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

Immunomodulatory derivative (IMiD) CC-4047,a new analog of thalidomide,directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However,there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047,while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors,including GATA-1,GATA-2,erythroid Kruppel-like factor (EKLF),and growth factor independence-1B (Gfi-1b),were down-regulated in CC-4047-treated CD34+ cells,while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha),C/EBPdelta,and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion,these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore,CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment. View Publication

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However,the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g.,the brain) is higher than for treatment of anemia. Notably,a dose-dependent risk of adverse effects has been associated with rhEPO administration,especially in high-risk groups,including polycythemia-hyperviscosity syndrome,hypertension,and vascular thrombosis. Of note,several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity,primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover,we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here,we demonstrate that rhEPO administration in the rat increases systemic blood pressure,reduces regional renal blood flow,and increases platelet counts and procoagulant activities. In contrast,carbamylated rhEPO,a heteromeric receptor-specific ligand that is fully tissue protective,increases renal blood flow,promotes sodium excretion,reduces injury-induced elevation in procoagulant activity,and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands,which appear to elicit fewer adverse effects,may be especially useful in clinical settings for tissue protection. View Publication

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

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

Because of the wide use of pesticides for domestic and industrial purposes,the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice,but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study,an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E,CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors,has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 micro M to 1000 micro M,and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand,human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC(50) values are 305.2 +/- 22.6 micro M [total erythroid colonies] and textgreater500 micro M [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors,as was found for the murine cells,emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose. View Publication

Multiple myeloma is a highly radiosensitive skeletal malignancy,but bone-seeking radionuclides have not yet found their place in disease management. We previously reported that the proteasome inhibitor PS-341 selectively sensitizes myeloma cells to the lethal effects of ionizing radiation. To extend these observations to an in vivo model,we combined PS-341 with the bone-seeking radionuclide 153-Sm-EDTMP. In vitro clonogenic assays demonstrated synergistic killing of myeloma cells exposed to both PS-341 and 153-Sm-EDTMP. Using the orthotopic,syngeneic 5TGM1 myeloma model,the median survivals of mice treated with saline,2 doses of PS-341 (0.5 mg/kg),or a single nonmyeloablative dose of 153-Sm-EDTMP (22.5 MBq) were 21,22,and 28 days,respectively. In contrast,mice treated with combination therapy comprising 2 doses of PS-341 (0.5 mg/kg),1 day prior to and 1 day following 153-Sm-EDTMP (22.5 MBq) showed a significantly prolonged median survival of 49 days (P textless .001). In addition to prolonged survival,this treatment combination yielded reduced clonogenicity of bone marrow-resident 5TGM1 cells,reduced serum myeloma-associated paraprotein levels,and better preservation of bone mineral density. Myelosuppression,determined by peripheral blood cell counts and clonogenicity assays of hematopoietic progenitors,did not differ between animals treated with 153-Sm-EDTMP alone versus those treated with the combination of PS-341 plus 153-Sm-EDTMP. PS-341 is a potent,selective in vivo radiosensitizer that may substantially affect the efficacy of skeletal-targeted radiotherapy in multiple myeloma. View Publication

Remarkably,apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice,but not other strains of mice,to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins,Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1,but not Bcl-2,Bcl-X(L),Bax,A1,or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand,Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis. View Publication

Erythropoietin (Epo) has been used in the treatment of anemia resulting from numerous etiologies,including renal disease and cancer. However,its effects are controversial and the expression pattern of the Epo receptor (Epo-R) is debated. Using in vivo lineage tracing,we document that within the hematopoietic and mesenchymal lineage,expression of Epo-R is essentially restricted to erythroid lineage cells. As expected,adult mice treated with a clinically relevant dose of Epo had expanded erythropoiesis because of amplification of committed erythroid precursors. Surprisingly,we also found that Epo induced a rapid 26% loss of the trabecular bone volume and impaired B-lymphopoiesis within the bone marrow microenvironment. Despite the loss of trabecular bone,hematopoietic stem cell populations were unaffected. Inhibition of the osteoclast activity with bisphosphonate therapy blocked the Epo-induced bone loss. Intriguingly,bisphosphonate treatment also reduced the magnitude of the erythroid response to Epo. These data demonstrate a previously unrecognized in vivo regulatory network coordinating erythropoiesis,B-lymphopoiesis,and skeletal homeostasis. Importantly,these findings may be relevant to the clinical application of Epo. View Publication