Impaired Granulocytic Differentiation In Vitro in Hematopoietic Cells Lacking Retinoic Acid Receptors α1 and γ

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 607-615 ◽  
Author(s):  
Jean Labrecque ◽  
Deborah Allan ◽  
Pierre Chambon ◽  
Norman N. Iscove ◽  
David Lohnes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Retinoic Acid ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Cell ◽  
Retinoic Acid Receptors ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Marrow Cells

Transcripts for the retinoic acid receptors (RARs) α1, α2, γ1, and γ2 were found in the granulocytic lineage (Gr-1+cells) through semiquantitative polymerase chain reaction (PCR) analysis. The screening of single cell cDNA libraries derived from hematopoietic progenitors also showed the presence of RARα and, to a lesser extent, RARγ transcripts in committed granulocyte (colony-forming unit-granulocyte [CFU-G]) or granulocyte-macrophage (CFU-GM) colony-forming cells. The contribution of RARα1 and γ to hematopoietic cell differentiation was therefore investigated in mice bearing targeted disruption of either one or both of these loci. Because RARγ and RARα1γ compound null mutants die shortly after birth, bone marrow cells were collected from fetuses at 18.5 days postcoitum (dpc) and evaluated for growth and differentiation in culture in the presence of Steel factor (SF), interleukin-3 (IL-3), and erythropoietin (Epo). The frequency of colony-forming cells from bone marrow populations derived from RARα1/γ double null mice was not significantly different from that of RARγ or RARα1 single nulls or from wild-type controls. In addition, the distribution of erythroid, granulocyte, and macrophage colonies was comparable between hematopoietic cells from all groups, suggesting that lineage commitment was not affected by the lack of RARα1 and/or RARγ. Colony cells were then harvested individually and evaluated by morphologic criteria. While terminal granulocyte differentiation was evident in wild-type cells and colonies from either single null mutant, colonies derived from RARα1−/−γ−/− bone marrow populations were blocked at the myelocyte and, to a lesser extent, at the metamyelocyte stages, whereas erythroid and macrophage differentiation was not affected. Together, these results indicate that both RARα1 and γ are required for terminal maturation in the granulocytic lineage in vitro, but appear to be dispensable for the early stages of hematopoietic cell development. Our results raise the possibility that in acute promyelocytic leukemia (APL), the different RARα fusion proteins cause differentiation arrest at a stage when further maturation requires not only RARα, but also RARγ. Finally, bone marrow cells appear to differentiate normally in vivo, suggesting an effective compensation mechanism in the RARα1/γ double null mice.

Impaired Granulocytic Differentiation In Vitro in Hematopoietic Cells Lacking Retinoic Acid Receptors α1 and γ

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 607-615 ◽  
Author(s):  
Jean Labrecque ◽  
Deborah Allan ◽  
Pierre Chambon ◽  
Norman N. Iscove ◽  
David Lohnes ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Retinoic Acid ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Cell ◽  
Retinoic Acid Receptors ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Marrow Cells

Abstract Transcripts for the retinoic acid receptors (RARs) α1, α2, γ1, and γ2 were found in the granulocytic lineage (Gr-1+cells) through semiquantitative polymerase chain reaction (PCR) analysis. The screening of single cell cDNA libraries derived from hematopoietic progenitors also showed the presence of RARα and, to a lesser extent, RARγ transcripts in committed granulocyte (colony-forming unit-granulocyte [CFU-G]) or granulocyte-macrophage (CFU-GM) colony-forming cells. The contribution of RARα1 and γ to hematopoietic cell differentiation was therefore investigated in mice bearing targeted disruption of either one or both of these loci. Because RARγ and RARα1γ compound null mutants die shortly after birth, bone marrow cells were collected from fetuses at 18.5 days postcoitum (dpc) and evaluated for growth and differentiation in culture in the presence of Steel factor (SF), interleukin-3 (IL-3), and erythropoietin (Epo). The frequency of colony-forming cells from bone marrow populations derived from RARα1/γ double null mice was not significantly different from that of RARγ or RARα1 single nulls or from wild-type controls. In addition, the distribution of erythroid, granulocyte, and macrophage colonies was comparable between hematopoietic cells from all groups, suggesting that lineage commitment was not affected by the lack of RARα1 and/or RARγ. Colony cells were then harvested individually and evaluated by morphologic criteria. While terminal granulocyte differentiation was evident in wild-type cells and colonies from either single null mutant, colonies derived from RARα1−/−γ−/− bone marrow populations were blocked at the myelocyte and, to a lesser extent, at the metamyelocyte stages, whereas erythroid and macrophage differentiation was not affected. Together, these results indicate that both RARα1 and γ are required for terminal maturation in the granulocytic lineage in vitro, but appear to be dispensable for the early stages of hematopoietic cell development. Our results raise the possibility that in acute promyelocytic leukemia (APL), the different RARα fusion proteins cause differentiation arrest at a stage when further maturation requires not only RARα, but also RARγ. Finally, bone marrow cells appear to differentiate normally in vivo, suggesting an effective compensation mechanism in the RARα1/γ double null mice.

scholarly journals Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling

Blood ◽  
2012 ◽  
Vol 119 (10) ◽  
pp. 2358-2367 ◽  
Author(s):  
Zbigniew Zasłona ◽  
Carlos H. Serezani ◽  
Katsuhide Okunishi ◽  
David M. Aronoff ◽  
Marc Peters-Golden
Keyword(s):  
Bone Marrow ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Protein Kinase A ◽  
Bone Marrow Cells ◽  
Lipid Mediator ◽  
Wild Type ◽  
Kinase A ◽  
Marrow Cells

Abstract Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation.

scholarly journals U2AF1(S34F) Mutant Hematopoietic Cells Require Expression of Wild-Type U2af1 for Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 941-941
Author(s):  
Brian Wadugu ◽  
Amanda Heard ◽  
Joseph Bradley ◽  
Matthew Ndonwi ◽  
Jin J Shao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rna Splicing ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Poly I:C ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Mutant Cells ◽  
Marrow Cells

Abstract Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease >1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

C/EBPε Plays a Role in Immunity By Regulating Trem-1 Expression in Granulocytes

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4100-4100
Author(s):  
Hyung Chan Suh ◽  
Tong Yin ◽  
Touati Benoukraf ◽  
Jonathan Said ◽  
Stephen Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Inflammatory Response ◽  
Binding Site ◽  
Binding Sites ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Luciferase Assay ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Marrow Cells

Abstract C/EBPepsilon (C/EBPε) knock-out (KO) mice have defects in granulocytic development and increased susceptibility to infection. We also observed a unique phenotype of C/EBPε KO mice showing prolonged lower grade, mild skin problems such as hair loss that progressed to severe wound problems; abdominal wall perforation to the peritoneum as well as mandibular skin perforation to the oral cavity, without significant clinical symptoms. The observation suggests inappropriate response to inflammation by loss of C/EBPε. To understand the functional defects of these granulocytes, we performed a systematic approach with ChIP-seq and microarray of bone marrow cells from C/EBPε KO mice. Duplicate ChIP-seq libraries were prepared from bone marrow cells of wild type and C/EBPε KO mice using two different C/EBPε specific antibodies. Analysis of pooled data identified 14,253 C/EBPε binding sites, and 12,865 of these binding sites overlapped with the two antibodies. Sixteen percent of C/EBPε binding sites were in the promoter regions within 1 kb of the transcriptional start sites. Direct canonical C/EBPε target genes were defined by 1.5 log2 fold or greater increase in expression in the Gr-1+/Mac-1+ cells of wild type bone marrow cells compared to those of the KO mice. 2,224 genes met this criteria including previously identified target genes, such as ltf, camp, ngp, of which 988 genes were bound by C/EBPε. C/EBPε bound to the promoter sites of 150 genes and the enhancer region of 838 genes. The other 1,236 up-regulated genes did not have binding of C/EBPε, suggesting their up-regulation was a secondary event during terminal granulocytic differentiation. Electromobility shift assay followed by antibody supershift confirmed that C/EBPε was able to bind to upstream regions of target genes such as lft, ngp, il-1b. Trem-1 is an amplifier of inflammatory response. Increased expression/activation of Trem-1 on neutrophil membranes takes part in cytokine production, phagocytosis, apoptosis, oxidative burst by cooperating with TLR4 in response to environmental conditions. Soluble TREM1 levels increase even in neutopenic patients, suggesting it is associated with emergent granulopoiesis in response to infection or inflammation. The expression of Trem-1 is regulated by NF-kB and PU.1. Trem-1 KO mice have reduced inflammation without defects in clearance of pathogens in mice. Considering previous reports demonstrating TLR4 increases transcription of C/EBP and potentiates transcriptional activity of C/EBP via NF-kB, regulatory loops between TLR4/NF-kB/C/EBP/Trem-1 may exist and regulate inflammatory response. ChIP-seq illustrated a C/EBPε binding site located 92 bp upstream from transcription start site of Trem-1 (GTTGTGAAAC). Microarray comparison of Trem-1 expression in Gr-1+/Mac-1+ bone marrow cells showed 5.3 log2 fold decreased gene expression in C/EBPε KO versus wild type mice. To confirm microarray data, quantitative PCR were performed with sorted Gr-1+ bone marrow cells from wild type and KO mice. The qPCR demonstrated that trem-1 expression of C/EBPε KO cells was 60% of wild type (p=0.001). A pGL3 basic luciferase vector with the first 291bp upstream of trem-1containing a putative C/EBPε binding site was cloned and co-transfected with 2 ug of C/EBPε expressing vector into 293T cells. The dual luciferase assay, normalized by renilla luciferase, showed 8 fold increase by C/EBPε (p<0.0001). In conclusion, we identified trem-1 as a downstream target of C/EBPε, which completes a positive regulatory loop of TLR4-NF-kB-C/EBPε-Trem1-TLR4 in response to inflammatory signal. Therefore, C/EBPε KO mice have an inappropriate inflammatory response by loss of the positive amplificatory loop, in addition to previously known defective clearance of pathogen by their inability to produce secondary granules. Disclosures No relevant conflicts of interest to declare.

scholarly journals A limited role for p16Ink4a and p19Arf in the loss of hematopoietic stem cells during proliferative stress

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 827-832 ◽  
Author(s):  
Lilia Stepanova ◽  
Brian P. Sorrentino
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Dominant Role ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Serial Transplantation ◽  
Marrow Cells

Abstract It has long been known that prolonged culture or serial transplantation leads to the loss of hematopoietic stem cells (HSCs); however, the mechanisms for this loss are not well understood. We hypothesized that expression of p16Ink4a or p19Arf or both may play a role in the loss of HSCs during conditions of enhanced proliferation, either in vitro or in vivo. Arf was not expressed in freshly isolated HSCs from adult mice but was induced in phenotypically primitive cells after 10 to 12 days in culture. When cultured bone marrow cells from either Arf–/– or Ink4a-Arf–/– mice were compared to wild-type cells in a competitive repopulation assay, no significant differences in HSC activity were seen. We then evaluated the role of p19Arf and p16Ink4a in the loss of HSCs during serial transplantation. Bone marrow cells from Ink4a-Arf–/–, but not Arf–/–, mice had a modestly extended life span and, on average, supported reconstitution of one additional recipient compared to wild-type cells. Mice given transplants of Ink4a-Arf–/–cells eventually did die of hematopoietic failure in the next round of transplantation. We conclude that mechanisms independent of the Ink4a-Arf gene locus play a dominant role in HSC loss during conditions of proliferative stress.

Gfi1-N382S Mutants from Human Severe Congenital Neutropenia Patients Function through a Transcriptional Dominant Negative Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1185-1185
Author(s):  
Adrian P. Zarebski ◽  
Avinash M. Baktula ◽  
Sudeep Basu ◽  
John O. Trent ◽  
H. Leighton Grimes
Keyword(s):  
Bone Marrow ◽  
Dna Binding ◽  
Bone Marrow Cells ◽  
Severe Congenital Neutropenia ◽  
Wild Type ◽  
Congenital Neutropenia ◽  
Murine Bone Marrow ◽  
Amino Terminal ◽  
Marrow Cells

Abstract The Growth factor independence 1 (Gfi1) zinc finger transcriptional repressor is a T cell leukemia oncoprotein that also plays a crucial role in granulopoiesis in both mice and humans. A single point mutation in the amino terminal SNAG repressor domain (P2A) is able to ablate both Gfi1 transcriptional repression activity and linked oncogenic activity in T lymphoctyes. Mice deleted for Gfi1 are lymphopenic, but also lack mature neutrophils. Gfi1−/− mice display a profound block to myeloid differentiation and abnormal promyelocytes accumulate in the blood. Humans with Severe Congenital Neutropenia (SCN) with heterozygous mutations in Gfi1 have similar abnormal promyelocytes. We introduced the SCN patient Gfi1N382S DNA-binding-deficient mutation into murine Gfi1 and overexpressed it in primary murine bone marrow cells. While expression of the wild type Gfi1 resulted almost exclusively in mature granulocyte differentiation, forced expression of the N382S mutant resulted almost exclusively in monocytic differentiation. Flow cytometric analysis revealed a population of N382S-expressing cells with markers of both monocytes and neutrophils resembling the atypical Gfi1−/− promyelocytes. To determine if mutation of the N382 residue is uniquely able to block Gfi1 function, we constructed a virtual model of Gfi1 zinc fingers 3, 4 and 5 interacting with DNA. The model revealed several possible protein-DNA interactions. In order to validate the model we mutated those residues to alanine and performed EMSA with in vitro transcribed/translated proteins. The same alanine substitution mutants were expressed in primary murine bone marrow and tested for their ability to control myelopoiesis. Lack of DNA binding in EMSA tightly correlated with impaired granulopoesis in our in vitro model, suggesting the necessity of intact DNA binding for proper Gfi1 function. These data suggested that the non-DNA binding mutants were able to inhibit repression by wild type endogenous Gfi1, perhaps through the sequestration of limiting corepressor proteins. The Gfi1P2A mutant is unable to repress transcription. We therefore tested the effect of Gfi1P2A expression on myelopoiesis and found that it blocked granulopoiesis equivalently to Gfi1N382S. To rigorously determine whether the titration of limiting corepressors was the cause of N382S neutropenia, we constructed a compound mutant containing both N382S and P2A and expressed it in primary murine bone marrow cells. Expression of Gfi1P2A-N382S had little effect on myelopoiesis. We conclude that SCN patients with heterozygous Gfi1 mutations have blocked granulopoiesis because the non-DNA binding mutant protein competes with the wild type allele for titratable associated cofactors.

Effects of the Homeodomain Gene Pitx2 on Self-Renewal of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 95-95 ◽  
Author(s):  
Hui Z. Zhang ◽  
Svetlana Rogulina ◽  
Wendy Chen ◽  
Barbara A. Degar ◽  
Bernard G. Forget
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Colony Forming Units ◽  
Marrow Cells

Abstract Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, is also a downstream target of genes important for hematopoiesis such as MLL and Wnt/Dvl/β-Catenin. We have previously reported that Pitx2 null hematopoietic stem cells (HSCs) can contribute to multi-lineage hematopoiesis under physiologic conditions. We have now carried out serial bone marrow transplantation experiments and demonstrated that after the 3rd round of serial transplantation, Pitx2 null cells reconstituted only 28.6% of the recipient hematopoietic cells as compared to 60% in the case of wild type cells (P<0.001). There were no Pitx2 null donor-derived cells in recipient mice after the 4th round of transplantation, whereas donor-derived chimerism was 57% with wild type cells (P<0.001), and 26% with Pitx2 +/− cells (P<0.001). Therefore, Pitx2 null HSCs have decreased self renewal capacity. To further study the function of Pitx2 in HSC, we constitutively overexpressed the Pitx2 gene in murine bone marrow cells following transduction using a MSCV/IRES/GFP retroviral vector, and analyzed the effects on hematopoiesis in vitro and in vivo. Bone marrow cells overexpressing Pitx2 were isolated on the basis of their GFP expression and analyzed for their colony forming ability in vitro. Retrovirally transduced bone marrow cells were also transplanted into lethally irradiated mice, and the transplanted mice were observed for long-term reconstitution. Colony-forming unit assays showed that Pitx2 overexpressing bone marrow cells, compared to control cells transduced with vector only, had increased numbers of GM colony forming units and reduced numbers of megakaryocytic colony forming units. Pitx2-overexpressing cells continued to form GM colonies after more than eight serial replatings. When these cells were cultured in liquid medium containing SCF, IL-3 and IL-6, they gave rise to cells that stained positively either for alpha naphthyl butyrate, indicating monocytic differentiation, or for peroxidase, indicating neutrophilic differentiation. The ability of these GM-colony forming cells to cause leukemia is currently under investigation. Long-term reconstitution of hematopoiesis in mice by Pitx2 over-expressing HSCs was demonstrated by identifying GFP positive multi-lineage peripheral blood cells four months following transplantation. One of these mice manifested leukemia at this time, as evidenced by a markedly elevated WBC count and other hematologic abnormalities. The leukemic WBCs had very high levels of GFP and Pitx2 expression and were shown to contain two retroviral integration sites, neither of which involved a known oncogene or overexpression of the gene at the integration site. Immunophenotyping by flow cytometry demonstrated that the majority of the leukemic cells were c-kit positive and expressed the megakaryocytic marker CD41, as well as the common myeloid progenitor marker, CD16/32. Some of the cells expressed the erythroid marker Ter119. The leukemic cells did not express any lymphoid markers, including CD3ε, B220, CD19, and IL7R3. This Pitx2-overexpression-associated leukemia was transplantable. Experiments are under way to characterize the leukemia initiating cells. Taken together, our results provide evidence that the homeodomain gene Pitx2 plays a role in the self-renewal of hematopoietic stem/progenitor cells.

scholarly journals Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1).

1996 ◽  
Vol 16 (8) ◽  
pp. 4566-4572 ◽  
Author(s):  
S L Lee ◽  
Y Wang ◽  
J Milbrandt
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
Zinc Finger ◽  
Bone Marrow Cells ◽  
Myeloid Cell ◽  
Hematopoietic Progenitor Cell ◽  
Wild Type ◽  
Granulocytic Differentiation ◽  
Macrophage Differentiation ◽  
Marrow Cells

The zinc finger protein NGFI-A (also called EGR1, Krox24, or zif268) is a candidate regulator of myeloid cell differentiation. Evidence supporting this hypothesis is twofold. First, NGFI-A antisense oligonucleotides prevent macrophage differentiation in HL-60 and U937 myeloid leukemia cell lines and in normal bone marrow cells. Second, enforced expression of NGFI-A blocks granulocytic differentiation and promotes macrophage differentiation in HL-60 cells and in the hematopoietic progenitor cell line 32D. We sought to determine the effect of NGFI-A deficiency on macrophage differentiation and function in vivo by examining native bone marrow cells from mice homozygous for a disrupted allele of NGFI-A derived from gene-targeted ES cells. Macrophages were observed in peripheral blood and several tissues, indicating that NGFI-A was not required for the formation of a variety of macrophage compartments. No differences in myeloid cell differentiation were observed between wild-type and NGFI-A-/- bone marrow cells cultured in the presence of macrophage, granulocyte-macrophage, or granulocyte colony-stimulating factor (M-CSF, GM-CSF, or G-CSF). Activation of NGFI-A-/- macrophages was comparable to that of wild-type macrophages as determined by nitric oxide production and increased cell surface expression of class II major histocompatibility complex molecules. Moreover, NGFI-A-/- mice showed no increased mortality or bacteria] burden when challenged with Listeria monocytogenes. Together, these results indicate that NGFI-A is not required for macrophage differentiation or activation.

scholarly journals Estrogen responsiveness of bone formation in vitro and altered bone phenotype in aged estrogen receptor-α-deficient male and female mice

2005 ◽  
Vol 152 (2) ◽  
pp. 301-314 ◽  
Author(s):  
Vilhelmiina Parikka ◽  
ZhiQi Peng ◽  
Teuvo Hentunen ◽  
Juha Risteli ◽  
Teresa Elo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Bone Marrow Cells ◽  
Type I ◽  
Wild Type ◽  
Male And Female ◽  
Female Mice ◽  
Bone Phenotype ◽  
Marrow Cells

Objective: Although the beneficial effects of estrogen on bone are well known, the roles of estrogen receptors (ERs) in mediating these effects are not fully understood. Methods: To study the effects of long-term ERα deficiency, bone phenotype was studied in aged ERα knockout (ERKO) mice. In addition, ERKO osteoclasts and osteoblasts were cultured in vitro. Design and results: Histomorphometric analysis showed that the trabecular bone volume and thickness were significantly increased and the rate of bone formation enhanced in both male and female ERKO mice in comparison to the wild-type animals. In ERKO males, however, the bones were thinner and their maximal bending strengths decreased. Consistent with previous reports, the bones of knockout mice, especially of female mice, were shorter than those of wild-type mice. In addition, the growth plates were totally absent in the tibiae of aged ERKO females, whereas the growth plate cartilages were detectable in wild-type females as well as in all the males. Analysis of cultured bone marrow cells from 10- to 12-week-old mice demonstrated that 17β-estradiol could stimulate osteoblastic differentiation of bone marrow cells derived from ERKO mice relatively to the same extent as those derived from wild-type mice. This was demonstrated by increases in synthesis of type I collagen, activity of alkaline phosphatase and accumulation of calcium in cultures. Total protein content was, however, reduced in ERKO osteoblast cultures. Conclusions: These results show altered bone phenotype in ERKO mice and demonstrate the stimulatory effect of estrogen on osteoblasts even in the absence of full-length ERα.

scholarly journals The SH3 Domain Contributes to BCR/ABL-Dependent Leukemogenesis In Vivo: Role in Adhesion, Invasion, and Homing

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 406-418 ◽  
Author(s):  
Tomasz Skorski ◽  
Malgorzata Nieborowska-Skorska ◽  
Pawel Wlodarski ◽  
Mariusz Wasik ◽  
Rossana Trotta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Sh3 Domain ◽  
In Vivo Studies ◽  
Collagen Type Iv ◽  
Wild Type ◽  
Type Iv

To determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (▵SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. ▵SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of ▵SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing ▵SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of ▵SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of α2 integrin by ▵SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/ membrane fraction, ▵SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.

Sign in / Sign up

Export Citation Format

Share Document