Evidence for An Anti-Cancer Barrier in a Mixed Lineage Leukemia Mouse Model in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3347-3347
Author(s):  
Sylvia Takacova ◽  
Jiri Bartek ◽  
Lucie Piterkova ◽  
Robert K. Slany ◽  
Vladimir Divoky
Keyword(s):  
Bone Marrow ◽  
Tumor Suppressor ◽  
Mouse Model ◽  
Peripheral Blood ◽  
Myeloid Cells ◽  
Mixed Lineage Leukemia ◽  
Cell Counts ◽  
Potential Tumor Suppressor

Abstract Mixed Lineage Leukemia (MLL) mutations identify a unique group of acute leukemias with distinct biological and clinical features. Although the role of MLL in leukemogenesis has been extensively studied, a precise mechanism regarding the leukemogenic potential of MLL mutations is not known. We generated a switchable MLL-ENL-ERtm mouse model, in which the MLL-ENL oncogene has been introduced by homologous recombination and is controlled by the endogenous MLL promoter, thus, expressed at physiological levels. Due to fusion with the estrogen receptor ligand binding domain (ERtm), the MLL-ENL-ERtm protein activity is dependent on continuous provision of tamoxifen or 4-hydroxytamoxifen. The MLL-ENL-ERtm mice have developed a myeloproliferative disorder (MPD) characterized by persistent mature neutrophilia after 484,5 +/− 75,68 days of latency on a tamoxifen diet, in association with high white cell counts in peripheral blood, splenomegaly and occasionally with anemia. Blood smears showed large numbers of mature myeloid elements consisting of 40–80% neutrophils (non-segmented forms in abundance), admixed with immature myeloid elements, 3–11% monocytes and 2–6% myeloblasts. The phenotype of MPD also involved myelomonocytic proliferation with 35% immature monocytic cells in one animal and severe anemia with increased numbers of immature erythroid cells in peripheral blood in another animal. Hematoxylin- and eosin-stained sections of the bone marrow from MLL-ENL-ERtm mice revealed expansion of myeloid cell population with no signs of progressive dysplasia. We observed massive infiltration of myeloid cells (positive for myeloperoxidase) into spleen with various degree of loss of normal splenic architecture depending on disease progression. FACS profiles of both bone marrow and spleen cells showed a typical pattern of granulocyte/macrophage/monocyte surface marker expression (CD34-CD43+Mac- 1+Gr-1+CD16/32+). In vitro evaluation of hematopoetic progenitors derived from bone marrow of leukemic mice at the terminal stage of the disease revealed decreased numbers of BFU-Es and increased numbers of CFU-GMs and CFU-Gs compared to matched controls. These results correlated with the expansion of the myelomonocytic and reduction of the erythroid compartment observed in the bone marrow of these animals. The average size (cellularity) of the mutant myeloid colonies was much smaller than the colonies derived from the wild-type controls, which could be caused by a partial block of terminal differentiation of myeloid progenitors in vitro. In vivo, MLL-ENL leads to expansion of differentiated myeloid cells in our model. High penetrance and long latency of leukemia in our model permits the study of early leukemia development. Our model revealed that MLL-ENL - induced myeloproliferation occurs as early as twelve weeks after MLL-ENL-ERtm activation in the bone marrow and infiltrates the spleen with a consequent decrease in lymphoid B220+CD19+IgM+ cells. Using the TUNEL assay on bone marrow sections, we observed induction of apoptosis in the highly proliferative bone marrow compartment compared to matched controls. These results suggest activation of a potential tumor suppressor mechanism by MLL-ENL in early stages of leukemia. We are currently investigating potential tumor suppressor pathways that might be involved in MLL-ENL - induced apoptosis in preleukemia.

A Novel Inhibitor of Mutant IDH1 Induces Differentiation in Vivo and Prolongs Survival in a Mouse Model of Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3598-3598 ◽  
Author(s):  
Anuhar Chaturvedi ◽  
Michelle Maria Araujo Cruz ◽  
Ramya Goparaju ◽  
Nidhi Jyotsana ◽  
Heike Baehre ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Medical School ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Leukemic Cells ◽  
Median Latency ◽  
Mutant Idh1

Abstract Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in patients with glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, cholangiocellular carcinoma and chondrosarcoma. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. We recently established an in vivo mouse model and investigated the function of mutant IDH1. By computational drug screening, we identified an inhibitor of mutant IDH1 (HMS-101), which inhibits mutant IDH1 cell proliferation, decreases R2HG levels in vitro, and efficiently blocks colony formation of AML cells from IDH1 mutated patients but not of normal CD34+ bone marrow cells. In the present study we investigated the effect of the inhibitor in our IDH1/HoxA9-induced mouse model of leukemia in vivo. To identify the maximally tolerated dose of HMS-101, we treated normal C57BL/6 mice with variable doses of HMS-101 for 9 days and measured the serum concentration. Mice receiving 0.5 mg and 1mg intraperitoneally once a day tolerated the drug well with mean plasma concentrations of 0.1 to 0.3 µM. To evaluate the effect of HMS-101 in the IDH1 mouse model, we transduced IDH1 R132C in HoxA9-immortalized murine bone marrow cells. Sorted transgene positive cells were then transplanted into lethally irradiated mice. After 5 days of transplantation, mice were treated with HMS-101 intraperitoneally for 5 days/week. The R/S-2HG ratio in serum was reduced 3-fold in HMS-101 treated mice after 8 weeks of treatment compared to control treated mice. HMS-101 or PBS treated mice had similar levels of transduced leukemic cells in peripheral blood at 2 and 6 weeks after transplantation. However, from week 6 to week 15 leukemic cells in peripheral blood decreased from 76% to 58, 63% to 29%, 67% to 7%, and 74% to 38% in 4/6 mice treated with HMS-101. In one mouse the percentage of leukemic cells was constant, and in one mouse it increased from week 6 to week 15 after transplantation. Leukemic cells increased constantly in peripheral blood until death in control treated mice. While the control cohort developed severe leukocytosis, anemia and thrombocytopenia around 8 to 10 weeks post transplantation, mice treated with HMS-101 still had normal WBC, RBC and platelet counts at 15 weeks after transplantation. Moreover, the HMS-101 treated mice had significantly more differentiated Gr1+CD11b+ cells in peripheral blood than control mice at 6 weeks and 15 weeks after transplantation and at death (P=.01). Morphologic evaluation of blood cells at 15 weeks or death from HMS-101 treated mice revealed a high proportion of mature neutrophils that were GFP positive and thus derived from IDH1 transduced cells, whereas control treated mice had monocytic morphology with a high proportion of immature cells. Importantly, HMS-101 treated mice survived significantly longer with a median latency of 87 days (range 80-118), whereas PBS-treated mice died with a median latency of 66 days (range 64-69) after transplantation (P<.001). Of note, HMS-101 was found to be specific for mutant IDH1, as mutant IDH2 cells were not preferentially inhibited over IDH2 wildtype cells in vitro. This data demonstrates that HMS-101 specifically inhibits R2HG-production of mutant IDH1 in vivo, inhibits proliferation, induces differentiation in leukemic cells, and thus prolongs survival of IDH1mutant leukemic mice. Therefore, HMS-101 - a novel inhibitor of mutant IDH1 - shows promising activity in vivo and warrants further development towards clinical use in IDH1 mutated patients. Disclosures Chaturvedi: Hannover Medical School: Patents & Royalties. Preller:Hannover Medical School: Patents & Royalties. Heuser:Hannover Medical School: Patents & Royalties.

scholarly journals Generation of Synthetic T-ALL By De Novo Transformation of Human Cord Blood Progenitors with a 4-Oncogene Cocktail

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3675-3675
Author(s):  
Manabu Kusakabe ◽  
Claire Shanna ◽  
Xuehai Wang ◽  
Catherine Jenkins ◽  
Vincenzo Giambra ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Human Cells ◽  
Post Transplant ◽  
Cell Counts ◽  
Flow Cytometric

Abstract Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of blood cancer that can arise in both children and adults. Numerous studies have explored the effects of putative T-ALL oncogenes in mouse models and have contributed significantly to our understanding of disease pathogenesis. Nonetheless, it is clear there are important differences between mouse and human cells, particularly with respect to cellular transformation, and additional work is therefore needed to generate more accurate models of human disease. We sought here to create human T-ALL in the lab from normal CB progenitors by lentiviral transduction with a combination of known T-ALL oncogenes. Methods: Human CD34+ hematopoietic progenitor cells were isolated from pooled cord blood by magnetic bead/flow cytometric sorting (MACS/FACS). Sorted cells were then transduced by lentiviral vectors encoding a combination of four known T-ALL oncogenes including activated NOTCH1. NOTCH1 virus was marked with a GFP reporter (N1/GFP) while the other three accessory oncogenes were marked with a Cherry reporter (3xOnc/Cherry). Transduced cells were cultured on OP9-DL1 stromal feeders briefly prior to transplantation into NOD/SCID-IL2Rg-null (NSG) mice to assess leukemogenesis, or for longer periods to study their behavior in vitro. Results: Initial transduction efficiencies were typically 3-5% for each virus with 1-2% doubly-transduced N1/GFP+, 3xOnc/Cherry+ cells (hereafter referred as 4xOnc cells). After 28 days culture in vitro, the 4xOnc population reproducibly expanded and outcompeted singly- and non-transduced populations, accounting for more than 70% of cells in mixed cultures. By absolute cell counts, non-transduced cells stopped expanding within the first few weeks; however, 4xOnc cells kept expanding even after 6 weeks of culture. To test leukemogenesis in vivo, CD45+ cells were FACS sorted after 10 days of culture on OP9-DL1 feeders (including doubly-, singly-, and non-transduced populations) and injected intrahepatically into NSG neonates. Engraftment of human cells was followed monthly by flow cytometry of peripheral blood. Engraftment of GFP+ Cherry+ 4xOnc cells was first detected 2 months after transplantation whereas no engraftment of singly- or non-transduced cells was detected. The level of engraftment was below 5% and did not increase substantially even after 6 months following transplantation. At day 203 post-transplant, the primary recipient was sacrificed and 4xOnc cells were recovered from bone marrow, spleen and thymus where the levels of engraftment were approximately 10%. 4xOnc cells from the primary recipient were then serially transplanted into secondary recipients. Engraftment of 4xOnc cells in secondary recipients was observed 5 weeks after transplant. Unlike the primary recipient, however, the percentage of 4xOnc cells in the peripheral blood of secondary recipients gradually increased and these animals developed clinically morbid disease by 20 weeks post-transplant. At the time of necropsy, splenomegaly, lymphadenopathy, and enlarged thymus were observed and the bone marrow contained 80-90% 4xOnc cells. By flow cytometric analyses, 4xOnc cells expressed CD2, CD3, CD7, CD38, and TdT supporting acute T-cell leukemia. Also, TCR gamma clonality assay was performed with genomic DNA from 4xOnc cells from secondary recipients and revealed of 5-7 distinct clonal populations. These in vitro and in vivo findings were observed with multiple experimental replicates and with different pools of cord blood. Conclusion: Our in vitro and in vivo results suggest that NOTCH1, in combination with 3 accessory oncogenes are sufficient to transform normal human blood cells into clonal T-ALL-like malignant cells. Although we cannot exclude the possibility of the spontaneous acquisition of additional co-operating genetic or epigenetic abnormalities, this model provides a significant step forward to reveal the mechanisms involved in human T-ALL pathogenesis. Disclosures No relevant conflicts of interest to declare.

Doxycycline Regulatable Expression of Cytidine Deaminase Mediates Myeloprotection and Avoids Lymphotoxicity in a Murine Transplant Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2054-2054
Author(s):  
Nico Lachmann ◽  
Sebastian Brennig ◽  
Nils Pfaff ◽  
Heiko Schermeier ◽  
Axel Schambach ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Myeloid Cells ◽  
Hematopoietic Cell ◽  
Significant Protection ◽  
Cell Compartments

Abstract Abstract 2054 Hematopoietic stem cells (HSCs) genetically modified to overexpress drug resistance genes have been advocated to overcome chemotherapy induced myelosuppression. In this context, we demonstrated that overexpression of hCDD from a constitutive spleen focus forming virus (SFFV)-derived promoter protects hematopoietic cells from Ara-C toxicity in vitro and in vivo. However, these studies also indicated substantial lymphotoxicity by high level constitutive CDD expression (Rattmann et al. Blood, 2006). To circumvent this problem, we now have established a Doxycycline (Dox)-inducible (TET-ON) CDD-expression system and have evaluated this system in murine in vitro and in vivo protection assays. In vitro CDD-mediated Ara-C resistance was evaluated in the hematopoietic cell line 32D as well as primary lineage negative (Lin-) clonogenic progenitor cells. In these studies cells were co-transduced with two lentiviral constructs expressing CDD (TET.CDD) or the reverse transactivator protein (rtTA3). In 32D cells, administration of 0.2 to 2.0 μg/ml Dox induced transgene expression to plateau levels within 24 hours. Rapid induction of the transgene also was observed at lower Dox concentrations, although exposure to 0.008 or 0.04 μg/ml led to notably reduced expression levels. Functionality of Dox-inducible hCDD expression was evaluated by exposing TET.CDD transgenic 32D cells to Ara-C. Transduced cells cultured in the presence of 2.0 μg/ml Dox proved completely resistant to Ara-C concentrations of up to 5000 nM, whereas eGFP- or not Dox treated control cells were susceptible to Ara-C exposure from 50 nM onwards. Again, hCDD-mediated drug resistance was dependent on the Dox dosage. When TET.CDD transduced 32D cells were exposed to Dox for 72h, marked protection from Ara-C was shown for Dox concentration of >0.2 μg/ml (LD50 > 2.000 nM Ara-C). After Dox withdrawal transgene expression remained detectable for at least three days. Similar protection was observed in primary hematopoietic cells and progenitor cell derived colonies were protected from Ara-C doses of 300 to 600 nM while untransduced control cells did not yield colony growth at doses of 50 nM Ara-C or higher. In vivo studies were performed by transplanting C57Bl/6 mice with Lin- cells from Rosa26-M2rtTA mice previously transduced with the TET.CDD or a control SIN lentiviral vector. Transgene expression was induced by Dox administration starting four weeks post transplantation. In this model Dox administration induced stable transgene expression in peripheral blood B, T, and myeloid cells peaking 15 days after start of administration and remaining detectable for 21 days after Dox withdrawal. No alterations in peripheral blood cell counts including the lymphocyte count were observed for up to eight weeks of Dox administration and also the relative contribution of gene modified cells to peripheral blood B, CD4+ or CD8+ T, and myeloid cells remained fairly constant during this time period. Even more important, a similar contribution of transduced cells was observed for the myeloid and the lymphoid cell compartment strongly arguing against a major lymphotoxicity. Also, no toxic effects of Dox-regulated hCDD expression was observed in other hematopoietic cell compartments including stem- or progenitor cells in various splenic, thymic and BM-derived hematopoietic cell compartments. Moreover, the TET.CDD vector conveyed significant protection against Ara-C (500 mg/kg, d1-4, i.v.) to the hematopoietic system as measured by granulocyte (0.26 +/−0.25 versus 0.8 +/−0, p=0.02) and platelet counts (584 +/−159 versus 883 +/−194, p=0.02) seven days after treatment. Furthermore, when the long term reconstitution potential of TET.CDD transduced Lin- cells was evaluated by secondary transplantation, robust, Dox-dependent transgenic eGFP expression was observed in peripheral blood B, CD4+ and CD8+ T, as well as myeloid cells of secondary recipients. Taken together, our data demonstrate efficient Dox-inducible hCDD expression in 32D and primary murine bone marrow cells in vitro as well as in a murine in vivo bone marrow transplant gene transfer model. Most importantly, in the latter model Dox-inducible CDD expression not only allowed for significant protection from Ara-C induced myelotoxicity but also abrogated the lymphotoxicity observed previously with high and constitutive hCDD expression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

scholarly journals Exosome-transported circRNA_0001236 enhances chondrogenesis and suppress cartilage degradation via the miR-3677-3p/Sox9 axis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Guping Mao ◽  
Yiyang Xu ◽  
Dianbo Long ◽  
Hong Sun ◽  
Hongyi Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Chondrogenic Differentiation ◽  
Cartilage Degradation ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Luciferase Reporter ◽  
Gene And Protein Expression

Abstract Objectives Aberrations in exosomal circular RNA (circRNA) expression have been identified in various human diseases. In this study, we investigated whether exosomal circRNAs could act as competing endogenous RNAs (ceRNAs) to regulate the pathological process of osteoarthritis (OA). This study aimed to elucidate the specific MSC-derived exosomal circRNAs responsible for MSC-mediated chondrogenic differentiation using human bone marrow-derived MSCs (hMSCs) and a destabilization of the medial meniscus (DMM) mouse model of OA. Methods Exosomal circRNA deep sequencing was performed to evaluate the expression of circRNAs in human bone marrow-derived MSCs (hMSCs) induced to undergo chondrogenesis from day 0 to day 21. The regulatory and functional roles of exosomal circRNA_0001236 were examined on day 21 after inducing chondrogenesis in hMSCs and were validated in vitro and in vivo. The downstream target of circRNA_0001236 was also explored in vitro and in vivo using bioinformatics analyses. A luciferase reporter assay was used to evaluate the interaction between circRNA_0001236 and miR-3677-3p as well as the target gene sex-determining region Y-box 9 (Sox9). The function and mechanism of exosomal circRNA_0001236 in OA were explored in the DMM mouse model. Results Upregulation of exosomal circRNA_0001236 enhanced the expression of Col2a1 and Sox9 but inhibited that of MMP13 in hMSCs induced to undergo chondrogenesis. Moreover, circRNA_0001236 acted as an miR-3677-3p sponge and functioned in human chondrocytes via targeting miR-3677-3p and Sox9. Intra-articular injection of exosomal circRNA_0001236 attenuated OA in the DMM mouse model. Conclusions Our results reveal an important role for a novel exosomal circRNA_0001236 in chondrogenic differentiation. Overexpression of exosomal circRNA_0001236 promoted cartilage-specific gene and protein expression through the miR-3677-3p/Sox9 axis. Thus, circRNA_0001236-overexpressing exosomes may alleviate cartilage degradation, suppressing OA progression and enhancing cartilage repair. Our findings provide a potentially effective therapeutic strategy for treating OA.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Transcription factor Gfi-1 induced by G-CSF is a negative regulator of CXCR4 in myeloid cells

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2276-2285 ◽  
Author(s):  
Maria De La Luz Sierra ◽  
Paola Gasperini ◽  
Peter J. McCormick ◽  
Jinfang Zhu ◽  
Giovanna Tosato
Keyword(s):  
Bone Marrow ◽  
Dna Sequences ◽  
Peripheral Blood ◽  
Cell Function ◽  
Myeloid Cell ◽  
Cxcr4 Expression ◽  
Negative Regulator ◽  
Hematopoietic Stem

The mechanisms underlying granulocyte-colony stimulating factor (G-CSF)–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood remain elusive. We provide evidence that the transcriptional repressor growth factor independence-1 (Gfi-1) is involved in G-CSF–induced mobilization of granulocytic lineage cells from the bone marrow to the peripheral blood. We show that in vitro and in vivo G-CSF promotes expression of Gfi-1 and down-regulates expression of CXCR4, a chemokine receptor essential for the retention of hematopoietic stem cells and granulocytic cells in the bone marrow. Gfi-1 binds to DNA sequences upstream of the CXCR4 gene and represses CXCR4 expression in myeloid lineage cells. As a consequence, myeloid cell responses to the CXCR4 unique ligand SDF-1 are reduced. Thus, Gfi-1 not only regulates hematopoietic stem cell function and myeloid cell development but also probably promotes the release of granulocytic lineage cells from the bone marrow to the peripheral blood by reducing CXCR4 expression and function.

scholarly journals Recombinant human megakaryocyte growth and development factor stimulates thrombocytopoiesis in normal nonhuman primates

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 54-59 ◽  
Author(s):  
AM Farese ◽  
P Hunt ◽  
T Boone ◽  
TJ MacVittie
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Growth And Development ◽  
Nonhuman Primates ◽  
Normal Male ◽  
Platelet Counts ◽  
Development Factor ◽  
Development In Vitro

Megakaryocyte growth and development factor (MGDF) is a novel cytokine that binds to the c-mpl receptor and stimulates megakaryocyte development in vitro and in vivo. This report describes the ability of recombinant human (r-Hu) MGDF to affect megakaryocytopoiesis in normal nonhuman primates. r-HuMGDF was administered subcutaneously to normal, male rhesus monkeys once per day for 10 consecutive days at dosages of 2.5, 25, or 250 micrograms/kg of body weight. Bone marrow and peripheral blood were assayed for clonogenic activity and peripheral blood counts were monitored. Circulating platelet counts increased significantly (P < .05) for all doses within 6 days of r-HuMGDF administration and reached maximal levels between day 12 and day 14 postcytokine administration. The 2.5, 25.0, and 250.0 micrograms/kg/d doses elicited peak mean platelet counts that were 592%, 670%, and 449% of baseline, respectively. Bone marrow-derived clonogenic data showed significant increases in the concentration of megakaryocyte (MEG)- colony-forming unit (CFU) and granulocyte-erythroid-macrophage- megakaryocyte (GEMM)-CFU, whereas that of granulocyte-macrophage (GM)- CFU and burst-forming unit-erythroid (BFU-e) remained unchanged during the administration of r-HuMGDF. These data show that r-HuMGDF is a potent stimulator of thrombocytopoiesis in the normal nonhuman primate.

Role of Il-2, Il-7, and IL-15 in T Cell Mediated Graft-vs-Leukemia Against Human Acute Lymphoblastic Leukemia in a NOD/scid Chimeric Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5256-5256
Author(s):  
Doug Cipkala ◽  
Kelly McQuown ◽  
Lindsay Hendey ◽  
Michael Boyer
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Mouse Model ◽  
Scid Mouse ◽  
In Vitro Cytotoxicity ◽  
Scid Mouse Model

Abstract The use of cytotoxic T-lymphocytes (CTL) has been attempted experimentally with various tumors to achieve disease control. Factors that may influence GVT include CTL cytotoxicity, ability to home to disease sites, and survival of T cells in the host. The objective of our study is to evaluate the GVL effects of human alloreactive CTL against ALL in a chimeric NOD/scid mouse model. CTL were generated from random blood donor PBMCs stimulated with the 697 human ALL cell line and supplemented with IL-2, -7, or -15. CTL were analyzed for in vitro cytotoxicity against 697 cells, phenotype, and in vitro migration on day 14. NOD/scid mice were injected with 107 697 ALL cells followed by 5x106 CTL. Mice were sacrificed seven days following CTL injection and residual leukemia was measured in the bone marrow and spleen via flow cytometry. The ratios of CD8/CD4 positive T cells at the time of injection were 46/21% for IL-2, 52/31% for IL-7, and 45/14% for IL-15 cultured CTL (n=13). Control mice not receiving CTL had a baseline leukemia burden of 2.01% and 0.15% in the bone marrow and spleen, respectively (n=15). Mice treated with IL-15 cultured CTL had a reduction in tumor burden to 0.2% (n=13, p=0.01) and 0.05% (n=13, p=0.01) in bone marrow and spleen, respectively. Those treated with IL-2 or IL-7 cultured CTL showed no significant difference in leukemia burden in either the bone marrow (IL-2 1.28%, Il-7 5.97%) or spleen (IL-2 0.4%, IL-7 0.33%). No residual CTL could be identified in the bone marrow or spleen at the time of sacrifice in any CTL group. CTL grown in each cytokine resulted in similar in vitro cytotoxicity at an effector:target ratio of 10:1 (IL-2 41.3%, IL-7 37.7%, IL-15 45.3%, n=12–15, p&gt;0.05 for all groups) and had statistically similar intracellular perforin and granzyme-B expression. In vitro CTL migration to a human mesenchymal stem cell line was greatest with IL-15 CTL (30.5%, n=4), followed by IL-7 CTL (18.9%, n=4), and least in IL-2 CTL (17.9%, n=4), though the differences were not significant. In vitro CTL migration was analyzed to an SDF-1α gradient as CXCR4/SDF-1α interactions are necessary for hematopoietic progenitor cell homing to the bone marrow. IL-15 cultured CTL showed the highest migration (48.8%, n=8) as compared to IL-2 (21.7%, n=6, p=0.048) or IL-7 CTL (35.9%, n=8, p&gt;0.05). However, surface expression of CXCR4 measured by flow cytometry was significantly higher in IL-7 CTL (89.4%, n=9) compared to IL-2 CTL (52.2%, n=9, p&lt;0.001) and IL-15 CTL (65.4%, n=10, p=0.002). Experiments are currently underway to further evaluate the role of CXCR4/SDF-1α in GVL. Preliminary in vivo experiments do not suggest any significant differences in CTL engraftment when evaluated at 24 hours post injection. Expression of the anti-apoptotic bcl-2 protein was greatest on IL-7 (MFI=5295, n=13) and IL-15 (MFI=4865, n=14) when compared to IL-2 CTL (MFI=3530, n=13, p=0.02 vs. IL-7, p=0.05 vs. IL-15), suggesting an increased in vivo survival ability. We hypothesize that IL-15 cultured CTL have greater GVL effects due to either higher in vivo survival, greater bone marrow homing efficiency, or both. Future experiments are planned to evaluate in vivo administration of IL-2 to enhance CTL survival in the host. In conclusion, IL-15 cultured CTL had significantly greater in vivo GVL effects compared to IL-2 and IL-7 CTL in the NOD/scid mouse model. This model can be utilized to evaluate the mechanism of T cell mediated GVL against ALL and potentially other human malignancies.

GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 374-374 ◽  
Author(s):  
Zhong-fa Yang ◽  
Karen Drumea ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
T Cell ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Development ◽  
Wild Type ◽  
Ets Domain

Abstract GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.

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