Key Points Abnormal signatures in TGF-β1 signaling gene expression were identified in spleen and marrow from the Gata1low model of MF. These signatures include abnormalities in individual gene (Id2, Stat1, mTOR) in spleen and of gene pathways (Smads and BMPs) in marrow.
All mice harboring the X-linked Gata1 low mutation in a predominantly CD1 background are born anemic and thrombocytopenic. They recover from anemia at 1 month of age but remain thrombocytopenic all their life and develop myelofibrosis, a syndrome similar to human idiopathic myelofibrosis, at 12 months. The effects of the genetic background on the myelofibrosis developed by Gata1 low mice was assessed by introducing the mutation, by standard genetic approaches, in the C57BL/6 and DBA/2 backgrounds and by analyzing the phenotype of the different mutants at 12 to 13 (by histology) and 16 to 20 (by cytofluorimetry) months of age. Although all the Gata1 low mice developed fibrosis at 12 to 13 months, variegations were observed in the severity of the phenotype expressed by mutants of different backgrounds. In C57BL/6 mice, the mutation was no longer inherited in a Mendelian fashion, and fibrosis was associated with massive osteosclerosis. Instead, DBA/2 mutants, although severely anemic, expressed limited fibrosis and osteosclerosis and did not present tear-drop poikilocytes in blood or extramedullary hemopoiesis in liver up to 20 months of age. We propose that the variegation in myelofibrosis expressed by
Recent evidence suggests that mutations in the IntroductionAmong the GATA family of transcription factors, 1 Gata1 exerts a specific role in the control of erythroid, 2 megakaryocytic, 3,4 eosinophil, 5 and mast 6 cell differentiation. Genetic alterations of this gene, however, are not only associated with X-linked inherited erythroid or megakaryocytic disorders, but are also found in acquired myeloproliferative disorders. Each mutation is associated with a specific abnormality: point mutations that abrogate the ability of the amino-terminal zinc finger domain of the protein to bind either DNA or Fog1, a partner of Gata1, are found in inherited disorders. [7][8][9][10] On the other hand, frame shift and splice mutations encoding GATA1s, a protein lacking the amino-terminal domain, are associated not only with impaired inherited erythropoiesis, 11,12 but are also found in patients with megakaryocytic leukemia in Down syndrome, 13,14 in newborns with transient myeloproliferative syndromes, 15 and in one adult patient with megakaryocytic leukemia. 16 These observations suggest that, in addition to its effect on terminal differentiation, Gata1 might control the biologic properties of hematopoietic progenitor cells, predisposing them to accumulate secondary mutations in a multistep leukemogenic process. However, direct proof for a possible function of Gata1 in progenitor cells has not been provided as yet.We had previously described that hematopoietic tissues from mice carrying the hypomorphic Gata1 low mutation contain high numbers (ϳ10%) of "unique" progenitor cells that generate colonies composed of erythroblasts, megakaryocytes, and mast cells. 6 Predicted by the stochastic model of hematopoietic commitment, 17 such a trilineage progenitor has not been isolated prospectively as yet from the marrow of normal mice. In fact, antigenic profiling has prospectively divided normal murine progenitors into myeloid-and mast cell-restricted. The myeloid-restricted ones are further divided into granulomonocytic progenitors (GMPs), megakaryocytic-erythroid progenitors (MEPs), and common myeloid progenitors (CMPs). 18 GMPs correspond to cells previously defined, by functional clonogenesis, as colony-forming cells that generate in 7 days granulocytic, monomacrophagic and granulomonocytic colonies (CFU-Gs, CFU-Ms, and CFU-GMs). MEPs, on the other hand, include cells once functionally defined as those that generate megakaryocytic or erythroid colonies either in 3 days (CFU-MKs day3 and CFU-Es) or 7 days (CFU-MKs day7 and BFUEs). CMPs were functionally defined as multilineage progenitor cells, that is, those that generate colonies of multiple lineages after 12 to 15 days either in vitro (CFU-mix) or in vivo (spleen colony-forming cells, CFU-Ss day12 ). Mast cells are localized in extramedullary sites where they engage themselves in the process of allergic response and in the immune reaction against parasites. 19,20 As all the other hematopoietic cells, they derive from progenitor cells present in the marrow (and in the spleen) of ...
Objective-To assess whether alterations in the SDF-1/CXCR4 occur in patients with primary myelofibrosis (PMF) and in Gata1 low mice, an animal model for myelofibrosis, and whether these abnormalities might account for increased stem/progenitor cell trafficking.Materials and Methods-In the mouse, SDF-1 mRNA levels were assayed in liver, spleen and marrow. SDF-1 protein levels were quantified in plasma and marrow and CXCR4 mRNA and protein levels were evaluated on stem/progenitor cells and megakaryocytes purified from the marrow. SDF-1 protein levels were also evaluated in plasma and in marrow biopsy specimens obtained from normal donors and PMF patients.Results-In Gata1 low mice, the plasma SDF-1 protein was 5-times higher than normal in younger animals. Furthermore, SDF-1 immuno-staining of marrow sections progressively increased with age. Similar abnormalities were observed in PMF patients. In fact, the plasma SDF-1 levels in PMF patients were significantly higher (by 2-fold) than normal (p<0.01) and SDF-1 immuno-staining of marrow biopsiy specimens demonstrated increased SDF-1 deposition in specific areas. In two of the patients, SDF-1 deposition was normalized by curative therapy with allogenic stem cell transplantation. Similarly to what already has been reported for PMF patients, the marrow from Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Gata1 low mice contained fewer CXCR4 pos CD117 pos cells and these cells expressed low levels of CXCR4 mRNA and protein. NIH Public AccessConclusion-Similar abnormalities in the SDF-1/CXCR4 axis are observed in PMF patients and in the Gata1 low mice model of myelofibrosis. We suggest that these abnormalities contribute to the increased stem/progenitor cell trafficking observed in this mouse model as well as patients with PMF.
Rigorously defined reconstitution assays developed in recent years have allowed recognition of the delicate relationship that exists between hematopoietic stem cells and their niches. This balance ensures that hematopoiesis occurs in the marrow under steady-state conditions. However, during development, recovery from hematopoietic stress and in myeloproliferative disorders, hematopoiesis occurs in extramedullary sites whose microenvironments are still poorly defined. The hypomorphic Gata1low mutation deletes the regulatory sequences of the gene necessary for its expression in hematopoietic cells generated in the marrow. By analyzing the mechanism that rescues hematopoiesis in mice carrying this mutation, we provide evidence that extramedullary microenvironments sustain maturation of stem cells that would be otherwise incapable of maturing in the marrow.
Blood transfusions have become indispensable to treat the anemia associated with a variety of medical conditions ranging from genetic disorders and cancer to extensive surgical procedures. In developed countries, the blood supply is generally adequate. However, the projected decline in blood donor availability due to population ageing and the difficulty in finding rare blood types for alloimmunized patients indicate a need for alternative red blood cell (RBC) transfusion products. Increasing knowledge of processes that govern erythropoiesis has been translated into efficient procedures to produce RBC ex vivo using primary hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. Although in vitro-generated RBCs have recently entered clinical evaluation, several issues related to ex vivo RBC production are still under intense scrutiny: among those are the identification of stem cell sources more suitable for ex vivo RBC generation, the translation of RBC culture methods into clinical grade production processes, and the development of protocols to achieve maximal RBC quality, quantity, and maturation. Data on size, hemoglobin, and blood group antigen expression and phosphoproteomic profiling obtained on erythroid cells expanded ex vivo from a limited number of donors are presented as examples of the type of measurements that should be performed as part of the quality control to assess the suitability of these cells for transfusion. New technologies for ex vivo erythroid cell generation will hopefully provide alternative transfusion products to meet present and future clinical requirements.
Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H(2)O(2) and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H(2)O(2). However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells.
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