Reintroduction of C/EBPα in leukemic CD34+ stem/progenitor cells impairs self-renewal and partially restores myelopoiesis

Schepers, Hein; Wierenga, Albertus T. J.; Gosliga, Djoke van; Eggen, Bart J. L.; Vellenga, Edo; Schuringa, Jan Jacob

doi:10.1182/blood-2006-10-052175

Cited by 42 publications

(34 citation statements)

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“…[30][31][32][33] Similarly, another transcription regulator, CEBPA, located at MDR 12p13.2 is a critical transcription factor that controls tissue specific gene expression and proliferation arrest. 34 Indeed, CEBPA acts as a tumor suppressor in a number of tumor types. 35 Regarding MM, CEBPA polymorphism has been described in 5% of MM patients.…”

Section: %mentioning

confidence: 99%

SNP-based mapping arrays reveal high genomic complexity in monoclonal gammopathies, from MGUS to myeloma status

et al. 2012

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Genetic events mediating transformation from premalignant monoclonal gammopathies (MG) to multiple myeloma (MM) are unknown. To obtain a comprehensive genomic profile of MG from the early to late stages, we performed high-resolution analysis of purified plasma cells from 20 MGUS, 20 smoldering MM (SMM) and 34 MM by high-density 6.0 SNP array. A progressive increase in the incidence of copy number abnormalities (CNA) from MGUS to SMM and to MM (median 5, 7.5 and 12 per case, respectively) was observed (P ¼ 0.006). Gains on 1q, 3p, 6p, 9p, 11q, 19p, 19q and 21q along with 1p, 16q and 22q deletions were significantly less frequent in MGUS than in MM. Although 11q and 21q gains together with 16q and 22q deletions were apparently exclusive of MM status, we observed that these abnormalities were also present in minor subclones in MGUS. Overall, a total of 65 copy number-neutral LOH (CNN-LOH) were detected. Their frequency was higher in active MM than in the asymptomatic entities (P ¼ 0.047). A strong association between genetic lesions and fragile sites was also detected. In summary, our study shows an increasing genomic complexity from MGUS to MM and identifies new chromosomal regions involved in CNA and CNN-LOH.

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Section: %mentioning

confidence: 99%

SNP-based mapping arrays reveal high genomic complexity in monoclonal gammopathies, from MGUS to myeloma status

et al. 2012

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Section: Lentiviral Virus Production and Infectionmentioning

confidence: 99%

“…AML CD34 ϩ blasts were transduced, as previously described. [30][31][32][33] Briefly, the cells were prestimulated for 4 hours in RPMI supplemented with 10% fetal calf serum (FCS), 20 ng/mL interleukin 3 (IL-3; Gist-Brocades), granulocyte-colony-stimulating factor (Rhone-Poulenc Rorer), and TPO, and afterward transduced on retronectin-coated plates in 3 consecutive rounds of 8 and 12 hours with lentiviral supernatants containing cytokines and polybrene, as indicated above.Ex vivo culture of primary cells, and colony-forming cell and long-term culture-initiating cell assays CB stroma-free cultures were propagated in either serum-free HPGM supplemented with SCF, Flt3L, and TPO (all 100 ng/mL), or Iscove modified Dulbecco medium (IMDM) supplemented with 10% FCS, IL-3 (10 ng/mL), and SCF (100 ng/mL). For the CB MS-5 coculture experiments and long-term culture-initiating cell (LTC-IC) assays, cells were grown in ␣ minimum essential medium (BioWhittaker) supplemented with 12.5% heat-inactivated FCS, 12.5% heat-inactivated horse serum (SigmaAldrich), penicillin and streptomycin, 2 mM glutamine, 57.2 M ␤-mercaptoethanol, and 1 M hydrocortisone (Sigma-Aldrich).…”

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confidence: 99%

Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Rizo

Olthof

Han

et al. 2009

Blood

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High expression of BMI1 in acute myeloid leukemia (AML) cells is associated with an unfavorable prognosis. Therefore, the effects of down-modulation of BMI1 in normal and leukemic CD34 ؉ AML cells were studied using a lentiviral RNA interference approach. We demonstrate that downmodulation of BMI1 in cord blood CD34 ؉ cells impaired long-term expansion and progenitor-forming capacity, both in cytokine-driven liquid cultures as well as in bone marrow stromal cocultures. In addition, long-term culture-initiating cell frequencies were dramatically decreased upon knockdown of BMI1, indicating an impaired maintenance of stem and progenitor cells. The reduced progenitor and stem cell frequencies were associated with increased expression of p14ARF and p16INK4A and enhanced apoptosis, which coincided with increased levels of intracellular reactive oxygen species and reduced FOXO3A expression. In AML CD34 ؉ cells, down-modulation of BMI1 impaired long-term expansion, whereby selfrenewal capacity was lost, as determined by the loss of replating capacity of the cultures. These phenotypes were also associated with increased expression levels of p14ARF and p16INK4A. Together our data indicate that BMI1 expression is required for maintenance and selfrenewal of normal and leukemic stem and progenitor cells, and that expression of IntroductionBMI1 is a member of the Polycomb group (PcG) genes, which are transcriptional repressors that play essential roles in the maintenance of appropriate gene expression during development. [1][2][3][4][5] Two distinct multiprotein PcG complexes have been identified, the Polycomb repression complex (PRC) 1 and PRC2. 1 The PRC2 complex is involved in initiation of silencing and contains histone deacetylases and methyltransferases that can methylate H3 lysine 9 and 27. 6 Deletion of PRC2 genes in mice results in embryonic lethality, emphasizing their importance in development. [7][8][9] PRC1 is implicated in stable maintenance of gene repression and recognizes the methylation marks set by PRC2. 10,11 Mice mutant for most PRC1 genes survive until birth as a result of partial functional redundancy provided by their homologues, but developmental defects do arise thereafter, as is, for example, the case in the hematopoietic compartment after deletion of BMI1. [12][13][14] Targeted deletion of Bmi1 has shown that although the numbers of fetal liver-derived hematopoietic stem cells (HSCs) are normal in these mice, their proliferative and self-renewal capacity is severely impaired. 15,16 In adult Bmi1-deficient mice, the HSCs are less frequent and display an impaired competitive repopulation capacity. 16,17 Gain-of-function studies demonstrated enhanced selfrenewal of murine HSCs and with a shift in balance toward more symmetric stem cell divisions. 17 We have demonstrated that constitutive expression of BMI1 in human cord blood (CB) cells results in prolonged maintenance of the stem cell pool and enhances self-renewal of human stem and progenitor cells. 18 BMI1 is a potent negative regulator of the IN...

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“…Lentiviral particles were made by transient transfection of 293T cells with the lentiviral expression vectors as described previously. 33 Isolated CD34 ϩ cells were cultured for 48 hours in serum-free medium (Lonza Netherlands) containing 100 ng/mL each of stem cell factor, thrombopoietin, and FLT-3L and subsequently transduced with the retroviral and lentiviral particles in 3 consecutive rounds as described previously. 33 Stromal coculture assays, CFC assays MS5 cocultures were performed as described previously.…”

Section: Cell Culture and Viral Transductionsmentioning

confidence: 99%

“…33 Isolated CD34 ϩ cells were cultured for 48 hours in serum-free medium (Lonza Netherlands) containing 100 ng/mL each of stem cell factor, thrombopoietin, and FLT-3L and subsequently transduced with the retroviral and lentiviral particles in 3 consecutive rounds as described previously. 33 Stromal coculture assays, CFC assays MS5 cocultures were performed as described previously. 19 Half of the cultures were removed weekly and analyzed by fluorescence-activated cell sorter (FACS) or sorted to perform colony-forming cell (CFC) assays or cytospins.…”

Section: Cell Culture and Viral Transductionsmentioning

confidence: 99%

Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation

Wierenga

Vellenga

Schuringa³

2010

Blood

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Previously, we have shown that overexpression of an activated mutant of signal transducer and activator of transcription-5 (STAT5) induces erythropoiesis, impaired myelopoiesis, and an increase in longterm proliferation of human hematopoietic stem/progenitor cells. Because GATA1 is a key transcription factor involved in erythropoiesis, the involvement of GATA1 in STAT5-induced phenotypes was studied by shRNA-mediated knockdown of GATA1. CD34 ؉ cord blood cells were double transduced with a conditionally active STAT5 mutant and a lentiviral vector expressing a short hairpin against GATA1. Erythropoiesis was completely abolished in the absence of GATA1, indicating that STAT5-induced erythropoiesis is GATA1-dependent. Furthermore, the impaired myelopoiesis in STAT5-transduced cells was restored by GATA1 knockdown. Interestingly, early cobblestone formation was only modestly affected, and long-term growth of STAT5-positive cells was increased in the absence of GATA1, whereby high progenitor numbers were maintained. Thus, GATA1 down-regulation allowed the dissection of STAT5-induced differentiation phenotypes from the effects on long-term expansion of stem/ progenitor cells. Gene expression profiling allowed the identification of GATA1-dependent and GATA1-independent STAT5 target genes, and these studies revealed that several proliferation-related genes were up-regulated by STAT5 independent of GATA1, whereas several erythroid differentiation-related genes were found to be GATA1 as well as STAT5 dependent. IntroductionSignal transducer and activator of transcription-5 (STAT5) is a member of a family of transcription factors that is composed of 7 genes (STAT1 to STAT6, with STAT5 being encoded by 2 genes, STAT5A and STATB). The pathways mediated by these transcription factors are involved in many processes in hematopoietic cells, including regulation of proliferation, antiapoptosis, differentiation, and self-renewal. 1,2 STAT transcription factors contain a conserved tyrosine residue in their SH2 domain, which on phosphorylation, gives rise to heterodimerization or homodimerization (or tetramerization 3 ) with another STAT molecule, leading to nuclear translocation. Constitutively activated STAT5 has been found in blast cells of up to 70% of acute myeloid leukemia cases. 4 Besides its function as a transcription factor, tyrosine phosphorylated STAT5 has also been described to interact with other partners to induce phosphoinositide-3 kinase activity, thereby contributing to leukemic transformation. 5 STAT transcription factors can be activated by growth factors via JAK kinases, present at the cytoplasmic domains of growth factor receptors, as well as by intrinsic kinase activity of certain membrane receptors or cytoplasmic tyrosine kinases. 6 STAT5 is expressed in a large variety of hematopoietic cells and involved in the signal transduction of many different growth factors and cytokines. Early acting cytokines, such as Fms-like tyrosine kinase 3 ligand (FLT-3L), thrombopoietin, and stem cell factor, all mediate at le...

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Reintroduction of C/EBPα in leukemic CD34+ stem/progenitor cells impairs self-renewal and partially restores myelopoiesis

Cited by 42 publications

References 32 publications

SNP-based mapping arrays reveal high genomic complexity in monoclonal gammopathies, from MGUS to myeloma status

SNP-based mapping arrays reveal high genomic complexity in monoclonal gammopathies, from MGUS to myeloma status

Repression of BMI1 in normal and leukemic human CD34+ cells impairs self-renewal and induces apoptosis

Down-regulation of GATA1 uncouples STAT5-induced erythroid differentiation from stem/progenitor cell proliferation

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