Notch signaling enhances survival and alters differentiation of 32D myeloblasts

The Notch transmembrane receptors play important roles in precursor survival and cell fate specification during hematopoiesis. To investigate the function of Notch and the signaling events activated by Notch in myeloid development, we expressed truncated forms of Notch1 or Notch2 proteins that either can or cannot activate the core binding factor 1 (CBF1) in 32D (clone 3) myeloblasts. 32D cells proliferate as blasts in the presence of the cytokines, GM-CSF or IL-3, but they initiate differentiation and undergo granulopoiesis in the presence of granulocyte CSF (G-CSF). 32D cells expressing constitutively active forms of Notch1 or Notch2 proteins that signal through the CBF1 pathway maintained significantly higher numbers of viable cells and exhibited less cell death during G-CSF induction compared with controls. They also displayed enhanced entry into granulopoiesis, and inhibited postmitotic terminal differentiation. In contrast, Notch1 constructs that either lacked sequences necessary for CBF1 binding or that failed to localize to the nucleus had little effect. Elevated numbers of viable cells during G-CSF treatment were also observed in 32D cells overexpressing the basic helix-loop-helix protein (bHLH), HES1, consistent with activation of the CBF1 pathway. Taken together, our data suggest that Notch signaling enhances 32D cell survival, promotes entry into granulopoiesis, and inhibits postmitotic differentiation through a CBF1-dependent pathway.     

Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling

Hematopoietic stem cells give rise to progeny that either self-renew in an undifferentiated state or lose self-renewal capabilities and commit to lymphoid or myeloid lineages. Here we evaluated whether hematopoietic stem cell self-renewal is affected by the Notch pathway. Notch signaling controls cell fate choices in both invertebrates and vertebrates by inhibiting certain differentiation pathways, thereby permitting cells to either differentiate along an alternative pathway or to self-renew. Notch receptors are present in hematopoietic precursors and Notch signaling enhances the in vitro generation of human and mouse hematopoietic precursors, determines T- or B-cell lineage specification from a common lymphoid precursor and promotes expansion of CD8(+) cells. Here, we demonstrate that constitutive Notch1 signaling in hematopoietic cells established immortalized, cytokine-dependent cell lines that generated progeny with either lymphoid or myeloid characteristics both in vitro and in vivo. These data support a role for Notch signaling in regulating hematopoietic stem cell self-renewal. Furthermore, the establishment of clonal, pluripotent cell lines provides the opportunity to assess mechanisms regulating stem cell commitment and demonstrates a general method for immortalizing stem cell populations for further analysis.     

Effects of histamine on immunophenotype and notch signaling in human HL-60 leukemia cells

Surface molecules are important biomarkers for cell proliferation and differentiation and play important roles in cell function and cell interaction. Notch is a transmembrane receptor that regulates developmental processes and cell-fate decision. Histamine is used as an adjunct to immunotherapy in myelogenous leukemia, and regulates hematopoietic cell development. Thus, we investigated the effects of histamine on immunophenotype and Notch signaling in human HL-60 leukemia cells. Histamine (0.1-10 microM) inhibited the colony-forming efficiency of HL-60 cells in a dose-dependent fashion and shifted the growth curve to the right. HL-60 cells were treated with histamine 0.1-1.0 microM for 6 days, and surface molecules were analyzed by flow cytometry. Histamine decreased CD49d positive cells by 74% while increasing CD31 positive cells by 53% as compared to controls. Histamine did not affect the expression of CD11b, CD14, CD34, CD44, CD54, CD49e, and CD62L. To examine Notch signaling in histamine-induced immunophenotype alterations in HL-60 cells, total RNA was isolated, purified, and subjected to real-time RT-PCR analysis. The expressions of Notch1, Notch4, the ligands Jagged1, Delta4, and the downstream hairy enhancer of split 1 gene (HES1) were not significantly altered by histamine. In summary, this study demonstrated that histamine inhibited HL-60 cell growth and regulated immunophenotypes of CD49d and CD31. These effects are not mediated through the Notch signaling.     

Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo

Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.     

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells

The Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6 h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells.     

Rbm15-Mkl1 Interacts with the Setd1b Histone H3-Lys4 Methyltransferase via a SPOC Domain That Is Required for Cytokine-Independent Proliferation

The Rbm15-Mkl1 fusion protein is associated with acute megakaryoblastic leukemia (AMKL), although little is known regarding the molecular mechanism(s) whereby this fusion protein contributes to leukemogenesis. Here, we show that both Rbm15 and the leukemogenic Rbm15-Mkl1 fusion protein interact with the Setd1b histone H3-Lys4 methyltransferase (also known as KMT2G). This interaction is direct and requires the Rbm15 SPOC domain and the Setd1b LSD motif. Over-expression of Rbm15-Mkl1 in the 6133 megakaryoblastic leukemia cell line, previously established by expression of the Rbm15-Mkl1 fusion protein in mice (Mercher et al., [2009] J. Clin. Invest. 119, 852-864), leads to decreased levels of endogenous Rbm15 and increased levels of endogenous Mkl1. These cells exhibit enhanced proliferation and cytokine-independent cell growth, which requires an intact Rbm15 SPOC domain that mediates interaction between the Rbm15-Mkl1 fusion protein and the Setd1b methyltransferase. These results reveal altered Setd1b complex function and consequent altered epigenetic regulation as a possible molecular mechanism that mediates the leukemogenic activity of the Rbm15-Mkl1 fusion protein in AMKL.