Distinct Metabolic Dependency of Normal and Leukemic Cells in a Mouse Model

Abstract Abstract 759 We hypothesized that metabolic differences between leukemia initiating cells and their normal counterparts represent a vulnerability in the leukemia initiating cell, which can be therapeutically exploited. To test this hypothesis, we used the MLL-AF9 acute myeloid leukemia (AML) model in mice. Actin-DsRed mouse bone marrow transduced with MLL-AF9 expressing retrovirus was used to produce serially transplantable leukemia. Leukemic granulocyte-monocyte precursors (L-GMPs), defined by others to be the leukemia initiating cells were flow sorted from secondary recipient mice and compared with normal GMPs (N-GMPs) from actin Ds-Red mice. Gene expression profiling, metabolomic profiling via liquid chromatography- mass spectrometry and an in vitro shRNA screen were used to identify metabolic pathways preferentially activated in leukemia initiating cells. Of 1574 defined metabolic enzymes, 44 were found to be differentially expressed between L-GMPs and their normal counterparts (N-GMPs). These together with 117 classic rate limiting enzymes were subjected to shRNA knockdown in vitro. Metabolomic profiling of both cell populations was used to corroborate findings from shRNA knockdowns. L-GMPs and N-GMPs were transduced with lentivirus expressing shRNAs of interest (5 shRNAs per gene) in a 384 well format, selected with puromycin and cultured for 72–96 hours in the presence of GFP-positive primary bone marrow stroma. The number of cells in each well at the end of this experiment was quantitated using an Image Xpress microscope. Genes, the knockdown of which by at least two independent shRNAs produced a two fold or more decrease in L-GMPs as compared to control wells and did not similarly decrease N-GMPs, were chosen for in vivo validation. Ten genes in the glycolysis pathway and TCA cycle, fatty acid metabolism and detoxification, and ketohexokinase were identified. Ketohexokinase, a rate-limiting enzyme in fructose metabolism was particularly potent and of interest given its potential to be exploited therapeutically. In vivo assessment of its relative ability to inhibit malignant versus normal hematopoietic cells is ongoing. These studies provide preliminary support for the hypothesis that specific metabolic circuits are differentially active in leukemia initiating cells in MLL-AF9 AML and may represent unique points of vulnerability that can be targeted therapeutically. Authors 1 and 2 contributed equally. Authors 3 and 4 contributed equally. Disclosures: No relevant conflicts of interest to declare.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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PML-RARα Deregulates an Unexpectedly Small Number of Genes in Pre-Leukemic Promyelocytes

Blood ◽

10.1182/blood.v120.21.3526.3526 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3526-3526

Author(s):

Coline M Gaillard ◽

Taku A Tokuyasu ◽

Emmanuelle Passegué ◽

Scott C. Kogan

Keyword(s):

Bone Marrow ◽

Fusion Protein ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Leukemic Cells ◽

Cytokine Signaling ◽

Proliferation Capacity ◽

Number Of Genes

Abstract Abstract 3526 Background: Acute Promyelocytic Leukemia (APL) is characterized by the accumulation in the blood and bone marrow of abnormal promyelocytes, which have the ability to transfer the disease to secondary recipients in animal models. The PML-RARα fusion protein is thought to be the primary abnormality implicated in the pathology, and is believed to prevent transcription of genes necessary for normal myeloid development and differentiation. Identifying PML-RARα targets is critical for understanding the road to leukemic transformation. However, such targets have so far been identified using cell line assays in vitro, murine cells differentiated into promyelocytes in vitro, or fully transformed murine or human leukemic cells. Focusing on the cell population in which the transforming potential is acquired, we describe here a novel strategy to identify the transcriptomic dysregulation induced by PML-RARα expression in maturing myeloid populations in vivo. Methods: We utilize a murine model of human APL in which the human PML-RARα fusion gene is expressed under the control of the MRP8 promoter, driving its expression in maturing myeloid populations. Those animals can be described as pre-leukemic since they eventually develop leukemia when additional mutations occur. Fresh bone marrows from normal (Fvb/n) or pre-leukemic (PML-RARα) animals were harvested. Using an improved cell surface antigen staining strategy and fluorescence-activated cell sorting, three populations of increasingly differentiated myeloid populations have been sorted (Granulocyte Macrophage Progenitor, Early promyelocyte and Late promyelocyte). RNA was extracted and submitted for whole-genome microarray analysis. In addition, we are using a variety of bioinformatics approaches to decipher the network of novel interactions driven by PML-RARα expression. Results: Markers used in our sorting strategy were validated in the dataset, including CD34 and Gr1. In the normal samples, markers of neutrophil maturation increased, largely as expected, and a number of early transcription factors decreased in an expected manner including Hoxa9 and Meis1. One remarkable finding was that despite the previously described ability of PML-RARα to regulate transcription from multiple sites in the genome, only a small number of genes were differentially impacted by the expression of this protein. Surprisingly, well-known regulators of myeloid differentiation that have been implicated in the retinoic acid responsiveness of APL including Sfpi1 (PU.1) and Cebpa were not differentially expressed. However, in pre-leukemic samples PML-RARα did cause decreased expression of multiple neutrophilic granule genes including Ltf, Mmp9 and Ngp. The gene most upregulated in the pre-leukemic samples was Spp1 which encodes the osteopontin phosphoprotein. Of interest, we identified the myeloid tumor suppressor Irf8 to be downregulated 5 fold in the presence of PML-RARα. To investigate the importance of IRF8 levels in APL initiation, we transplanted Irf8+/+ PML-RARα or Irf8+/− PML-RARα bone marrow into irradiated recipients. Despite the potential for decreased expression of IRF8 to contribute to APL, we observed no difference. This result does not confirm a role for IRF8 in APL pathogenesis, but further investigations are needed to exclude such a role. Bioinformatics studies highlighted enrichment in cell cycle-related genes upon PML-RARα expression, suggesting a possible difference in the proliferation capacity of the pre-leukemic cells, which is currently under investigation. Conclusions: We found that in vivo the transcriptome was only modestly dysregulated by the presence of PML-RARα. These observations open up new questions on the role of the fusion protein in pathogenesis: How does PML-RARα prime pre-leukemic cells for full transformation? How do secondary events allow an initiated cell to advance to a fully transformed state? Such questions are currently being investigated, with a special interest on looking at the cooperation between PML-RARα and activated cytokine signaling in leukemia initiation. Disclosures: No relevant conflicts of interest to declare.

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The Role of microRNA-155 in Mouse Models of MLL -AML

Blood ◽

10.1182/blood.v126.23.2446.2446 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2446-2446

Author(s):

Anna Staffas ◽

Edith Schneider ◽

Linda Fogelstrand ◽

Linda Röhner ◽

Michael Heuser ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Therapeutic Target ◽

Conflicts Of Interest ◽

Micro Rna ◽

Blood Analysis ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Fusion Genes

Abstract Background: Genetic rearrangements that fuse the mixed lineage leukemia (MLL) gene, now termed KMT2A, to one of a variety of partners are seen in 5% - 20% of acute myeloid leukemia (AML). MLL -fusions are especially common in childhood AML and many of them are associated with poor prognosis. The MLL -fusions perturb transcription through different mechanisms and they are often associated with high expression of the transcription factors HOXA9 and MEIS1. Based on a micro-RNA screen in an AML mouse model mimicking the step-wise development of aggressive AML we have found that concurrent Hoxa9 and Meis1 overexpression is associated with upregulation of micro-RNA-155 (miR-155). Expression of miR-155 was also found to be higher in bone marrow samples from patients with MLL- AML compared with bone marrow from healthy donors (p <0.001), as were the expression of HOXA9 and MEIS1 (p <0.05). In lymphomas, miR-155 plays a pivotal role as an oncogene. It is frequently upregulated in samples from lymphoma patients and a mouse model of lymphoma showed a certain degree of miR-155-addiction which could be targeted by miR-155 inhibitors. Despite the differences in the pathobiology of AML and lymphoma, the upregulation of miR-155 in AML with high HOXA9 and MEIS1 expression may indicate miR-155 as a relevant therapeutic target also in MLL -AML. Methods: To test the importance of miR-155 and its potency as a drug target in MLL -AML we used a miR-155 knock-out mouse model (miR-155-/-) (Thai et al, Science, 2007). MLL -fusion genes of varying leukemic potential; MLL-AF5 (KMT2A-AFF4), MLL-ENL (KMT2A-MLLT1), MLL-AF9 (KMT2A-MLLT3) were retrovirally expressed in miR-155-/- mouse bone marrow (mbm) cells and in wild-type mbm cells (miR-155+/+). Results: In concordance with the previous findings in human AML patient samples, miR-155+/+ cells expressing MLL-AF5, MLL-ENL, or MLL-AF9 showed upregulation of miR-155 (p < 0.05). Also, Hoxa9 and Meis1 transcripts were increased (p<0.05). Interestingly, the magnitude of upregulation of both miR-155 and Meis1 correlated with the degree of aggressiveness based on disease latency and survival observed in these leukemia models with highest upregulation in MLL-ENL and MLL-AF9 and lowest in MLL-AF5 (p<0.05). Expression of the MLL-fusion genes in miR-155-/- mbm cells resulted in similar induction of Hoxa9 and Meis1 expression as in miR-155+/+ mbm cells, indicating that miR-155 is downstream of the Hoxa9/Meis1 axis. To determine the leukemic potential in vivo, we transplanted recipient mice with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-ENL or MLL-AF9. Engraftment of leukemic cells, based on peripheral blood analysis, did not differ between mice transplanted with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-fusions. Also, disease development induced by MLL-AF9 and MLL-ENL (4-8 weeks and 10-32 weeks, respectively) was similar in mice transplanted with miR-155-/- mbm cells and mice transplanted with miR-155+/+ mbm cells. In accordance with the in vivo results, functional studies in vitro showed that the proliferative capacity and colony forming ability of MLL -fusion expressing cells were similar in miR-155+/+ mbm cells and miR-155-/- mbm cells, indicating that miR-155 is not essential for MLL-ENL- or MLL-AF9-induced leukemic transformation. Conclusions: In summary, miR-155 is upregulated in MLL-AML in both mice and man, seemingly through an MLL>HOXA9/MEIS1>miR-155 axis. Since absence of miR-155 does not alter the leukemic potential induced by MLL-AF9 or MLL-ENL, miR-155 may contribute to, but is not pivotal for MLL leukemogenesis. We therefore conclude that miR-155 is not a therapeutic target in MLL- AML. Disclosures No relevant conflicts of interest to declare.

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Modeling Growth Of Pediatric T-ALL In Vivo and In Vitro: Clinical Meaning and Activation Of The NFkB Pathway

Blood ◽

10.1182/blood.v122.21.2571.2571 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2571-2571

Author(s):

Sandrine Poglio ◽

Xavier Cahu ◽

Benjamin Uzan ◽

Hélène Lapillonne ◽

Thierry Leblanc ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Stromal Cells ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Nsg Mice ◽

Nfkb Activation ◽

Two Systems

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the proliferation of T-cell precursors in various sites, such as thymus, bone marrow, blood, lymph nodes or central nervous system. As T-ALL cells alone do not successfully grow in vitro, xenografts of T-ALL cells into NOD/scid/IL-2R null (NSG) mice and long-term co-cultures of T-ALL cells with stromal cells have been developed to study the biology of T-ALL cells (Armstrong et al, Blood, 2009). However, the growth of T-ALL cells in these two systems is highly variable across T-ALL samples. Moreover, the clinical relevance of both assays and, except for NOTCH pathway activation, the molecular pathways involved in successful in vivo and in vitro growths are still elusive. The aim of this work was to determine the relationships between clinical, biological and molecular characteristics of human T-ALL at diagnosis and the growth of T-ALL in these two systems. Human T-ALL blood samples were collected at diagnosis from pediatric or young adult patients with T-ALL. 50,000 T-ALL cells were intravenously injected into NSG mice. Mouse bone marrow samples were collected every 3-4 weeks from day 35 to day 210 post-transplant. Leukemic engraftment was monitored using flow cytometry measuring the % of human CD45+CD7+ leukemic cells. Time to leukemic engraftment (TTL) was defined as the time between T-ALL injection and the detection of ≥20% leukemic cells in at least one mouse. In vitro co-culture growth assay consisted in plating 200,000 cells on MS5 or MS5-DL1 (Armstrong, Blood, 2009) and count every 7 days up to 28 days. A total of 36 samples were tested of which 22 (61%) engrafted into mice. Global median TTL was 82 days (range, 36-121) defining short (TTL<82 days) and long or no engraftment (TTL>82 days) TTL groups. Patient gender, age, mediastinal involvement or abnormal karyotype had no significant impact on TTL. A trend for a shorter TTL was observed for T-ALL samples with a white blood cell count (WBC) > median WBC = 146 G/L (p =0.06). Samples containing more than 20% of TCRαβ or CD8 positive cells exhibited increased incidence of engraftment (p = 0.049 and p=0.04 respectively) whereas CD34, CD1a, CD4 or sCD3 markers were not significantly correlated with TTL. Unlike samples with TLX1, TLX3 overexpression or NOTCH/FBXW7 mutations, samples with SIL-TAL1 deletion exhibited a shorter TTL (p = 0.0004). The 2-year progression free survival of “short TTL” patients was 72% vs 70% for patients with “longer TTL” or no engraftment (p=0.38). T-ALL samples for which growth could be achieved on MS5 cells also displayed a shorter TTL. To unravel molecular mechanisms involved in the growth of leukemic cells in these two systems, micro-arrays were performed for 8 “short TTL” T-ALL versus 8 “long TTL or no engraftment” T-ALL. 346 genes were differentially express in short TTL samples compared to long/no TTL samples (P<0.05, fold change: 1.5). As expected, most of genes up-regulated in short TTL group were implicated in cell cycle function enhancing the commitment of cells to S/M phases. Analysis of regulated networks revealed that several indirect modulators of NFkB (MAL, AhR and CYLD) were significantly up/down regulated in short TTL patient samples resulting in NFkB activation. Overall, T-ALL with SIL-TAL1 deletion display an increased ability to engraft into NSG mice, in accordance with increased WBC in T-ALL patients. Contrary to B-ALL, shorter TTL is not associated with poor prognosis in T-ALL. Moreover, NSG engraftment and co-culture on stromal cells are well correlated. A shorter TTL seems to be associated with an increased leukemic proliferation through NFkB activation. Disclosures: No relevant conflicts of interest to declare.

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A MODEL to STUDY the Function of NPM-MLF1 IN MYELODYSPLASIA.

Blood ◽

10.1182/blood.v116.21.1571.1571 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1571-1571

Author(s):

Wen-hsin Lee

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Dna Damage Response ◽

Conflicts Of Interest ◽

Mouse Bone Marrow ◽

Self Renewal ◽

Damage Response ◽

Elevated Expression

Abstract Abstract 1571 Myelodysplastic syndromes (MDS) are bone marrow disorders characterized by ineffective haematopoiesis and peripheral cytopenia(s) with frequent evolution to acute myeloid leukemia (AML). Apoptosis is significantly deregulated in early MDS whereas advanced MDS is characterized by deregulation of DNA damage response. As MDS proceeds to AML, the ratio of apoptosis to proliferation decreases, resulting in clonal outgrowth of abnormal cells. The t(3;5)(q25;q34) translocation, creating the NPM-MLF1 fusion, has been found as a sole cytogenetic abnormality in MDS. It is recurrent, with poor prognosis but the precise mechanism through which NPM-MLF1 induces malignant transformation remains unknown. We aimed to model this disease in vitro and in vivo by expressing NPM-MFL1 in mouse bone marrow hematopoietic progenitor cells (HPCs) and analyzing any changes in HPC self-renewal and response to DNA damage. NPM-MLF1 did not impair haematopoiesis in vitro and in vivo. FLT3/ITD was frequently associated with NPM mutant in AML patients; however, NPM-MLF1 did not collaborate with FLT3/ITD in our system. To recapitulate NPM hemizygosity in t(3;5)-MDS patients, we have expressed NPM-MLF1 in HPCs derived from Npm+/− mice. A transient increase in the self-renewal of the NPM-MLF1-expressing Npm+/− HPCs was seen. These cells did not exhibit enhanced proliferation as confirmed by growth curve and analysis of DNA synthesis. Interestingly, unlike control cells, NPM-MLF1-expressing Npm+/− HPCs showed prolonged self-renewal ability in vitro, and an elevated expression of c-Myc, Hoxa9, Hoxa10 and Meis1 genes. In addition to altering HPC self-renewal, NPM-MLF1 was also found to modulate their DNA damage response. This study suggests that the ability of NPM-MLF1 to maintain HPC self-renewal and impaired DNA damage responses may favour the accumulation and outgrowth of the aberrant HPCs, contributing to the abnormal haematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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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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Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow

10.1101/2021.08.06.455158 ◽

2021 ◽

Author(s):

Zixian Liu ◽

Jinhong Wang ◽

Miner Xie ◽

Peng Wu ◽

Yao Ma ◽

...

Keyword(s):

Bone Marrow ◽

Single Cell ◽

Pcr Analysis ◽

Mouse Bone Marrow ◽

Hematopoietic Stem ◽

Colony Assay ◽

Megakaryocyte Progenitors ◽

Cell Colony

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in C57BL/6 mice. Single-cell transplantation and single-cell colony assay showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average eight megakaryocytes per colony) than did previously reported megakaryocyte progenitors (MkPs). Single-cell RNA-sequencing supported that these MgPs lie between HSCs and MkPs along the megakaryocyte differentiation pathway. Single-cell colony assay and single-cell RT-PCR analysis suggested the coexpression of CD41 and Pf4 is associated with megakaryocyte colony-forming activity. Single-cell colony assay of a small number of cells generated from single HSCs in culture suggested that MgPs are not direct progeny of HSCs. In this study, we propose a differentiation model in which HSCs give rise to MkPs through MgPs.

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Two classes of murine granulocyte progenitor cells expressed in plasma clot diffusion chamber cultures

Blood ◽

10.1182/blood.v59.4.838.838 ◽

1982 ◽

Vol 59 (4) ◽

pp. 838-843 ◽

Cited By ~ 6

Author(s):

HN Steinberg ◽

PL Page ◽

SH Robinson

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Cytosine Arabinoside ◽

Diffusion Chamber ◽

Velocity Sedimentation ◽

Mouse Bone Marrow ◽

Plasma Clot ◽

Residual Concentration

Abstract Two distinct classes of granulocyte progenitor cells present in normal mouse bone marrow are expressed sequentially in the vivo plasma clot diffusion chamber culture system. By several criteria, progenitor cells giving rise to granulocyte colonies on day 4 of culture (CFU-D4) are different from those giving rise to colonies on day 7 (CFU-D7). These differences include: cell cycle activity as measured by in vitro incubation with cytosine arabinoside, residual concentration in the bone marrow after in vivo treatment of donor mice with cytosine arabinoside or methotrexate, resistance to osmotic lysis, size as determined by velocity sedimentation, and the morphology of the granulocyte colonies to which these cells give rise. The CFU-D7 appears to represent an earlier progenitor cell than the CFU-D4 in the differentiation pathway of the granulocyte and is analagous in many respects to the BFU-E in the erythroid pathway.

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Pasteurella multocida Toxin Activates Human Monocyte-Derived and Murine Bone Marrow-Derived Dendritic Cells In Vitro but Suppresses Antibody Production In Vivo

Infection and Immunity ◽

10.1128/iai.73.1.413-421.2005 ◽

2005 ◽

Vol 73 (1) ◽

pp. 413-421 ◽

Cited By ~ 22

Author(s):

Kenneth C. Bagley ◽

Sayed F. Abdelwahab ◽

Robert G. Tuskan ◽

George K. Lewis

Keyword(s):

Bone Marrow ◽

Dendritic Cells ◽

Phospholipase C ◽

Cholera Toxin ◽

Pasteurella Multocida ◽

Human Monocyte ◽

Mouse Bone Marrow ◽

Dependent Manner

ABSTRACT Pasteurella multocida toxin (PMT) is a potent mitogen for fibroblasts and osteoblastic cells. PMT activates phospholipase C-β through Gqα, and the activation of this pathway is responsible for its mitogenic activity. Here, we investigated the effects of PMT on human monocyte-derived dendritic cells (MDDC) in vitro and show a novel activity for PMT. In this regard, PMT activates MDDC to mature in a dose-dependent manner through the activation of phospholipase C and subsequent mobilization of calcium. This activation was accompanied by enhanced stimulation of naïve alloreactive T cells and dominant inhibition of interleukin-12 production in the presence of saturating concentrations of lipopolysaccharide. Surprisingly, although PMT mimics the activating effects of cholera toxin on human MDDC and mouse bone marrow-derived dendritic cells, we found that PMT is not a mucosal adjuvant and that it suppresses the adjuvant effects of cholera toxin in mice. Together, these results indicate discordant effects for PMT in vitro compared to those in vivo.

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Cell surface peptidase CD26/DPPIV mediates G-CSF mobilization of mouse progenitor cells

Blood ◽

10.1182/blood-2002-12-3893 ◽

2003 ◽

Vol 101 (12) ◽

pp. 4680-4686 ◽

Cited By ~ 144

Author(s):

Kent W. Christopherson ◽

Scott Cooper ◽

Hal E. Broxmeyer

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Mouse Bone Marrow ◽

Peptidase Activity ◽

Hematopoietic Stem ◽

Migratory Response ◽

Stromal Cell Derived Factor ◽

Marrow Cells

AbstractCXC ligand 12 (CXCL12; also known as stromal cell–derived factor 1α/SDF-1α) chemoattracts hematopoietic stem and progenitor cells (HSCs/HPCs) and is thought to play a crucial role in the mobilization of HSCs/HPCs from the bone marrow. CD26 (dipeptidylpeptidase IV [DPPIV]) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N-terminus of polypeptide chains. CD26 has the ability to cleave CXCL12 at its position-2 proline. We found by flow cytometry that CD26 is expressed on a subpopulation of normal Sca-1+c-kit+lin— hematopoietic cells isolated from mouse bone marrow, as well as Sca-1+c-kit—lin— cells, and that these cells possess CD26 peptidase activity. To test the functional role of CD26 in CXCL12-mediated normal HSC/HPC migration, chemotaxis assays were performed. The CD26 truncated CXCL12(3-68) showed an inability to induce the migration of sorted Sca-1+c-kit+lin— or Sca-1+c-kit—lin— mouse marrow cells compared with the normal CXCL12. In addition, CXCL12(3-68) acts as an antagonist, resulting in the reduction of migratory response to normal CXCL12. Treatment of Sca-1+c-kit+lin— mouse marrow cells, and myeloid progenitors within this population, or Sca-1+c-kit—lin— cells with a specific CD26 inhibitor, enhanced the migratory response of these cells to CXCL12. Finally, to test for potential in vivo relevance of these in vitro observations, mice were treated with CD26 inhibitors during granulocyte colony-stimulating factor (G-CSF)–induced mobilization. This treatment resulted in a reduction in the number of progenitor cells in the periphery as compared with the G-CSF regimen alone. This suggests that a mechanism of action of G-CSF mobilization involves CD26.

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