination of imatinib and nilotinib is highly efficacious in these models,indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted. View Publication

Metachromatic leukodystrophy (MLD) is a demyelinating lysosomal storage disorder for which new treatments are urgently needed. We previously showed that transplantation of gene-corrected hematopoietic stem progenitor cells (HSPCs) in presymptomatic myeloablated MLD mice prevented disease manifestations. Here we show that HSC gene therapy can reverse neurological deficits and neuropathological damage in affected mice,thus correcting an overt neurological disease. The efficacy of gene therapy was dependent on and proportional to arylsulfatase A (ARSA) overexpression in the microglia progeny of transplanted HSPCs. We demonstrate a widespread enzyme distribution from these cells through the CNS and a robust cross-correction of neurons and glia in vivo. Conversely,a peripheral source of enzyme,established by transplanting ARSA-overexpressing hepatocytes from transgenic donors,failed to effectively deliver the enzyme to the CNS. These results indicate that the recruitment of gene-modified,enzyme-overexpressing microglia makes the enzyme bioavailable to the brain and makes therapeutic efficacy and disease correction attainable. Overall,our data provide a strong rationale for implementing HSPC gene therapy in MLD patients. View Publication

Mice lacking the zinc finger transcriptional repressor protein GFI-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Furthermore,Gfi-1(-/-) mice are highly susceptible to bacterial infection. Interestingly,Gfi-1(-/-) myeloid cells overexpress target genes of the PU.1 transcription factor such as the macrophage colony-stimulating factor receptor and PU.1 itself. We therefore determined whether GFI-1 modulates the transcriptional activity of PU.1. Our data demonstrate that GFI-1 physically interacts with PU.1,repressing PU.1-dependent transcription. This repression is functionally significant,as GFI-1 blocked PU.1-induced macrophage differentiation of a multipotential hematopoietic progenitor cell line. Retroviral expression of GFI-1 in primary murine hematopoietic progenitors increased granulocyte differentiation at the expense of macrophage differentiation. We interbred Gfi-1(+/-) and PU.1(+/-) mice and observed that heterozygosity at the PU.1 locus partially rescued the Gfi-1(-/-) mixed myeloid lineage phenotype,but failed to restore granulocyte differentiation. Our data demonstrate that GFI-1 represses PU.1 activity and that lack of this repression in Gfi-1(-/-) myeloid cells contributes to the observed mixed lineage phenotype. View Publication

The kinase inhibitor imatinib mesylate targeting the oncoprotein Bcr-Abl has revolutionized the treatment of chronic myeloid leukemia (CML). However,even though imatinib successfully controls the leukemia in chronic phase,it seems not to be able to cure the disease,potentially necessitating lifelong treatment with the inhibitor under constant risk of relapse. On a molecular level,the cause of disease persistence is not well understood. Initial studies implied that innate features of primitive progenitor cancer stem cells may be responsible for the phenomenon. Here,we describe an assay using retroviral insertional mutagenesis (RIM) to identify genes contributing to disease persistence in vivo. We transplanted mice with bone marrow cells retrovirally infected with the Bcr-Abl oncogene and subsequently treated the animals with imatinib to select for leukemic cells in which the proviral integration had affected genes modulating the imatinib response. Southern blot analysis demonstrated clonal outgrowth of cells carrying similar integration sites. Candidate genes located near the proviral insertion sites were identified,among them the transcription factor RUNX3. Proviral integration near the RUNX3 promoter induced RUNX3 expression,and Bcr-Abl-positive cell lines with stable or inducible expression of RUNX1 or RUNX3 were protected from imatinib-induced apoptosis. Furthermore,imatinib treatment selected for RUNX1-expressing cells in vitro and in vivo after infection of primary bone marrow cells with Bcr-Abl and RUNX1. Our results demonstrate the utility of RIM for probing molecular modulators of targeted therapies and suggest a role for members of the RUNX transcription factor family in disease persistence in CML patients. 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

Neurotrophins (NTs) and their receptors play a key role in neurogenesis and survival. The TRK (tropomyosin-related kinase) receptor protein tyrosine kinases (TRKA,TRKB,TRKC) are high-affinity NT receptors that are expressed in a variety of human tissues. Their role in normal and malignant hematopoiesis is poorly understood. In a prospective study involving 94 adult patients we demonstrate for the first time cell-surface expression of the 3 TRKs and constitutive activation in blasts from patients with de novo or secondary acute leukemia. At least one TRK was expressed in 55% of the analyzed cases. We establish a clear correlation between the TRK expression pattern and FAB classification. Although only few point mutations were found in TRK sequences by reverse-transcriptase-polymerase chain reaction (RT-PCR),we observed coexpression of BDNF (ligand for TRKB) in more than 50% of TRKB(+) cases (16/30). Activation of TRKA or TRKB by NGF and BDNF,respectively,efficiently rescued murine myeloid cells from irradiation-induced apoptosis. Coexpression of TRKB/BDNF or TRKA/NGF in murine hematopoietic cells induced leukemia. Moreover,activation of TRKs was important for survival of both human and murine leukemic cells. Our findings suggest that TRKs play an important role in leukemogenesis and may serve as a new drug target. View Publication

Alcohol abuse predisposes the host to bacterial infections. In response to bacterial infection,the bone marrow hematopoietic activity shifts toward granulocyte production,which is critical for enhancing host defense. This study investigated the hematopoietic precursor cell response to bacteremia and how alcohol affects this response. Acute alcohol intoxication was induced in BALB/c mice 30 min before initiation of Escherichia coli bacteremia. Bacteremia caused a significant increase in the number of bone marrow lineage (lin(-))-c-kit(+)Sca-1(+) cells. Marrow lin(-)c-kit(+)Sca-1(+) cells isolated from bacteremic mice showed an increase in CFU-granulocyte/macrophage activity compared with controls. In addition to enhanced proliferation of lin(-)c-kit(+)Sca-1(+) cells as reflected by BrdU incorporation,phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells primarily accounted for the rapid increase in marrow lin(-)c-kit(+)Sca-1(+) cells following bacteremia. Bacteremia increased plasma concentration of TNF-alpha. Culture of marrow lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells with murine rTNF-alpha for 24 h caused a dose-dependent increase in conversion of these cells to lin(-)c-kit(+)Sca-1(+) cells. Sca-1 mRNA expression by the cultured cells was also up-regulated following TNF-alpha stimulation. Acute alcohol intoxication inhibited the increase in the number of lin(-)c-kit(+)Sca-1(+) cells in the bone marrow after E. coli infection. Alcohol impeded the increase in BrdU incorporation into marrow lin(-)c-kit(+)Sca-1(+) cells in response to bacteremia. Alcohol also suppressed the plasma TNF-alpha response to bacteremia and inhibited TNF-alpha-induced phenotypic inversion of lin(-)c-kit(+)Sca-1(+)Sca-1(-) cells in vitro. These data show that alcohol inhibits the hematopoietic precursor cell response to bacteremia,which may serve as one mechanism underlying the impaired host defense in alcohol abusers with severe bacterial infections. View Publication

The core-binding factor (CBF) is a master regulator of developmental and differentiation programs,and CBF alterations are frequently associated with acute leukemia. The role of the CBF member RUNX2 in hematopoiesis is poorly understood. Genetic evidence suggests that deregulation of Runx2 may cause myeloid leukemia in mice expressing the fusion oncogene Cbfb-MYH11. In this study,we show that sustained expression of Runx2 modulates Cbfbeta-smooth muscle myosin heavy chain (SMMHC)-mediated myeloid leukemia development. Expression of Runx2 is high in the hematopoietic stem cell compartment and decreases during myeloid differentiation. Sustained Runx2 expression hinders myeloid progenitor differentiation capacity and represses expression of CBF targets Csf1R,Mpo,Cebpd,the cell cycle inhibitor Cdkn1a,and myeloid markers Cebpa and Gfi1. In addition,full-length Runx2 cooperates with Cbfbeta-SMMHC in leukemia development in transplantation assays. Furthermore,we show that the nuclear matrix-targeting signal and DNA-binding runt-homology domain of Runx2 are essential for its leukemogenic activity. Conversely,Runx2 haplo-insufficiency delays the onset and reduces the incidence of acute myeloid leukemia. Together,these results indicate that Runx2 is expressed in the stem cell compartment,interferes with differentiation and represses CBF targets in the myeloid compartment,and modulates the leukemogenic function of Cbfbeta-SMMHC in mouse leukemia. View Publication

Loss of neurofibromin or interferon consensus sequence binding protein (Icsbp) leads to a myeloproliferative disorder. Transcription of NF1 is directly controlled by ICSBP. It has been postulated that loss of NF1 expression resulting from loss of transcriptional activation by ICSBP contributes to human hematologic malignancies. To investigate the functional cooperation of these 2 proteins,we have established Icsbp-deficient mice with Nf1 haploinsufficiency. We here demonstrate that loss of Icsbp and Nf1 haploinsufficiency synergize to induce a forced myeloproliferation in Icsbp-deficient mice because of an expansion of a mature myeloid progenitor cell. Furthermore,Nf1 haploinsufficiency and loss of Icsbp contribute synergistically to progression of the myeloproliferative disorder toward transplantable leukemias. Leukemias are characterized by distinct phenotypes,which correlate with progressive genetic abnormalities. Loss of Nf1 heterozygosity is not mandatory for disease progression,but its occurrence with other genetic abnormalities indicates progressive genetic alterations in a defined subset of leukemias. These data show that loss of the 2 tumor suppressor genes Nf1 and Icsbp synergize in the induction of leukemias. 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

Phenotypic markers associated with human hematopoietic stem cells (HSCs) were developed and validated using uncultured cells. Because phenotype and function can be dissociated during culture,better markers to prospectively track and isolate HSCs in ex vivo cultures could be instrumental in advancing HSC-based therapies. Using an expansion system previously shown to increase hematopoietic progenitors and SCID-repopulating cells (SRCs),we demonstrated that the rhodamine-low phenotype was lost,whereas AC133 expression was retained throughout culture. Furthermore,the AC133(+)CD38(-) subpopulation was significantly enriched in long-term culture-initiating cells (LTC-IC) and SRCs after culture. Preculture and postculture analysis of total nucleated cell and LTC-IC number,and limiting dilution analysis in NOD/SCID mice,showed a 43-fold expansion of the AC133(+)CD38(-) subpopulation that corresponded to a 7.3-fold and 4.4-fold expansion of LTC-ICs and SRCs in this subpopulation,respectively. Thus,AC133(+)CD38(-) is an improved marker that tracks and enriches for LTC-IC and SRC in ex vivo cultures. View Publication

Gfi-1B is a transcriptional repressor that is crucial for erythroid differentiation: inactivation of the GFI1B gene in mice leads to embryonic death due to failure to produce differentiated red cells. Accordingly,GFI1B expression is tightly regulated during erythropoiesis,but the mechanisms involved in such regulation remain partially understood. We here identify HMGB2,a high-mobility group HMG protein,as a key regulator of GFI1B transcription. HMGB2 binds to the GFI1B promoter in vivo and up-regulates its trans-activation most likely by enhancing the binding of Oct-1 and,to a lesser extent,of GATA-1 and NF-Y to the GFI1B promoter. HMGB2 expression increases during erythroid differentiation concomitantly to the increase of GfI1B transcription. Importantly,knockdown of HMGB2 in immature hematopoietic progenitor cells leads to decreased Gfi-1B expression and impairs their erythroid differentiation. We propose that HMGB2 potentiates GATA-1-dependent transcription of GFI1B by Oct-1 and thereby controls erythroid differentiation. View Publication