transforming-growth-factor-beta and Leukemia--Myeloid

The role of transforming growth factor-β (TGFβ) signaling in embryological development and tissue homeostasis has been thoroughly characterized. Its canonical downstream cascade is well known, even though its true complexity and other non-canonical pathways are still being explored. TGFβ signaling has been described as an important pathway involved in carcinogenesis and cancer progression. In the hematopoietic compartment, the TGFβ pathway is an important regulator of proliferation and differentiation of different cell types and has been implicated in the pathogenesis of a diverse variety of bone marrow disorders. Due to its importance in hematological diseases, novel inhibitors of this pathway are being developed against a number of hematopoietic disorders, including myelodysplastic syndromes (MDS). In this review, we provide an overview of the TGFβ pathway, focusing on its role in hematopoiesis and impact on myeloid disorders. We will discuss therapeutic interventions with promising results against MDS.

Topics: Animals; Cell Differentiation; Cell Transformation, Neoplastic; Clinical Studies as Topic; Disease Susceptibility; Drug Development; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Molecular Targeted Therapy; Myelodysplastic Syndromes; Signal Transduction; Transforming Growth Factor beta

TGFbeta is well known for its inhibitory effect on cell cycle G1 checkpoint kinases. However, its role in the control of pRb-E2F complexes is not well established. TGFbeta inhibits phosphorylation of pRb at several serine and threonine residues and regulates the association of E2F transcription factors with pRb family proteins. Recent studies found that predominantly E2F-4, p130, and histone deacetylase (HDAC) are found to bind to corresponding E2F-responsive promoters in G0/G1 phase. As cells progress through mid-G1, p130-E2F4 complex are replaced by p107-E2F4 followed by activators E2F1, 2, and 3. pRb was not detectable in the promoters containing the E2F-responsive site in cycling cells but was associated with E2F4-p130 complexes or E2F4-p107 complexes during G0/G1 phase. In human myeloid leukemia cell line, MV4-11, TGFbeta upregulated pRb-E2F-4 and p130-E2F-4, and downregulated p107-E2F-4 complexes. However, pRB-E2F1 and pRb-E2F3 complexes were found in proliferating cells but not in TGFbeta arrested G1 cells. In addition, electrophoretic gel mobility shift assay (EMSA) could not detect pRb-E2F DNA-binding activities either in S or G1 phase but exhibited the existence of p107-E2F4 in proliferating cells and p130-E2F4 complexes in TGFbeta-arrested G1 cells, respectively. Our data suggest that p107 and p130, but not pRb, and the repressor E2F, but not activator E2Fs, play a critical role in regulating E2F-responsive gene expression in TGFbeta-mediated cell cycle control in human myeloid leukemia cells.

Topics: Cell Cycle; Cell Line, Tumor; E2F Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid; Retinoblastoma Protein; Signal Transduction; Transforming Growth Factor beta

The EVI1 gene codes for a zinc finger transcription factor with important roles both in normal development and in leukemogenesis. Transcriptional activation of this gene through chromosome rearrangements or other, yet to be identified mechanisms leads to particularly aggressive forms of human myeloid leukemia. In vitro as well as in animal model systems, EVI1 affected cellular proliferation, differentiation, and apoptosis in cell type specific ways. Retroviral integrations into the EVI1 locus provided cells with increased abilities to engraft, survive, and proliferate in bone marrow transplantation experiments. Experimental overexpression of EVI1 by itself was insufficient to cause leukemia in animal model systems, but it cooperated with other genes in this process. This review summarizes the currently available experimental evidence for the proposed biochemical and biological functions of this important oncogene.

Topics: Animals; Cell Proliferation; Chromosomes; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid; MDS1 and EVI1 Complex Locus Protein; Mice; Myelodysplastic Syndromes; Oncogenes; Protein Structure, Tertiary; Proto-Oncogenes; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Zinc Fingers

Transforming growth factor-beta (TGF-beta) is a potent growth inhibitor of various cell types including hematopoietic cells. Two receptors, TGFbetaRI and TGFbetaRII, govern the interaction between the cell and the TGF-beta ligand. Primary binding of the ligand occurs with the RII receptor, promoting formation of a heterodimer with RI and activation of signaling. This induces transient association of Smad proteins with the receptors. Smad 3 and 4 may be involved in the TGF-beta-induced G(1) arrest. TGF-beta(1) down-regulates G(1) and G(2) cyclin-dependent kinases (cdks) and cyclins in terms of both kinase activity and protein amount. TGF- beta (1) also inhibits phosphorylation of the product of the retinoblastoma tumor suppressor gene (pRb) at multiple serine and threonine residues in human myeloid leukemia cells. The underphosphorylated pRb associates with transcription factor E2F-4 in G(1) phase, whereas the phosphorylated pRb mainly binds to E2F-1 and E2F-3. Because TGF-beta(1) up-regulates p130(pRb family member)/E2F-4 complex formation and down-regulates p107(pRb family member)/E2F-4 complex formation, with E2F-4 levels remaining constant, these results suggest that E2F-4 is switched from p107 to pRb and p130 when cells exit from the cell cycle and arrest in G(1) by the action of TGF-beta(1). The "cdk inhibitor" p27 is both a positive and a negative regulator of TGF-beta(1)-mediated cell cycle control. Although TGF-beta(1) has been reported to be a selected inhibitor of normal primitive hematopoietic stem cells, TGF-beta inhibits both primitive and more differentiated myeloid leukemia cell lines.

Topics: Cell Cycle; Hematopoiesis; Humans; Leukemia, Myeloid; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

TGFbeta1 is a potent growth inhibitor of both primitive and more differentiated human myeloid leukemic cells. The extent of the growth inhibitory response to TGFbeta varies with cell type, and is not linked to stages of differentiation of cell lines. Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGFbeta1-mediated growth inhibition of human MV4-11 myeloid leukemia cells. Both G1-phase and G2-phase cyclins and cdks participate in the regulation of TGFbeta1-mediated growth inhibition of MV4-11 cells. By both depressing cdk2 synthesis and up-regulating cyclin E-associated p27, TGFbeta1 may magnify its inhibitory efficiency. TGFbeta1 also rapidly inhibits phosphorylation of pRb at several serine and threonine residues. The underphosphorylated pRb associates with E2F-4 in G1 phase, whereas the phosphorylated pRb mainly binds to E2F-1 and E2F-3 in proliferating MV4-11 cells. Since TGFbeta1 upregulates p130/E2F-4 complex formation and downregulates p107/E2F-4 complex formation, with E2F-4 levels remaining constant, our results suggest that E2F-4 is switched from p107 to pRb and p130 when cells exit from the cell cycle and arrest in G1 by TGFbeta1. In summary, TGFbeta1 inhibits growth of human myeloid leukemic cells through multiple pathways, whereas the "cdk inhibitor" p27 is both a positive and negative regulator.

Topics: CDC2-CDC28 Kinases; Cell Cycle; Cell Cycle Proteins; Cell Division; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Gene Expression Regulation, Leukemic; Growth Inhibitors; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Macromolecular Substances; Microtubule-Associated Proteins; Neoplasm Proteins; Neoplastic Stem Cells; Nuclear Proteins; Peptide Elongation Factor 2; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proteins; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Myelodysplastic syndromes (MDS) are clonal disorders, which frequently undergo leukemic transformation. It was recently shown that the promoter of the p15INK4b but not the p16INK4a gene is frequently and selectively hypermethylated in MDS. The p15INK4b gene is a cyclin dependent kinase inhibitor gene, which is actively transcribed after TGFbeta exposure. Methylation of the p15INK4b gene is significantly correlated with blastic bone marrow involvement, and sequential analyses have shown that methylation increases with disease evolution toward AML. These data strongly suggest that p15INK4b gene methylation is a mechanism allowing leukemic cells to escape to inhibitory signals from the bone marrow environment, however the exact role of p15INK4b gene methylation in disruption of the signal mediated by TGFbeta remains to be investigated.

Topics: Acute Disease; Animals; Antimetabolites, Antineoplastic; Azacitidine; Bone Marrow; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 9; Clinical Trials, Phase II as Topic; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinases; Decitabine; Disease Progression; DNA Methylation; Genes, p16; Genes, Tumor Suppressor; Hematopoiesis; Humans; Leukemia, Myeloid; Mice; Myelodysplastic Syndromes; Neoplasm Proteins; Precancerous Conditions; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Transforming Growth Factor beta; Tumor Suppressor Proteins

Topics: Apoptosis; Cell Division; Hematopoiesis; Humans; Leukemia, Myeloid; Ornithine Decarboxylase; Proto-Oncogene Proteins c-myc; Transforming Growth Factor beta

Natural regulatory T cells (nTregs) ensure the control of self-tolerance and are currently used in clinical trials to alleviate autoimmune diseases and graft-versus-host disease after hematopoietic stem cell transfer. Based on CD39/CD26 markers, blood nTreg analysis revealed the presence of five different cell subsets, each representing a distinct stage of maturation. Ex vivo added microenvironmental factors, including IL-2, TGFβ, and PGE2, direct the conversion from naive precursor to immature memory and finally from immature to mature memory cells, the latest being a no-return stage. Phenotypic and genetic characteristics of the subsets illustrate the structural parental maturation between subsets, which further correlates with the expression of regulatory factors. Regarding nTreg functional plasticity, both maturation stage and microenvironmental cytokines condition nTreg activities, which include blockade of autoreactive immune cells by cell-cell contact, Th17 and IL-10 Tr1-like activities, or activation of TCR-stimulating dendritic cell tolerization. Importantly, blood nTreg CD39/CD26 profile remained constant over a 2-y period in healthy persons but varied from person to person. Preliminary data on patients with autoimmune diseases or acute myelogenous leukemia illustrate the potential use of the nTreg CD39/CD26 profile as a blood biomarker to monitor chronic inflammatory diseases. Finally, we confirmed that naive conventional CD4 T cells, TCR-stimulated under a tolerogenic conditioned medium, could be ex vivo reprogrammed to FOXP3 lineage Tregs, and further found that these cells were exclusively committed to suppressive function under all microenvironmental contexts.

Topics: Apyrase; Autoimmune Diseases; CD4-Positive T-Lymphocytes; Cellular Microenvironment; Cytokines; Dendritic Cells; Dinoprostone; Dipeptidyl Peptidase 4; Forkhead Transcription Factors; Humans; Interleukin-10; Interleukin-2; Leukemia, Myeloid; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

Activation of SMAD-independent p44/42 MAPK (ERK1/2) signalling by TGFbeta has been recently reported in various cell types. However, the mechanisms for the linkage between the SMAD-dependent and -independent pathways are poorly understood. In this study, we investigated whether TGF-beta activates the ERK pathway and how TGFbeta communicates with the MAP kinase signals induced by a mitogen, in human myeloid leukaemia cells.. TGFbeta dramatically suppressed proliferation of MV4-11 and TF-1 cells without detectable phosphorylation of ERK1/2 and MEK1/2 for the duration of 48 h, as detected by MTT assay and Western blot analysis, respectively. In contrast, GM-CSF induced rapid and transient phosphorylation of MEK1/2 and ERK1/2 and up-regulated cell proliferation. Both GM-CSF-induced ERK1/2 activation and cell proliferation were significantly inhibited by TGFbeta. GM-CSF also induced transient phosphorylation of the p85 subunit of PI3-kinase. Corresponding to this change, phosphorylated p85 was found to bind to the GM-CSF receptor-alpha subunit, as detected by immunoprecipitation and Western blot analysis. PD98059, a selective inhibitor of MEK, blocked GM-CSF-induced phosphorylation of MEK and ERK but not p85. However, TGFbeta and LY294002, a potent inhibitor of PI3-kinase, significantly inhibited phosphorylation of both p85 and ERK1/2.. These studies thus indicate that TGFbeta does not activate the ERK pathway but turns off the GM-CSF-induced ERK signal via inhibition of the PI3-kinase-Akt pathway, in these human leukaemia cells.

Topics: Blotting, Western; Cell Line, Tumor; Cell Proliferation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunoprecipitation; Leukemia, Myeloid; Mitogen-Activated Protein Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta

Transforming growth-interacting factor (TGIF) is a homeobox transcriptional repressor that has been implicated in holoprosencephaly and various types of cancer. TGIF is expressed in hematopoietic stem cells and modulates TGF-beta and retinoic acid (RA) signaling, both of which play an important role in hematopoiesis. We recently reported that TGIF's levels correlate inversely with survival in patients with acute myelogenous leukemia. Here we present the first direct evidence of a role for TGIF in myelopoiesis. We used short hairpin RNA interference to define the effects of TGIF knockdown on proliferation and differentiation of myeloid leukemia-derived cell lines. Decreased TGIF expression resulted in reduced proliferation and differentiation and lower expression of CEBPbeta, CEBPepsilon, PU.1 and RUNX1, key myeloid transcription factors. Furthermore, TGF-beta signaling was increased and RA signaling was decreased. Further insights into the molecular basis of TGIF's effects were provided by a genome-wide chromatin immunoprecipitation-based elucidation of TGIF target genes. Together, these data suggest that TGIF has an important role myelopoiesis and may regulate the balance between proliferation and differentiation. Reduced TGIF expression could tip the balance toward quiescence thus providing progenitor as well as hematopoietic stem cells protection from anti-cycle agents.

Topics: Animals; Apoptosis; Cell Cycle; Cell Differentiation; Cell Proliferation; Gene Knockdown Techniques; HL-60 Cells; Homeodomain Proteins; Humans; Leukemia, Myeloid; Mice; Myelopoiesis; Repressor Proteins; RNA Interference; Signal Transduction; Transcription Factors; Transforming Growth Factor beta; Tretinoin

Transforming Growth Factor-beta (TGFbeta) is known to be a negative regulator of G1 cyclin/cdk activity. It is not clear whether TGFbeta has any effect on G2 checkpoint kinases. We have found that TGFbeta downregulated the expression of several G2 checkpoint kinases including cdc2, cyclin B1, and cdc25c without causing cell accumulation in G2/M phases in two human leukemia cell lines. The inhibition was time-dependent with a maximal inhibition being observed by 24h for cyclin B1 and cdc2 and by 48h for cdc25c. The inhibition was not a result of G1 arrest but a direct effect of TGFbeta which downregulates their expression at mRNA level. In proliferating cells, there was a significant formation of cdc2-pRb complexes, which was decreased to 30% of control levels by 48h after initiating TGFbeta treatment. Cdc2 showed a marked kinase activity on GST-Rb protein in proliferating cells detected by in vitro kinase assay, which was downregulated in response to TGFbeta. In addition, TGFbeta caused a rapid and transient dephosphorylation of cdc2 (Tyr15) and cdc25c (Ser216) for about 2-3h before a dramatic decrease of both molecules by 48h. Taken together, our data suggest that TGFbeta has a direct inhibitory effect on G2 checkpoint kinases, which is regulated at mRNA level. The transient activation of cdc2 and cdc25c and subsequent inhibition of cdc2, cyclin B1, and cdc25c could amplify TGFbeta-induced G1 arrest and growth inhibition.

Topics: CDC2 Protein Kinase; cdc25 Phosphatases; Cell Cycle Proteins; Cell Line, Tumor; Cyclin B; Cyclin B1; G2 Phase; Humans; Leukemia, Myeloid; Recombinant Fusion Proteins; Retinoblastoma Protein; Transforming Growth Factor beta

The translocation t(8;21) yields the leukemic fusion gene AML1/MTG8 and is associated with 10%-15% of all de novo cases of acute myeloid leukemia. We demonstrate the efficient and specific suppression of AML1/MTG8 by small interfering RNAs (siRNAs) in the human leukemic cell lines Kasumi-1 and SKNO-1. siRNAs targeted against the fusion site of the AML1/MTG8 mRNA reduce the levels of AML1/MTG8 without affecting the amount of wild-type AML1. These data argue against a transitive RNA interference mechanism potentially induced by siRNAs in such leukemic cells. Depletion of AML1/MTG8 correlates with an increased susceptibility of both Kasumi-1 and SKNO-1 cells to tumor growth factor beta(1) (TGF beta(1))/vitamin D(3)-induced differentiation, leading to increased expression of CD11b, macrophage colony-stimulating factor (M-CSF) receptor, and C/EBP alpha (CAAT/enhancer binding protein). Moreover, siRNA-mediated AML1/MTG8 suppression results in changes in cell shape and, in combination with TGF beta(1)/vitamin D(3), severely reduces clonogenicity of Kasumi-1 cells. These results suggest an important role for AML1/MTG8 in preventing differentiation, thereby propagating leukemic blast cells. Therefore, siRNAs are promising tools for a functional analysis of AML1/MTG8 and may be used in a molecularly defined therapeutic approach for t(8;21)-positive leukemia.

Topics: Acute Disease; CCAAT-Enhancer-Binding Protein-alpha; CD11b Antigen; Cell Differentiation; Cell Size; Cholecalciferol; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 8; Core Binding Factor Alpha 2 Subunit; Drug Design; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid; Neoplasm Proteins; Oncogene Proteins, Fusion; Receptor, Macrophage Colony-Stimulating Factor; RNA Interference; RNA, Messenger; RNA, Small Interfering; RUNX1 Translocation Partner 1 Protein; Transcription Factors; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Translocation, Genetic; Tumor Cells, Cultured; Tumor Stem Cell Assay

Angiogenesis contributes to disease progression in solid and hematopoietic malignancies, and endoglin (CD105), a component of the transforming growth factor (TGF)-beta receptor complex, is a powerful marker of neovascularization. Elevated amounts of soluble CD105 (sCD105) have been recently identified in selected solid tumors but no data are available on sCD105 in hematopoietic malignancies. Therefore, levels of sCD105 were investigated in sera of patients with acute myeloid leukemia (AML) (n = 10) or chronic myeloproliferative disorders (CMD) (n = 28), and correlated with those of soluble TGF-beta(1) (sTGF-beta(1)). Dot blot assay detected higher amounts of sCD105 (P < 0.05) both in AML (4.34 +/- 2.62 OD/mm(2)) and in CMD (3.71 +/- 2.09 OD/mm(2)) patients than in healthy subjects (n = 14, 2.38 +/- 1.18 OD/mm(2)). Instead, enzyme-linked immunosorbent assay (ELISA) identified (P < 0.05) lower and higher levels of sTGF-beta(1) in AML (32,017 +/- 1,900 pg/ml) and CMD (60,700 +/- 19,200 pg/ml) patients, respectively, compared to healthy individuals (n = 11, 47,173 +/- 5,443 pg/ml). In essential thrombocythemia (ET) patients with thrombotic episodes, levels of sCD105 were lower (P < 0.05) compared to patients without thrombotic complications, and inversely correlated with those of sTGF-beta(1) (r = 0.94). Conversely, amounts of sCD105 directly correlated with levels of sTGF-beta(1) (r = 0.74) in ET patients without thrombotic events. Our results show that high levels of sCD105 are present in myeloid malignancies that are characterized by a high cellular proliferation rate, and suggest that an altered balance between sCD105 and sTGF-beta(1) might favor disease progression and clinical complications.

Topics: Acute Disease; Adolescent; Adult; Aged; Aged, 80 and over; Antigens, CD; Chronic Disease; Endoglin; Female; Humans; Leukemia, Myeloid; Male; Middle Aged; Myeloproliferative Disorders; Receptors, Cell Surface; Solubility; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Cell Adhesion Molecule-1

The hematopoietic supporting abilities are known to be impaired in marrow stromal layers developed from patients with acute myeloid leukemia (AML). In this study, fibroblast growth factor-4 (FGF-4), epidermal growth factor (EGF) or transforming growth factor-beta1 (TGF-beta1) were studied to see whether these growth factors can modify the functional development of leukemic stromal layers. Adherent stromal layers from 13 patients with AML and from six non-leukemic controls were established with 3ng/ml of FGF-4, EGF or TGF-beta1. Established stromal layers were washed three times and irradiated, followed by recharge of allogenic peripheral CD34 positive cells as an indicator of supportive function. Progenitor-outputs into supernatant were evaluated at biweekly interval with colony-forming assay until 6 weeks. The results showed that both leukemic and non-leukemic stromal cells established with FGF-4, but not with EGF, showed significantly higher progenitor cell-outputs compared with control stromal cells. By contrast, stromal cells developed with TGF-beta1 showed significantly lower progenitor cell-outputs compared with control. These differences were significant at later than 4 weeks after the recharge of indicator cells, suggesting that the stromal layer developed with EGF or TGF-beta1 preferentially affected the primitive progenitors rather than committed ones. These results indicate that FGF-4 and TGF-beta1 differentially affect the functional development of leukemic as well as of normal stromal layers.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Bone Marrow; Cell Communication; Cell Count; Cell Culture Techniques; Cell Division; Coculture Techniques; Epidermal Growth Factor; Female; Fibroblast Growth Factor 4; Fibroblast Growth Factors; Growth Substances; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Male; Middle Aged; Proto-Oncogene Proteins; Stromal Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1

Loss of transforming growth factor (TGF) beta signaling has been implicated in malignant transformation of various tissues. To investigate a potential role of Smad4 in acute myeloid leukemia (AML), the expression of Smad4 was determined in blast cells from AML patients. Western analysis of nuclear extracts of nine AML samples indicated the absence of Smad4 protein in two cases. Smad4 RT-PCR analysis of these cases indicated normal Smad4 mRNA expression, and sequencing of one of these cases revealed no mutations as compared to wild type Smad4. Next, it was investigated whether Smad4 protein from these AML cases was subject to proteolytic degradation by incubating cell extracts of these Smad4-negative AML cells with extracts from COS-7 cells in which a tagged Smad4 was overexpressed. Inhibitor studies indicated that the extracts of AML blasts lacking Smad4 possessed a serine-dependent proteolytic activity, capable of degrading Smad4. Transfection studies using an SBE containing reporter construct as well as RT-PCR analysis of endogenous TGFbeta1 responsive genes indicated that the AML blasts were still able to respond to TGFbeta1, despite the observed degradation of Smad4. It was, therefore, concluded that the degradation of Smad4 was possibly AML subtype-dependent, in vitro phenomenon, occurring during the preparation of nuclear and cellular extracts despite the addition of a protease inhibitor cocktail. The results indicate that care should be taken when interpreting data obtained from protein expression studies using AML blast cells.

Topics: Acute Disease; Blood Cells; Blotting, Western; Cell Extracts; DNA-Binding Proteins; Humans; Leukemia, Myeloid; Peptide Hydrolases; Reverse Transcriptase Polymerase Chain Reaction; Smad4 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta

We studied the effects of transforming growth factor on proliferation of cultured smooth muscle cells from human aortic intima and proliferation and differentiation of human leukemia THP-1 promonocytes. Transforming growth factor inhibited proliferation of these cells, but stimulated differentiation of THP-1 cells. Therefore, transforming growth factor probably modulates proliferation and differentiation of smooth muscle cells and monocytes/macrophages involved in the pathogenesis of atherosclerotic damages.

Topics: Aorta; Cell Differentiation; Cell Division; Cells, Cultured; Humans; Leukemia, Myeloid; Monocytes; Muscle, Smooth, Vascular; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tunica Intima

Cells from patients with MDS-derived AML display heterogeneous proliferative responses to transforming growth factor beta (TGF beta). We analyzed growth inhibition and SMAD2 phosphorylation by TGF beta in CD34+ cells from nine patients, as compared to normal controls. While TGF beta consistently inhibited thymidine incorporation of normal cells (41% of control, P < 0.05), cells from patients with AML were growth-inhibited in only four of seven cases (40%), whereas TGF beta stimulated thymidine incorporation in the three other samples (166%). Remarkably, TPO reverted the stimulatory effect of TGF beta to profound growth inhibition. Upon exposure to TGF beta, SMAD2 protein was phosphorylated in normal CD34+ cells (n = 3), CD34+ leukemic blasts from all examined patients with AML (n = 4), and in the myeloid leukemic cell lines M-07e and HEL. TGF beta inhibited TPO-mediated thymidine incorporation, cell proliferation and survival in all samples analyzed. In M-07e cells and CD34+ cells from healthy donors, this inhibition was enhanced by an antagonist of JAK2 (AG490), but not a MEK-1 antagonist (PD098059). Conversely, in CD34+ cells from a patient with AML, both AG490 and PD098059 significantly enhanced TGF beta-mediated suppression of TPO-induced thymidine incorporation. Thus, in MDS-derived AML, altered responses to TGF beta may be due to defects downstream of SMAD2 and may involve MAPK activation.

Topics: Acute Disease; Adult; Antigens, CD34; Cell Division; Disease Progression; DNA Replication; DNA-Binding Proteins; DNA, Neoplasm; Enzyme Inhibitors; Flavonoids; Hematopoietic Stem Cells; Humans; Interleukin-3; Janus Kinase 2; Leukemia, Myeloid; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase Kinases; Myelodysplastic Syndromes; Neoplasm Proteins; Neoplastic Stem Cells; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Reverse Transcriptase Polymerase Chain Reaction; Smad2 Protein; Thrombopoietin; Thymidine; Trans-Activators; Transforming Growth Factor beta; Tyrphostins

Topics: Acute Disease; Carrier Proteins; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Disease Progression; DNA Methylation; DNA-Binding Proteins; Gene Expression Regulation; Gene Expression Regulation, Leukemic; Gene Silencing; Genes, Tumor Suppressor; Humans; Leukemia, Myeloid; MDS1 and EVI1 Complex Locus Protein; Myelodysplastic Syndromes; Neoplasm Proteins; Promoter Regions, Genetic; Proto-Oncogenes; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Tumor Suppressor Proteins

In this report we have studied the mechanism by which Transforming Growth Factor beta (TGF beta) inhibits growth of human myeloid leukemia cell lines. TGF beta 1 arrested cells in G1 phase and significantly downregulated the expression of cyclin D2, cyclin D3, cdk4, cyclin A, and cdk2. The downregulation of the molecules resulted in approximately 50-90% decrease of the molecule-dependent kinase activity, varying with each molecule. Although treatment of cells with TGF beta 1 up-regulated accumulation of p27(kip1) in both nucleus and cytoplasm, the association of the p27(kip1) with cdk2, cyclin A, cyclin D2, cyclin D3, and cdk4 was markedly down-regulated, suggesting that p27(kip1) is not responsible for the downregulation of the kinase activity. In contrast, TGF beta 1 upregulated cyclin E-associated p27(kip1) with no effect on the expression of cyclin E. p27(kip1)-immunodepletion upregulated cyclin E-dependent kinase activity by more than 10-fold in TGF beta 1-treated cells but not in proliferating cells; whereas immunodepletion of p27(kip1) from cdk2-immunoprecipitates markedly downregulated cdk2 kinase activity in the lysates extracted from both proliferating and TGF beta-treated cells. Consistent with this observation, TGF beta 1 and p27(kip1) antisense cDNA had a synergistic or additive inhibitory effect on cdk2 but not cyclin E-dependent kinase activity. Our data suggest that (1) TGF beta 1-mediated growth inhibition is accomplished through multiple pathways and (2) p27(kip1) has opposing effects on cdk2 and cyclin E activity in response to TGF beta 1.

Topics: CDC2-CDC28 Kinases; Cell Cycle Proteins; Cell Division; Cyclin D; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Down-Regulation; G1 Phase; Humans; Leukemia, Myeloid; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Retinoblastoma Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tumor Suppressor Proteins

Transforming growth factor beta (TGFbeta)1 induced dephosphorylation of pRb at multiple serine and threonine residues including Ser249/Thr252, Thr373, Ser780, and Ser807/811 in MV4-11 cells. Likewise, TGFbeta1 caused the dephosphorylation of p130, while inhibiting accumulation of p107 protein. Phosphorylated pRb was detected to bind E2F-1 and E2F-3, which appears to be a major form of pRb complexes in actively cycling cells. TGFbeta1 significantly downregulated pRb-E2F-1 and pRb-E2F-3 complexes as a result of inhibition of E2F-1 and E2F-3. In contrast, complexes of E2F-4 with pRb and with p130 were increased markedly upon TGFbeta1 treatment, whereas p107 associated E2F-4 was dramatically decreased. In agreement with these results, p130-E2F-4 DNA binding activity was dominant in TGFbeta1 treated cells, whereas p107-E2F-4 DNA binding activity was only found in proliferating cells. Our data strongly suggest that inhibition of E2Fs and differential regulation of pRb family-E2F-4 complexes are linked to TGFbeta1-induced growth inhibition. E2F-4 is switched from p107 to p130 and pRb when cells are arrested in G1 phase by TGFbeta1.

Topics: Blotting, Western; Carrier Proteins; Cell Cycle; Cell Cycle Proteins; Cell Division; Cell Line; DNA; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; E2F3 Transcription Factor; E2F4 Transcription Factor; Gene Expression Regulation; Humans; Leukemia, Myeloid; Macromolecular Substances; Myeloid Cells; Nuclear Proteins; Phosphoproteins; Phosphorylation; Precipitin Tests; Protein Binding; Proteins; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; Retinoblastoma-Like Protein p107; Retinoblastoma-Like Protein p130; Serine; Threonine; Time Factors; Transcription Factor DP1; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Transforming growth factor beta (TGF-beta) has been shown to be a specific inhibitor of early human myeloid progenitors. We show here that TGF-beta1 potentially inhibited not only the growth of primitive but also more mature myeloid leukemic cells. Surprisingly, those apparently more mature progenitor cells, such as MV4-11 and Mo7e cells, are very sensitive to the action of TGF-beta. The addition of TGF-beta1 to liquid cultures of these cells significantly inhibited their proliferation, with as much as 72% inhibition of growth of MV4-11 cells. The suppressive effect by TGF-beta1 was not reversed or prevented by granulocyte-macrophage colony-stimulating factor or interleukin 3 used to promote cell growth in TF-1a and MV4-11 cells. TGF-beta1 completely abolished the clonal growth of MV4-11 cells in soft agar and inhibited Mo7e, KG-1, K562, TF-1, and TF-1a colony growth by 99%, 90%, 63%, 53%, and 43%, respectively. The cells treated with TGF-beta1 showed progressive accumulation in the G1 phase of cell cycle. Maximal G1 arrest (93%) was observed in MV4-11 cells. Using anti-retinoblastoma protein (pRb) and anti-specific phosphorylated-pRb antibodies, we demonstrated that TGF-beta1 greatly inhibited pRb phosphorylation at serine 795 in MV4-11 and Mo7e cells. Taken together, our data suggest that the sensitivity of myeloid leukemic progenitor cells to growth inhibition by TGF-beta may not be inversely correlated with their maturation stage, and the inhibition of the cells appeared to be linked to the suppression of pRb phosphorylation at serine 795.

Topics: Cell Differentiation; Cell Division; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Humans; Leukemia, Myeloid; Phosphorylation; Proto-Oncogene Proteins; Retinoblastoma Protein; S Phase; Serine; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay

Fas-Fas ligand (FasL) interactions play a significant role in the immune privilege status of certain cell populations, and several cytokines and growth factors can modulate their expression. When a FasL-expressing cell binds a Fas-bearing immune cell, it triggers its death by apoptosis. In this study, we demonstrate that normal human endometrial epithelial but not stromal cells express FasL. Moreover, we showed that macrophage-conditioned media induced FasL expression by endometrial stromal cells in a dose-dependent manner. To elucidate which macrophage product was responsible for the up-regulation of FasL, endometrial stromal cell cultures were treated with the macrophage products platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta1, and basic fibroblast growth factor (bFGF). The first two (which are known to be elevated in the peritoneal fluid of women with endometriosis) induced a dose-dependent up-regulation of FasL expression, which was specifically inhibited by the antibody. Interestingly, bFGF (which is not elevated in peritoneal fluid of women with endometriosis) did not induce any response. These results suggest that the pro-inflammatory nature of the peritoneal fluid of women with endometriosis induces the FasL expression by regurgitated endometrial cells, and signals Fas-mediated cell death of activated immune cells. This could be a mechanism for endometrial cells to escape immune surveillance, implant and grow.

Topics: Cell Differentiation; Cells, Cultured; Choriocarcinoma; Culture Media, Conditioned; Endometrium; Fas Ligand Protein; Female; Fibroblast Growth Factor 2; Gene Expression Regulation; Humans; Leukemia, Myeloid; Ligands; Macrophages; Membrane Glycoproteins; Platelet-Derived Growth Factor; Pregnancy; Stromal Cells; Transforming Growth Factor beta; Tumor Cells, Cultured; Uterine Neoplasms

To characterize the TGF-beta1 response of monocytic leukemia cells, we analyzed the effects of TGF-beta1 on cell proliferation, differentiation, and apoptosis of human monoblastic U937 cells. Treatment of cells with TGF-beta1 in the absence of growth factors significantly enhanced cell viability. Flow cytometric analysis of DNA content and CD14 expression revealed that TGF-beta1 does not affect cell proliferation and differentiation. Consistent with these results was the finding that no transcriptional induction of Cdk inhibitors such as p21Waf1, p15Ink4b, and p27Kip1 was detected following TGF-beta1 treatment. Interestingly, however, pretreatment of TGF-beta1 significantly inhibited Fas-, DNA damage-, and growth factor deprivation-induced apoptosis. This antiapoptotic effect was totally abrogated by anti-TGF-beta1 antibody. Quantitative RT-PCR analysis demonstrated a dose- and time-dependent transcriptional up-regulation of Bcl-X(L), suggesting its implication in the TGF-1-mediated antiapoptotic pathway. We also observed elevated expression of c-Fos and PTEN/MMAC1. But, no detectable change was recognized in expression of c-Jun, Fas, Fadd, Fap-1, Bcl-2, and Bax. Taken together, our study shows that TGF-beta1 enhancement of cellular viability is associated with its antiapoptotic effect, which may result from the transcriptional up-regulation of Bcl-X(L).

Topics: Apoptosis; bcl-X Protein; Cell Cycle; Cell Differentiation; Cell Division; Cell Survival; DNA; DNA Damage; fas Receptor; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Humans; Leukemia, Myeloid; Lipopolysaccharide Receptors; Neoplasm Proteins; Phosphoric Monoester Hydrolases; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase; Receptors, Antigen, T-Cell; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tumor Suppressor Proteins; U937 Cells; Up-Regulation

Signals from transforming growth factor-beta (TGF-beta), a bifunctional regulator of the proliferation of hematopoietic progenitor cells, have been recently shown to be transduced by five novel human genes related to a Drosophila gene termed MAD (mothers against the decapentaplegic gene). We showed by reverse transcriptase polymerase chain reaction that the RNA from one homologue gene, Smad5, was present in the immortalized myeloid leukemia cell lines, KG1 and HL60, in bone marrow mononuclear and polymorphonuclear cells, as well as in purified CD34+ bone marrow cells. Therefore, we studied the role of this gene in the regulation of human hematopoiesis by TGF-beta. TGF-beta1 and TGF-beta2 significantly inhibited myeloid, erythroid, megakaryocyte, and multilineage colony formation as assayed in semisolid culture systems. The levels of Smad5 mRNA in CD34+ cells were decreased by antisense but not sense oligonucleotides to Smad5. Preincubation of CD34+ marrow cells with two sense oligonucleotides to Smad5 did not reverse the inhibitory effects of TGF-beta on hematopoietic colony formation. However, preincubation with two antisense oligonucleotides to Smad5 reversed the inhibitory effects of TGF-beta. These data show that the Smad5 gene is involved in the signaling pathway by which TGF-beta inhibits primitive human hematopoietic progenitor cell proliferation and that Smad5 antisense oligonucleotides can interrupt this signal.

Topics: Cell Division; Cell Lineage; Colony-Forming Units Assay; Depression, Chemical; DNA-Binding Proteins; Hematopoiesis; Hematopoietic Stem Cells; HL-60 Cells; Humans; Leukemia, Myeloid; Oligonucleotides, Antisense; Phosphoproteins; Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad5 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor-beta1 is a pleiotropic cytokine involved in a variety of biological processes in both transformed and normal cells, including regulation of cellular proliferation and differentiation; its predominant action on hematopoietic cells is to inhibit cell growth. We used growth factor-dependent cell lines to assess TGF-beta1 effects on human myeloid leukemia cell growth. While four lines were completely or predominantly resistant, TGF-beta1 inhibited effectively, albeit to various extents, the growth of 12 other cell lines. This effect was dose dependent and specific, because a neutralizing anti-TGF-beta1 antibody prevented TGF-beta1-induced growth suppression. In the present system, basic fibroblast growth factor, known as an antagonist of TGF-beta1 counteracting its inhibitory effects, did not abrogate the suppressive effects of TGF-beta1. Other growth-stimulatory cytokines negated the TGF-beta1-induced inhibition in several cell lines, again to various extents. When proliferation was enhanced by growth-promoting cytokines (e.g. granulocyte-macrophage colony-stimulating factor, GM-CSF, stem cell factor, SCF, or PIXY-321), some previously TGF-beta1-sensitive cell lines acquired cellular resistance toward TGF-beta1-mediated growth suppression, whereas four other cell lines remained susceptible to TGF-beta1 growth inhibition despite possible counteraction by other cytokines. Thus, three growth response patterns to TGF-beta1 were seen: (1) constitutive resistance; (2) factor-dependent relative resistance; and (3) sensitivity to growth inhibition indifferent to counteracting cytokines. In the latter case, TGF-beta1 did not downregulate expression of one specific growth factor receptor. These studies indicate that human myeloid leukemia cells, represented here by leukemia cell lines as model systems, exhibit heterogeneous growth responses to TGF-beta1; its inhibitory effects can be modulated or completely alleviated by positive antagonistic cytokines. The availability of TGF-beta1-susceptible and -refractory cell lines allows for detailed investigations on the mechanisms of these regulatory pathways, the nature of TGF-beta1-resistance, and the possible contribution of acquired TGF-beta1-resistance to disease progression.

Topics: Cell Division; Cytokines; Fibroblast Growth Factor 2; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Inhibitors; Humans; Interleukin-3; Leukemia, Myeloid; Recombinant Fusion Proteins; Stem Cell Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

The t(3;21)(q26;q22) chromosomal translocation associated with blastic crisis of chronic myelogenous leukemia results in the formation of the AML1/Evi-1 chimeric protein, which is thought to play a causative role in leukemic transformation of hematopoietic cells. Here we show that AML1/Evi-1 represses growth-inhibitory signaling by transforming growth factor-beta (TGF-beta) in 32Dcl3 myeloid cells. The activity of AML1/Evi-1 to repress TGF-beta signaling depends on the two separate regions of the Evi-1 portion, one of which is the first zinc finger domain. AML1/Evi-1 interacts with Smad3, an intracellular mediator of TGF-beta signaling, through the first zinc finger domain, and represses the Smad3 activity, as Evi-1 does. We also show that suppression of endogenous Evi-1 in leukemic cells carrying inv(3) restores TGF-beta responsiveness. Taken together, AML1/Evi-1 acts as an inhibitor of TGF-beta signaling by interfering with Smad3 through the Evi-1 portion, and both AML1/Evi-1 and Evi-1 repress TGF-beta-mediated growth suppression in hematopoietic cells. Thus, AML1/Evi-1 may contribute to leukemogenesis by specifically blocking growth-inhibitory signaling of TGF-beta in the t(3;21) leukemia.

Topics: Animals; Cell Division; Chromosomes, Human, Pair 21; Chromosomes, Human, Pair 3; Core Binding Factor Alpha 2 Subunit; COS Cells; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid; MDS1 and EVI1 Complex Locus Protein; Proto-Oncogene Proteins; Proto-Oncogenes; Recombinant Fusion Proteins; Smad3 Protein; Trans-Activators; Transcription Factors; Transfection; Transforming Growth Factor beta; Translocation, Genetic

With promonocytic leukemia cell line THP-1 cells as an experimental material, the present paper described the proliferation, differentiation and maturation of these cells into m phi-like cells when they were treated with rhTGF-beta 1. Both cell number count and 3H-TdR uptake experiments indicated that rhTGF-beta 1 obviously inhibited the proliferation of THP-1 cells, and the inhibiting effect was related to its concentration. At the same time, the changes in the mode of cell growth and morphology occurred. The cells changed gradually from suspensive into adherent state and formed two groups of cell populations. The number of adherent cells formed was dependent on the concentration and duration of the treatment of rhTGF-beta 1. Therefore, based on the degree of inhibition of cell proliferation and the number of adherent cells with different rhTGF-beta 1 concentrations in a trial experiment, 1.25 ng/ml rhTGF-beta 1 was chosen as the dose in other experiments. From scanning electronmicroscopic observation, it was found that the external morphology of rhTGF-beta 1 treated THP-1 cells gradually transformed into typical macrophage-like cells. Concomitantly, their subcellular organelles also became progressively matured, with primary lysosomes typical for early M phi in 72 h and secondary lysosomes and phagosomes for mature M phi in 120 h of induction, as observed with transmission electron microscope. The ANAE activity, NBT reduction and phagocytosis of differentiated adherent cells were higher than those of control cells and suspensive cells. Specific anti-human TGF-beta-neutralizing mAb could completely block the differentiation of THP-1 cells into M phi-like cells. To sum up, from the results of the studies on cell morphology, growth mode, ultrastructures, phagocytosis, enzyme activation and TGF-beta 1 mAb blocking of induction and differentiation, it is clear that rhTGF-beta 1 can induce THP-1 cells to differentiate and mature into M phi-like cells, with the parallel development of cytoplasmic organoids, phenotype variation and the gaining of phagocytosis activity etc. Concordantly, rhTGF-beta 1 made the M phi-like cells to an activated state as they became matured during the induced differentiation.

Topics: Cell Transformation, Neoplastic; Humans; Leukemia, Myeloid; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta 1 (TGF-beta 1), a product of marrow stromal cells, inhibits the proliferation and differentiation of hematopoietic progenitor cells within the hematopoietic microenvironment. Steel factor (SF), also a product of marrow stromal cells, is an essential positive regulator of hematopoiesis in vivo. TGF-beta 1 has been shown to repress human and murine leukemic cell and murine lin- bone marrow mononuclear cell expression of the receptor for SF (c-kit). We speculated that TGF-beta 1 might exert its inhibitory effect on hematopoiesis in part by decreasing SF/c-kit interactions. Therefore, we tested the hypothesis that TGF-beta 1 inhibits both stromal cell expression of SF and hematopoietic progenitor cell expression of c-kit. We measured stromal cell expression of SF protein and hematopoietic progenitor cell expression of membrane-bound c-kit before and after exposure to recombinant human TGF-beta 1. Both stromal cell expression of SF protein and hematopoietic progenitor cell expression of c-kit protein were inhibited 50% to 80% by TGF-beta 1. Using Northern blot and ribonuclease protection assays, we determined that TGF-beta 1 repressed stromal cell SF mRNA, but did not alter SF transcript stability. TGF-beta 1 was also found to repress c-kit mRNA in human leukemic myeloblasts as well as in normal lin- hematopoietic progenitor cells. In contrast with its effect on SF mRNA, TGF-beta 1 accelerated the degradation of c-kit mRNA. We conclude that TGF-beta 1 inhibits stromal cell production of SF by repression of SF gene transcription and represses hematopoietic progenitor cell expression of c-kit by decreasing the stability of c-kit transcripts. Taking into account the importance of SF and c-kit in maintaining steady-state hematopoiesis in vivo, the dual effect of TGF-beta 1 on both SF and c-kit gene expression is likely to be one of the major mechanisms by which TGF-beta 1 inhibits hematopoiesis in vivo.

Topics: Acute Disease; Bone Marrow; Cells, Cultured; Connective Tissue; Depression, Chemical; Exons; Fibroblasts; Gene Expression Regulation; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid; Leukocytes, Mononuclear; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-kit; Receptor Protein-Tyrosine Kinases; Receptors, Colony-Stimulating Factor; Recombinant Proteins; RNA, Messenger; Stem Cell Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Inappropriate expression of genes involved in cell proliferation can result in altered regulation of apoptosis, a process of programmed cell death. Since B-myb has recently been implicated in the cell cycle progression we wanted to examine its role in the apoptotic process. For this purpose we used transforming growth factor beta 1 (TGF-beta 1)-treated M1 myeloid leukemia cell lines that continuously express murine B-myb. It was found that in cells overexpressing B-myb, TGF-beta 1-induced apoptosis was accelerated as assessed by cell viability and DNA fragmentation into nucleosomal fragments. A DNA ladder was detected after 24 h of TGF-beta 1 treatment in these cells, whereas it was not detected until after 36 h in the parental M1 cells. It was further determined by Northern blot analysis that this higher sensitivity of B-myb overexpressing clones was not due to a change in the expression of TGF-beta receptor type I or in the kinetics of the regulation of c-myc, c-myb, bcl-2, and/or bax.

Topics: Animals; Apoptosis; Blotting, Northern; Cell Cycle Proteins; Cell Line; Cell Survival; DNA-Binding Proteins; DNA, Neoplasm; Genes, myc; Leukemia, Myeloid; Mice; Nucleosomes; Oncogenes; Protein Biosynthesis; Receptors, Transforming Growth Factor beta; Retroviridae; Trans-Activators; Transcription Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

The protease gamma-glutamyl transpeptidase (gamma-GT) activity was detected at the surface of human blood granulocytes and monocytes and myeloblastic HL-60 and monoblastic U937 leukemia cell lines using an enzymatic assay (cleavage of gamma-glu-p-nitroanilide and inhibition by the specific irreversible inhibitor of gamma-GT, i.e., acivicin). Flow cytometric analysis of gamma-GT expression and detection of a 2.4-kb gamma-GT mRNA species by Northern blot analysis confirmed the presence of gamma-GT in cells of the monocytic-granulocytic lineage. Differentiation of HL-60, U937 cells, and blood monocytes along the macrophage pathway or granulocytic maturation of HL-60 cells was accompanied by an increase in gamma-GT mRNA levels without modulation of cell surface gamma-GT activity and protein. When added to leukemic cell cultures, acivicin produced a dose- and time-dependent inhibitory growth effect associated with the induction of morphological features characteristic of macrophage maturation and enhanced surface expression of phenotypic markers CD11b and CD71 characteristic of monocyte development. When cultured in the presence of acivicin, freshly isolated monocytes also underwent characteristic changes in morphology and antigenic phenotype (increase in CD71 and HLA-DR class II) consistent with their differentiation into macrophages. In parallel, a marked production of latent transforming growth factor (TGF)-beta was observed in supernatants of cells cultured with acivicin, although TGF-beta 1 mRNA species were expressed in these cells at a level almost similar to that in unstimulated cell cultures. Moreover, acivicin-treated cells still differentiated into macrophages in the presence of a neutralizing antibody to TGF-beta 1/beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Differentiation; Cell Membrane; Cells, Cultured; Cellular Senescence; gamma-Glutamyltransferase; Humans; Leukemia, Monocytic, Acute; Leukemia, Myeloid; Macrophages; Transforming Growth Factor beta; Tumor Cells, Cultured

Urokinase receptors are distributed on surfaces of many cell types where they are thought to focus plasminogen-dependent proteolysis important to migration and tissue remodeling to the immediate pericellular space. In addition to its well characterized role in proteolysis, urokinase receptor binding per se promotes the adhesiveness of leukemic cell lines exposed to differentiating cytokines in vitro. We sought to determine if a serum or matrix component is involved in urokinase-dependent adhesion. We now report that cytokine-stimulated human myelomonocytic cells express a divalent cation- and Arg-Gly-Asp-independent high affinity receptor for urea-purified vitronectin (Kd < 10 nM). Soluble native vitronectin does not effectively bind to the receptor, while cellular adhesion was noted to both urea-purified and native vitronectin when adsorbed to plastic. The activity of this receptor is tightly coupled to urokinase receptor occupancy. Urokinase receptor binding thus induces selective and reversible cellular adhesion to the matrix form of vitronectin. Because transfer of vitronectin-bound plasminogen activator inhibitor type 1 to urokinase promotes rapid turnover of receptor-bound enzyme, these results illuminate a novel binding cycle by which urokinase receptor occupancy coordinately regulates cellular adhesiveness and pericellular proteolysis.

Topics: Adenocarcinoma; Amino Acid Sequence; Blood Proteins; Cations, Divalent; Cell Adhesion; Cell Line; Culture Techniques; Cytokines; Edetic Acid; Glycoproteins; Humans; Leukemia, Myeloid; Molecular Sequence Data; Oligopeptides; Receptors, Cell Surface; Receptors, Urokinase Plasminogen Activator; Transforming Growth Factor beta; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator; Vitronectin

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of growth factor-stimulated hematopoiesis in normal and leukemic conditions. Using the factor-dependent myelogenous leukemia cell lines GF-D8 and Mo7, we show that TGF-beta interferes with stem cell factor (SCF)-induced proliferation by downmodulating c-jun gene expression. The ability of SCF to induce accumulation of c-jun transcripts was abolished when TGF-beta was present in culture. Transcriptional nuclear run-on assays indicated that TGF-beta relieved the capacity of SCF to enhance the transcriptional rate of the c-jun gene. Deletion analysis of the c-jun promoter furthermore showed that SCF was activating the c-jun promoter via the NF-jun transcription factor. Gel mobility shift assays showed that SCF increased the binding activity of NF-jun to its recognition site within 5 to 15 minutes. Binding activity peaked at 1 hour after exposure to SCF and declined to starting levels within 4 hours. The ability of SCF to enhance NF-jun binding activity was also dose-dependent in the range of 5 to 100 ng/mL. Exposure of GF-D8 and Mo7 cells to TGF-beta before the addition of SCF antagonized SCF-induced NF-jun binding. Moreover, whereas SCF was capable of functionally activating a heterologous promoter containing the NF-jun binding site, pretreatment of GF-D8 cells with TGF-beta abolished transcriptional activation of this heterologous promoter. These findings indicate that SCF-mediated activation of c-jun via NF-jun is crucial for the SCF-inducible proliferative response and is inhibited by TGF-beta. In additional experiments, the antisense technique was used. Treatment of GF-D8 and Mo7 cells with an antisense oligodeoxyribonucleotide directed against the translation initiation site of c-jun abolished the capacity of SCF to induce a proliferative response, whereas sense and nonsense oligomers had no effect. Taken together, our data indicate that the counteracting modulation of the binding activity of NF-jun by SCF and TGF-beta regulates the expression of the c-jun gene and thereby the proliferative state of the GF-D8 and Mo7 target.

Topics: Binding Sites; Cell Division; DNA; Gene Expression; Genes, jun; Hematopoietic Cell Growth Factors; Humans; Leukemia, Myeloid; Nuclear Proteins; Oligonucleotides, Antisense; Promoter Regions, Genetic; Proto-Oncogene Proteins c-jun; Recombinant Proteins; Stem Cell Factor; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

The human myeloid leukemia cell lines HL-60, U-937, and THP-1 were used to analyze the alterations of transforming growth factor beta (TGF-beta) during hematopoietic cell growth and differentiation. Differentiation of these cell lines was induced by the phorbol ester phorbol 12-myristate 13-acetate, tumor necrosis factor alpha or by retinoic acid. Northern hybridization analyses indicated that the basal levels of TGF-beta 1, latent TGF-beta binding protein, and type II TGF-beta receptor (T beta IIR) mRNAs were low in untreated cells. Major increases of these mRNAs were observed in cells treated with phorbol 12-myristate 13-acetate, with maximal induction after 12-72 h of stimulation. Retinoic acid and tumor necrosis factor alpha elevated significantly only the expression of T beta IIR mRNA. TGF-beta 1 induced its receptor mRNA in HL-60 and U937-1SF cells but not in THP-1 cells. These changes in gene expression were related to the differentiation of myeloid leukemia cells. Affinity labeling with 125I-TGF-beta 1 indicated that type I TGF-beta receptor was coregulated with T beta IIR. Types I and II receptors were coprecipitated by T beta IIR-specific antibodies. Differentiation of myeloid cells induced secretion of latent TGF-beta 1 protein, as shown by immunoblotting, but significant changes in the levels of active TGF-beta were not observed. These results indicate that the genes involved in TGF-beta signal transduction are coordinately up-regulated during myeloid differentiation.

Topics: Amino Acid Sequence; Animals; Antibodies; Base Sequence; Cell Differentiation; Cell Line; Cloning, Molecular; DNA Primers; Fibrosarcoma; Gene Expression; Humans; Kinetics; Leukemia, Myeloid; Leukemia, Promyelocytic, Acute; Lung; Mink; Molecular Sequence Data; Polymerase Chain Reaction; Receptors, Transforming Growth Factor beta; Recombinant Fusion Proteins; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

To understand the relationship between transforming growth factor beta-1 (TGF-beta 1) and the integrin profile presented by chronic myeloid leukemia cells, we have studied, using Northern analysis, the expression of TGF-beta 1 messenger RNA (TGF-beta mRNA) in myeloid cell lines and in patients with acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In addition we determined the positivity for alpha 4 and alpha 5 integrin molecules in those cells using specific monoclonal antibodies and flow cytometry. CML patients (N = 3) presented mean values of alpha 4 and alpha 5 higher (alpha 4: 60 +/- 20%; alpha 5: 70 +/- 41%) than AML (N = 10) blast cells (alpha 4: 25 +/- 23%; alpha 5: 18 +/- 16%). Northern analysis revealed an almost four-fold higher expression of TGF-beta mRNA in K562 (derived from a patient with chronic myeloid leukemia) compared to the myeloblastic cell line HL60. The highest TGF-beta mRNA levels were seen in the U937 lineage. CML leukemic cells (N = 3) showed high TGF-beta mRNA levels comparable to the levels expressed by K562 which was paralleled by high beta 1 integrin mRNA. AML blast cells presented a variable degree of expression of TGF-beta mRNA when compared to HL60. One patient with acute megakaryoblastic leukemia (FAB subtype M7), usually associated with myelofibrosis, presented the highest TGF-beta mRNA levels. We conclude that studying TGF-beta 1 and its mechanisms of action will help in understanding fibrosis in leukemic patients, and perhaps to design treatments for such conditions.

Topics: Humans; Integrins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid; Transforming Growth Factor beta; Tumor Cells, Cultured

For growth, ML-1 human myeloblastic leukemia cells require insulin-like growth factor 1 together with transferrin, whereas for differentiation they depend upon transforming growth factor beta in combination with transferrin. As shown in this study, growth stimulation is accompanied by c-myb expression, whereas initiation of differentiation results in the cessation of c-myb expression through premature termination of transcription in the first intron of the myb gene. Growth factor-stimulated c-myb elongation was found to correlate with an elevated level of nuclear c-ets-1 protein and with increased binding of this protein to an 18-base pair sequence in intron 1 of the c-myb gene containing the putative regulatory element PEA 3. In contrast, differentiation factor-initiated ML-1 cell maturation was accompanied by a very low level of nuclear c-ets-1 protein, by the inability to detect binding of the protein to the 18-base pair sequence, and by the cessation of c-myb expression. These results show a correlation to exist between c-ets-1 binding to intron 1 of the c-myb gene and c-myb expression. The mechanism underlying this correlation is under further study.

Topics: Base Sequence; Cell Differentiation; Cell Division; DNA; Humans; Insulin-Like Growth Factor I; Introns; Leukemia, Myeloid; Molecular Sequence Data; Nuclear Proteins; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Proto-Oncogene Proteins c-myb; RNA, Messenger; Transcription Factors; Transcriptional Activation; Transferrin; Transforming Growth Factor beta; Tumor Cells, Cultured

Cell numbers are regulated by a balance among proliferation, growth arrest, and programmed cell death. A profound example of cell homeostasis, controlled throughout life, is the complex process of blood cell development, yet little is understood about the intracellular mechanisms that regulate blood cell growth arrest and programmed cell death. In this work, using transforming growth factor beta 1 (TGF beta 1)-treated M1 myeloid leukemia cells and genetically engineered M1 cell variants, the regulation of growth arrest and apoptosis was dissected. Blocking of early expression of MyD118, a novel differentiation primary response gene also shown to be a primary response gene induced by TGF beta 1, delayed TGF beta 1-induced apoptosis, demonstrating that MyD118 is a positive modulator of TGF beta 1-mediated cell death. Elevated expression of bcl-2 blocked the TGF beta 1-induced apoptotic pathway but not growth arrest induced by TGF beta 1. Deregulated expression of either c-myc or c-myb inhibited growth arrest and accelerated apoptosis, demonstrating for the first time that c-myb plays a role in regulating apoptosis. In all cases, the apoptotic response was correlated with the level of MyD118 expression. Taken together, these findings demonstrate that the primary response gene MyD118 and the c-myc, c-myb, and bcl-2 proto-oncogenes interact to modulate growth arrest and apoptosis of myeloid cells.

Topics: Animals; Antigens, Differentiation; Apoptosis; Cell Division; Cell Line; Clone Cells; Gene Expression Regulation, Neoplastic; Genes, myc; Genetic Variation; Kinetics; Leukemia, Experimental; Leukemia, Myeloid; Mice; Polymerase Chain Reaction; Protein-Tyrosine Kinases; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myb; Proto-Oncogenes; Transforming Growth Factor beta; Tumor Cells, Cultured

M1 clone S6 myeloid leukemic cells do not express detectable p53 protein. When stably transfected with a temperature-sensitive mutant of p53, these cells undergo rapid cell death upon induction of wild-type (wt) p53 activity at the permissive temperature. This process has features of apoptosis. In a number of other cell systems, wt p53 activation has been shown to induce a growth arrest. Yet, wt 53 fails to induce a measurable growth arrest in M1 cells, and cell cycle progression proceeds while viability is being lost. There exists, however, a relationship between the cell cycle and p53-mediated death, and cells in G1 appear to be preferentially susceptible to the death-inducing activity of wt p53. In addition, p53-mediated M1 cell death can be inhibited by interleukin-6. The effect of the cytokine is specific to p53-mediated death, since apoptosis elicited by serum deprivation is refractory to interleukin-6. Our data imply that p53-mediated cell death is not dependent on the induction of a growth arrest but rather may result from mutually incompatible growth-regulatory signals.

Topics: Animals; Apoptosis; Cell Cycle; Cell Division; Cell Survival; Culture Media, Serum-Free; G1 Phase; Genes, myc; Interleukin-6; Leukemia, Myeloid; Mice; RNA, Messenger; Time Factors; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Transforming growth factor-beta 1 (TGF-beta 1) induces cell death in myeloid leukemia by apoptosis. In the M1 myeloid leukemia, this induction of apoptosis was inhibited by granulocyte colony-stimulating factor (G-CSF) or interleukin-6 (IL-6) and to a lesser extent by IL-1 alpha. IL-3 and stem cell factor/mast cell growth factor (SCF) showed only a marginal effect, and granulocyte-macrophage and macrophage CSFs (GM-CSF and M-CSF, respectively) were inactive. The induction of apoptosis by TGF-beta 1 in a different myeloid leukemia (7-M12) was inhibited by GM-CSF and IL-3 but not by the other cytokines. In the absence of TGF-beta 1, both M1 and 7-M12 leukemic cells were independent of hematopoietic cytokines for cell viability and growth. The cytotoxic compounds vincristine, vinblastine, adriamycin, cytosine arabinoside, cycloheximide, and sodium azide, some of which are used in cancer chemotherapy, induced cell death by apoptosis in both leukemias. As with TGF-beta 1, apoptosis induced by these cytotoxic compounds was inhibited by GM-CSF (7-M12 leukemia) and by G-CSF or IL-6 (M1 leukemia). Cyclosporine A decreased cell multiplication in M1 cells without inducing apoptosis, and G-CSF and IL-6 inhibited the cytostatic effect of cyclosporine A. It is suggested that the clinical use of cytokines to correct therapy-associated myelosuppression should be carefully timed to avoid protection of malignant cells from the cytotoxic action of the therapeutic compounds.

Topics: Animals; Antineoplastic Agents; Apoptosis; Azides; Cell Survival; Cycloheximide; Cytarabine; Cytokines; DNA Damage; Dose-Response Relationship, Drug; Doxorubicin; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Cell Growth Factors; Interferon Type I; Interleukin-1; Interleukin-3; Interleukin-6; Leukemia, Experimental; Leukemia, Myeloid; Mice; Recombinant Proteins; Sodium Azide; Stem Cell Factor; Transforming Growth Factor beta; Tumor Cells, Cultured; Vincristine

Transforming growth factor-Beta (TGF-beta) is a potent growth inhibitor for several cell types including epithelial cells and hematopoietic progenitor cells. Using a human promonocytic leukemia cell line, THP-1, we have shown that TGF-beta inhibits their proliferation and promotes differentiation into cells exhibiting macrophage-like properties. Therefore, a key question is whether TGF-beta influences the expression of genes associated with proliferation and/or growth inhibition. TGF-beta treatment of THP-1 cells results in downregulation of expression of c-myc. We also observe that TGF-beta 1-treated cells express reduced levels of the cell cycle regulated histone, H2B, but express elevated levels of an RNA splicing variant of this histone that has been observed to be upregulated in growth inhibited and terminally differentiated cells. In addition, a nuclear protein associated with senescence and withdrawal of cells from the cell cycle, statin, is also expressed by THP-1 cells in response to TGF-beta 1 treatment. These results suggest that TGF-beta 1 is capable of inducing expression of specific nuclear proteins associated with differentiation and/or cessation of proliferation that may result in changes in nuclear organization and altered gene expression. Such changes in nuclear organization may be incompatible with continued proliferation of the cells.

Topics: Cell Adhesion; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Gene Expression Regulation, Neoplastic; Genes, myc; Growth Inhibitors; Histones; Humans; Leukemia, Myeloid; Peptide Elongation Factor 1; Proteins; RNA Splicing; Transforming Growth Factor beta; Tumor Cells, Cultured

Proliferation of the human monocytic leukemia cell line JOSK-I is inhibited by transforming growth factor-beta (TGF-beta). Growth inhibition by TGF-beta was not due to either a toxic effect or to induction of differentiation. TGF-beta induced a cell cycle arrest at late G1 phase and was not found to be inhibitory to JOSK-I cells in S phase or G2/M. This G1 cell cycle arrest was associated with an accumulation of the unphosphorylated form of the retinoblastoma susceptibility gene product (Rb) in good correlation with inhibition of DNA synthesis. In contrast to the effects of TGF-beta, two other agents which induced a G1 arrest of JOSK-I cells had a different effect on Rb. Aphidicolin blocked cells at G1/S but could not reduce Rb phosphorylation as great as that seen with TGF-beta. 12-O-Tetradecanoylphorbol-13-acetate, an inducer of differentiation, did reduce Rb phosphorylation, but not until 72 h, when differentiation had already occurred. The identities of the Rb kinases are unknown, but recent evidence suggests that the cdc2 gene product could participate in Rb phosphorylation. Although cdc2 mRNA and total protein levels were not affected, TGF-beta inhibited the rate of translation and kinase activity of cdc2 in JOSK-I cells. These results suggest that growth inhibition of hematopoietic cells by TGF-beta is linked to suppression of Rb phosphorylation to retain Rb in an unphosphorylated, growth-inhibitory state. The suppression of Rb phosphorylation is suggested to be mediated through inhibition of cdc2 kinase activity by TGF-beta.

Topics: Aphidicolin; Blotting, Northern; Cell Cycle; Cell Division; Cell Line; DNA Replication; Genes, Retinoblastoma; HLA-DR Antigens; Humans; Kinetics; Leukemia, Myeloid; Phosphorylation; Receptors, Transferrin; Retinoblastoma Protein; RNA, Neoplasm; Tetradecanoylphorbol Acetate; Thymidine Kinase; Transforming Growth Factor beta

The viability of normal bone marrow myeloid precursor cells induced by interleukin-6 (IL-6) or IL-1 alpha and the ability of IL-6 and IL-1 alpha to induce the formation of colonies of granulocytes, macrophages, or megakaryocytes in densely seeded bone marrow cultures was suppressed by transforming growth factor-beta 1 (TGF-beta 1). Induction of normal bone marrow colony formation by IL-3 was much less sensitive to TGF-beta 1, and there was little or no effect of TGF-beta 1 on colony formation induced by macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage CSF (GM-CSF). In different clones of myeloid leukemic cells, TGF-beta 1 suppressed differentiation induced with IL-6, IL-1 alpha, or lipopolysaccharide (LPS), but did not suppress differentiation induced with IL-3 or GM-CSF. The effect of TGF-beta 1 on differentiation of the leukemic cells can be dissociated from its effect on cell growth. TGF-beta 1 suppressed the production of IL-6 in normal bone marrow cells cultured with IL-1 alpha and the production of IL-6 and GM-CSF in leukemic cells cultured with IL-1 alpha or LPS. The suppression of IL-6 production can explain the suppression by TGF-beta 1 of the effects of IL-1 alpha and LPS that are mediated by IL-6. TGF-beta 1 also suppressed differentiation in clones of myeloid leukemic cells induced with differentiation factor/leukemia inhibitory factor and tumor necrosis factor. In different leukemic clones TGF-beta 1 suppressed or enhanced induction of differentiation with dexamethasone. The results show that TGF-beta 1 can selectively control the activity of different molecular regulators of normal and leukemic hematopoiesis.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Cell Division; Cell Line; Cells, Cultured; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Interleukin-1; Interleukin-6; Leukemia, Myeloid; Lipopolysaccharides; Mice; Transcription Factors; Transforming Growth Factor beta