Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow

In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.

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Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow

Blood ◽

10.1182/blood.v82.12.3580.3580 ◽

1993 ◽

Vol 82 (12) ◽

pp. 3580-3591 ◽

Cited By ~ 68

Author(s):

N Falla ◽

Vlasselaer Van ◽

J Bierkens ◽

B Borremans ◽

G Schoeters ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Collagen Matrix ◽

Collagen Type I ◽

Nodule Formation ◽

Mouse Bone Marrow ◽

Type I ◽

Murine Bone Marrow

Abstract In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.

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Expression of human adenosine deaminase in murine hematopoietic cells

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5116-5125.1988 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5116-5125

Author(s):

J W Belmont ◽

G R MacGregor ◽

K Wager-Smith ◽

F A Fletcher ◽

K A Moore ◽

...

Keyword(s):

Bone Marrow ◽

Adenosine Deaminase ◽

High Titer ◽

Virus Production ◽

Marrow Transplant ◽

Mouse Bone Marrow ◽

Regulation Of Expression ◽

Murine Bone Marrow

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

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CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v110.11.1224.1224 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1224-1224

Author(s):

Jerry C. Cheng ◽

Dejah Judelson ◽

Kentaro Kinjo ◽

Jenny Chang ◽

Elliot Landaw ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Mouse Bone Marrow ◽

T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

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Type I interferons produced by dendritic cells promote their phenotypic and functional activation

Blood ◽

10.1182/blood.v99.9.3263 ◽

2002 ◽

Vol 99 (9) ◽

pp. 3263-3271 ◽

Cited By ~ 332

Author(s):

Maria Montoya ◽

Giovanna Schiavoni ◽

Fabrizio Mattei ◽

Ion Gresser ◽

Filippo Belardelli ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

T Cell ◽

Bone Marrow Cells ◽

Type I Interferons ◽

Type I ◽

Type I Ifn ◽

Type I Ifns

Abstract Resting dendritic cells (DCs) are resident in most tissues and can be activated by environmental stimuli to mature into potent antigen-presenting cells. One important stimulus for DC activation is infection; DCs can be triggered through receptors that recognize microbial components directly or by contact with infection-induced cytokines. We show here that murine DCs undergo phenotypic maturation upon exposure to type I interferons (type I IFNs) in vivo or in vitro. Moreover, DCs either derived from bone marrow cells in vitro or isolated from the spleens of normal animals express IFN-α and IFN-β, suggesting that type I IFNs can act in an autocrine manner to activate DCs. Consistent with this idea, the ability to respond to type I IFN was required for the generation of fully activated DCs from bone marrow precursors, as DCs derived from the bone marrow of mice lacking a functional receptor for type I IFN had reduced expression of costimulatory and adhesion molecules and a diminished ability to stimulate naive T-cell proliferation compared with DCs derived from control bone marrow. Furthermore, the addition of neutralizing anti–IFN-α/β antibody to purified splenic DCs in vitro partially blocked the “spontaneous” activation of these cells, inhibiting the up-regulation of costimulatory molecules, secretion of IFN-γ, and T-cell stimulatory activity. These results show that DCs both secrete and respond to type I IFN, identifying type I interferons as autocrine DC activators.

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Influence of Epinastine Hydrochloride, an -Receptor Antagonist, on the Function of Mite Allergen-Pulsed Murine Bone Marrow-Derived Dendritic Cells In Vitro and In Vivo

Mediators of Inflammation ◽

10.1155/2009/738038 ◽

2009 ◽

Vol 2009 ◽

pp. 1-8 ◽

Cited By ~ 2

Author(s):

Ken-Zaburo Oshima ◽

Kazuhito Asano ◽

Ken-Ichi Kanai ◽

Miyuki Suzuki ◽

Harumi Suzaki

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Immune Responses ◽

Clinical Status ◽

Mouse Bone Marrow ◽

Dc Functions ◽

Receptor Antagonists ◽

Murine Bone Marrow

There is established concept that dendritic cells (DCs) play essential roles in the development of allergic immune responses. However, the influence of receptor antagonists on DC functions is not well defined. The aim of the present study was to examine the effect of epinastine hydrochloride (EP), the most notable histamine receptor antagonists in Japan, onDermatophagoides farinae (Der f)-pulsed mouse bone marrow-derived DCs in vitro and in vivo. EP at more than 25 ng/mL could significantly inhibit the production of IL-6, TNF- and IL-10 fromDer f-pulsed DCs, which was increased byDer fchallenge in vitro. On the other hand, EP increased the ability ofDer f-pulsed DCs to produce IL-12. Intranasal instillation ofDer f-pulsed DCs resulted in nasal eosinophilia associated with a significant increase in IL-5 levels in nasal lavage fluids.Der f-pulsed and EP-treated DCs significantly inhibited nasal eosinophila and reduced IL-5. These results indicate that EP inhibits the development of Th2 immune responses through the modulation of DC functions and results in favorable modification of clinical status of allergic diseases.

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Meis1-mediated apoptosis is caspase dependent and can be suppressed by coexpression of HoxA9 in murine and human cell lines

Blood ◽

10.1182/blood-2004-03-0802 ◽

2005 ◽

Vol 105 (3) ◽

pp. 1222-1230 ◽

Cited By ~ 16

Author(s):

Peter J. Wermuth ◽

Arthur M. Buchberg

Keyword(s):

Bone Marrow ◽

Bone Marrow Cells ◽

Cell Types ◽

Selective Advantage ◽

Myeloid Differentiation ◽

Homeodomain Protein ◽

Murine Bone Marrow ◽

Differentiation Pathways

AbstractCoexpression of the homeodomain protein Meis1 and either HoxA7 or HoxA9 is characteristic of many acute myelogenous leukemias. Although Meis1 can be overexpressed in bone marrow long-term repopulating cells, it is incapable of mediating their transformation. Although overexpressing HoxA9 alone transforms murine bone marrow cells, concurrent Meis1 overexpression greatly accelerates oncogenesis. Meis1-HoxA9 cooperation suppresses several myeloid differentiation pathways. We now report that Meis1 overexpression strongly induces apoptosis in a variety of cell types in vitro through a caspase-dependent process. Meis1 requires a functional homeodomain and Pbx-interaction motif to induce apoptosis. Coexpressing HoxA9 with Meis1 suppresses this apoptosis and provides protection from several apoptosis inducers. Pbx1, another Meis1 cofactor, also induces apoptosis; however, coexpressing HoxA9 is incapable of rescuing Pbx-mediated apoptosis. This resistance to apoptotic stimuli, coupled with the previously reported ability to suppress multiple myeloid differentiation pathways, would provide a strong selective advantage to Meis1-HoxA9 coexpressing cells in vivo, leading to leukemogenesis.

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Genotoxicity Assessment of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

International Journal of Toxicology ◽

10.1080/10915810500366922 ◽

2005 ◽

Vol 24 (6) ◽

pp. 427-434 ◽

Cited By ~ 9

Author(s):

Gunda Reddy ◽

Gregory L. Erexson ◽

Maria A. Cifone ◽

Michael A. Major ◽

Glenn J. Leach

Keyword(s):

Bone Marrow ◽

World War Ii ◽

Environmental Protection Agency ◽

Bone Marrow Cells ◽

Metabolic Activation ◽

Maximum Tolerated Dose ◽

Mouse Bone Marrow ◽

Mouse Lymphoma

Hexahydro-1,3,5-trinitro-1,3,5-triazine, a polynitramine compound, commonly known as RDX, has been used as an explosive in military munitions formulations since World War II. There is considerable data available regarding the toxicity and carcinogenicity of RDX. It has been classified as a possible carcinogen (U.S. Environmental Protection Agency, Integrated Risk Information System, 2005, www.epa.gov/IRIS/subst/0313.htm ). In order to better understand its gentoxic potential, the authors conducted the in vitro mouse lymphoma forward mutation and the in vivo mouse bone marrow micronucleus assays. Pure RDX (99.99%) at concentrations ranging from 3.93 to 500 μg/ml showed no cytotoxicity and no mutagenicity in forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, with and without metabolic activation. This finding was also confirmed by repeat assays under identical conditions. In addition, RDX did not induce micronuclei in mouse bone marrow cells when tested to the maximum tolerated dose of 250 mg/kg in male mice. These results show that RDX was not mutagenic in these in vitro and in vivo mammalian systems.

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Clonal growth of murine pre-B colony-forming cells and their targeted infection by a retroviral vector: dependence on interleukin-7

Blood ◽

10.1182/blood.v75.5.1132.bloodjournal7551132 ◽

1990 ◽

Vol 75 (5) ◽

pp. 1132-1138

Author(s):

DE Williams ◽

AE Namen ◽

DY Mochizuki ◽

RW Overell

Keyword(s):

Bone Marrow ◽

Clonal Growth ◽

Bone Marrow Cells ◽

Lymphoid Cells ◽

Mouse Bone Marrow ◽

Retroviral Infection ◽

Murine Bone Marrow ◽

Interleukin 7 ◽

B Lymphoid

The cDNA for interleukin-7 (IL-7) was recently isolated from a stromal cell line derived from a long-term B-lymphoid culture. We report that purified recombinant murine IL-7 can promote the clonal growth in semi- solid culture of a subpopulation of cells expressing the B220 surface antigen from normal murine bone marrow. These colony-forming cells (CFC- Pre-B) give rise to colonies of 20 to 1,000 cells after 7 days in culture. Morphologic examination of cells within the colonies showed a characteristic lymphoid morphology, and histochemical examination demonstrated an absence of markers associated with granulocyte, macrophage, eosinophil, or megakaryocyte differentiation, as well as an absence of hemoglobinization (indicative or erythroid differentiation). IL-7 was found to specifically enhance the infection of CFC-Pre-B but not CFU-GM when the cytokine was present during a 48-hour co- cultivation period between irradiated, retrovirus-producing psi 2 clones and normal mouse bone marrow cells. In contrast, IL-3 enhanced the infection of CFU-GM but not CFC-Pre-B. Thymidine suiciding studies suggest that this targeted infection is due to specific induction of cycling of CFC-Pre-B by IL-7 and CFU-GM by IL-3. These data demonstrate that IL-7 can target retroviral infection into a specific subpopulation of early B-lymphoid cells (CFC-Pre-B), and that IL-7 cannot directly promote the in vitro clonal growth of myeloid committed progenitor cells (ie, CFU-GM).

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A comparative study of the effects of genistein, estradiol and raloxifene on the murine skeletal system.

Acta Biochimica Polonica ◽

10.18388/abp.2009_2458 ◽

2009 ◽

Vol 56 (2) ◽

Cited By ~ 15

Author(s):

Leszek Sliwiński ◽

Joanna Folwarczna ◽

Barbara Nowińska ◽

Urszula Cegieła ◽

Maria Pytlik ◽

...

Keyword(s):

Mechanical Properties ◽

Bone Marrow ◽

Mrna Expression ◽

Bone Marrow Cells ◽

Bone Mineralization ◽

Mouse Bone Marrow ◽

Skeletal System ◽

Mouse Bone Marrow Cells

Genistein, a major phytoestrogen of soy, is considered a potential drug for prevention and treatment of postmenopausal osteoporosis. The aim of the present study was to compare the effects of genistein, estradiol and raloxifene on the skeletal system in vivo and in vitro. Genistein (5 mg/kg), estradiol (0.1 mg/kg) or raloxifene hydrochloride (5 mg/kg) were administered daily by a stomach tube to mature ovariectomized Wistar rats for 4 weeks. Bone mass, mineral and calcium content, macrometric parameters and mechanical properties were examined. Also the effects of genistein, estradiol and raloxifene (10(-9)-10(-7) M) on the formation of osteoclasts from neonatal mouse bone marrow cells and the activity of osteoblasts isolated from neonatal mouse calvariae were compared. In vivo, estrogen deficiency resulted in the impairment of bone mineralization and bone mechanical properties. Raloxifene but not estradiol or genistein improved bone mineralization. Estradiol fully normalized the bone mechanical properties, whereas genistein augmented the deleterious effect of estrogen-deficiency on bone strength. In vitro, genistein, estradiol and raloxifene inhibited osteoclast formation from mouse bone marrow cells, decreasing the ratio of RANKL mRNA to osteoprotegerin mRNA expression in osteoblasts. Genistein, but not estradiol or raloxifene, decreased the ratio of alkaline phosphatase mRNA to ectonucleotide pyrophosphatase phosphodiesterase 1 mRNA expression in osteoblasts. This difference may explain the lack of genistein effect on bone mineralization observed in ovariectomized rats in the in vivo study. Concluding, our experiments demonstrated profound differences between the activities of genistein, estradiol and raloxifene towards the osseous tissue in experimental conditions.

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