hes1-protein--human and Leukemia--Promyelocytic--Acute

Notch proteins determine cell fate decisions in the development of diverse tissues. Notch has been initially found in T-ALL but its role has been also studied in myelopoiesis and myeloid leukemias. Studies in different model systems have led to a widespread controversy as to whether Notch promotes or blocks myeloid differentiation. In this work, we evaluated the influence of Notch activation on leukemic cell differentiation along the monocytic and myelocytic pathway induced by phorbol 12-myristate 13-acetate (PMA) or all-trans retinoic acid (ATRA). We observed that differentiation of the human myeloblastic cell line HL-60 can be retarded or blocked by Delta/Notch interaction. ATRA induces complete remission in patients with acute promyelocytic leukemia, but it cannot completely eliminate the leukemic clone and to be effective it should be combined with chemotherapy. Our findings suggest that Notch signaling may contribute to the incomplete elimination of the leukemic cells after PMA or ATRA treatment and the blockage of Notch pathway may be beneficial in the treatment of myeloid leukemia. © 2014 International Society for Advancement of Cytometry.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Granulocyte Precursor Cells; HL-60 Cells; Homeodomain Proteins; Humans; Jurkat Cells; Leukemia, Promyelocytic, Acute; Mice; Myeloid Cells; Precursor T-Cell Lymphoblastic Leukemia-Lymphoma; Receptor, Notch1; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription Factor HES-1; Tretinoin; U937 Cells

The Notch signaling pathway has been implicated in the development of several leukemia and lymphoma. In order to investigate the relationship between Notch signaling and acute myeloid leukemia (AML), in this study, we expressed a recombinant Notch ligand protein, the DSL domain of the human Jagged1 fused with GST (GST-Jag1). GST-Jag1 could activate Notch signaling in the human promyelocytic leukemia cell line HL60, as shown by a reporter assay and the induced expression of Notch effector gene Hes1 and Hes5. However, GST-Jag1 had no effect on the proliferation and survival of HL60 cells. HL60 cells expressed both Notch ligands and receptors, and had a potential of reciprocal stimulation of Notch signaling between cells. We, therefore, blocked Notch signaling in cultured HL60 cells using a gamma-secretase inhibitor (GSI). We found that GSI inhibited the proliferation of HL60 cells significantly by blocking the cell-cycle progression in the G1 phase. Furthermore, GSI induced remarkably apoptosis of HL60 cells. These changes in GSI-treated HL60 cells correlated with the down-regulation of c-Myc and Bcl2, and the low phosphorylation of the Rb protein. These results suggested that reciprocal Notch signaling might be necessary for the proliferation and survival of AML cells, possibly through the maintenance of the expression of c-Myc and Bcl2, as well as the phosphorylation of the Rb protein.

Topics: Amyloid Precursor Protein Secretases; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cell Cycle; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; HeLa Cells; HL-60 Cells; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Leukemia, Promyelocytic, Acute; Membrane Proteins; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Receptors, Notch; Recombinant Fusion Proteins; Repressor Proteins; Retinoblastoma Protein; Serrate-Jagged Proteins; Signal Transduction; Time Factors; Transcription Factor HES-1; Transfection

Integrin-linked kinase (ILK) directly interacts with beta integrins and phosphorylates Akt in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. In this study, we examined the functional role of ILK activation in leukemic and bone marrow stromal cells on their direct contact. Coculture of leukemic NB4 cells with bone marrow-derived stromal mesenchymal stem cells (MSC) resulted in robust activation of multiple signaling pathways, including ILK/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducers and activators of transcription 3 (STAT3), and Notch1/Hes. Blockade of PI3K or ILK signaling with pharmacologic inhibitors LY294002 or QLT0267 specifically inhibited stroma-induced phosphorylation of Akt and glycogen synthase kinase 3beta, suppressed STAT3 and ERK1/2 activation, and decreased Notch1 and Hes1 expression in leukemic cells. This resulted in induction of apoptosis in both leukemic cell lines and in primary acute myelogenous leukemia samples that was not abrogated by MSC coculture. In turn, leukemic cells growing in direct contact with bone marrow stromal elements induce activation of Akt, ERK1/2, and STAT3 signaling in MSC, accompanied by significant increase in Hes1 and Bcl-2 proteins, which were all suppressed by QLT0267 and LY294002. In summary, our results indicate reciprocal activation of ILK/Akt in both leukemic and bone marrow stromal cells. We propose that ILK/Akt is a proximal signaling pathway critical for survival of leukemic cells within the bone marrow microenvironment. Hence, disruption of these interactions by ILK inhibitors represents a potential novel therapeutic strategy to eradicate leukemia in the bone marrow microenvironment by simultaneous targeting of both leukemic cells and activated bone marrow stromal cells.

Topics: Basic Helix-Loop-Helix Transcription Factors; Bone Marrow Cells; Cell Communication; Cell Survival; Chromones; Coculture Techniques; Enzyme Activation; Homeodomain Proteins; Humans; Janus Kinases; Leukemia, Promyelocytic, Acute; Mesenchymal Stem Cells; Mitogen-Activated Protein Kinase Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptors, Notch; Signal Transduction; STAT3 Transcription Factor; Stromal Cells; Transcription Factor HES-1