Lack of the Transcription Factor NFAT (Nuclear Factor of Activated T cells) c2 in Hematopoietic Progenitor Cells Results in Profound Hematological Abnormalities in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1296-1296
Author(s):  
Laleh S. Arabanian ◽  
Michael Haase ◽  
Ivonne Habermann ◽  
Malte von Bonin ◽  
Claudia Waskow ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Extramedullary Hematopoiesis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Hematological Abnormalities

Abstract Abstract 1296 Understanding the transcriptional mechanisms that control hematopoiesis and the interaction between hematopoietic stem cells and the bone marrow microenvironment in vivo is of considerable interest. We have previously shown that aged mice lacking the transcription factor NFATc2 develop bone marrow hypoplasia, anemia, and extramedullary hematopoiesis in spleen and liver. The proliferation and differentiation of NFATc2-deficient hematopoietic progenitor cells (HPC) ex vivo, however, was found to be intact. It remained therefore unclear whether the disturbed hematopoiesis in NFATc2-deficient mice was caused by the hematopoietic or the stroma component of the bone marrow hematopoietic niche. In the current study we dissected the relative contribution of hematopoietic and stroma cells to the phenotype of the NFATc2-deficent mice by transplanting immunomagnetically purified NFATc2-deficient (ko) HPCs to lethally irradiated wildtype (wt) mice, and vice versa. After a posttransplantation period of 6–8 months, peripheral blood, bone marrow as well as spleen and liver of the transplanted animals were analyzed and compared to wt and ko mice transplanted with control cells. Transplantation of NFATc2-deficient HPCs into wt recipients (ko → wt) induced similar hematological abnormalities as those occurring in non-transplanted ko mice or in ko mice transplanted with ko cells (ko → ko). Compared to wt mice transplanted with wt cells (wt → wt), ko → wt mice showed evidence of anemia, thrombocytopenia and a significantly reduced number of hematopoietic cells in their bone marrow. Likewise, ko → wt mice developped clear signs of extramedullary hematopoiesis in spleen and liver, which was not the case in wt → wt control animals. Our data demonstrate for the first time, that NFAT transcription factors directly regulate the intrinsic function of hematopoietic progenitor cells in vivo. The transcriptional targets for NFAT in these cells are yet unknown and are the focus of further investigations. Disclosures: No relevant conflicts of interest to declare.

Flt3 Ligand Negatively Regulates In Vitro Migration, Intracellular Signaling Activated by SDF-1α/CXCR4 and In Vivo Homing of Hematopoietic Progenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2674-2674
Author(s):  
Seiji Fukuda ◽  
Hal E. Broxmeyer ◽  
Louis M. Pelus
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Flt3 Ligand ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Hematopoietic Stem

Abstract The Flt3 receptor tyrosine kinase (Flt3) is expressed on primitive normal and transformed hematopoietic cells and Flt3 ligand (FL) facilitates hematopoietic stem cell mobilization in vivo. The CXC chemokine SDF-1α(CXCL12) attracts primitive hematopoietic cells to the bone marrow microenvironment while disruption of interaction between SDF-1α and its receptor CXCR4 within bone marrow may facilitate their mobilization to the peripheral circulation. We have previously shown that Flt3 ligand has chemokinetic activity and synergistically increases migration of CD34+ cells and Ba/F3-Flt3 cells to SDF-1α in short-term migration assays; this was associated with synergistic phosphorylation of MAPKp42/p44, CREB and Akt. Consistent with these findings, over-expression of constitutively active ITD (internal tandem duplication) Flt3 found in patients with AML dramatically increased migration to SDF-1α in Ba/F3 cells. Since FL can induce mobilization of hematopoietic stem cells, we examined if FL could antagonize SDF-1α/CXCR4 function and evaluated the effect of FL on in vivo homing of normal hematopoietic progenitor cells. FL synergistically increased migration of human RS4;11 acute leukemia cells, which co-express wild-type Flt3 and CXCR4, to SDF-1α in short term migration assay. Exogenous FL had no effect on SDF-1α induced migration of MV4-11 cells that express ITD-Flt3 and CXCR4 however migration to SDF-1α was partially blocked by treatment with the tyrosine kinase inhibitor AG1296, which inhibits Flt3 kinase activity. These results suggest that FL/Flt3 signaling positively regulates SDF-1α mediated chemotaxis of human acute leukemia cells in short-term assays in vitro, similar to that seen with normal CD34+ cells. In contrast to the enhancing effect of FL on SDF-1α, prolonged incubation of RS4;11 and THP-1 acute myeloid leukemia cells, which also express Flt3 and CXCR4, with FL for 48hr, significantly inhibited migration to SDF-1α, coincident with reduction of cell surface CXCR4. Similarly, prolonged exposure of CD34+ or Ba/F3-Flt3 cells to FL down-regulates CXCR4 expression, inhibits SDF-1α-mediated phosphorylation of MAPKp42/p44, CREB and Akt and impairs migration to SDF-1α. Despite reduction of surface CXCR4, CXCR4 mRNA and intracellular CXCR4 in Ba/F3-Flt3 cells were equivalent in cells incubated with or without FL, determined by RT-PCR and flow cytometry after cell permeabilization, suggesting that the reduction of cell surface CXCR4 expression is due to accelerated internalization of CXCR4. Furthermore, incubation of Ba/F3-Flt3 cells with FL for 48hr or over-expression of ITD-Flt3 in Ba/F3 cells significantly reduced adhesion to VCAM1. Consistent with the negative effect of FL on in vitro migration and adhesion to VCAM1, pretreatment of mouse bone marrow cells with 100ng/ml of FL decreased in vivo homing of CFU-GM to recipient marrow by 36±7% (P<0.01), indicating that FL can negatively regulate in vivo homing of hematopoietic progenitor cells. These findings indicate that short term effect of FL can provide stimulatory signals whereas prolonged exposure has negative effects on SDF-1α/CXCR4-mediated signaling and migration and suggest that the FL/Flt3 axis regulates hematopoietic cell trafficking in vivo. Manipulation of SDF-1α/CXCR4 and FL/Flt3 interaction could be clinically useful for hematopoietic cell transplantation and for treatment of hematopoietic malignancies in which both Flt3 and CXCR4 are expressed.

Sociology of Normal Stem and Progenitor Cells in CML Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1234-1234
Author(s):  
Robert S Welner ◽  
Giovanni Amabile ◽  
Deepak Bararia ◽  
Philipp B. Staber ◽  
Akos G. Czibere ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Progenitor Cells ◽  
Quiescent State ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

Abstract Abstract 1234 Specialized bone marrow (BM) microenvironment niches are essential for hematopoietic stem and progenitor cell maintenance, and recent publications have focused on the leukemic stem cells interaction and placement within those sites. Surprisingly, little is known about how the integrity of this leukemic niche changes the normal stem and progenitor cells behavior and functionality. To address this issue, we started by studying the kinetics and differentiation of normal hematopoietic stem and progenitor cells in mice with Chronic Myeloid Leukemia (CML). CML accounts for ∼15% of all adult leukemias and is characterized by the BCR-ABL t(9;22) translocation. Therefore, we used a novel SCL-tTA BCR/ABL inducible mouse model of CML-chronic phase to investigate these issues. To this end, BM from leukemic and normal mice were mixed and co-transplanted into hosts. Although normal hematopoiesis was increasingly suppressed during the disease progression, the leukemic microenvironment imposed distinct effects on hematopoietic progenitor cells predisposing them toward the myeloid lineage. Indeed, normal hematopoietic progenitor cells from this leukemic environment demonstrated accelerated proliferation with a lack of lymphoid potential, similar to that of the companion leukemic population. Meanwhile, the leukemic-exposed normal hematopoietic stem cells were kept in a more quiescent state, but remained functional on transplantation with only modest changes in both engraftment and homing. Further analysis of the microenvironment identified several cytokines that were found to be dysregulated in the leukemia and potentially responsible for these bystander responses. We investigated a few of these cytokines and found IL-6 to play a crucial role in the perturbation of normal stem and progenitor cells observed in the leukemic environment. Interestingly, mice treated with anti-IL-6 monoclonal antibody reduced both the myeloid bias and proliferation defects of normal stem and progenitor cells. Results obtained with this mouse model were similarly validated using specimens obtained from CML patients. Co-culture of primary CML patient samples and GFP labeled human CD34+CD38- adult stem cells resulted in selective proliferation of the normal primitive progenitors compared to mixed cultures containing unlabeled normal bone marrow. Proliferation was blocked by adding anti-IL-6 neutralizing antibody to these co-cultures. Therefore, our current study provides definitive support and an underlying crucial mechanism for the hematopoietic perturbation of normal stem and progenitor cells during leukemogenesis. We believe our study to have important implications for cancer prevention and novel therapeutic approach for leukemia patients. We conclude that changes in cytokine levels and in particular those of IL-6 in the CML microenvironment are responsible for altered differentiation and functionality of normal stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

scholarly journals Increased Hematopoietic Stem Cells/Hematopoietic Progenitor Cells Measured as Endogenous Spleen Colonies in Radiation-Induced Adaptive Response in Mice (Yonezawa Effect)

Dose-Response ◽  
2018 ◽  
Vol 16 (3) ◽  
pp. 155932581879015 ◽  
Author(s):  
Bing Wang ◽  
Kaoru Tanaka ◽  
Yasuharu Ninomiya ◽  
Kouichi Maruyama ◽  
Guillaume Varès ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adaptive Response ◽  
Underlying Mechanism ◽  
Hematopoietic Progenitor Cells ◽  
X Rays ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Different Types ◽  
Radiation Induced

The existence of radiation-induced adaptive response (AR) was reported in varied biosystems. In mice, the first in vivo AR model was established using X-rays as both the priming and the challenge doses and rescue of bone marrow death as the end point. The underlying mechanism was due to the priming radiation-induced resistance in the blood-forming tissues. In a series of investigations, we further demonstrated the existence of AR using different types of ionizing radiation (IR) including low linear energy transfer (LET) X-rays and high LET heavy ion. In this article, we validated hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs) measured as endogenous colony-forming units-spleen (CFU-S) under AR inducible and uninducible conditions using combination of different types of IR. We confirmed the consistency of increased CFU-S number change with the AR inducible condition. These findings suggest that AR in mice induced by different types of IR would share at least in part a common underlying mechanism, the priming IR-induced resistance in the blood-forming tissues, which would lead to a protective effect on the HSCs/HPCs and play an important role in rescuing the animals from bone marrow death. These findings provide a new insight into the mechanistic study on AR in vivo.

scholarly journals Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 801-807 ◽  
Author(s):  
T Leemhuis ◽  
D Leibowitz ◽  
G Cox ◽  
R Silver ◽  
EF Srour ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Polymerase Chain Reaction Analysis ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.

Targeting the IRF2 Transcription Factor To Inhibit Leukaemic Cell Growth.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1425-1425
Author(s):  
Alla Dolnikov ◽  
Ailyn Choo ◽  
Patricia Palladinetti ◽  
Toby Passioura ◽  
Geoff Symonds ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Cell Growth ◽  
Progenitor Cells ◽  
Growth Suppression ◽  
Hematopoietic Progenitor Cells ◽  
Leukemia Cells ◽  
Hematopoietic Progenitor ◽  
Down Regulation ◽  
Hematopoietic Stem ◽  
Ras Genes

Abstract Activating mutations of the Ras genes occur at high frequency in acute myeloid leukemia (AML). We have previously shown that expression of mutant N-ras(N-rasm) in murine hematopoietic stem cells is sufficient to induce a myeloid malignancy that resembles human AML(Mackenzie et al. Blood, 1999, 93, 2043–2056). In a ’humanised’ NOD/SCID mouse model N-rasm induced a pre-leukemic condition characterised by myeloid proliferation of human hematopoietic progenitor cells in the bone marrow of recipient mice (Shen et al. Exp. Hematol., 2004, 32: 852–860). Even though Ras usually acts as a dominant transforming oncogene, in primary cells and some cancer cell lines, Ras inhibits cell growth. We have previously shown that ectopic expression of N-rasm in leukemia U937 and K562 cells leads to growth suppression (Passioura et al. Cancer Res. 2005, 65, 797–804). The expression profile induced by N-rasm in these cells included the up-regulation of transcription factor Interferon Regulatory Factor1 (IRF1) and activation of cdk inhibitor p21WAF. IRF1 was previously defined as a tumour suppressor, and as such is a target of oncogenic mutations in AML. Antisense suppression of IRF1 prevented N-rasm induced growth suppression and up-regulation of p21WAF1. These results defined a novel tumour suppressive response to oncogenic N-rasm in leukemia cells. A retroviral cDNA library screen for genes that counteract N-rasm-induced growth suppression identified the gene for the Interferon Regulatory Factor2 (IRF2), and as confirmation of the screen, over-expression of IRF2 in leukemia U937 cells acted to inhibit N-rasm-induced growth suppression (Passioura et al. Oncogene. 2005; 24: 7327–36). IRF2 is known for its oncogenic properties and can antagonise IRF1-mediated tumour suppression. In addition, IRF2 is often up-regulated in primary leukemia samples. Here we show that IRF2 gene suppression using RNA interference acts to suppress the growth of leukemia TF-1 cells bearing N-ras mutation in codon 61 and expressing high levels of IRF1 and IRF2 and low level of p21Waf1. IRF2 down-regulation confirmed at RNA (quantitative RT-PCR) and protein (Western analysis) levels resulted in up-regulation of p21Waf1 and G2/M- rather than G1/S-growth arrest. In addition, increased polyploidisation that results from discoordinated DNA synthesis in mitotically arrested cells, was observed. In addition, IRF2-down-regulation significantly reduced clonogenic growth of the leukemic blasts. Cell growth of normal hematopoietic progenitor cells that express low levels of both IRF1 and IRF2, however, was not affected by IRF2 targeting. IRF2 targeting is currently being examined in primary AML samples in an animal model of AML. We suggest that IRF2 suppression can be used for ex vivo purging of leukemia cells in the autologous stem cell transplantation setting. To the best of our knowledge, specific IRF2 inhibition in cancer cells as a potential therapeutic approach has not been tested to date. IRF2 suppression may prove to be a novel therapeutic target for leukemia therapy.

A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide.

1991 ◽  
Vol 9 (3) ◽  
pp. 478-490 ◽  
Author(s):  
D A Scheinberg ◽  
D Lovett ◽  
C R Divgi ◽  
M C Graham ◽  
E Berman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Phase I ◽  
Progenitor Cells ◽  
Phase I Trial ◽  
Whole Body ◽  
Hematopoietic Progenitor Cells ◽  
Target Cells ◽  
Hematopoietic Progenitor

Ten patients with myeloid leukemias were treated in a phase I trial with escalating doses of mouse monoclonal antibody (mAb) M195, reactive with CD33, a glycoprotein found on myeloid leukemia blasts and early hematopoietic progenitor cells but not on normal stem cells. M195 was trace-labeled with iodine-131 (131I) to allow detailed pharmacokinetic and dosimetric studies by serial sampling of blood and bone marrow and whole-body gamma-camera imaging. Total doses up to 76 mg were administered safely without immediate adverse effects. Absorption of M195 onto targets in vivo was demonstrated by biopsy, pharmacology, flow cytometry, and imaging; saturation of available sites occurred at doses greater than or equal to 5 mg/m2. The entire bone marrow was specifically and clearly imaged beginning within hours after injection; optimal imaging occurred at the lowest dose. Bone marrow biopsies demonstrated significant dose-related uptake of M195 as early as 1 hour after infusion in all patients, with the majority of the dose found in the marrow. Tumor regressions were not observed. An estimated 0.33 to 1.0 rad/mCi 131I was delivered to the whole body, 1.1 to 6.1 rad/mCi was delivered to the plasma, and up to 34 rad/mCi was delivered to the red marrow compartment. 131I-M195 was rapidly modulated, with a majority of the bound immunoglobulin G (IgG) being internalized into target cells in vivo. These data indicate that whole bone marrow ablative doses of 131I-M195 can be expected. The rapid, specific, and quantitative delivery to the bone marrow and the efficient internalization of M195 into target cells in vivo also suggest that the delivery of other isotopes such as auger or alpha emitters, toxins, or other biologically important molecules into either leukemia cells or normal hematopoietic progenitor cells may be feasible.

The RNA Editing Enzyme ADAR1 Is Required for Survival of Differentiating Hematopoietic Progenitor Cells in Adult Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1395-1395
Author(s):  
Feng Xu ◽  
Qingde Wang ◽  
Hongmei Shen ◽  
Hui Yu ◽  
Yanxin Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Rna Editing ◽  
Cell Population ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Low Level ◽  
Adult Mice

Abstract Adenosine Deaminases Acting on RNA (ADAR) are RNA-editing enzymes converting adenosine residues into inosine (A-to-I) in many double-stranded RNA substrates including coding and non-coding sequences as well as microRNAs. Disruption of the ADAR1 gene in mice results in fetal liver, but not yolk sac, defective erythropoiesis and death at E11.5 (Wang Q et al, Science 2000). Subsequently, a conditional knockout mouse model confirmed these findings and showed massively increased cell death in the affected organs (Wang Q et al, JBC 2004). However, the actual impact of ADAR1 absence on definitive or adult hematopoiesis has not been examined. To define the role of ADAR1 in adult hematopoiesis, we first examined the expression of ADAR1 in different hematopoietic stem/progenitor cell subsets isolated from bone marrow by real-time RT-PCR. ARAR1 was present in hematopoietic stem cells (HSCs) at relatively low level and increased in hematopoietic progenitor cells (HPCs). A series of functional hematopoietic assays were then undertaken. A conditional deletion of ADAR1 was achieved by transducing Lin− or Lin−cKit+ bone marrow cells from ADAR1-lox/lox mice with a MSCV retroviral vector co-expressing Cre and GFP. PCR analysis confirmed the complete deletion of ADAR1 in the transduced cells within 72 hours after the transduction. This system allowed us to evaluate the acute effect of ADAR1 deletion in a specific hematopoietic cell population. Following 4 days of in vitro culture after transduction, the absolute number of Lin− Sca1+ cells in the Cre transduced group was similar to the input number; however the differentiating Lin+ cells significantly decreased whereas both the Lin−Sca1+ and Lin+ cells in the vector (MSCV carrying GFP alone) transduced group increased during culture. Moreover, the colony forming cell (CFC) assay showed much fewer and smaller colonies that contained dead cells from the gene deleted group as compared to those from the control group (p<0.001). The TUNEL assay showed a dramatic increase of apoptosis in the Lin+ population but not in the Lin− cells. Given the mixed genetic background of the ADAR1-lox/lox mice, repopulation of the transduced hematopoietic cells in vivo was examined in immunodeficient mice. Sublethally irradiated (3.5 Gy) NOD/SCID-γcnull recipient were transplanted with either 1.5 × 105 Cre or vector transduced Lin− ADAR1-lox/lox cells. Multi-lineage engraftment in peripheral blood was monitored monthly. While the vector transduced cells were able to constitute more than 90% in multiple lineages of the peripheral blood at 1 to 3 months, Cre-transduced cells were virtually undetectable at all the time points (n=9 to 13, p<0.001). A similar result was found in the hematopoietic organs, including the bone marrow, spleen and thymus. Interestingly, however, the Lin−Sca1+cKit+ cell population was preserved in the Cre transduced group despite the very low level of total donor-derived cells in the bone marrow (n=6 to 7, p<0.01). Consistently, the single cell culture experiment demonstrated that there was no significant difference between ADAR−/− and wild-type HSCs in terms of survival and division during the first 3 days of culture. Taken together, our current study demonstrates nearly absolute requirement of ADAR1 for hematopoietic repopulation in adult mice and it is also suggested that ADAR1 has a preferential effect on the survival of differentiating progenitor cells over more primitive cells.

Identification of Highly Clonal Cells in CD34+ and Unselected Bone Marrow Samples From Patients with Myelodysplastic Syndrome Using a Sensitive Quantitative PCR Approach.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1760-1760
Author(s):  
Maximilian Mossner ◽  
Daniel Nowak ◽  
Ouidad Benlasfer ◽  
Jana Reins ◽  
Olaf Joachim Hopfer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Progenitor Cells ◽  
Quantitative Pcr ◽  
X Chromosome ◽  
Hematologic Malignancies ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1760 Poster Board I-786 Myelodysplastic syndromes (MDS) are clonal hematologic malignancies with molecular defects most probably arising in the hematopoietic stem or progenitor compartment. However, due to a frequent lack of trackable cytogenetic aberrations in a large proportion of MDS patients the capability to monitor the manifestation and origin of malignant MDS clones remains limited. To elucidate clonal dominance in a given cell population, the analysis of skewed X-chromosome inactivation patterns in females, based on the methylation analysis of X-chromosomal HUMARA alleles has been used widely. However, this method has several technical and biological drawbacks as methylation changes can be induced with increasing age leading to inaccuracy of the method in this context. Recently, the application of a quantitative PCR-based method to accurately detect single nucleotide polymorphism (SNP) allele-specific RNA transcription from the X-chromosome (Swierczek et al, Blood, 2008) has shown robust results for reliable calculation of X-chromosome allelic ratios. In our study we employed this method to assess clonality in CD34+ selected and unselected bone marrow cells derived from MDS patients and provide evidence for distinct clonal manifestations of MDS clones in hematopoietic progenitor cells of all analysed MDS samples as compared to healthy controls. Bone marrow (BM) cells were obtained from patients with MDS (IPSS-low/int-1-risk n=9, IPSS-int-2/high-risk n=9) after informed consent. Immunomagnetic selection of CD34+ cells was performed from the BM samples of MDS patients (IPSS-low/int-1-risk n=8, IPSS-int-2/high-risk n=10) and age related healthy donors (n=6) served as controls for normalization. Genomic DNA SNP genotyping using Taqman SNP Genotyping Assays (Applied Biosystems, Foster City, CA) was carried out in order to screen for informative clonality marker genes located on the X-chromosome, namely BTK, FHL1, IDS and MPP1. RNA transcripts from different alleles were quantified using SNP/allele-specific primers in a Taqman based quantitative PCR approach. Individual allelic ratios were calculated as previously described. Clonality values were assigned to 0 % according to an allelic ratio of 50/50 (polyclonal) up to 100 % for a ratio of 100/0 (clonal). All clonality values are presented as mean. Our analyses revealed a remarkably elevated proportion of clonal cells in all purified CD34+ cells from MDS low/int-1-risk (88 %) and MDS int-2/high-risk patients (98 %) compared to the cells from healthy donors (16 %, p<0.001). The degree of clonality in unselected BM samples was similarly increased in MDS low/int-1-risk (74 %) and MDS int-2/high-risk specimen (87 %, both p<0.001 as compared to controls). However, whereas all purified CD34+ samples from MDS patients appeared to be highly clonal, 2 of 9 (22 %) of the MDS low/int-1-risk samples exhibited distinctively lower clonality in unselected BM cells with values comparable to the healthy control group. Furthermore, we observed nearly identical high clonality values in 12 paired BM/CD34+ MDS samples, except for 1 of 6 MDS low/int-1-risk samples with significantly lower clonality in the unselected bone marrow as compared to purified CD34+ cells of the same patient. Our observation of specific clonality in both unselected bone marrow and purified CD34+ cells of MDS patients as compared to healthy controls underlines the proliferative manifestation of malignant MDS clones even in early hematopoietic progenitor cells. Furthermore, the high degree of clonality in all purified CD34+ cells suggests a clonal involvement of not only myeloid but also lymphoid lineages. Interestingly, we also identified 2 patients harboring polyclonal cells in the unselected bone marrow. In these cases differentiating cells in the bone marrow may be sustained by residual healthy hematopoietic progenitor cells. The determination whether patients with this constellation have a different clinical prognosis from patients with clonality of the complete bone marrow may be interesting to pursue. In summary, determination of clonality levels in distinct cell populations of hematologic malignancies using quantitative PCR appears to be highly suitable for monitoring the manifestation and origin of a malignant hematopoietic stem/precursor cell. Disclosures No relevant conflicts of interest to declare.

Oxidative Stress-Mediated Activation of AKT/mTOR Signaling Pathway Leads to Myeloproliferative Syndrome in FoxO3 Null Mice: A Role for Lnk Adaptor Protein

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 509-509 ◽  
Author(s):  
Safak Yalcin ◽  
Sathish Kumar Mungamuri ◽  
Dragan Marinkovic ◽  
Xin Zhang ◽  
Wei Tong ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Hematopoietic Progenitor Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem

Abstract Reactive oxygen species (ROS) are toxic byproducts of oxidative metabolism implicated in many debilitating human disorders including hematological malignancies and aging. ROS are also generated by growth factors and cytokine stimulation and play critical functions in normal cellular signaling. However, not much is known of how ROS impact physiological processes in normal and diseased states. We and others have recently shown critical functions for box (O) family of forkhead transcription factors (Fox)O in the regulation of physiological ROS in primitive hematopoietic cells. In particular, FoxO3 has emerged as the principal FoxO whose regulation of ROS is essential for the maintenance of hematopoietic stem cell pool. Although FoxO3’s activity is constitutively repressed by several oncoproteins that play critical roles in myeloproliferative disorders the role of FoxO3 in the regulation of primitive hematopoietic progenitors remains elusive. FoxO’s function is restrained by AKT serine threonine protein kinase. AKT supports growth, survival and proliferation by promoting inhibition of FoxO and activation of the mammalian target of rapamycin (mTOR) and its downstream target p70 S6 Kinase (S6K) through phosphorylation. We demonstrate that loss of FoxO3 leads to a myeloproliferative-like syndrome characterized by leukocytosis, splenomegaly, enhanced generation of primitive progenitors including colony-forming-unit-spleen (CFU-S) in hematopoietic organs and hypersensitivity of hematopoietic progenitor cells to cytokines in FoxO3 null mice. These findings were intriguing since we had not found FoxO3 null hematopoietic stem cells to exhibit enhanced cycling in vivo or to generate excessive hematopoietic progenitors ex vivo (Yalcin et al., JBC, 2008). To investigate the mechanism of enhanced myeloproliferation, we interrogated cytokine-mediated activation of signaling pathways in freshly isolated FoxO3 null versus wild type bone marrow cells enriched for hematopoietic progenitors. To our surprise we found that stimulation with cytokines including IL-3 led to hyperphosphorylation of AKT, mTOR and S6K but not STAT5 proteins in FoxO3 null as compared to wild type cytokine-starved hematopoietic progenitors. In agreement with these results, in vivo administration of the mTOR inhibitor rapamycin resulted in significant reduction of FoxO3 null- but not wild type-derived CFU-Sd12 in lethally irradiated hosts. These unexpected results suggested that AKT/mTOR signaling pathway is specifically overactivated as part of a feedback loop mechanism and mediates enhanced generation of FoxO3 null primitive multipotential hematopoietic progenitors in vivo. We further showed that phosphorylation of AKT/mTOR/S6K is highly sensitive to ROS scavenger N-Acetyl-Cysteine (NAC) in vivo and ex vivo in both wild type and FoxO3 null primitive hematopoietic progenitors indicating that ROS are involved in cytokine signaling in primary hematopoietic progenitor cells. Interestingly, in vivo administration of NAC normalized the number of FoxO3 null-derived CFU-Sd12 in lethally irradiated hosts without any impact on wild type CFU-Sd12 strongly suggesting that ROS mediate specifically enhanced generation of primitive hematopoietic progenitors in FoxO3 null mice. In this context, we were surprised to find similar levels of ROS concentrations in FoxO3 mutant as compared to control hematopoietic progenitors. Thus, we asked whether the increase in FoxO3 null primitive hematopoietic progenitor compartment is due to an increase sensitivity of cytokine signaling to ROS as opposed to increased ROS build up per se in these cells. In search for a mechanism we found the expression of Lnk, a negative regulator of cytokine signaling, to be highly reduced in FoxO3 null primitive hematopoietic progenitor cells. We further demonstrated that retroviral reintroduction of Lnk but not vector control in FoxO3 null primitive bone marrow cells reduced significantly the number of FoxO3 null-derived CFU-Sd12in vivo. Collectively, these results suggest that reduced expression of Lnk hypersensitizes FoxO3-deficient hematopoietic progenitors to ROS generated by cytokine signaling leading to myeloproliferation. These cumulative findings uncover a mechanism by which deregulation of cellular sensitivity to physiological ROS leads to hematopoietic malignancies specifically in disorders in which FoxO play a role.

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