Abstract 19419: Anti Atherogenic Effects of Micro RNA - 21

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Rihab E Hamed-Berair ◽  
Abhinav Agarwal ◽  
Marcin Wysoczynski ◽  
Srinivas D Sithu ◽  
Nalinie S Wickramasinghe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Interferon Gamma ◽  
Bone Marrow Cells ◽  
Plasma Cholesterol ◽  
Oxidized Ldl ◽  
White Blood Cells ◽  
Micro Rna ◽  
Chimeric Mice ◽  
Wild Type ◽  
Late Apoptosis

Micro RNA-21 (miR-21), an evolutionary conserved micro RNA has been implicated in the pathogenesis of restenosis, myocardial infarction and heart failure. However, little is known about the role of miR-21 in atherosclerosis. Our data show that in vitro, LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-3-fold (P<0.05) and down regulated its target protein PDCD4 in bone marrow derived macrophages (BMDM). Feeding the LDL receptor-knockout (LDLR-KO) mice with western diet (WD, 8-20 weeks) increased the abundance of miR-21 in BMDM by 1.5-fold (P<0.05). Basally, BMDM isolated from miR-21-KO mice showed induction of TNF alpha, interferon gamma, M-CSF, RANTES, IP10 and LIF by (1.5-3.0-fold); increased early and late apoptosis (2-3-fold, P<0.05); and induced PDCD4 and PTEN. We also observed 40% decrease in the survival of F4/80+ cells during differentiation of bone marrow derived cells isolated from miR-21-KO mice. Stimulation of miR-21-KO BMDM with interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86) and decreased IL-10 formation as compared with WT BMDM. Staurosporin and oxidized lipids derived aldehyde 4-hydroxynonenal significantly increased both early and late apoptosis of miR-21-KO BMDM (2-4-fold, P<0.05). This was accompanied by increased cleavage of caspase 3. Characterization of miR-21-KO mice showed 30% decrease in white blood cells and neutrophils in KO mice. However, levels of circulating immune cells and common progenitor cells in bone marrow of miR-21-KO mice were comparable with wild type mice. Transplantation of bone marrow cells from miR-21-KO into LDLR-KO mice, followed by 12 weeks of WD increased the lesion formation (1.7-fold, P<0.05), apoptosis (3-fold, P<0.05) and necrosis (1.6-fold, P<0.05) in the aortic valve of the chimeric mice. This was accompanied by increased accumulation of macrophages in the non-necrotic areas of the lesion and decrease in lesional smooth muscle cells. Plasma cholesterol levels, and lesional collagen and T-cell levels in the miR-21 chimeric mice were comparable with wild type chimeric mice. Collectively, these data suggest that miR-21 prevents atherosclerosis by inhibiting macrophage apoptosis, necrosis and inflammation.

Abstract 263: Role of Micro RNA-21 in Atherosclerosis

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Rihab E Hamed-Berair ◽  
Srinivas D Sithu ◽  
Nalinie Wickramasinghe ◽  
Jasmit Shah ◽  
Abhinav Agawral ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Oxidized Ldl ◽  
Ldl Receptor ◽  
Micro Rna ◽  
Limited Information ◽  
Micro Rnas ◽  
Late Apoptosis ◽  
Apoptosis And Inflammation

Micro RNAs (miR) are short non-coding RNAs that regulate several genes under pathophysiological conditions. Accumulating evidence suggest the involvement of miR in atherogenesis. However, limited information is available about atherogenic miR and the underling mechanisms by which miR affect atherogenesis. Our data shows that 12 weeks of western diet (WD) in LDL receptor-knockout (LDLR-KO) mice upregulated 99 and downregulated 50 miR in the aorta. Among the 41 differentially expressed miR associated with macrophage inflammation and apoptosis, expression of micro RNA-21 (miR-21) was increased by 1.4-fold (P<0.05). WD also increased the expression of miR-21 by 1.5-fold in bone marrow derived macrophages (BMDM). In vitro , LDL, oxidized LDL, acetylated LDL and LPS induced miR-21 by 2-3-fold (P<0.05) and down regulated its target protein PDCD4 in BMDM. Basally, miR-21 deficient BMDM showed increased secretion of IL-6, IL-9 and CXCL-2,-3,-4, and -10 (P<0.05)); and increased early and late apoptosis (2-3-fold, P<0.05). We also observed 40% decrease in the survival of F4/80+ cells during differentiation of bone marrow derived cells isolated from miR-21-KO mice. Stimulation of miR-21-KO BMDM with LPS significantly increased the activation of NF-κB and enhanced the secretion of several pro-inflammatory cytokines including TNFα, IL-6, IL-12 and CXCL-2 (2-10 fold; P<0.05); interferon gamma+LPS polarized the macrophages to pro-inflammatory M1 phenotype (increased expression of CD11c and CD86). Staurosporin and oxidized lipids derived aldehyde 4-hydroxynonenal significantly increased both early and late apoptosis of miR-21-KO BMDM (2-4-fold, P<0.05). This was accompanied by increased cleavage of caspase -3, -7 and -9. Transplantation of bone marrow cells from miR-21-KO into LDLR-KO mice, followed by 12 weeks of WD increased the lesion formation (1.7-fold, P<0.05), apoptosis (3-fold, P<0.05) and necrosis (1.6-fold, P<0.05) in the aortic valve of the chimeric mice. Collectively, these data suggest that miR-21 prevents atherosclerosis, at least in parts, by preventing macrophage apoptosis and inflammation.

scholarly journals Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2148-2159 ◽  
Author(s):  
Harshal H. Nandurkar ◽  
Lorraine Robb ◽  
David Tarlinton ◽  
Louise Barnett ◽  
Frank Köntgen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Null Mutation ◽  
Wild Type ◽  
Normal Numbers ◽  
Interleukin 11 ◽  
Marrow Cells

Abstract Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

Impaired neutrophil maturation in truncated murine G-CSF receptor–transgenic mice

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 2990-2995 ◽  
Author(s):  
Tetsuo Mitsui ◽  
Sumiko Watanabe ◽  
Yoshihiro Taniguchi ◽  
Sachiyo Hanada ◽  
Yasuhiro Ebihara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Acute Myelocytic Leukemia ◽  
Wild Type ◽  
Maturation Arrest ◽  
Mutant Receptor ◽  
Cell Counts ◽  
Marrow Cells

Abstract Severe congenital neutropenia (SCN) is a hematopoietic disorder characterized by neutropenia in peripheral blood and maturation arrest of neutrophil precursors in bone marrow. Patients with SCN may evolve to have myelodysplastic syndrome or acute myelocytic leukemia. In approximately 20% of SCN cases, a truncation mutation is found in the cytoplasmic region of the granulocyte colony-stimulating factor receptor (G-CSFR). We then generated mice carrying murine wild-type G-CSFR and its mutants equivalent to truncations at amino acids 718 and 731 in human G-CSFR, those were reported to be related to leukemic transformation of SCN. Although numbers of peripheral white blood cells, red blood cells, and platelets did not differ among mutant and wild-type G-CSFR transgenic (Tg) mice, both of the mutant receptor Tg mice had one third of peripheral neutrophil cell counts compared with wild-type receptor Tg mice. The mutant receptor Tg mice also showed impaired resistance to the infection with Staphylococcus aureus. Moreover, bone marrow of these Tg mice had an increased percentage of immature myeloid cells, a feature of SCN. This maturation arrest was also observed in in vitro cultures of bone marrow cells of truncated G-CSFR Tg mice under G-CSF stimulation. In addition, clonal culture of bone marrow cells of the truncated G-CSFR Tg mice showed the hypersensitivity to G-CSF in myeloid progenitors. Our Tg mice may be useful in the analysis of the role of truncated G-CSFR in SCN pathobiology.

scholarly journals Adult Mice With Targeted Mutation of the Interleukin-11 Receptor (IL11Ra) Display Normal Hematopoiesis

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2148-2159 ◽  
Author(s):  
Harshal H. Nandurkar ◽  
Lorraine Robb ◽  
David Tarlinton ◽  
Louise Barnett ◽  
Frank Köntgen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Cells ◽  
White Blood Cells ◽  
Null Mutation ◽  
Wild Type ◽  
Normal Numbers ◽  
Interleukin 11 ◽  
Marrow Cells

Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the α-chain (IL-11Rα). Two genes potentially encode the IL-11Rα: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11Rα, we have generated mice with a null mutation of IL11Ra (IL11Ra−/−) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra−/− bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra−/− mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra−/− mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra−/− and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra−/− and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.

scholarly journals Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

1998 ◽  
Vol 331 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Masafumi YOSHIMURA ◽  
Yoshito IHARA ◽  
Tetsuo NISHIURA ◽  
Yu OKAJIMA ◽  
Megumu OGAWA ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Adherent Cell ◽  
Wild Type ◽  
Spleen Cells ◽  
Bisecting Glcnac ◽  
Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

scholarly journals INCIDENCE AND PREVALENCE OF ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) IN KASHMIRI POPULACE (NORTH INDIA)

2021 ◽  
Vol 9 (5) ◽  
pp. 1349-1354
Author(s):  
Fozia Mohammad ◽  
◽  
Arshad A. Pandith ◽  
Mithilesh Kumar ◽  
Aabid Koul ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Rural Areas ◽  
Urban Areas ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
White Blood Cells ◽  
North India ◽  
Cell Phenotype ◽  
Age Group ◽  
Mixed Phenotype

Background: The most frequent cancer of the childhood is acute lymphoblastic leukaemia (ALL). It is the blood and bone marrow cancer affecting white blood cells. It is caused by errors in the DNA in the bone marrow cells. Our goal was to evaluate the prevalence of ALL in Kashmiri populace. Methods: The study in the hindsight was initiated for ALL patients registered between early 2018up to late 2019 to investigate its frequency and prevalence. Results: Overall from 74 ALL patients, based on gender, 44 (59%) were males and 30 (41%) were females. Based on age, 53 (72%) were in the age group of ≤18 years while 21 (28%) were in the age group of >18. Based on immunophenotypes 69 (93%) were of Pre B-cell phenotype, 3 (4%) belonged to T-cell phenotype while 2 (3%) were of mixed phenotype. Based on demography, 10 (14%) were from urban areas while as 64 (86%) were from rural areas of Kashmir region. Conclusions: Although the prevalence of ALL in this region is very high, but gender has no significance while age and dwelling has significance on its overall frequency and significance.

scholarly journals Retroviral-Mediated Gene Transfer of gp91phox Into Bone Marrow Cells Rescues Defect in Host Defense Against Aspergillus fumigatus in Murine X-Linked Chronic Granulomatous Disease

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 41-48 ◽  
Author(s):  
Helga Björgvinsdóttir ◽  
Chunjin Ding ◽  
Nancy Pech ◽  
Mary A. Gifford ◽  
Ling Lin Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Aspergillus Fumigatus ◽  
Chronic Granulomatous Disease ◽  
Host Defense ◽  
Bone Marrow Cells ◽  
Superoxide Production ◽  
Granulomatous Disease ◽  
Wild Type ◽  
Marrow Cells

Abstract The X-linked form of chronic granulomatous disease (X-CGD), an inherited deficiency of the respiratory burst oxidase, results from mutations in the X-linked gene for gp91phox, the larger subunit of the oxidase cytochrome b. The goal of this study was to evaluate the impact of retroviral-mediated gene transfer of gp91phox on host defense against Aspergillus fumigatus in a murine model of X-CGD. Retrovirus vectors constructed using the murine stem cell virus (MSCV) backbone were used for gene transfer of the gp91phox cDNA into murine X-CGD bone marrow cells. Transduced cells were transplanted into lethally irradiated syngeneic X-CGD mice. After hematologic recovery, superoxide production, as monitored by the nitroblue tetrazolium (NBT) test, was detected in up to ≈80% of peripheral blood neutrophils for at least 28 to 35 weeks after transplantation. Neutrophil expression of recombinant gp91phox and superoxide production were significantly less than wild-type neutrophils. However, 9 of 9 mice with ≈50% to 80% NBT+ neutrophils after gene transfer did not develop lung disease after respiratory challenge with 150 to 500 A fumigatus spores, doses that produced disease in 16 of 16 control X-CGD mice. In X-CGD mice transplanted with mixtures of wild-type and X-CGD bone marrow, ≥5% wild-type neutrophils were required for protection against A fumigatus challenge. These data suggest that expression of even low levels of recombinant gp91phox can substantially improve phagocyte function in X-CGD, although correction of very small percentage of phagocytes may not be sufficient for protection against A fumigatus.

New Role of the Regulatory Gene SOX2 in Hematopoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4195-4195
Author(s):  
Elena Levantini ◽  
Francesca Bertolotti ◽  
Francesco Cerisoli ◽  
Anna L. Ferri ◽  
Elisa Brescia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Blood Cell ◽  
Molecular Mechanisms ◽  
Bone Marrow Cells ◽  
Regulatory Gene ◽  
Embryonic Stem ◽  
Mutant Mice ◽  
Wild Type ◽  
Cell Production

Abstract Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.

An Accelerated Incidence of T-Cell Malignancies in a Mouse Model of XSCID Gene Therapy Is Associated with an Expanded Progenitor Cell Pool in the Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 197-197 ◽  
Author(s):  
Yan Shou ◽  
Lilia Stepanova ◽  
Brian P. Sorrentino
Keyword(s):  
Risk Factors ◽  
Gene Therapy ◽  
Bone Marrow ◽  
T Cell ◽  
Insertional Mutagenesis ◽  
Bone Marrow Cells ◽  
Suppressor Gene ◽  
Wild Type ◽  
Gfp Expression ◽  
T Cell Lymphomas

Three patients in the French X-SCID gene therapy trial have developed T-cell lymphomas associated with integration of a γ c-expressing oncoretroviral vector into the LMO2 locus. These occurrences have raised important questions regarding the safety of gene therapy for hematopoietic diseases: 1) are there unique risk factors for XSCID gene therapy that increase the risk of insertional mutagenesis; 2) does deregulated expression of the vector-encoded γ c gene contribute to transformation; 3) what other genetic lesions may contribute to transformation; 4) can safer vectors be designed that result in lower levels of cellular gene activation? To address these questions, we generated a mouse model of XSCID gene therapy in which both the Arf tumor suppressor gene and the γ c gene were ablated. Bone marrow cells from these Arf −/−, γ c−/− mice were transduced with a MSCV-γ c-ires-GFP vector or with a control MSCV-GFP vector. These transduced cells were then transplanted into lethally-irradiated, CD45.1+ wild-type mice. After 1 year of follow-up, 13 out of 15 mice in γ c-transplanted group developed lymphomas in which 12 were T-cell lymphomas and 1 was a B-cell lymphoma. All of these lymphomas except one were highly positive for GFP expression and were derived from transplanted donor cells. In contrast, there were only 3 lymphomas in the MSCV-GFP control group, all of which lacked GFP expression and two of which were derived from recipient hematopoietic cells. Southern blot analysis of lymphoma cells in the γ c-group demonstrated that the lymphomas were clonally-derived. Ligation-mediated PCR analysis showed integrations near or within established proto-oncogenes in 8 cases demonstrating that T-cell transformation was associated with potential insertional oncogene activation. To examine whether the X-SCID background was essential to the increased transformation rate, a second transplant experiment was performed in which bone marrow cells from ARF−/−, γ c+/+ mice were transduced with a MFG-γ c vector. Only 4 out of 18 mice developed lymphomas at 54 weeks indicating that the XSCID background was required for accelerated transformation and that there was no direct effect from deregulated γ c expression per se. We hypothesized that γ c gene deletion could lead to an increase in the number of early progenitors with a resultant increase in target cells susceptible to insertional mutagenesis. Flow cytometry analysis indeed revealed that XSCID mice had a 5-fold increase in the number of Lin−, c-kit+, Sca1+ progenitor cells in the bone marrow relative to wild-type mice, suggesting potential expansion of common lymphocyte progenitors was present. Our results show that unique risk factors exist for gene therapy-induced transformation in XSCID suggesting that the risk for gene therapy in other hematopoietic disorders may be significantly less. One unique risk factor is likely to be an expansion of early progenitors resulting from loss of γ c gene function. Loss of tumor suppressor gene function is likely to be a required secondary event; a hypothesis that is consistent with the relatively long latency for tumor development in patients. Lastly, our animal model should now allow us to test vector safety modifications such as the use of self-inactivation vectors and chromatin insulators and could define a new γ c vector suitable for use in XSCID gene therapy.

A Conserved Mechanism of Transcription Factor Competition Controls Hematopoietic Progenitor Self-Renewal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 91-91
Author(s):  
Shane R. Horman ◽  
Chinamenveni S. Velu ◽  
Tristan Bourdeau ◽  
Avinash Baktula ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Conditional Deletion

Abstract An intrinsic mechanism of self-renewal is critical for the maintenance of hematopoietic stem cells (HSC), but this HSC function is extinguished during differentiation of progenitors. Here we show that the self-renewal capacity of hematopoietic progenitor cells is regulated through physical competition for occupancy of select DNA binding sites. Initially, we found that conditional deletion of the Growth factor independent-1 (Gfi1) gene results in the accumulation of abnormally persistent myeloid progenitors in vivo. Specifically, while germline Gfi1 deletion induces defective HSC self renewal and a block to granulopoiesis, we find that conditional deletion of Gfi1 induces a severe but transient block to neutrophil development with repopulation of the bone marrow by the remaining wild type HSC within 8 weeks post deletion. However, even though normal levels of granulocyte colony forming units (G-CFU) returned by 8 weeks post deletion, an abnormal Gfi1−/− myeloid progenitor remained in the bone marrow in vivo. Subsequently, we find in vitro that both wild-type bone marrow cells expressing Gfi1-dominant-negative mutants, and Gfi1−/− Lin- bone marrow contain cells that replate indefinitely. We hypothesized that Gfi1 is critical to extinguish self renewal in hematopoietic progenitors. In seemingly unrelated work, we discovered antagonism between the drosophila orthologs of Gfi1 and the Hoxa9/Pbx1/Meis1 transcription factor complex during drosophila embryo segmentation. We extended our drosophila findings to discover that a subset of mammalian DNA regulatory sequences encode DNA binding sites for both Gfi1 and Hoxa9/Pbx1/Meis1. These DNA sequences are able to bind either factor, and function as a molecular switch. Interestingly, composite Gfi1/ Hoxa9/Pbx1/Meis1 binding sites are present in the regulatory regions of the gene encoding Hoxa9. We note that Gfi1 expression is normally induced, while Hoxa9 expression is down-regulated, during the transition from common myeloid progenitor (CMP) to the granulocyte-monocyte progenitor (GMP). CMP have greater self renewal potential than GMP. Conditional deletion of Gfi1 in sorted CMP or GMP both increases Hoxa9 expression and generates progenitors capable of replating indefinitely in vitro. Thus, Gfi1 is critical to limit self renewal in these progenitors. Deregulated Hoxa9 expression or activity appears pivotal to this new Gfi1-null phenotype, because Gfi1 dominant-negative mutants immortalize wild-type (or Hoxa7−/−) but not Hoxa9−/− bone marrow cells in vitro. An abnormal gain of self-renewal can unleash the leukemic potential of progenitor cells. We find that both limiting Gfi1 gene dosage and expression of Gfi1 dominant-negative mutants significantly increases Nup98-Hoxa9-mediated colony formation. In contrast, forced expression of Gfi1 prevents Nup98-Hoxa9 immortalization. Notably, the expression of Hoxa9 (independent of cases with Nup98-Hoxa9 fusions) has been reported to be of significant prognostic value in human acute myeloid leukemia. In conclusion, Gfi1 and the Hoxa9/Pbx1/Meis1 complex compete to control the expression of genes (such as Hoxa9) which are critical to extinguish self renewal and limit the leukemogenic potential of hematopoietic progenitors. The antagonism between these transcription factor complexes is conserved from drosophila segment formation to mammalian hematopoietic progenitor biology.

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