Infection of bone marrow cells in vitro with FLV: Effects on stem cell proliferation, differentiation and leukemogenic capacity

Cell ◽  
1977 ◽  
Vol 12 (2) ◽  
pp. 355-364 ◽  
Author(s):  
T Dexter
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Stem Cell Proliferation ◽  
Marrow Cells

scholarly journals NR2F6 (EAR-2) Is a Novel Leukemia Oncogene Whose Cellular Function Is to Regulate Terminal Differentiation of Erythrocytes at the Proerythroblast Stage

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1337-1337
Author(s):  
Christine Victoria Ichim ◽  
Dzana Dervovic ◽  
David Koos ◽  
Marciano D. Reis ◽  
Alden Chesney ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Bone Marrow Cells ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Over Expression ◽  
Marrow Cells

Abstract The leukemia stem cell model suggests that elucidation of the genes that regulate growth ability within the leukemia cell hierarchy will have important clinical relevance. We showed that the expression of NR2F6 (EAR-2), is greater in clonogenic leukemia single cells than in leukemia cells that do not divide, and that this gene is over-expressed in patients with acute myeloid leukemia and myelodysplastic syndrome. In vivo, overexpression of EAR-2 using a retroviral vector in a chimeric mouse model leads to a condition that resembles myelodysplastic syndrome with hypercellular bone marrow, increased blasts, abnormal localization of immature progenitors, morphological dysplasia of the erythroid lineage and a competitive advantage over wild-type cells, that eventually leads to AML in a subset of the mice, or after secondary-transplantation. Interestingly, animals transplanted with bone marrow that over-expresses EAR-2 develop leukemia that is preceded by expansion of the stem cell compartment in the transplanted mice—suggesting that EAR-2 is an important regulator of hematopoietic stem cell differentiation. Here we report that over-expression of EAR-2 also has a profound effect on the differentiation of erythroid progenitor cells both in vitro and in vivo. Studies of the roles of EAR-2 in normal primary bone marrow cells in vitro showed that overexpression of EAR-2 profoundly impaired differentiation along the erythroid lineage. EAR-2 over-expressing bone marrow cells formed 40% fewer BFU-E colonies, but had greatly extended replating capacity in colony assays. While knockdown of EAR-2 increased the number of cells produced per BFU-E colony 300%. Normal mice transplanted with grafts of purified bone marrow cells that over-expressed EAR-2 developed a rapidly fatal leukemia characterized by pancytopenia, enlargement of the spleen, and infiltration of blasts into the spleen, liver and peripheral blood. Sick animals had profound reduction of peripheral blood cell counts, particularly anemia with a 55% reduction in hemoglobin levels. Anemia was evident even on gross inspection of the blood and the liver in EAR-2 overexpressing animals. Analysis of the leukemic cells revealed an erythroblastic morphology, with the immunophenotype lineageneg, CD71high, TER119med. Hence, we wondered weather EAR-2 caused leukemia by arresting erythroid progenitor cell differentiation. Examination of the bone marrow of pre-leukemic animals showed a four-fold increase in cells with a pro-erythroblastic immunophenotype (CD71highTER119med , region I), and a four-fold decrease in orthochromatophilic erythroblasts (CD71lowTER119high , region IV). We observed no change in the numbers of basophilic erythroblasts (CD71highTER119high , region II) or late basophilic and polychromatophilic erythroblasts (CD71medTER119high, region III). These data suggests that over-expression of EAR-2 blocks erythroid cell differentiation at the pro-erythroblastic stage. Since EAR-2 over-expressing recipients died within 4 week, we wanted to definitively test whether animals had compromised radioprotection. We showed that decreasing the size of the bone marrow graft, reduced survival of the EAR-2 over-expressing cohort by a week, but had no effect on control animals proving that EAR-2 over-expression has a profound effect on erythropoietic reconstitution in vivo. Mechanistically, we show that DNA binding is necessary for EAR-2 function, and that EAR-2 functions in an HDAC-dependent manner, regulating expression of several genes. Pre-leukemic pro-erythroblastic cells (CD71highTER119med) that over-expressed EAR-2 had lower expression of genes involved in erythroid differentiation such as GATA1, EBF1, inhibitor of NFKB (NFKBia), ETV6, CEBP/a, LMO2, and Nfe2, and increased expression of GATA2, GLI1, ID1 and PU.1 than GFP control pro-erythroblasts. These data establish that EAR-2 is a novel oncogene whose cellular function is to regulate terminal differentiation of erythroid cells at the proerythroblastic (CD71highTER119med) stage by deregulating gene expression necessary for erythroid differentiation. Disclosures Ichim: Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding. Koos:Entest BioMedical: Employment, Equity Ownership, Patents & Royalties, Research Funding.

ATRA and the RARα Specific Agonist, NRX195183, Have Opposing Effects on the In Vitro Clonogenicity of Pre-Leukemic Murine AML1-ETO Bone Marrow Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 999-999
Author(s):  
Lynette C.Y. Chee ◽  
Jean Hendy ◽  
Louise Purton ◽  
Grant A. McArthur
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Sustained Remission ◽  
Self Renewal ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Abstract 999 All-trans retinoic acid (ATRA) is used successfully to treat acute promyelocytic leukemia (APML), however, to date it has not shown promise in treating other AML subtypes. ATRA has been shown to enhance hematopoietic stem cell (HSC) self-renewal (requiring RARγ activation) but promotes differentiation of myeloid progenitors likely through RARα activation. We hypothesized that (1) the lack of success of ATRA in treating other AML subtypes may be due to the potential ability of ATRA to enhance self-renewal of the leukemic stem cell and (2) the use of a specific RARα agonist may have more promise in enhancing AML differentiation. We therefore compared the effects of pharmacological levels (1μM) of ATRA and an RARα-specific agonist, NRX195183, on bone marrow cells harvested from a Cre-inducible conditional AML1-ETO (AE) knock-in murine model. AE cells cultured for 2 weeks with ATRA showed significant reductions in the proportions of mature myeloid cells (Gr1brightCD11b+) by fluorescence activated cell sorting (FACS) (DMSO: 14.2±4.3%, ATRA: 4.0±1.6%, p=0.04, n=4). By 4 weeks of culture, ATRA-treated AE cells had increased blast and reduced maturing myeloid cell proportions (Blasts %: DMSO 70.2 ± 3.0, ATRA 95.3 ± 1.2, p=0.08; Intermediate %: DMSO 14.3 ± 2.6, ATRA 3.8 ± 1.0, p=0.01; Neutrophils %: DMSO 2.3± 1.0, ATRA 0.3 ± 0.2, p=0.07, n=6). Furthermore, ATRA potentiated the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells: DMSO 505.8±337.0, ATRA 4394±388.9, p=0.001; n=6). In contrast, AE cells cultured for 2 weeks with NRX195183 showed significant increases in the proportions of mature myeloid cells by FACS (DMSO: 15.8±3.5%, NRX195183 26.7±3.0%, p=0.03; n=5). By 4 weeks of culture, NRX195183-treated AE cells had decreased blast and increased maturing myeloid cell proportions (Blasts %: DMSO 82.4±3.0, NRX195183 58.8±9.1, p=0.03; Intermediate %: DMSO 14.5±2.5, NRX195183 29.0±6.8, p=0.07; Neutrophils %: DMSO 1.6±0.8, NRX195183 8.2±4.7 p=ns; DMSO n=8, NRX195183 n=5). Moreover, NRX195183 reduced the clonogenicity of the AE cells after 5 weeks of treatment in vitro (Mean±SEM for colony #/ 5×104 cells DMSO 554.8±252.6, NRX195183 82.6±61.6, p=0.05; n=8). Short-term in vivo transplants of fetal liver cells overexpressing the truncated AE gene, AE9a, into sublethally irradiated recipients revealed similar findings in the NRX195183-treated mice with a decrease in blasts and an increase in mature neutrophils in the peripheral blood on morphological analysis after 4 weeks of treatment (Blasts x106/ml: DMSO 3.1±1.0, NRX195183 0.9±0.3, p=0.08; Neutrophils x106/ml: DMSO 0.5±0.1, NRX195183 0.8±0.1, p=0.04; DMSO n=16, NRX195183 n=11). Taken together, these findings support a model whereby ATRA promotes self-renewal of leukemic blasts whilst NRX195183 has the opposing effect. To understand the mechanism by which ATRA promotes self-renewal in AE cells, we performed genome-wide gene expression analyses on the ATRA- versus control-treated AE cells. This revealed 16 differentially upregulated genes after 24 hours of treatment. Using Ingenuity Pathway Analysis, the top scoring network in the ATRA-treated AE cells was cell-to-cell signalling and interaction (p=1.1E-7-2.4E-3), lipid metabolism (p=2.3E-7-2.0E-3) and small molecule biochemistry (p=2.3E-7-2.1E-3); SERPINE1 and BMP2 were the genes with the highest connectivity within the network interacting with molecules known for their roles in tumorigenesis, including AKT, NF-kβ complex and TGFβ1. SERPINE1 upregulation has been shown to be RARα-mediated whilst BMP2 has been shown to be a RARγ-regulated gene. Interestingly, the specific RARγ agonist, NRX204723, had no effect on the clonogenic potential of these AE progenitors thus raising the hypothesis that both RARα and RARγ activation are required to promote self-renewal of the AE progenitors. Further studies using both RARα/RARγ agonists are warranted to assess if the ATRA effects on AE cells are phenocopied. Collectively, these findings reveal the contrasting roles of specific RARα activation in promoting loss of self-renewal ability and enhancing differentiation in the AE cells whilst ATRA promotes clonogenicity of these cells. This has potential significant implications in AML treatment as specific RARα agonists may be beneficial in improving the efficacy of current treatment modalities to achieve sustained remission in other AML subtypes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Susceptibility of AML to Chloroquine Therapy Is Independent of Autophagy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1262-1262 ◽  
Author(s):  
Xiaoyi Chen ◽  
Jason Clark ◽  
Jun-lin Guan ◽  
Ashish R Kumar ◽  
Yi Zheng
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Bone Marrow Cells ◽  
Leukemia Cells ◽  
Specific Targeting ◽  
Xenograft Mice ◽  
Autophagy Activity ◽  
Marrow Cells

Abstract Introduction: autophagy is a self-recycling and "waste disposal" process that maintains cellular homeostasis. Recently, the autophagy mechanism has evolved as a therapeutic target in cancer treatment due to commonly seen high autophagy activity in cancer cells and its potential role in chemoresistance. To date, inhibition of autophagy has shown therapeutic benefits in several types of solid tumors. However, whether autophagy can be a potentially effective target in AML therapy remains unclear. Here we have used autophagy gene targeted mouse models and drug inhibitors to examine the potential benefits and limitations of autophagy targeting in the treatment of AML. Methods: MLL-AF9 (MA9) oncogene transduced Atg5f/fCreER and Atg5f/fMxCre Lin- mouse bone marrow cells were used for in vitro and in vivo experiments, respectively. Autophagy activity was determined by biochemical Western Blotting and immunofluorescent microscopy against LC3 and electron microscopy (EM). Deletion of FIP200, a gene that is indispensable for both canonical and alternative autophagy pathways, was carried out similarly to further confirm the effect of autophagy-specific targeting on MA9 leukemia. Chloroquine (CQ), a late stage autophagy and lysosome inhibitor, was used at 10μM to 25μM dosages in vitro. Combinatory effects of CQ with chemotherapy, including Doxorubicin (DA) and Cytarabine (AraC), were also analyzed. CQ was also administered to mice through i.p., at 50mg/Kg, bid X 4 days. Leukemia burden, cell survival and CBC counts were analyzed after drug treatment. Results: Primary and clonal MA9 leukemia cells showed a significantly higher level of autophagy flux than normal bone marrow cells. As expected, Atg5-/- MA9 cells showed defective LC3II formation and higher p62 accumulation upon CQ treatment. However, Atg5 deletion did not cause detectable defects in proliferation or survival, or altered ROS or mitochondria levels in leukemia cells. Surprisingly, Atg5-/- MA9 leukemia cells showed reduced sensitivity to either DA or AraC treatment. Transplantation experiments showed that Atg5 deletion in vivo did not reduce leukemia burden in the bone marrow or prolong the survival of the leukemic mice, although it decreased WBC counts in peripheral blood. When examined by EM, no obvious ultrastructural difference was observed between Atg5+/+ and Atg5-/- leukemia cells and both could form endolysosomes upon CQ blockage. Although FIP200 deletion in MA9 leukemia cells caused p62 accumulation at the basal state, similar functional effects were seen as in the case of Atg5 deletion. When compared to wild type controls, FIP200-/- MA9 leukemia cells did not show any proliferation or survival disadvantage, changes in ROS accumulation or mitochondrial level. FIP200 deletion also failed to sensitize leukemia cells to chemotherapy. Finally, CQ independently suppressed leukemia cell proliferation and induced apoptosis, but it did not distinguish Atg5+/+ vs Atg5-/- or FIP200+/+ vs FIP200-/- MA9 leukemia cells in sensitivity. CQ also showed a combinatory effect with DA or AraC in inhibiting MA9 cell proliferation. Treatment of MA9 leukemia xenograft mice with CQ greatly improved anemia in the mice (P<0.01), and we are currently examining the potential effects of CQ on survival of the leukemia bearing mice. Conclusions: MA9 leukemia cells contain a high basal autophagy activity. However, autophagy-specific targeting, either through FIP200-deletion which abolishes autophagy initiation, or via Atg5-deletion which prevents autophagosome membrane elongation, does not affect the survival and proliferation of MA9 leukemia cells nor does it prolong survival of MA9 leukemia xenograft mice. These results suggest that targeting autophagy alone will unlikely produce therapeutic benefit in AML. Loss of Atg5or FIP200 does not sensitize leukemia cells to chemotherapy, further reducing the potential value of targeting autophagy in a combinatory chemotherapy scheme. Mechanistically, neither Atg5 nor FIP200 is required for proper endolysosome-formation or lysosomal degradation in leukemia cells. While CQ displayed an apparent anti-leukemic effect, it acts through an autophagy independent pathway. CQ has a combinatory effect with conventional chemotherapy drugs and may be a useful treatment regimen for MA9 mediated and other types of AML. Disclosures No relevant conflicts of interest to declare.

scholarly journals Selective Migration of Subpopulations of Bone Marrow Cells along an SDF-1α and ATP Gradient

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Michael Laupheimer ◽  
Anna Skorska ◽  
Jana Große ◽  
Gudrun Tiedemann ◽  
Gustav Steinhoff ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Stem Cell Marker ◽  
Stem Cell Markers ◽  
Hematopoietic Stem ◽  
Spontaneous Migration ◽  
Marrow Cells

Both stem cell chemokine stromal cell-derived factor-1α (SDF-1α) and extracellular nucleotides such as adenosine triphosphate (ATP) are increased in ischemic myocardium. Since ATP has been reported to influence cell migration, we analysed the migratory response of bone marrow cells towards a combination of SDF-1 and ATP. Total nucleated cells (BM-TNCs) were isolated from bone marrow of cardiac surgery patients. Migration assays were performed in vitro. Subsequently, migrated cells were subjected to multicolor flow cytometric analysis of CD133, CD34, CD117, CD184, CD309, and CD14 expression. BM-TNCs migrated significantly towards a combination of SDF-1 and ATP. The proportions of CD34+ cells as well as subpopulations coexpressing multiple stem cell markers were selectively enhanced after migration towards SDF-1 or SDF-1 + ATP. After spontaneous migration, significantly fewer stem cells and CD184+ cells were detected. Direct incubation with SDF-1 led to a reduction of CD184+ but not stem cell marker-positive cells, while incubation with ATP significantly increased CD14+ percentage. In summary, we found that while a combination of SDF-1 and ATP elicited strong migration of BM-TNCs in vitro, only SDF-1 was responsible for selective attraction of hematopoietic stem cells. Meanwhile, spontaneous migration of stem cells was lower compared to BM-TNCs or monocytes.

The Role of Annexin II in Stem Cell Homing to Endosteal Osteoblasts - Molecular Dissection of the Hematopoietic Stem Cell Niche.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 589-589
Author(s):  
Russell S. Taichman ◽  
Younghun Jung ◽  
Jingcheng Wang ◽  
Junhui Song ◽  
Jianhua H. Wang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Annexin Ii ◽  
Cell Homing ◽  
Binding Partner ◽  
Stem Cell Homing ◽  
Marrow Cells

Abstract HSC differentiation occurs in direct proximity to osteoblasts (OBs) with in the bone marrow cavity. Using a novel cell blotting system in which OB membranes were run on non-denaturing discontinuous gels and blotted with labeled HSC cell lines, two major adhesive bands were identified. The two proteins were identified as annexin II monomers and multimers. Immunohistochemistry and laser capture and microarray studies demonstrated that annexin II is preferentially expressed at endosteal surfaces and endothelial cells. Antibody studies against annexin II blocked the adhesion of HSC to OB in vitro. siRNA knockdown and over-expression studies of annexin II resulted in either reduced or enhanced binding characteristics, respectively, as expected. These findings were verified using primary human CD34+ bone marrow cells and human OBs where the binding of the progenitor cells and CFU-activities were linked with annexin II. Peptide mapping demonstrated that the majority of the HSC binding activity resides in the N-terminal aspect of the peptide. To assess the role of annexin II in hematopoietic reconstitution, lethally irradiated mice were transplanted with bone marrow cells in the presence or absence of antibody to annexin II, antibody to the p11 annexin II binding partner, or isotype matched controls. Additional groups included use of the N-terminal p1-12 annexin II synthetic peptide or control. Animals that did not receive any marrow cells died within 14 days. Of the mice transplanted with 2x10^5 bone marrow cells, 1/8 animals died at day 11, representing an 80% survival rate. In contrast, when mice were transfused with the same number of marrow cells together with antibody to annexin II, none of the mice survived; the control treated animals survived (n = 8). Equivalent antibody doses against the annexin II binding partner p11 decreased the survival rate of the treated animals compared to IgG controls. However this treatment was less effective in inhibiting survival compared to the antibody to annexin II. Like wise, the survival of mice treated with two different doses of the p1-12 peptides at two different cell concentrations was significantly relative to the groups treated with a scrambled peptide control. These data suggest that the N-terminal twelve amino acids of annexin II facilitate stem cell homing to the marrow. Together these in vitro and in vivo data suggest a novel role for annexin II in the marrow microenvironment.

scholarly journals The effect of stem cell proliferation regulators demonstrated with an in vitro assay

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 196-201 ◽  
Author(s):  
IB Pragnell ◽  
EG Wright ◽  
SA Lorimore ◽  
J Adam ◽  
M Rosendaal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Clonogenic Assay ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Stem Cell Proliferation ◽  
Stem Cell Regulation

Abstract Spleen colony formation after transplantation of bone marrow cells into irradiated mice has been used as an assay for hematopoietic stem cells (CFU-S), but has serious limitations intrinsic to an in vivo assay. In this report we describe experiments using an in vitro clonogenic assay that is especially suitable for studies of stem cell regulation as defined growth factors and normal untreated bone marrow can be used. We have demonstrated that the colony-forming cells have proliferative properties in common with CFU-S and respond to specific proliferation regulators previously detected using the spleen colony assay.

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