transforming-growth-factor-beta and Leukemia--Megakaryoblastic--Acute

Acute megakaryocytic leukemia (AMKL) is one of the rarest sub-types of acute myeloid leukemia (AML). AMKL is characterized by high proliferation of megakaryoblasts and myelofibrosis of bone marrow, this disease is also associated with poor prognosis. Previous analyses have reported that the human megakaryoblastic cells can be differentiated into cells with megakaryocyte (MK)-like characteristics by phorbol 12-myristate 13-acetate (PMA). However, little is known about the mechanism responsible for regulating this differentiation process. We performed long non-coding RNA (lncRNA) profiling to investigate the differently expressed lncRNAs in megakaryocyte blast cells treated with and without PMA and examined those that may be responsible for the PMA-induced differentiation of megakaryoblasts into MKs. We found 30 out of 90 lncRNA signatures to be differentially expressed after PMA treatment of megakaryoblast cells, including the highly expressed JPX lncRNA. Further, in silico lncRNA-miRNA and miRNA-mRNA interaction analysis revealed that the JPX is likely involved in unblocking the expression of TGF-β receptor (TGF-βR) by sponging oncogenic miRNAs (miR-9-5p, miR-17-5p, and miR-106-5p) during MK differentiation. Further, we report the activation of TGF-βR-induced non-canonical ERK1/2 and PI3K/AKT pathways during PMA-induced MK differentiation and ploidy development. The present study demonstrates that TGF-βR-induced non-canonical ERK1/2 and PI3K/AKT pathways are associated with PMA-induced MK differentiation and ploidy development; in this molecular mechanism, JPX lncRNA could act as a decoy for miR-9-5p, miR-17-5p, and miR-106-5p, titrating them away from TGF-βR mRNAs. Importantly, this study reveals the activation of ERK1/2 and PI3K/AKT pathway in PMA-induced Dami cell differentiation into MK. The identified differentially expressed lncRNA signatures may facilitate further study of the detailed molecular mechanisms associated with MK development. Thus, our data provide numerous targets with therapeutic potential for the modulation of the differentiation of megakaryoblastic cells in AMKL.

Topics: Carcinogenesis; Cell Differentiation; Cells, Cultured; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Megakaryoblastic, Acute; MAP Kinase Signaling System; Megakaryocytes; MicroRNAs; Phorbol Esters; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta

Chemokine stromal cell-derived factor-1 (SDF-1) is expressed by bone marrow (BM) stromal cells and plays key roles in BM cell migration. Modulation of its expression could affect the migratory capacity of cells trafficking the BM, such as hematopoietic progenitor and leukemic cells. Transforming growth factor-beta1 (TGF-beta1) is present in the BM environment and constitutes a pivotal molecule controlling BM cell proliferation and differentiation. We used the BM stromal cell line MS-5 as a model to investigate whether SDF-1 expression constitutes a target for TGF-beta1 regulation and its functional consequences. We show here that TGF-beta1 down-regulates SDF-1 expression, both at the mRNA level, involving a decrease in transcriptional efficiency, and at the protein level, as detected in lysates and supernatants from MS-5 cells. Reduction of SDF-1 in supernatants from TGF-beta1-treated MS-5 cells correlated with decreased, SDF-1-dependent, chemotactic, and transendothelial migratory responses of the BM model cell lines NCI-H929 and Mo7e compared with their responses to supernatants from untreated MS-5 cells. In addition, supernatants from TGF-beta1-exposed MS-5 cells had substantially lower efficiency in promoting integrin alpha4beta1-mediated adhesion of NCI-H929 and Mo7e cells to soluble vascular cell adhesion molecule-1 (sVCAM-1) and CS-1/fibronectin than their untreated counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1-dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to sVCAM-1 when supernatants from TGF-beta1-treated MS-5 cells were used compared with supernatants from untreated cells. These data indicate that TGF-beta1-controlled reduction in SDF-1 expression influences BM cell migration and adhesion, which could affect the motility of cells trafficking the bone marrow.

Topics: 3T3 Cells; Animals; Bone Marrow Cells; Cell Adhesion; Cell Movement; Chemokine CXCL12; Chemokines, CXC; Down-Regulation; Gene Expression; Humans; Leukemia, Megakaryoblastic, Acute; Mice; Multiple Myeloma; RNA, Messenger; Stromal Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Thrombopoietin (TPO) and transforming growth factor-beta(1) (TGF-beta(1)) have been shown to exert opposite effects on proliferation and megakaryocytic differentiation of hematopoietic cells. To determine whether TGF-beta(1) interferes directly with TPO-induced signal transduction in hematopoietic cells, we compared the regulatory effects in the TPO-responsive cell lines Mo-7e and HEL.. The cells were stimulated by 100 ng/mL TPO and/or 100 ng/mL TGF-beta1 and analyzed for proliferation (3H thymidine incorporation), viability (trypan blue exclusion), and protein expression and phosphorylation (Western blot).. TPO enhanced the proliferation of Mo-7e cells as determined by 3H-thymidine incorporation, whereas TGF-beta1 suppressed baseline cell growth and antagonized the proliferative effect of TPO. TPO-induced proliferation also was reduced by a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway (PD098059), which inhibits activation of the MAPK extracellular signal-regulated kinases (ERK) ERK1 and ERK2, and AG490, an inhibitor of Janus kinase-2, which completely blocked TPO-induced proliferation. As demonstrated by Western blotting, TGF-beta1 reduced the TPO-stimulated ERK1/ERK2 and STAT5 phosphorylation in Mo-7e and HEL cells. This effect was completely reversed by preincubation with a tyrosine phosphatase inhibitor (Na3VO4), which suggests that TGF-beta1 activated a phosphatase. Although STAT3 also was activated by TPO, STAT3 activation remained unaltered by TGF-beta1.. Taken together, these data suggest that TGF-beta1 modulates TPO-mediated effects on megakaryocytic proliferation by interfering with TPO-induced signal transduction, particularly by reducing the activities of MAPK ERK1/ERK2 and STAT5.

Topics: Cell Differentiation; Cell Division; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Erythroid Precursor Cells; Flavonoids; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Leukemia, Megakaryoblastic, Acute; MAP Kinase Signaling System; Megakaryocytes; Milk Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Neoplastic Stem Cells; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; STAT3 Transcription Factor; STAT5 Transcription Factor; Thrombopoietin; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tyrphostins; Vanadates

To determine whether platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1) are involved in organ fibrosis in patients with transient myeloproliferative disorder (TMD) in Down syndrome, the expression of PDGF and TGF-beta 1 mRNA in blast cells of TMD was investigated using real-time quantitative reverse transcription polymerase chain reaction. Blasts and liver tissue from TMD patients with hepatic fibrosis showed a significantly elevated expression of PDGF gene. The expression of TGF-beta 1 gene was higher in TMD and acute megakaryoblastic leukaemia than in the control group. These results suggest that PDGF in combination with TGF-beta 1 plays a role in organ fibrosis of TMD.

Topics: Adolescent; Adult; Down Syndrome; Female; Gene Expression; Humans; Leukemia, Megakaryoblastic, Acute; Liver Cirrhosis; Male; Myeloproliferative Disorders; Platelet-Derived Growth Factor; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Thrombopoietin (TPO) is a recently characterized growth and differentiation factor for megakaryocytes and platelets exerting its effects via the receptor MPL. We examined the expression of MPR on the cell surface of a panel of 43 myelomonocytic, erythroid and megakaryocytic leukemia cell lines and 21 primary acute myeloid leukemia (AML) cases by flow cytometry. With few exceptions MPL was found on all 32 erythroid/megakaryocytic cell lines and on all 11 growth factor-dependent myelomonocytic cell lines, albeit at variable percentages and intensities per cell population (with a 10% cut-off level for positivity still 30/43 cell lines scored as MPL positive). The majority of the primary AML samples (including all seven M6/M7 cases) expressed the MPL protein regardless of the morphological and immunological subtype (13/21 cases had >10% MPL-positive cells). Recombinant TPO overexpressed in hamster cells induced a mitogenic response in seven cell lines (one growth factor-independent and six factor-dependent lines) and in 3/21 AML specimens (two AML M2, one AML M7) as measured by 3H-thymidine incorporation. Expression of MPL clearly did not correlate with response to TPO. For further detailed studies of the interaction of TPO with other cytokines we used the AML M7-derived M-07e cells as an informative indicator cell line for which both murine and human TPO acted as a very potent mitogen in a dose-dependent fashion (3- to 11-fold proliferation increase relative to medium alone). This growth factor-dependent cell line which is normally cultured in conditioned medium containing several cytokines could be grown in long-term culture supplemented only with TPO. Co-incubation of M-07e with various cytokines and TPO showed additive proliferative effects for interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and synergistic responses for stem cell factor (SCF), interferon (IFN)-alpha, and to a lesser extent for IFN-gamma and tumor necrosis factor (TNF)-alpha. Erythropoietin (EPO), IL-1, IL-6, IL-11 and leukemia inhibitory factor (LIF), know as megakaryocytic maturation-inducing molecules, were not substantially effective, neither singly nor in combination with TPO, with regard to cell growth. Transforming growth factor (TGF)-beta1 antagonized the inductive effect of TPO on M-07e cell growth. Addition of TPO to cultures of megakaryocytic cell lines failed to significantly alter the ploidy distribution and the differentiation marker immuno

Topics: Adult; Aged; Animals; Base Sequence; Cell Division; Child, Preschool; Cricetinae; Cytokines; Female; Humans; Leukemia; Leukemia, Megakaryoblastic, Acute; Leukemia, Myeloid, Acute; Male; Mice; Middle Aged; Molecular Sequence Data; Neoplasm Proteins; Proto-Oncogene Proteins; Receptors, Cytokine; Receptors, Thrombopoietin; Thrombopoietin; Transforming Growth Factor beta; Tumor Cells, Cultured

The actions of transforming growth factor beta (TGF beta) and erythropoietin (Epo) were studied using normal erythroid progenitors from fetal rat liver and spleen at 18, 19 and 20 days. rhTGF beta 1 inhibited the growth of late BFUe colonies significantly at each age and in both organs in methylcellulose cultures containing 2 U/ml rhEpo. There was no significant inhibition of CFUe proliferation, except for spleen CFUe at 18 days, suggesting different CFUe sensitivities to growth factors at a given fetal age, 18 days, in liver and spleen. The colorimetric MTT assay was used to examine the inhibition of the growth of human leukemic UT-7 cells by TGF beta 1. TGF beta 1 inhibited the proliferation of UT-7 cells in cultures without Epo at 24 h and in cultures with Epo at 24 and 72 h. The specific binding of [125I]Epo to UT-7 surface was decreased by TGF beta 1 without any change in non-specific binding. TGF beta 1 also inhibited the expression of Epo-receptors on UT-7 cells, without changing receptor affinity. The inhibition of hematopoietic progenitor cell growth by TGF beta could involve altering the cell surface expression of growth factor receptors.

Topics: Animals; Cell Division; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Gestational Age; Humans; Leukemia, Megakaryoblastic, Acute; Liver; Neoplasm Proteins; Neoplastic Stem Cells; Rats; Rats, Wistar; Receptors, Erythropoietin; Recombinant Proteins; Spleen; Transforming Growth Factor beta

Acute megakaryoblastic leukemia (AMKL) manifesting myelofibrosis and tumor formation in the liver with marked increase of reticulin is described. The megakaryoblastic nature of the leukemic cells of the bone marrow and the hepatic tumor nodule was established by positive immunohistochemical stains for CD41a and CD41b on frozen tissue sections. Immunolocalization of the platelet-derived growth factor (PDGF) protein and transforming growth factor (TGF)-beta protein also was demonstrated in the leukemic cells of the bone marrow and the hepatic tumor. Further, the deposition of fibronectin that has been known as the ligand of CD41a molecule and collagen types I and IV were recognized in the extracellular matrix of the bone marrow and the hepatic tumor. These results suggest that specific expression of growth factor proteins by the leukemic cells may selectively regulate the fibrosis of the bone marrow as well as the tumor formation of AMKL. The expression of adhesion molecules and growth factor proteins by the leukemic cells and the deposition of extracellular matrix are discussed in relation to the myelofibrosis as well as the tumor-forming nature of AMKL.

Topics: Collagen; Fibroblast Growth Factor 2; Fibronectins; Growth Substances; Humans; Immunophenotyping; Laminin; Leukemia, Megakaryoblastic, Acute; Male; Membrane Glycoproteins; Middle Aged; Platelet-Derived Growth Factor; Transforming Growth Factor beta

Platelet protein makeup is determined during transformation of megakaryoblasts to mature megakaryocytes, the immediate precursor of circulating platelets. To better understand the molecular mechanisms of megakaryocyte formation, gene expression was characterized by Northern analysis and RNA fingerprinting of cultured human CHRF-288 megakaryoblastic cells undergoing phorbol ester-stimulated megakaryocytic differentiation or serum-stimulated megakaryoblast proliferation. Protooncogenes c-fos and c-jun were coordinately upregulated in both proliferating and differentiating cells, whereas c-myc transcripts were upregulated during proliferation only. In contrast, mRNAs for transforming growth factor-beta 1 (TGF-beta 1) and thromboxane receptors were coordinately upregulated during differentiation but differentially regulated during proliferation. RNA fingerprinting revealed multiple transcripts specific to either proliferating or differentiated cells. Three of these were identified by homology to known DNA sequence: CDw44 adhesion molecule (upregulated during differentiation), glutathione sulfhydryl peroxidase (downregulated during differentiation), and plectin cytoskeletal protein (upregulated during differentiation). Thus, although megakaryoblast proliferation and megakaryocyte differentiation both involve DNA and protein synthesis, each growth response is characterized by a distinct pattern of gene expression.

Topics: Base Sequence; Cell Differentiation; Cell Division; Cell Line; DNA Primers; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, jun; Genes, myc; Humans; Infant; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Molecular Sequence Data; Proto-Oncogenes; Receptors, Thromboxane; RNA, Messenger; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured; Up-Regulation

The current hypothesis for the pathogenesis of myelofibrosis involves the intramedullary release of growth factors from defective or abnormal megakaryocytes. We describe a case of an acute micromegakaryocytic leukaemia, in a patient with chronic myelofibrosis, that provides additional evidence for this concept. The micromegakaryocytes, which reached 223 x 10(9)/l, were characterized morphologically by both light and electron microscopy, immunocytochemically and by platelet peroxidase activity. The cells were shown to have a mature cytoplasm, containing alpha granules and the associated proteins; vWF:Ag, fibrinogen, fibronectin and protein S. DNA analysis, by both a Seescan Solitaire Plus image analysis system and flow cytometry, revealed nuclear immaturity, with 92% of cells being diploid. Serum markers of connective tissue synthesis, namely carboxy terminal peptide of procollagen I (PICP), procollagen terminal peptide III (PIIIP) and laminin all increased significantly following transformation and were associated with an increase in platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta). These observations support the current hypothesis for bone marrow fibrosis formation and provide, for the first time, a link between in vivo growth factor release, bone marrow stromal turnover and megakaryocyte mass. In addition, the release of biologically active TGF-beta may explain both the increased fibronectin and angiogenesis characteristic of myelofibrotic bone marrow.

Topics: Female; Humans; Laminin; Leukemia, Megakaryoblastic, Acute; Megakaryocytes; Microscopy, Electron; Middle Aged; Peptide Fragments; Platelet-Derived Growth Factor; Primary Myelofibrosis; Procollagen; Transforming Growth Factor beta