Pbx-1 Is a Direct Target of Evi-1 in Hematopoietic Stem/Progenitors and Leukemic Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1192-1192
Author(s):  
Munetake Shimabe ◽  
Susumu Goyama ◽  
Motoshi Ichikawa ◽  
Yoichi Imai ◽  
Tsuyoshi Takahashi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Candidate Genes ◽  
Zinc Finger ◽  
Target Genes ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Direct Target

Abstract The ecotropic viral integration site-1 (Evi-1) gene was first identified as a common locus of retroviral integration in murine leukemia. In humans, Evi-1 is located on chromosome 3q26, and rearrangements on chrmosome 3q26 often activate Evi-1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Patients with these karyotypes are characterized by the elevated platelet count and lack of response to antileukemic therapy. Elevated Evi-1 expression occurs with high frequency in AML patients without 3q26 abnormalities, and is also associated with unfavorable outcomes. Thus, Evi-1 is one of the key factors that predict poor survival in leukemia patients. Evi-1 is a member of the SET/PR domain family of transcription factors and it contains a total of 10 zinc finger motifs organized in two discrete domains, comprising 7 and 3 repeats respectively, which have distinct DNA binding specificities. Recently, we generated Evi-1-mutant mice and showed that Evi-1 plays an essential role in proliferation of both hematopoietic stem cells (HSCs) and transformed leukemic cells. Furthermore, we identified candidate target genes of Evi-1 using gene expression profiling analysis in HSCs combined with the gene expression data of AML samples. These genes include Gata1, Gata2, Angpt1, Mpl, Jag2, Pbx-1, Setbp1 and Itga2b. In this study, we first analyzed relative gene expression of these candidate genes in control- or Evi-1-transduced hematopoietic stem/progenitors (c-Kit+ cells). Among these candidate genes, Evi-1 up-regulates transcription of Pbx-1, Mpl, Setbp1 and Itga2b. Next, we cloned 5 ′ up-stream genomic regions of these four genes into the pGL-4 luciferase reporter vector, and found that Evi-1 increased luciferase activity of Pbx-1 reporter construct in COS7 cells. Deletion of reporter constructs revealed that Evi-1 binds to -0.5kb upstream of the transcription start site of Pbx-1. We then examined the transcription activity of a series of Evi-1 mutants and found that both the first and second zinc finger domains are required for the Pbx-1 up-regulation. Furthermore, chromatin immunoprecipitation (ChIP) analysis revealed that Evi-1 directly binds to the promoter region of Pbx-1. We next evaluated a role for Pbx-1 in Evi-1-induced myeloid transformation. Bone marrow progenitors transduced with Evi-1 showed sustained colony formation in the serial replating assay. After establishment of sustained clonogenic activity following more than three rounds of replating in methylcellulose medium, the cells were transduced with control or Pbx-1-shRNA. Interestingly, reduction of Pbx-1 levels through RNAi-mediated knockdown significantly inhibited Evi-1-induced transformation. In contrast, knockdown of Pbx-1 did not impair bone marrow transformation by transcription factor E2A-hepatic leukemia factor (E2A-HLF), suggesting that Pbx-1 is specifically, as opposed to generally, required for maintenance of transformation mediated by Evi-1. Taken together, these results indicate that Pbx-1 is one of the direct target genes of Evi- 1 in hematopoietic cells, and aberrant Pbx-1 expression is responsible, at least in part, for the oncogenic activity of Evi-1. Because Pbx-1 is known as a critical regulator of hematopoietic stem cells and leukemia development, the Evi-1-Pbx-1 pathway may be a key modulator of stem cell activity in normal and malignant hematopoiesis.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4836-4836
Author(s):  
Gyeongsin Park ◽  
Michael Heuser ◽  
Tobias Berg ◽  
R. Keith Humphries
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Leukemic Cell ◽  
Fixed Number ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

Ott1(Rbm15)-Deficient Hematopoietic Stem Cells Are Unable to Maintain Quiescence During Replicative Stress and Display Features of Premature Aging,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3433-3433
Author(s):  
Nan Xiao ◽  
Kaushal Jani ◽  
Jonathan L Jesneck ◽  
Glen D Raffel
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Failure ◽  
Gene Set Enrichment Analysis ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Replicative Stress

Abstract Abstract 3433 With age, hematopoietic stem cells (HSCs) have numerical expansion, skewing towards myeloid development, loss of lymphoid potential, an underlying pro-inflammatory state and loss of self-renewal potential thus severely limiting responses to hematopoietic stress, ultimately leading to bone marrow failure. The mechanisms and pathways responsible for these changes in aged HSCs are incompletely understood. Using a conditional allele of Ott1, a gene originally isolated as the 5' fusion partner in t(1;22) acute megakaryocytic leukemia, we previously found a global regulatory role for the gene in hematopoiesis. Deletion of Ott1 in adult mice utilizing Mx1-cre recapitulated certain aspects of aging hematopoiesis including increased Lin−Sca1+c-Kit+ (LSK) population, myeloid expansion and decreased lymphopoiesis. The LSK compartment was further characterized using SLAM and CD34/Flk2 markers and demonstrated normal levels of LT-HSCs and increased ST-HSCs. Despite sufficient LT-HSC numbers, Ott1-deleted bone marrow was unable to competitively or non-competitively repopulate irradiated recipients. To exclude a homing or engraftment effect, Ott1flox/null Mx1-cre bone marrow was transplanted with competitor then excised post-engraftment. The rapid loss of the Ott1-deficient graft demonstrated Ott1 is required for maintenance under competitive stress. In contrast, primary mice undergoing Ott1 excision lived a normal lifespan and were able to maintain sufficient hematopoiesis although with a partial reduction in bone marrow clonagenicity showing loss of Ott1 is not limiting under steady state conditions. To test the HSC requirement for Ott1 under replicative stress, Ott1 knockout mice were challenged with 5-fluorouracil (5-FU). Ott1-deleted mice treated with 5-FU displayed delayed peripheral blood neutrophil recovery and showed accelerated bone marrow failure. Cell cycle analysis of steady state Ott1 knockout HSCs showed a similar profile to wild type controls, however, after 5-FU treatment, the G0 fraction was dramatically reduced. The G0 fraction is associated with the quiescent, self-renewing HSC population, therefore, Ott1 is required for maintaining HSC quiescence during replicative stress but not steady state hematopoiesis. To more specifically assess whether the functional hematopoietic changes seen after loss of Ott1 were accompanied by alterations in known aging-associated pathways, Gene Set Enrichment Analysis comparing Ott1-deleted HSCs in steady state to aged HSCs was performed and showed a highly enriched gene expression signature (NES 2.02 p<0.0001). Physiologic sequelae of HSC aging were observed after Ott1 excision including activation of NFκβ, elevation of reactive oxygen species (ROS), increase in DNA damage (γH2A.X levels) and activation of p38Mapk. Although ROS was elevated under steady state conditions, neither apoptosis, senescence or proliferation was significantly different from wild type control HSCs. Furthermore, anti-oxidant treatment with N-acetyl-cysteine was unable to rescue the HSC maintenance defect of the Ott1 knockout, signifying additional requirements in HSCs for Ott1 beyond regulation of ROS. An observed increase of mitochondrial mass in Ott1-deleted HSCs suggests an upstream function for Ott1 in metabolic control, potentially contributing to ROS generation or degradation. In summary, we have demonstrated an essential role for Ott1 in maintaining HSC quiescence during replicative stress and shown loss of Ott1 leads to the acquisition of key gene expression patterns and pathophysiologic changes associated with aging. These data suggest Ott1 functions in part to oppose specific consequences of aging in the hematopoietic compartment. Ott1 and Ott1-dependent pathways therefore represent a potential therapeutic target to prevent the morbidity and mortality arising from age-related defects in hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

p53 Co-Activator Aspp1 Induces Apoptosis in Damaged Hematopoietic Stem Cells and Prevents Malignant Transformation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 603-603 ◽  
Author(s):  
Masayuki Yamashita ◽  
Eriko Nitta ◽  
Toshio Suda
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Dna Damage ◽  
Hematopoietic Stem Cells ◽  
Hematological Malignancies ◽  
Aberrant Methylation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Apoptotic Genes

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Mutation accumulation in HSCs precedes the development of leukemia and lymphoma, and these “pre-leukemic HSCs” can survive after chemotherapy, contributing to the relapse of the disease. Thus, understanding for the DNA damage response at a HSC level is a matter of critical importance for lifelong hematopoiesis, yet the protection mechanism for HSCs from DNA damage accumulation remains to be elucidated. During our study on the response of HSCs to ionizing radiation (IR), we have detected higher responsiveness of HSCs to DNA damage compared with committed progenitor cells: higher p53 activation was observed in HSC-enriched LSK (Lin-Sca1+cKit+) cells and LT-HSCs (CD150+CD41-CD48-LSK) than in myeloid progenitor-enriched LKS- cells. Of note, when treated with 4 Gy IR, LSK cells exhibited stronger upregulation of pro-apoptotic genes Bax, Noxa and Puma compared with LKS- cells, whereas upregulation of survival-contributing p21 and Mdm2 genes was comparable between the two populations. Corresponding to such characteristic behavior, we have identified apoptosis-stimulating protein of p53 1 (Aspp1) as a novel specific regulator of HSCs that provides HSCs with high sensitivity to apoptosis. We found that mRNA and protein of Aspp1 were specifically detected in LSK cells and LT-HSCs. To uncover the roles of Aspp1 in the regulation of HSCs, we evaluated HSCs of adult Aspp1 knockout (KO) mice. These mutant mice exhibited a major increase in the absolute number of LSK cells (1.5-fold; P<0.05) and LT-HSCs (2-fold; P<0.0005). Furthermore, self-renewal capacity of Aspp1-null HSCs was significantly enhanced as measured by serial competitive bone marrow (BM) transplantation assays (P<0.01). To assess the cause of enhanced self-renewal of Aspp1-null HSCs, we examined gene expression profile of Aspp1-null LSK cells before and after BM transplantation using multiplex quantitative RT-PCR array. Aspp1-null LSK cells showed higher expression of multiple quiescence-related genes including Tek, Mpl and Ndn. In line with this, Ki67 staining revealed that Aspp1-null LSK cells showed resistance to the loss of quiescence after serial BM transplantation (P<0.01), and Aspp1 KO mice showed accelerated recovery of peripheral blood and BM when treated with a single dose of 5-FU (P<0.05). Moreover, when serially transplanted or subjected to 4 Gy IR in vivo, Aspp1-null LSK cells exhibited higher resistance to apoptosis which was detected as decreased proportion of Annexin V-positive cells (P<0.05). Gene expression analysis consistently revealed that the induction of pro-apoptotic genes Bax, Noxa and Puma was impaired in irradiated Aspp1-null LSK cells. As a result of the reduced apoptosis, Aspp1-null LSK cells exhibited the tendency to retain persistent DNA damage after genotoxic stress as assessed by γH2AX and 53BP1 foci (chi-square test, P<0.05). Importantly, by breeding Aspp1 KO mice with Mx1-Cre mice and p53flox/flox mice, we verified that Aspp1 synergized with p53 to regulate self-renewal and genomic integrity of HSCs beyond its canonical p53-dependent function. Aspp1 loss further enhanced self-renewal capacity of HSCs in a p53-null background when assayed by serial BM transplantation (P<0.05). Likewise, Aspp1 deficiency further accentuated the accumulation of DNA damage after IR exposure in the absence of p53 (P<0.05). Consequently, whereas approximately half of the recipients receiving p53-null LSK cells died of thymic lymphoma, the recipient mice transplanted with LSK cells deficient for both Aspp1 and p53 were 100% lethal within 6 months after BM transplantation (log-rank test, P<0.01). These mice succumbed to hematological malignancies, mostly T-cell acute lymphoblastic lymphoma and leukemia (ALL) (88%) but also B-cell (6%) and myeloid (6%) malignancies. Taken together, our study demonstrates that Aspp1 attenuates HSC quiescence and induces apoptosis in damaged HSCs, in both p53-dependent and -independent manners, thereby inhibiting the development of leukemia and lymphoma in conjunction with p53 in HSCs. As loss of Aspp1 expression due to aberrant methylation of its promoter has already been proven to be an independent poor prognosis factor in ALL patients, Aspp1 may be a potential target for stem cell-directed therapy of leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Menin regulates the function of hematopoietic stem cells and lymphoid progenitors

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1661-1669 ◽  
Author(s):  
Ivan Maillard ◽  
Ya-Xiong Chen ◽  
Ann Friedman ◽  
Yuqing Yang ◽  
Anthony T. Tubbs ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Protein A ◽  
Suppressor Gene ◽  
Hematopoietic Stem ◽  
Lymphoid Progenitors ◽  
Functional Defect ◽  
B Lymphoid

Abstract Men1 is a tumor suppressor gene mutated in endocrine neoplasms. Besides its endocrine role, the Men1 gene product menin interacts with the mixed lineage leukemia (MLL) protein, a histone H3 lysine 4 methyltransferase. Although menin and MLL fusion proteins cooperate to activate Homeobox (Hox) gene expression during transformation, little is known about the normal hematopoietic functions of menin. Here, we studied hematopoiesis after Men1 ablation. Menin loss modestly impaired blood neutrophil, lymphocyte, and platelet counts. Without hematopoietic stress, multilineage and myelo-erythroid bone marrow progenitor numbers were preserved, while B lymphoid progenitors were decreased. In contrast, competitive transplantation revealed a marked functional defect of long-term hematopoietic stem cells (HSC) in the absence of menin, despite normal initial homing of progenitors to the bone marrow. HoxA9 gene expression was only modestly decreased in menin-deficient HSCs. These observations reveal a novel and essential role for menin in HSC homeostasis that was most apparent during situations of hematopoietic recovery, suggesting that menin regulates molecular pathways that are essential during the adaptive HSC response to stress.

Reversible Expansion of Hematopoietic Stem Cells (HSC) and CML-Like Disease in Transgenic Mice Expressing BCR-ABL under the Control of the SCL 3′ Enhancer.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 715-715
Author(s):  
Steffen Koschmieder ◽  
Berthold Goettgens ◽  
Pu Zhang ◽  
Tajhal Dayaram ◽  
Kristin Geary ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgenic Mice ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Molecular Mechanisms ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Double Transgenic Mice

Abstract Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Using the tet-off system, we have generated double-transgenic mice in which BCR-ABL is expressed under the control of the murine SCL 3′ enhancer, which targets expression to the vast majority of HSC and progenitors. After induction of BCR-ABL, all mice developed progressive chronic neutrophilia and leukocytosis (20–40 K/ul), and the animals died or were sacrificed in moribund condition within 58+/−28 days. Upon necropsy, bone marrow granulocytic hyperplasia, splenomegaly as well as organ infiltration by leukemic cells (liver, kidney, lung, small intestine, skin) were found. In addition, 31% of the mice subsequently developed ALL or lymphomas. BCR-ABL mRNA and protein expression were demonstrated in the affected organs. Expression of the transactivating transgene tTA was high in HSC, CMP, and CLP, but low in GMP and MEP, as assessed by real-time PCR, suggesting that the SCL 3′ enhancer indeed directed BCR-ABL expression to the most primitive hematopoietic cells within the bone marrow. The percentage of HSC in the bone marrow was expanded 7- and 26-fold in double-transgenic as compared to single-transgenic or wild-type control mice within 12 and 21 days, respectively, after BCR-ABL induction. GMP were increased 2- and 3-fold while the number of CMP was decreased 2-fold after 12 days but was increased 1.5-fold after 21 days. MEP were decreased 3-fold at both time points. In keeping with these results, the percentage of Ter-119 positive erythroid cells was decreased while the percentage of Gr-1 positive granulocytic cells was increased in the bone marrow. To assess reversibility of the phenotype, we readministered tetracycline to abrogate BCR-ABL expression. Double-transgenic mice showed rapid clinical improvement, reversion of neutrophilia and leukocytosis, normalization of Gr-1/Mac-1 positive cells in the peripheral blood and spleen, and reversion of splenomegaly. In addition, in mice that had developed lymphoblastic disease, readministration of tetracycline led to disappearance of lymphomas and of B220/BP-1 positive lymphoblastic cells in the peripheral blood. Furthermore, expansion of the HSC compartment in the bone marrow was also reversible, and the percentage of HSC decreased to levels observed in control mice. Repeated induction of BCR-ABL expression by removal of tetracycline led to reappearance of the myeloid and lymphoid phenotype. Again, the disease was reversible, and none of the animals relapsed while on tetracycline, suggesting that the phenotype remained completely dependent on the expression of the oncogene. In conclusion, we present a model of BCR-ABL mediated CML-like disease with expansion of phenotypic hematopoietic stem cells and myeloid progenitor cells in the bone marrow. The target cell population in this model closely resembles the origin of transformation in patients with CML, allowing for in vivo monitoring of early molecular mechanisms of BCR-ABL transformation. We are currently studying the function of the expanded HSC and progenitor cells in transplantation experiments.

Hypoxia-Adapted CML Cells Are More Primitive Population and Are Eradicated by Glyoxalase-1 Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2166-2166 ◽  
Author(s):  
Miki Takeuchi ◽  
Shinya Kimura ◽  
Junya Kuroda ◽  
Eishi Ashihara ◽  
Makoto Kawatani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Cell Lines ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Nog Mice

Abstract Abstract 2166 Poster Board II-143 Normal hematopoietic stem cells reside in the epiphyses of the bone marrow that is a low oxygenated area and are protected against ROS-induced DNA damages. Recent study showed that the hypoxic environment plays a crucial role not only in maintaining stem cells but also in tumorigenesis. Hypoxia induces dramatical changes in cell characteristics including cell cycle quiescence, self renewal potency, and shift in energy production from an aerobic to anaerobic pathway, and it induces resistance to a variety of cell death stimuli. Chronic myelogenous leukemia (CML) is a disorder of hematopoietic stem cells caused by the constitutive activation of the Bcr-Abl tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have led to marked improvement in prognosis of CML patients. However, CML cells could not be eradicated completely by TKI alone because quiescent CML stem cells are less sensitive to such molecular target drugs. Therefore, we speculate that the adaptation of leukemic cells to hypoxia in the bone marrow niche alters their characteristics contributing to minimal residual disease. We first evaluated the oxygen levels of engrafted leukemic cells by pimonidazol (hypoxia specific marker) staining.We transplanted K562 cells to the bone marrow of NOD/SCID/gcnull (NOG) mice and found that those cells engrafted and survived in the epiphysis of the bone marrow where O2 concentrations are less than 1.3%. Then, we generated two hypoxia-adapted (HA) CML subclones from K562 and KCL22 by cultivating under 1.0% O2, and were denoted as K562/HA and KCL22/HA, respectively. Both cell lines survived and proliferated continuously for years under 1.0% O2 conditions, although their growth was slower than that of their parental counterparts under 20% O2 conditions. Interestingly, HA-CML cells exhibited several unique characteristics compared to their parental cells. First, these HA cells showed higher transplantation efficacy in NOG mice. The transplanted HA cells grow more rapidly in vivo than the parental cells and mice transplanted with HA cells died earlier. Next, the percentage of G0 fractions in K562 and K562/HA cells were 0.87 ± 0.58 % and 4.9 ± 2.1 %, respectively, indicating that K562/HA cells included more quiescent fractions than the parental K562. Hoechst staining analysis confirmed that HA cell lines include more SP (side population) fractions than their parental cells, indicating that HA cells contains more dormant cells. We next examined the signaling pathway of HA cell lines. Despite the unchanged levels of AKT, STAT, and ERK phosphrylation, BCR-ABL phsophrylation was suppressed in HA cells. Both of HA cell lines showed higher expression of b-catenin which is considered essential for survival and self-renewality of CML stem cells. Furthermore, HA cells were less sensitive to TKIs (imatinib, dasatinib, and bafetinib) and chemotherapeutic agents (daunorubicin and busulfan). Taken together, our HA cell lines have characteristics of more primitive CML cell populations resistant to cytotoxic agents. We next examined the energy metabolites such as adenosine triphosphate (ATP), glucose consumption, and lactate production in HA cells. The amounts of ATP in K562/HA and KCL22/HA cells decreased, whereas glucose consumption and lactate production increased compared with those in their parental cell lines. These findings indicate that ATP production of HA cells depends on glycolysis. Furthermore, we found higher expression and kinase activity of Glyoxalase-1 (Glo-I). Glo-I is an enzyme that detoxifies glycolysis-specific cytocidal byproducts in glycolytis system. Glo-I inhibitors such as S-p-bromobenzylglutathione cyclopentyl diester (BBGC), 2-crotonyloxymethyl-4,5,6-trihydroxycylohex-2-enone (COTC), and methyl-gerfelin were much more cytotoxic against HA-CML cells than their parental cells in vitro. Notably, when K562/HA-transplanted mice were treated with 100 mg/kg/day BBGC for 8 days, the treated mice survived longer than the untreated mice (Figure 1). These findings suggest that Glo-1 plays an important role in primitive CML cells survival under hypoxia. In conclusion, Glo-1 is a novel attractive target against hypoxia-adapted primitive CML cells in the bone marrow milieu. Investigation of hypoxia-specific pathways and roles on CML cells could develop novel therapeutic approach targeting TKIs resistance. Disclosures: No relevant conflicts of interest to declare.

Dysregulation of Hematopoietic Stem Cells with Age.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. sci-2-sci-2
Author(s):  
Margaret A. Goodell
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Time Course ◽  
Regional Analysis ◽  
Gene Clusters ◽  
Hematopoietic Stem ◽  
Effects Of Aging ◽  
The Impact

Aging is an inexorable process marked by an accumulation of traits that ultimately limit the normal function of the organism. Stem cells have long been of interest to the study of aging due to their potential to rejuvenate somatic tissues. However, several groups have now shown that stem cells themselves are subject to the effects of aging. In the hematopoietic system, stem cells markedly decline in function with time, and display characteristic behaviors such as skewing of differentiation toward myeloid development. Current studies focus on both the broad effects of aging on stem cells, and also on the role of specific genes in the aging process. Our laboratory has used gene expression profiling to examine the global gene expression changes that occur in purified hematopoietic stem cells in a time course of aging in mice. Of around 14,000 genes profiled, we identified ~1500 that were age-induced and ~1600 that were age-repressed. Genes associated with the stress response, inflammation, and protein aggregation dominated the upregulated expression profile, while the downregulated profile was marked by genes involved in the preservation of genomic integrity and chromatin remodeling. Regional analysis suggested dysregulation of many gene clusters rather than alterations of a small number of aging-specific genes. These studies suggest that hematopoietic stem cells are subject to extensive epigenetic changes over time. This epigenetic dysregulation lays a fertile ground for secondary events that may enhance the impact of genetic changes, thus driving both functional attenuation and the increased propensity for neoplastic transformation with age. The influence of the aging environment on stem cells is only now being explored. The composition of the bone marrow changes with age, and this can clearly exert effects on the types and proportions of progenitors. Furthermore, environmental conditions that typify the aging state, such as chronic inflammation, can directly impact the stem cells. A broad view of aging bone marrow environment and stem cells, as well as current molecular data addressing these issues, will be presented.

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