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

The next-generation immunotherapy can only be effective if researchers have an in-depth understanding of the function and regulation of Treg cells in antitumor immunity combined with the discovery of new immunity targets. This can enhance clinical efficacy of future and novel therapies and reduces any adverse reactions arising from the latter. This review discusses tumor treatment strategies using regulatory T (Treg) cell therapy in a Tumor Microenvironment (TME). It also discusses factors affecting TME instability as well as relevant treatments to prevent future immune disorders. It is prognosticated that PD-1 inhibitors are risky and their adverse effects should be taken into account when they are administered to treat Acute Myeloid Leukemia (AML), lung adenocarcinoma, and prostate adenocarcinoma. In contrast, Treg molecular markers FoxP3 and CD25 analyzed here have stronger expression in almost all kinds of cancers compared with normal people. However, CD25 inhibitors are more effective compared to FoxP3 inhibitors, especially in combination with TGF-β blockade, in predicting patient survival. According to the data obtained from the Cancer Genome Atlas, we then concentrate on AML immunotherapy and discuss different therapeutic strategies including anti-CD25/IL-2, anti-CTLA-4, anti-IDO, antityrosine kinase receptor, and anti-PI3K therapies and highlight the recent advances and clinical achievements in AML immunotherapy. In order to prognosticate the risk and adverse effects of key target inhibitors (namely against CTLA-4, FoxP3, CD25, and PD-1), we finally analyzed and compared the Cancer Genome Atlas derived from ten common cancers. This review shows that Treg cells are strongly increased in AML and the comparative review of key markers shows that Tregbased immunotherapy is not effective for all kinds of cancer. Therefore, blocking CD25(+)FoxP3(+) Treg cells is suggested in AML more than other kinds of cancer; meanwhile, Treg markers studied in other cancers have also great lessons for AML immunotherapy.

Topics: Forkhead Transcription Factors; Humans; Immunotherapy; Leukemia, Myeloid, Acute; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment

Regulatory T-cells (Tregs) are a very important subtype of lymphocytes when it comes to self-control in the human immunological system. Tregs are decisive not only in the protection against destruction of own tissues by autoimmune immunocompetent cells but also in the immunological answer to developing cancers. On the other hand, Tregs could be responsible for the progression of acute and chronic leukemias. In our study, we review publications available in the PUMED database concerning acute leukemia, with a particular emphasis on child's leukemias. The percentage of regulatory T-lymphocytes in peripheral blood and bone marrow was elevated compared to those in healthy individuals and correlated with progressive disease. Regulatory T-cells taken from children diagnosed with leukemia showed a higher suppressive capability, which was confirmed by detecting elevated levels of secreted IL-10 and TGF-beta. The possibility of pharmacological intervention in the self-control of the immunological system is now under extensive investigation in many human cancers. Presumably, Treg cells could be a vital part of targeted therapies. Routine Treg determination could be used to assess the severity of disease and prognosis in children with acute lymphoblastic leukemia. This proposition results from the fact that in some studies, higher percentage of Treg cells in peripheral blood was demonstrated. However, observations confirming these facts are scarce; thus, extrapolating them to the population of children with hematological malignancies needs to be verified in additional studies.

Topics: Disease Progression; Flow Cytometry; Forkhead Transcription Factors; Humans; Immunophenotyping; Interleukin-10; Leukemia, Myeloid, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Acute myeloid leukemia (AML) is the most common hematological malignancy in adults, characterized by distorted proliferation and development of myeloid cells and their precursors in blood and bone marrow. Impressive biologic advances have increased our understanding of leukemogenesis, however, little is known about the pathogenic events which lead to the initiation and progression of AML. T helper type 17 (Th17) cells are a unique subset of CD4+ T cells. They play important roles in the pathogenesis of many diseases, including inflammatory diseases, autoimmune diseases, and cancers. A range of cytokines, such as interleukin (IL)-23, transforming growth factor-beta (TGF-beta), IL-1beta, IL-6, IL-17, IL-22, and IL-21, have been shown related to Th17 cells. Some researchers have reported that the levels of Th17 and its related cytokines were different between normal cells and malignant AML cells, suggesting that Th17 might be involved in AML pathogenesis. In this review, we summarize current progress in the mechanisms of Th17 related cytokines in AML pathogenesis.

Topics: Animals; Cell Differentiation; Cytokines; Humans; Interleukin-17; Interleukin-1beta; Interleukin-23; Interleukin-6; Interleukins; Leukemia, Myeloid, Acute; Mice; Th17 Cells; Transforming Growth Factor beta

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; DNA-Binding Proteins; Humans; Leukemia, Myeloid, Acute; MicroRNAs; Prognosis; THP-1 Cells; Transforming Growth Factor beta

To investigate the expression and significance of regulatory T cells (Tregs), FoxP3 and transforming growth factor-β (TGF-β) in different phase of chronic myeloid leukemia (CML).. Peripheral blood of 73 CML patients in Department of Hematology, Heze Municipal Hospital from March 2018 to March 2021 were collected. According to patient's period in CML, they were divided into ND CML group (newly diagnosed), CP CML group (chronic period), and BP CML group (blast phase). The percentage of Tregs, expression level of. The abnormal distribution of Treg subset in different phases of CML and the significant increase of the expression level of. Treg、. 分析调节性T细胞（Treg）、叉头状转录因子P3（FoxP3 ）和转化生长因子-β（TGF-β）在慢性髓系白血病（CML）不同时期中的表达及意义。.. 收集2018年3月-2021年3月在菏泽市立医院血液内科就诊的73例CML患者的外周血，根据患者所处的CML不同时期，将患者分为初诊组、慢性期组和急变期组，应用流式细胞术检测患者Treg水平，RT-qPCR检测. Treg细胞亚群在CML不同时期中的异常分布和. 去甲氧柔红霉素联合大剂量阿糖胞苷巩固治疗首次缓解的老年急性髓系白血病的疗效分析.. 评估去甲氧柔红霉素联合大剂量阿糖胞苷作为老年急性髓系白血病（AML）患者缓解后治疗方案的疗效及安全性。.. 对2017年11月至2021年6月纳入的24例年龄≥60岁的初次诱导缓解的AML患者进行去甲氧柔红霉素联合大剂量阿糖胞苷的巩固化疗（去甲氧柔红霉素10 mg/m

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Blast Crisis; Cytarabine; Female; Forkhead Transcription Factors; Humans; Idarubicin; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Male; Middle Aged; Remission Induction; Retrospective Studies; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The present study is designed to investigate the expressions of microRNA-143-3p (miR-143-3p) and Lysine acetyltransferase 6A (KAT6A) in acute myeloid leukemia (AML) samples and AML cell lines and to explore the possible effects and underlying mechanisms of miR-143-3p on the proliferation of AML cells. The expressions of miR-143-3p and KAT6A in AML samples and cell lines were detected by RT-qPCR assay. CCK-8 and flow cytometry were performed to evaluate the role of KAT6A in viability of AML cells. EdU assay was performed to determine the effects of KAT6A on proliferation of AML cells. Western blot analysis was utilized to assess the impacts of KAT6A on proliferation-related protein expressions of AML cells. ELISA assay was adopted to illustrate the influence of KAT6A on inflammatory responses of AML cells. In addition, the relationship between KAT6A and miR-143-3p was predicted by ENCORI and miRWalk, and confirmed by dual-luciferase reporter assay. Moreover, the effects of KAT6A on the proliferation of AML cells mediated with miR-143-3p were carried out by rescue experiment. The expression of KAT6A was significantly upregulated, while miR-134-4p was downregulated both in the AML tissues and in AML cell lines. In addition, the silence of KAT6A significantly inhibited the viability of AML cells. Besides, KAT6A silencing notably suppressed the proliferation of AML cells and reduced the protein expressions of Ki-67 and PCNA. Knockdown of KAT6A notably decreased the expression levels of IL-1β, TNF-α and IL-6, and increased the expression levels of TGF-β and IL-10. Moreover, overexpression of miR-143-3p repressed viability and proliferation of AML cells and overexpression of KAT6A partially reversed the inhibitory effects of miR-143-3p mimic on viability and proliferation of AML cells. miR-143-3p/KAT6A played an essential role in the viability and proliferation of AML cells.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gene Knockdown Techniques; Histone Acetyltransferases; Humans; Interleukins; Leukemia, Myeloid, Acute; MicroRNAs; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Up-Regulation

Topics: Animals; Hepatic Stellate Cells; Hepatocytes; Leukemia, Myeloid, Acute; Limonins; Liver; Liver Cirrhosis; Mice; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1

The modeling of chimeric antigen receptor (CAR) T cell therapies has been mostly focused on immunodeficient models. However, there are many advantages in studying CAR-T cell biology in an immunocompetent setting. We generated a fully murine CAR targeting CD105 (endoglin), a component of the TGFβ receptor expressed on the surface of certain solid tumors and acute leukemias. CD105-targeted CAR-T cells can be grown from various murine backgrounds, tracked

Topics: Animals; Endoglin; Humans; Leukemia, Myeloid, Acute; Mice; Receptors, Chimeric Antigen; T-Lymphocytes; Transforming Growth Factor beta

Heterogeneity of leukemia stem cells (LSCs) is involved in their collective chemoresistance. To eradicate LSCs, it is necessary to understand the mechanisms underlying their heterogeneity. Here, we aimed to identify signals responsible for heterogeneity and variation of LSCs in human acute myeloid leukemia (AML). Monitoring expression levels of endothelial cell-selective adhesion molecule (ESAM), a hematopoietic stem cell-related marker, was useful to detect the plasticity of AML cells. While healthy human hematopoietic stem/progenitor cells robustly expressed ESAM, AML cells exhibited heterogeneous ESAM expression. Interestingly, ESAM

Topics: Cell Line, Tumor; Cell Proliferation; Hematopoietic Stem Cells; Humans; Leukemia, Myeloid, Acute; Neoplastic Stem Cells; Signal Transduction; Transforming Growth Factor beta

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy, whose immunological mechanisms are still partially uncovered. Regulatory B cells (Bregs) and CD4+ regulatory T cells (Tregs) are subgroups of immunoregulatory cells involved in modulating autoimmunity, inflammation, and transplantation reactions. Herein, by studying the number and function of Breg and Treg cell subsets in patients with AML, we explored their potential role in the pathogenesis of AML. Newly diagnosed AML patients, AML patients in complete remission, and healthy controls were enrolled. Flow cytometry was used to detect percentages of Bregs and Tregs. ELISA was conducted to detect IL-10 and TGF-

Topics: Adolescent; Adult; Aged; Aged, 80 and over; B-Lymphocytes, Regulatory; Case-Control Studies; Female; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Humans; Interleukin-10; Leukemia, Myeloid, Acute; Lymphocyte Count; Male; Middle Aged; RNA, Messenger; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Up-Regulation; Young Adult

Patients who receive allogeneic hematopoietic stem cell transplantation may experience oral complications due to chronic graft-versus-host disease (cGVHD). The manifestations may include progressive sclerosis-like changes that may involve various body sites, including the oropharynx.. We present two cGVHD cases of oropharyngeal fibrotic changes that affected functions that were treated with photobiomodulation (PBM) therapy. These case reports suggest that PBM therapy represents an additional, innovative approach affecting discrete phases in cGVHD-associated fibrotic changes.. We discuss these observations in the context of currently understood molecular mechanisms, especially induction of transforming growth factor beta and NFκB that appear to be counter-intuitive to their known roles in matrix synthesis and inflammation that contribute to tissue fibroses. The clinical benefit noted in the two cases presented clearly indicates that there are distinct mechanistic and biological insights in the regulation of these molecular pathways in determining therapeutic efficacy with PBM therapy.

Topics: Adult; Child; Chronic Disease; Female; Fibrosis; Follow-Up Studies; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Leukemia, Myeloid, Acute; Low-Level Light Therapy; Male; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Radiotherapy Dosage; Sampling Studies; Time Factors; Transforming Growth Factor beta; Treatment Outcome

Mutations in ASXL1 and SETBP1 genes have been frequently detected and often coexist in myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We previously showed that coexpression of mutant ASXL1 and SETBP1 in hematopoietic progenitor cells induced downregulation of TGFβ pathway genes and promoted the development of MDS/AML in a mouse model of bone marrow transplantation. However, whether the repression of TGFβ pathway in fact contributes to leukaemogenesis remains unclear. Moreover, mechanisms for the repression of TGFβ pathway genes in ASXL1/SETBP1-mutated MDS/AML cells have not been fully understood. In this study, we showed that expression of a constitutively active TGFβ type I receptor (ALK5-TD) inhibited leukaemic proliferation of MDS/AML cells expressing mutant ASXL1/SETBP1. We also found aberrantly reduced acetylation of several lysine residues on histone H3 and H4 around the promoter regions of multiple TGFβ pathway genes. The histone deacetylase (HDAC) inhibitor vorinostat reversed histone acetylation at these promoter regions, and induced transcriptional derepression of the TGFβ pathway genes. Furthermore, vorinostat showed robust growth-inhibitory effect in cells expressing mutant ASXL1, whereas it showed only a marginal effect in normal bone marrow cells. These data indicate that HDAC inhibitors will be promising therapeutic drugs for MDS and AML with ASXL1 and SETBP1 mutations.

Topics: Acetylation; Animals; Carrier Proteins; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Histones; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C57BL; Mutation; Nuclear Proteins; Repressor Proteins; Signal Transduction; Smad Proteins; Survival Rate; Transcriptional Activation; Transforming Growth Factor beta; Vorinostat

Homoharringtonine (HHT) has long and widely been used in China for the treatment of acute myeloid leukemia (AML), the clinical therapeutic effect is significant but the working mechanism is poorly understood. The purpose of this study is to screen the possible target for HHT with virtual screening and verify the findings by cell experiments. Software including Autodock, Python, and MGL tools were used, with HHT being the ligand and proteins from PI3K-Akt pathway, Jak-stat pathway, TGF-β pathway and NK-κB pathway as the receptors. Human AML cell lines including U937, KG-1, THP-1 were cultured and used as the experiment cell lines. MTT assay was used for proliferation detection, flowcytometry was used to detect apoptosis and cell cycle arrest upon HHT functioning, western blotting was used to detect the protein level changes, viral shRNA transfection was used to suppress the expression level of the target protein candidate, and viral mRNA transfection was used for over-expression. Virtual screening revealed that smad3 from TGF-β pathway might be the candidate for HHT binding. In AML cell line U937 and KG-1, HHT can induce the Ser423/425 phosphorylation of smad3, and this phosphorylation can subsequently activate the TGF-β pathway, causing cell cycle arrest at G1 phase in U937 cells and apoptosis in KG-1 cells, knockdown of smad3 can impair the sensitivity of U937 cell to HHT, and over-expression of smad3 can re-establish the sensitivity in both cell lines. We conclude that smad3 is the probable target protein of HHT and plays an important role in the functioning mechanism of HHT.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; G1 Phase Cell Cycle Checkpoints; Harringtonines; Homoharringtonine; Humans; Leukemia, Myeloid, Acute; Phosphorylation; RNA Interference; RNA, Small Interfering; Smad3 Protein; THP-1 Cells; Transforming Growth Factor beta; U937 Cells

Mutations in ASXL1 are frequent in patients with myelodysplastic syndrome (MDS) and are associated with adverse survival, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) is not fully understood. Recently, it has been found that deletion of Asxl1 or expression of C-terminal-truncating ASXL1-MTs inhibit myeloid differentiation and induce MDS-like disease in mice. Here, we find that SET-binding protein 1 (SETBP1) mutations (SETBP1-MT) are enriched among ASXL1-mutated MDS patients and associated with increased incidence of leukemic transformation, as well as shorter survival, suggesting that SETBP1-MT play a critical role in leukemic transformation of MDS. We identify that SETBP1-MT inhibit ubiquitination and subsequent degradation of SETBP1, resulting in increased expression. Expression of SETBP1-MT, in turn, inhibited protein phosphatase 2A activity, leading to Akt activation and enhanced expression of posterior Hoxa genes in ASXL1-mutant cells. Biologically, SETBP1-MT augmented ASXL1-MT-induced differentiation block, inhibited apoptosis and enhanced myeloid colony output. SETBP1-MT collaborated with ASXL1-MT in inducing acute myeloid leukemia in vivo. The combination of ASXL1-MT and SETBP1-MT activated a stem cell signature and repressed the tumor growth factor-β signaling pathway, in contrast to the ASXL1-MT-induced MDS model. These data reveal that SETBP1-MT are critical drivers of ASXL1-mutated MDS and identify several deregulated pathways as potential therapeutic targets in high-risk MDS.

Topics: Adult; Animals; Apoptosis; Carrier Proteins; Cell Differentiation; Cell Transformation, Neoplastic; Gene Expression Regulation, Leukemic; HEK293 Cells; HL-60 Cells; Homeodomain Proteins; Humans; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C57BL; Mutation; Myelodysplastic Syndromes; Nuclear Proteins; Protein Phosphatase 2; Proteolysis; Proto-Oncogene Proteins c-akt; Repressor Proteins; Signal Transduction; Survival Analysis; Transforming Growth Factor beta; Ubiquitination

Polyglutamylation of antifolates catalyzed by folylpoly-γ-glutamate synthetase (FPGS) is essential for their intracellular retention and cytotoxic activity. Hence, loss of FPGS expression and/or function results in lack of antifolate polyglutamylation and drug resistance. Members of the TGF-β/Smad signaling pathway are negative regulators of hematopoiesis and deregulation of this pathway is considered a major contributor to leukemogenesis. Here we show that FPGS gene expression is inversely correlated with the binding of a Smad4/Ets-1 complex to exon12 of FPGS in both acute lymphoblastic leukemia cells and acute myeloid leukemia blast specimens. We demonstrate that antifolate resistant leukemia cells harbor a heterozygous point mutation in exon12 of FPGS which disrupts FPGS activity by abolishing ATP binding, and alters the binding pattern of transcription factors to the genomic region of exon12. This in turn results in the near complete silencing of the wild type allele leading to a 97% loss of FPGS activity. We show that exon12 is a novel intragenic transcriptional regulator, endowed with the ability to drive transcription in vitro, and is occupied by transcription factors and chromatin remodeling agents (e.g. Smad4/Ets-1, HP-1 and Brg1) in vivo. These findings bear important implications for the rational overcoming of antifolate resistance in leukemia.

Topics: Adult; Aged; Base Sequence; Binding Sites; Cell Line, Tumor; Chromobox Protein Homolog 5; Chromosomal Proteins, Non-Histone; DNA Helicases; DNA Methylation; Drug Resistance, Neoplasm; Female; Folic Acid Antagonists; Gene Silencing; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Nuclear Proteins; Peptide Synthases; Polymorphism, Single Nucleotide; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Protein Binding; Proto-Oncogene Protein c-ets-1; Sequence Analysis, DNA; Smad4 Protein; Transcription Factors; Transcription, Genetic; Transforming Growth Factor beta; Young Adult

Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFβ1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFβ1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G0 state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFβ-neutralizing antibody 1D11 abrogated rhTGFβ1 induced cell cycle arrest. Blocking TGFβ with 1D11 further enhanced cytarabine (Ara-C)-induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFβ by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.

Topics: Animals; Antibodies, Neutralizing; Antineoplastic Agents; Apoptosis; Benzylamines; Bone Marrow; Cell Line, Tumor; Cell Movement; Cell Survival; Chemokine CXCL12; Coculture Techniques; Cyclams; Cytarabine; Female; Heterocyclic Compounds; Humans; Leukemia, Myeloid, Acute; Mice, SCID; Neoplasm Transplantation; Receptors, CXCR4; Transforming Growth Factor beta; Tumor Microenvironment

Immunosuppression in acute myeloid leukemia (AML) is an important mechanism of tumor escape. CD200, as an immunosuppressive molecule, is overexpressed in some hematological malignancies and it has also been shown to be an independent prognostic factor in AML. In the current study, simultaneous CD200 expression and Foxp3(+) regulatory T cell levels were investigated in Iranian patients with AML by flow cytometry. We also assessed the effect of CD200-CD200R blockade on Th1 and T-reg cytokine production and T cell proliferation in autologous AML- and monocyte-DC mixed lymphocyte reactions (MLRs). ELISA assay was performed to detect IL-2, IL-12, IFN-γ, IL-10, and TGF-β production in MLR supernatants. Expression of Foxp3, IL-10, and TGF-β mRNAs in MLRs were detected by real-time PCR. Our results demonstrated significant overexpression of CD200 (P = 0.001) in association with higher frequencies of Foxp3(+) T cells in AML patients (r = 0.8, P < 0.001). Blocking of CD200-CD200R interaction demonstrated a significant decrease in TGF-β and IL-10 expression in AML-DC MLRs and a significant increase in IL-12 and IFN-γ expression in monocyte-DC MLRs. Elevated T cell levels with lower Foxp3 intensity was also shown in CD200-CD200R-blocked MLRs. Expression of IL-10 mRNA declined significantly only in AML-DC MLRs where CD200-CD200R interaction was blocked and the same result was observed for TGF-β and Foxp3 mRNA in both AML- and monocyte-DC MLRs. These data present a significant role for CD200 in suppressing anti-tumor immune response through stimulation of regulatory mechanisms in AML patients and suggest that CD200 may have a prognostic value in this malignancy and its blockade may be used as a target for AML immunotherapy.

Topics: Adolescent; Adult; Aged; Antigens, CD; Antigens, Surface; Cell Proliferation; Cells, Cultured; Dendritic Cells; Disease Progression; Female; Forkhead Transcription Factors; Humans; Interferon-gamma; Interleukin-10; Interleukin-12; Interleukin-2; Leukemia, Myeloid, Acute; Lymphocyte Activation; Male; Middle Aged; Orexin Receptors; Receptors, Cell Surface; RNA, Messenger; T-Lymphocytes, Regulatory; Th1 Cells; Transforming Growth Factor beta; Tumor Escape; Up-Regulation; Young Adult

Epidermal Langerhans cells (LC) and dermal interstitial dendritic cells (IDC) were found to express the ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP; ABCG2). Also, low BCRP expression was present on CD34(+) blood DC precursors and expression was increased upon their differentiation to LC. The CD34(+) acute myeloid leukemia-derived DC cell line MUTZ3 can be cultured into LC or IDC, depending on the cytokine cocktail used. Introduction of functional BCRP in MUTZ3 progenitor cells through retroviral transduction resulted in the emergence of typical LC-characteristics in IDC cultures; the majority of cells remained negative for the IDC-specific C-type lectin DC-SIGN, but rather displayed enhanced expression of the LC-specific C-type lectin Langerin and characteristic high expression levels of CD1a. BCRP-induced skewing toward LC-like differentiation coincided with early RelB expression in 'IDC', derived from MUTZ3-BCRP, and depended on endogenous transforming growth factor beta (TGF-β) production. Intriguingly, cellular BCRP localization differed between skin LC and IDC, and a more cytoplasmic BCRP localization, as observed in primary skin LC, seemed to relate to LC-like differentiation in IDC cultures upon BCRP introduction in MUTZ3 progenitors. Together these data support a role for BCRP in preferential LC differentiation from CD34(+) myeloid DC progenitors.

Topics: Antigens, CD1; Antigens, CD34; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Dendritic Cells; Female; Hematopoietic Stem Cells; Humans; Langerhans Cells; Lectins, C-Type; Leukemia, Myeloid, Acute; Neoplasm Proteins; Skin; Transcription Factor RelB; Transforming Growth Factor beta

Activating mutations in fms-like tyrosine kinase-3 (FLT3) are frequent in acute myeloid leukemia and represent both a poor prognostic feature and a therapeutic target. We have identified a previously unrecognized downstream effect of FLT3 activation, namely up-regulation of the homeodomain genes, DLX1 and DLX2.. MV4;11 cells with FLT3-internal tandem duplication mutation, RS4;11 cells with wild-type FLT3 and blasts from patients with acute myeloid leukemia were used to pursue the relation between FLT3, DLX1/2 and transforming growth factor-β (TGFβ). Real-time quantitative reverse transcriptase polymerase chain reaction, western blot and reverse-phase protein array were performed to detect changes in gene and protein expression. RNA interference and MTS assays were used to study the interaction of PKC412, FLT3 inhibitor and TGFβ1.. A direct relationship between FLT3 activity and DLX1/2 expression was revealed by both inhibition and up-regulation of FLT3 signaling in MV4;11 and RS4;11 cell lines, respectively, in isolated blast cells from patients with acute myeloid leukemia, and in reverse-phase protein array assays of samples from patients with acute myeloid leukemia. Mechanistically, the link between FLT3 and DLX1 expression appears to involve MAPK signaling through the ERK and JNK pathways. To determine whether elevated DLX1 had a functional consequence, we explored the reported inhibition by DLX1 on TGFβ/Smad signaling. Indeed, TGFβ responses were blunted by FLT3 activation in a DLX1-dependent manner and FLT3 inhibition resulted in a time-dependent increase in nuclear phospho-Smad2.. These findings suggest that alterations in DLX1/2 contribute to the biological consequences of FLT3 activation.

Topics: Antigens, Surface; Cell Line, Tumor; Cell Nucleus; fms-Like Tyrosine Kinase 3; Gene Expression Regulation, Leukemic; Gene Silencing; Homeodomain Proteins; Humans; Leukemia, Myeloid, Acute; Phosphorylation; Protein Kinase Inhibitors; Signal Transduction; Smad2 Protein; Staurosporine; Transcription Factors; Transforming Growth Factor beta; Up-Regulation

We studied leukemic stem cells (LSCs) in a Smad4(-/-) mouse model of acute myelogenous leukemia (AML) induced either by the HOXA9 gene or by the fusion oncogene NUP98-HOXA9. Although Hoxa9-Smad4 complexes accumulate in the cytoplasm of normal hematopoietic stem cells and progenitor cells (HSPCs) transduced with these oncogenes, there is no cytoplasmic stabilization of HOXA9 in Smad4(-/-) HSPCs, and as a consequence increased levels of Hoxa9 is observed in the nucleus leading to increased immortalization in vitro. Loss of Smad4 accelerates the development of leukemia in vivo because of an increase in transformation of HSPCs. Therefore, the cytoplasmic binding of Hoxa9 by Smad4 is a mechanism to protect Hoxa9-induced transformation of normal HSPCs. Because Smad4 is a potent tumor suppressor involved in growth control, we developed a strategy to modify the subcellular distribution of Smad4. We successfully disrupted the interaction between Hoxa9 and Smad4 to activate the TGF-β pathway and apoptosis, leading to a loss of LSCs. Together, these findings reveal a major role for Smad4 in the negative regulation of leukemia initiation and maintenance induced by HOXA9/NUP98-HOXA9 and provide strong evidence that antagonizing Smad4 stabilization by these oncoproteins might be a promising novel therapeutic approach in leukemia.

Topics: Animals; Apoptosis; Blotting, Western; Bone Marrow Transplantation; Cell Nucleus; Cell Transformation, Neoplastic; Chromatin Immunoprecipitation; Cytoplasm; Flow Cytometry; Hematopoietic Stem Cells; Homeodomain Proteins; Humans; Immunoprecipitation; Leukemia, Myeloid, Acute; Mice; Mice, Inbred C57BL; Mice, Knockout; Nuclear Pore Complex Proteins; Oncogene Proteins, Fusion; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad4 Protein; Transforming Growth Factor beta

The leaves of Artocarpus tonkinensis are used in Vietnamese traditional medicine for treatment of arthritis, and the compound maesopsin 4-O-β-D-glucoside (TAT-2), isolated from them, inhibits the proliferation of activated T cells. Our goal was to test the anti-proliferative activity of TAT-2 on the T-cell leukemia, Jurkat, and on the acute myeloid leukemia, OCI-AML. TAT-2 inhibited the growth of OCI-AML (and additional acute myeloid leukemia cells) but not Jurkat cells. Growth inhibition was shown to be due to inhibition of proliferation rather than increase in cell death. Analysis of cytokine release showed that TAT-2 stimulated the release of TGF-β, yet TGF-β neutralization did not reverse the maesopsin-dependent effect. Gene expression profiling determined that maesopsin modulated 19 identifiable genes. Transcription factor CP2 was the gene most significantly modulated. Real-time PCR validated that up-regulation of sulphiredoxin 1 homolog (SRXN1), hemeoxygenase 1 (HMOX1), and breast carcinoma amplified sequence 3 (BCAS3) were consistently modulated.

Topics: Artocarpus; Benzofurans; Cell Death; Cell Growth Processes; Cell Line, Tumor; DNA-Binding Proteins; Dose-Response Relationship, Drug; Gene Expression; Gene Expression Profiling; Glucosides; Heme Oxygenase-1; HL-60 Cells; Humans; Jurkat Cells; Leukemia, Myeloid, Acute; Leukemia, T-Cell; Male; Middle Aged; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Oxidoreductases Acting on Sulfur Group Donors; T-Lymphocytes; Transcription Factors; Transforming Growth Factor beta; U937 Cells; Up-Regulation

An unsupervised multi-strategy approach has been developed to identify informative genes from high throughput genomic data. Several statistical methods have been used in the field to identify differentially expressed genes. Since different methods generate different lists of genes, it is very challenging to determine the most reliable gene list and the appropriate method. This paper presents a multi-strategy method, in which a combination of several data analysis techniques are applied to a given dataset and a confidence measure is established to select genes from the gene lists generated by these techniques to form the core of our final selection. The remainder of the genes that form the peripheral region are subject to exclusion or inclusion into the final selection. This paper demonstrates this methodology through its application to an in-house cancer genomics dataset and a public dataset. The results indicate that our method provides more reliable list of genes, which are validated using biological knowledge, biological experiments, and literature search. We further evaluated our multi-strategy method by consolidating two pairs of independent datasets, each pair is for the same disease, but generated by different labs using different platforms. The results showed that our method has produced far better results.

Topics: Animals; Artificial Intelligence; Cell Transformation, Neoplastic; Computational Biology; Databases, Genetic; Decision Trees; Gene Expression Profiling; Genomics; Humans; Leukemia, Myeloid, Acute; Mice; Oligonucleotide Array Sequence Analysis; Transforming Growth Factor beta

T helper type 17 (Th17) cells, which represent a novel subset of CD4(+) T cells, play an active role in inflammatory and autoimmune diseases. Recent studies have also suggested that they have an impact on solid tumours. However, the nature of Th17 cells in haematological malignancies remains unknown. In this study, we investigated Th17 cell frequency and secretion of related cytokines in patients with acute myeloid leukaemia (AML). First, we found that Th17 cell frequencies were increased significantly in peripheral blood samples from untreated patients with AML, compared with those from healthy volunteers. Moreover, increased interleukin (IL)-17 concentrations accompanied the increased Th17 cell frequencies in these patients. These results suggest that Th17 cells may play a role in the pathogenesis of AML. Secondly, we found that the increased Th17 cell frequencies were reduced when patients achieved complete remission after chemotherapy, suggesting that measurement of Th17 cell frequencies may have clinical value in the evaluation of therapeutic effect. In addition, we found that IL-6 and transforming growth factor (TGF)-beta1 concentrations increased in the untreated patients and that IL-6 concentrations showed a positive correlation with the frequencies of Th17 cells, suggesting that IL-6 may play an important role in Th17 cell differentiation in patients with AML.

Topics: Adult; Antineoplastic Agents; Female; Flow Cytometry; Humans; Interleukin-17; Interleukin-6; Leukemia, Myeloid, Acute; Male; Middle Aged; Remission Induction; T-Lymphocyte Subsets; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta

To investigate the regulatory mechanisms of angiogenesis in the development of myelodysplastic syndromes (MDS) and its progression to overt leukaemia (OL), bone marrow samples from control, paired samples from MDS patients before and after transformation to OL (MDS --> OL) and de novo acute myeloid leukaemia (AML) were analysed. Immunohistochemical staining showed a significant increase of bone marrow microvascular density (MVD) in MDS and de novo AML compared with controls. Surprisingly, in MDS, MVD significantly decreased upon transformation to OL, which was also significantly lower than the MVD of de novo AML. This evidence was strengthened by the pattern of angiogenic mediator gene expression, confirming the importance of various angiogenic mediators including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumour necrosis factor alpha (TNFalpha), hepatocyte growth factor (HGF) and the angiopoietin family of mediators (Ang-1 and Ang-2) as well as the receptors for angiogenic mediators, such as VEGF receptor 2 (VEGFR2) and the tyrosine kinase receptor, TIE2. By contrast, the anti-angiogenic mediator, transforming growth factor-beta (TGFbeta) exhibited significantly higher expression in the bone marrow of MDS --> OL, indicating the importance of this cytokine as the suppressive factor of angiogenesis in MDS. These findings indicate that the bone marrow microenvironment in MDS --> OL and de novo AML differs remarkably, suggesting the different efficacy of anti-angiogenic therapy between de novo AML and leukaemia secondary to MDS.

Topics: Aged; Angiopoietin-1; Angiopoietin-2; Bone Marrow; Disease Progression; Female; Fibroblast Growth Factor 2; Hepatocyte Growth Factor; Humans; Immunohistochemistry; Leukemia; Leukemia, Myeloid, Acute; Male; Microcirculation; Middle Aged; Myelodysplastic Syndromes; Neovascularization, Pathologic; Receptor, TIE-2; RNA, Messenger; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

N-MYC encodes a basic helix-loop-helix/leucine zipper (bHLH/LZ) transcription factor that is frequently overexpressed in human neuroblastoma. N-MYC overexpression has also been reported in human acute myeloid leukemias (AML), which we show here is a frequent event. Myeloid cells in N-Myc-overexpressing mouse bone marrow hyperproliferate but those in c-MYC-overexpressing bone marrow do not. The NH(2)-terminal transactivation domain, nuclear localization signal, and bHLH/LZ domain of N-Myc are essential for this effect. Microarray analysis revealed 969 differentially expressed genes between N-Myc- and c-MYC-overexpressing myeloid cells. N-Myc-overexpressing cells showed decreased transforming growth factor beta signaling and increased c-Jun-NH(2)-kinase signaling, both of which are associated with proliferation and leukemic transformation of myeloid cells. Mice transplanted with bone marrow expressing wild-type N-Myc developed clonal and transplantable AML after approximately 1 month; those transplanted with bone marrow expressing mutant N-Myc did not. Twist, a known suppressor of the p19Arf/p53 pathway, was up-regulated in all tumors. These results show that N-Myc overexpression is highly oncogenic in mouse myeloid cells and suggest that N-MYC up-regulation contributes to human myeloid leukemogenesis.

Topics: Animals; Bone Marrow Cells; Cell Proliferation; Cell Separation; Cell Transformation, Neoplastic; Gene Expression Regulation, Leukemic; JNK Mitogen-Activated Protein Kinases; Leukemia, Myeloid, Acute; Mice; Myeloid Cells; Oligonucleotide Array Sequence Analysis; Protein Structure, Tertiary; Proto-Oncogene Proteins c-myc; Signal Transduction; Transforming Growth Factor beta

In this issue of the Journal, Soucek et al. challenge the assumption that increased functional granulocytic maturation of HL-60, an ATRA-responsive acute myeloid leukemia cell line devoid of the APL-specific PML-RARalpha fusion protein, results in more rapid or more sustained cell death. In this model cell line, the authors demonstrate that TGFbeta1, a well-known haemopoietic growth factor, enhances retinoid-dependent cyto-differentiation and growth arrest while inhibiting apoptosis. Concomitantly, treatment of HL-60 cells with the combination of TGFbeta1 and the retinoid partially suppresses ATRA-dependent induction of TRAIL. This is a death receptor ligand of the TNF family implicated in the paracrine mechanism underlying the apoptotic action of ATRA in APL blasts The protein activates the death-receptor-dependent or extrinsic apoptotic pathway, which is associated with caspase-8 activation. Down-regulation of TRAIL is correlated to an increase in the levels of the anti-apoptotic c-FLIP(L) and Mcl-1 proteins that are likely to be involved in the suppression of caspase-8 activation and apoptosis.

Topics: Acute Disease; Apoptosis; Apoptosis Regulatory Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Caspases; Cell Differentiation; Cell Proliferation; Granulocytes; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Myeloid, Acute; Membrane Glycoproteins; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; TNF-Related Apoptosis-Inducing Ligand; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Recent research indicates that TGF-beta and type II receptor (TbetaR-II) play an important role in the pathogenesis of tumor. A high frequency of abnormalities in TbetaR-II has been demonstrated in various cancers. To identify the mutation of TbetaR-II in patients with acute leukemia, the bone marrow samples from 6 patients with acute leukemia and 11 normal individuals as control were detected by long-range RT-PCR. To detect a deletion in sequence of the TbetaR-IIgene, the PCR products were cloned to T vector and then sequenced. The results showed that there was existance of the isoform of TbetaR-II in 2 cases out of 6 patients with acute leukemia. These two patients had more poor prognosis than others. In conclusion, there was the isoform of TbetaR-II in partial patients with acute leukemia, and the isoform may be related with prognosis.

Topics: Adult; Base Sequence; Female; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; Molecular Sequence Data; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prognosis; Protein Isoforms; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Cotylenin A, which has been isolated as a plant growth regulator, potently induces the differentiation of human myeloid leukemia cells. Treatment of HL-60 cells with a combination of transforming growth factor (TGF)-beta and 1alpha, 25-dihydroxyvitamin D(3) (VD3) resulted in increased differentiation compared to separate treatments, but TGF-beta did not affect the cotylenin A-induced differentiation of HL-60 cells. It is possible that the signal transduction pathway used by cotylenin A for inducing the differentiation of leukemia cells is the same as that used by TGF-beta. However, cotylenin A did not affect the expression of TGF superfamily or Smad genes in HL-60 cells. Treatment with neutralizing anti-TGF-beta antibody or an inhibitor of TGF-beta signaling did not inhibit cotylenin A-induced differentiation, although VD3-induced differentiation was significantly suppressed by these treatments. The subcellular distribution of Smad3 was also unaffected by cotylenin A. These results suggest that the cotylenin A-induced differentiation of leukemia cells is independent of the TGF-beta signaling system, although TGF-beta acts as an autocrine mediator of the growth arrest and differentiation of leukemia cells induced by VD3 and other inducers.

Topics: Cell Differentiation; Cell Line, Tumor; Diterpenes; Gene Expression Regulation, Neoplastic; HL-60 Cells; Humans; Immunohistochemistry; Leukemia, Myeloid, Acute; Models, Biological; RNA, Messenger; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; U937 Cells

Disruption of transforming growth factor-beta (TGFB1/TGF-beta) signaling contributes to the formation of human hematological malignancies. Smad4, a tumor suppressor, functions as an essential intracellular signal transducer of the TGF-beta signaling pathway. Recent studies have demonstrated that some tumor-derived mutations of Smad4 are associated with protein instability; however, the precise mechanism by which mutated Smad4 proteins undergo rapid degradation remains to be elucidated. A missense mutation of the SMAD4 gene in the Mad homology 1 (MH1) domain (c.305C>T, Pro102Leu) and one frameshift mutation resulting in termination in the Mad homology 2 (MH2) domain (c.1447_1448insAATA, Delta483-552) have been identified in acute myelogenous leukemia. It is not known whether protein instability of these SMAD4 mutants is one of the contributors to TGF-beta signaling disruption in acute myelogenous leukemia. Here we report that these two acute myelogenous leukemia-derived SMAD4 mutants are degraded rapidly when compared to their wild-type counterpart. We have demonstrated that both mutated proteins exhibit enhanced polyubiquitination (or polyubiquitylation) and proteasomal degradation. Importantly, we found that beta-transducin-repeat-containing protein 1 (beta-TrCP1), an F-box protein in the ubiquitin E3 ligase Skp1-Cullin-F-box protein (SCF) complex, directly interacts with and acts as a critical determinant for degradation of both mutated SMAD4 proteins. In addition, small interference RNA (siRNA)-triggered endogenous beta-TrCP1 suppression increased the protein expression level of both overexpressed SMAD4 mutants and endogenous mutated SMAD4 protein in acute myelogenous leukemia cells. These data suggest that mutated SMAD4 proteins undergo rapid degradation in acute myelogenous leukemia cells via SCF(beta-TrCP1) E3 ligase-mediated protein ubiquitination (or ubiquitylation) and subsequent proteasomal degradation.

Topics: Animals; beta-Transducin Repeat-Containing Proteins; Chlorocebus aethiops; COS Cells; Frameshift Mutation; Humans; Leukemia, Myeloid, Acute; Mutation, Missense; Proteasome Endopeptidase Complex; Protein Structure, Tertiary; Protein Transport; RNA Interference; RNA, Small Interfering; Signal Transduction; SKP Cullin F-Box Protein Ligases; Smad4 Protein; Transforming Growth Factor beta; Ubiquitin

Loss of transforming growth factor (TGF)-beta signaling has been implicated in malignant transformation of various tissues. Smad4 plays a central role in the signal transduction of TGF-beta. Deletion or mutation of Smad4 has been described in a number of cancers. This study was aimed to investigate a potential role of Smad4 in leukemia including its expression and location in blast cells. The mononuclear cells were separated from bone marrow of leukemia patients. The samples, blast cells of which were more than 90% in mononuclear cells, were selected. The expression and location of Smad4 protein were analyzed by immunohistochemistry methods. The results showed that the Smad4 protein located mainly in nucleus, part of this protein located in cytoplasma, the expressions of Smad4 were not detected in 6 out of 9 ALL patients, in 7 out of 24 AML patients and in 1 out of 2 CML patients; these leukemia patients, in whose cells the expression of Smad4 was not detected, included one L1 and one L3, four L2, one M0, one M1, two M2a, one M3a, one M4b, one M6 and one CML. In conclusion, the Smad4 protein was mainly in nucleus, the deletion or functional change of Smad4 may related with the pathogenesis of human AML.

Topics: Humans; Leukemia, Myeloid, Acute; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta

The receptors for transforming growth factor beta (TGF-beta) and their signaling intermediates make up an important tumor-suppressor pathway. The role of one of these intermediates--Smad3--in the pathogenesis of lymphoid neoplasia is unknown.. We measured Smad3 messenger RNA (mRNA) and protein in leukemia cells obtained at diagnosis from 19 children with acute leukemia, including 10 with T-cell acute lymphoblastic leukemia (ALL), 7 with pre-B-cell ALL, and 2 with acute nonlymphoblastic leukemia (ANLL). All nine exons of the SMAD3 gene (MADH3) were sequenced. Mice in which one or both alleles of Smad3 were inactivated were used to evaluate the role of Smad3 in the response of normal T cells to TGF-beta and in the susceptibility to spontaneous leukemogenesis in mice in which both alleles of the tumor suppressor p27Kip1 were deleted.. Smad3 protein was absent in T-cell ALL but present in pre-B-cell ALL and ANLL. No mutations were found in the MADH3 gene in T-cell ALL, and Smad3 mRNA was present in T-cell ALL and normal T cells at similar levels. In mice, the loss of one allele for Smad3 impairs the inhibitory effect of TGF-beta on the proliferation of normal T cells and works in tandem with the homozygous inactivation of p27Kip1 to promote T-cell leukemogenesis.. Loss of Smad3 protein is a specific feature of pediatric T-cell ALL. A reduction in Smad3 expression and the loss of p27Kip1 work synergistically to promote T-cell leukemogenesis in mice.

Topics: Adult; Animals; Cell Cycle Proteins; Child; Cyclin-Dependent Kinase Inhibitor p27; DNA-Binding Proteins; Exons; Gene Deletion; Gene Expression; Humans; Interleukin-2; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Myeloid, Acute; Leukemia, T-Cell; Mice; Mice, Knockout; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Transforming Growth Factor beta; RNA, Messenger; Sequence Analysis, DNA; Signal Transduction; Smad3 Protein; T-Lymphocytes; Trans-Activators; Transforming Growth Factor beta; Tumor Suppressor Proteins

To explore the possible cross-talk between the IL-6 and TGF-beta1 pathways in AML blast cells, the effect of TGF-beta1 pretreatment on IL-6-induced STAT3 tyrosine phosphorylation was studied. A reduction of STAT3 tyrosine phosphorylation after TGF-beta1 pretreatment was observed in four out of 40 AML cases (10%), although all of the AML cases responded to TGF-beta1 by means of SMAD3 translocation. The reduced IL-6-mediated STAT3 tyrosine phosphorylation after pre-treatment with TGF-beta1 was associated with apoptosis and coincided with the degradation of certain cellular proteins, including JAK1 and -2 and Tyk2, without affecting the ERK expression and phosphorylation. Furthermore, treatment of AML blasts with the cytostatic agent VP16, as an alternative way to induce apoptosis, resulted in a similar degree of degradation of JAK kinases and concomitant reduction of IL-6-mediated STAT3 tyrosine phosphorylation. Although degradation of JAK kinases could be rescued by incubating the cells with the pan-caspase inhibitor Z-VAD-fmk, the attenuating effect of TGF-beta1 treatment on the STAT3 tyrosine phosphorylation was still partly present. It was shown that in AML cells cultured in the presence of Z-VAD-fmk, TGF-beta1 pretreatment resulted in a reduction of JAK1 phosphorylation upon IL-6 stimulation. Expression of SOCS1 and -3 could be ruled out as a possible cause of reduced JAK1 phosphorylation levels in the investigated AML case.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carrier Proteins; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Down-Regulation; Electrophoretic Mobility Shift Assay; Epithelial Cells; Etoposide; Humans; Interleukin-6; Intracellular Signaling Peptides and Proteins; Janus Kinase 1; Leukemia, Myeloid, Acute; Phosphorylation; Protein Transport; Protein-Tyrosine Kinases; Repressor Proteins; Signal Transduction; Smad3 Protein; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling Proteins; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured; Tyrosine

The Smad family proteins are critical components of the transforming growth factor (TGF)-beta signaling pathway. TGF-beta is a multipotent cytokine that elicits many biological functions. In particular, TGF-beta exhibits effects on the cell cycle manifested by G1-phase arrest, differentiation, or apoptosis of several target cells, suggesting that disruption of TGF-beta signaling pathway could be involved in cancer formation. Here we show one missense mutation of the Smad4 gene in the MH1 domain (P102L) and one frame shift mutation resulting in termination in the MH2 domain (Delta(483 - 552)) in acute myelogeneous leukemia. Both of the mutated Smad4 proteins lack transcriptional activities. Concomitant expression of the P102L mutant with wild-type Smad4 inactivates wild-type Smad4 through inhibiting its DNA-binding ability. The Delta(483 - 552) mutant blocks nuclear translocation of wild-type Smad4 and thus disrupts TGF-beta signaling. This is the first report showing that mutations in the Smad4 gene are associated with the pathogenesis of acute myelogeneous leukemia and the obtained results should provide useful insights into the mechanism whereby disruption of TGF-beta signaling pathway could lead to acute myelogeneous leukemia. Oncogene (2001) 20, 88 - 96.

Topics: Animals; Cell Division; Cell Transformation, Neoplastic; COS Cells; DNA; DNA-Binding Proteins; Enzyme Activation; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Genetic Vectors; Growth Inhibitors; HL-60 Cells; Humans; Jurkat Cells; Leukemia, Myeloid, Acute; Mutation; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Protein Binding; Recombinant Fusion Proteins; Serine Proteinase Inhibitors; Signal Transduction; Smad3 Protein; Smad4 Protein; Subcellular Fractions; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Tumor Cells, Cultured

The products of the tumor suppressor genes are considered to function as specific inhibitors of tumor cell growth. In this communication, we present evidence to show that these proteins inhibit tumor cell proliferation by participating in the activation of tumor cell differentiation. The ML-1 human myeloblastic leukemia cells used in this study proliferate when treated with insulin-like growth factor I and transferrin but differentiate to monocytes when exposed to tumor necrosis factor alpha or transforming growth factor beta1, or to macrophage-like cells when treated with both these cytokines. Initiation of proliferation but not of differentiation was followed by a 20- to 25-fold increase in the nuclear level of the DNA polymerase-associated processivity factor PCNA and of the proliferation-specific transcription factor E2F1. In contrast, induction of differentiation but not of proliferation was followed by a 25- to 30-fold increase in the nuclear level of the tumor suppressor proteins p53 (wild type), pRb, and p130/Rb2 and of the p53-dependent cyclin kinase inhibitor p21/Cip1. p53 and p21/Cip1, respectively, inhibit the expression and activation of PCNA, whereas p130 and pRb, respectively, inhibit the expression and activation of E2F1. As a result, G1-S-associated DNA and mRNA synthesis is inhibited, growth uncoupled from differentiation, and maturation enabled to proceed. Where this function of the tumor suppressor proteins is impaired, the capacity for differentiation is lost, which leads to the sustained proliferation that is characteristic of the cancer cell.

Topics: Carrier Proteins; Cell Cycle Proteins; Cell Differentiation; Cell Division; Cell Nucleus; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytokines; DNA-Binding Proteins; E2F Transcription Factors; E2F1 Transcription Factor; Genes, Tumor Suppressor; Humans; Insulin-Like Growth Factor I; Leukemia, Myeloid, Acute; Macrophages; Monocytes; Phosphoproteins; Proliferating Cell Nuclear Antigen; Proteins; Retinoblastoma Protein; Retinoblastoma-Binding Protein 1; Retinoblastoma-Like Protein p130; Transcription Factor DP1; Transcription Factors; Transferrin; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53

To investigate the mechanism by which TGF beta 1 inhibits the growth of leukemic cells and the significance of serum TGF beta 1 level in acute leukemia(AL) patients.. RT-PCR was used to detect gene expression of cytokines including IL-1, IL-3, IL-6, TGF beta 1 and their receptors (R) in leukemic and normal cells. Leukemic colony assay was carried out to determine the effect of TGF beta 1 on the growth of leukemic cells and the serum TGF beta 1 level in 33 AL patients were measured by ELISA.. TGF beta 1 was found to significantly inhibit the proliferation of leukemia cells of HL-60, K562 and DAMI cell lines. Furthermore, a variety of leukemia cell lines including HEL, HL-60, K562, U937, DAMI, MEG-01, HUT78 and CA were found to express mRNAs for TGF beta 1 and its receptor. In addition, TGF beta 1 was found to be able to evidently down-regulate the expression of mRNAs for IL-6, IL-6R, IL-3, GM-CSFR in DAMI cells and, interestingly, up-regulate TGF beta 1 gene expression in the cells per se before the cells showed DNA fragmentation, a molecular hallmark for cell apoptosis. It was shown that serum TGF beta 1 levels were significantly decreased in leukemic patients, restored to normal in the patients achieved complete remission, and tended to decrease again in the recurrent patients.. 1. TGF beta 1 may act as an inhibitory autocrine factor in the proliferation of leukemia cells; 2. the mechanism by which TGF beta 1 inhibits the growth of leukemia cells involves its down-regulating expression of positive cytokines and receptors and up-regulating TGF beta 1 expression in leukemia cells per se; 3. serum TGF beta 1 is a valuable parameter in monitoring the prognosis of leukemia and weakened control of TGF beta 1 on leukemia cells may play an important role in the pathogenesis of AL.

Topics: Adult; Apoptosis; Female; Humans; Leukemia, Myeloid, Acute; Male; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Transforming Growth Factor beta1

We have compared the effects of AcSDKP, Thymosin beta4 (Tbeta4), MIP1alpha and TGFbeta on acute myeloid leukemia (AML) and B-lineage acute lymphoid leukemia (B-ALL) cells using liquid cultures in the presence of GM-CSF, IL-3 and SCF for AML cells and IL-3 and IL-7 for ALL cells. Each molecule was added daily and cell proliferation was evaluated on day 3 by thymidine incorporation. Whereas TGFbeta was found inhibitory in all the AML and B-ALL cases studied, MIP1alpha was inhibitory in 6/12 AML cases and had no effect on B-ALL cells. AcSDKP and Tbeta4 showed an inhibitory effect in a few cases but only at high doses which were inactive on normal cells. Thus, our study not only confirms the effect of TGFbeta, MIP1alpha and AcSDKP on AML cells but also provides new data concerning their effect on B-ALL and the possible inhibitory effect of AcSDKP at high doses. Furthermore, we show for the first time the effect of Tbeta4 on leukemic cells. Altogether, our data indicate differences of sensitivity of leukemic cells to negative regulators, some leukemias being inhibited by one or several of these molecules whereas others were unresponsive to all used. The clinical relevance of these observations still remains to be determined.

Topics: Adolescent; Adult; Aged; Burkitt Lymphoma; Cell Division; Chemokine CCL4; Child; Child, Preschool; Female; Humans; Infant; Leukemia, Myeloid, Acute; Macrophage Inflammatory Proteins; Male; Middle Aged; Oligopeptides; Thymosin; Transforming Growth Factor beta; Tumor Cells, Cultured

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

We evaluated the effects of transforming growth factor-beta 1 (TGF-beta 1) on the growth of hematopoietic progenitors in normal donors and in patients with hematologic malignancies now designed as clonal disorders of multipotential stem cells. TGF-beta 1 at 80 pM exhibited differential effects on the normal hematopoietic progenitors when cells were stimulated with different growth factors, such as G-CSF, GM-CSF, interleukin-3 (IL-3), or stem cell factor (SCF). The suppressive effect by TGF-beta 1 was increased for growth with GM-CSF, IL-3, and SCF, and growth with G-CSF was unaffected in hematologic malignancies, TGF-beta 1 suppression for growth with G-CSF was increased for essential thrombocythemia (ET) and polycythemia vera; chronic myelogenous leukemia (CML) in chronic phase; CML in accelerated phase; CML in myeloid crisis; myelodysplastic syndrome (MDS) in refractory anemia; MDS in refractory anemia with an excess of blasts; and acute myeloblastic leukemia (AML). In CML-myeloid crisis and AML, TGF-beta 1 almost completely abolished the growth, with some patient-to-patient variation. The mean ED50s for the growth of leukemic blast progenitors were 1.6, 1.2, 0.7, and 0.2 pM in the presence of G-CSF, GM-CSF, IL-3, and SCF, respectively, c-myc and c-myb antisense oligonucleotides significantly suppressed the growth of leukemic blast progenitors, but not that of clonogenic cells from normal donors and patients with ET. We also demonstrated that TGF-beta 1 inhibits mRNA expression by AML blasts for c-myc and/or c-myb. When the data are taken together, growth suppression by TGF-beta 1 appears to increase with the progression of clonal evolution in hematologic malignancies.

Topics: Base Sequence; Blast Crisis; Bone Marrow; Cell Division; Colony-Forming Units Assay; Gene Expression; Genes, myc; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Stem Cells; Interleukin-3; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Molecular Sequence Data; Oligodeoxyribonucleotides; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Proto-Oncogene Proteins c-myc; Recombinant Proteins; Reference Values; RNA, Messenger; Transforming Growth Factor beta

C-myb and c-ets-1 have variously been demonstrated to function as protooncogenes. Using a human leukemic cell line, ML-1, we have examined the mechanism by which these genes participate in establishing the sustained proliferation mode that is characteristic of the transformed cell. In the absence of serum, ML-1 cells were found to require IGF-1 and transferrin (TF) for growth and TGF-beta or TNF-alpha plus TF for differentiation. Upon administration of the growth factors, c-myb expression increased within 60 min, whereas after addition of the differentiation factors c-myb expression ceased completely within 3 hr. A correlation was found to exist between the level of c-ets-1 protein in the cells, the extent to which that protein is bound to intron I of the myb gene and the amount of c-myb mRNA that is expressed. Upon administration of growth factors, a sizable increase in the intracellular, and particularly, in the intranuclear level of c-ets-1 protein was observed, whereas a pronounced decrease in the level of this protein occurred after exposure to the differentiation factors. These data demonstrated that the level at which an oncogene-specified transcription factor is expressed can affect the expression of other target oncogenes involved in the regulation of cell proliferation. Stimulated expression of such transcription factor can then lead to the continuous proliferation cycle characteristic of the cancer cell.

Topics: Blotting, Western; Cell Nucleus; Cytoplasm; Gene Expression Regulation, Neoplastic; Humans; Insulin-Like Growth Factor I; Leukemia, Myeloid, Acute; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Proto-Oncogene Proteins c-myb; Proto-Oncogenes; Transcription Factors; Transcription, Genetic; Transferrin; Transforming Growth Factor beta; Tumor Cells, Cultured

In acute myeloblastic leukemia (AML), the T cell response and cytotoxic activity are impaired at time of diagnosis due to not-yet-identified soluble immunosuppressing factors. The inhibition of autologous antileukemic immune response by these factors may support immunosurveillance of AML. A well-known inhibitor of lymphokine-activated killer (LAK) cell activity is transforming growth factor-beta 1 (TGF-beta 1). To evaluate the possible significance of TGF-beta 1 for the impaired cytotoxic activity in AML at time of diagnosis, we looked for the TGF-beta 1-specific mRNA, for the production and release of TGF-beta 1, and for its relevance for immunosuppressing activities in AML. In the culture supernatants of 18 investigated AMLs, we detected various amounts of TGF-beta protein. The TGF-beta 1 and TGF-beta 2 protein concentrations were 105 pg/mL (< 50-240 pg/mL) and 32 pg/mL (< 2-91 pg/mL), respectively. In 13 of 15 patients, the leukemic blasts expressed TGF-beta 1 mRNA. To exclude possible interferences with contaminating mononuclear cells (MNC), the data were confirmed by analysis of sorted blast cells and leukemic cell lines. All investigated leukemic cell lines expressed TGF-beta 1 protein and mRNA. The culture supernatants of AMLs inhibited LAK activity strongly in a dose-dependent manner. The inhibition of cytotoxicity could be restored by the addition of neutralizing TGF-beta 1 antibodies. The data suggest TGF-beta 1 to be a relevant factor for the inhibition of cytotoxic activities in AMLs.

Topics: Adult; Aged; Base Sequence; Blotting, Southern; Female; Gene Expression; Humans; Immune Tolerance; Killer Cells, Lymphokine-Activated; Leukemia, Myeloid, Acute; Male; Middle Aged; Molecular Sequence Data; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured

Hematopoiesis is a profound example of cell homeostasis that is regulated throughout life. This process requires the participation of many factors, including positive and negative regulators of growth and differentiation, that determine survival, growth stimulation, differentiation, functional activation, and programmed cell death. Understanding the effects of multiple stimuli on specific cells at the molecular and cellular level is crucial towards understanding how the population of blood cells maintains a homeostatic state. Two appropriate stimuli for analysis, both of which are found in bone marrow, are differentiation-inducing cytokines, which induce terminal differentiation associated with growth arrest, ultimately culminating in programmed cell death, and transforming growth factor-beta 1 (TGF-beta 1), which induces rapid growth arrest and apoptosis of hematopoietic cells. Previously, we have shown, using M1 myeloblastic leukemic cells as a model system, that differentiation-inducing cytokines induce terminal differentiation associated with growth arrest and, only after 5 to 7 days, apoptosis, whereas TGF-beta 1 induces rapid growth arrest and apoptosis. In this report, we show that M1 myeloid leukemic cells treated concomitantly with the differentiation inducer interleukin-6 and TGF-beta 1 undergo terminal differentiation, in which modulators of the MyD118 gene product, previously shown to be a positive regulator of TGF-beta 1-induced apoptosis, are implicated to play a role in protecting the cells from TGF-beta 1-induced apoptosis. Furthermore, using M1 cell variants blocked at different stages after induction of differentiation, including M1myb and M1myc, as well as conditionally blocked M1mycer, it has been shown that the dominance of interleukin-6 to TGF-beta 1-induced apoptosis is dependent on the progression of the differentiation program. Further studies with M1 and the genetically engineered M1 cell variants will be instrumental towards molecularly dissecting the interaction of hematopoietic differentiation with a variety of apoptotic pathways.

Topics: Animals; Apoptosis; Cell Adhesion; Cell Cycle; Cell Differentiation; Cell Division; Cell Line; Estradiol; Humans; Interleukin-6; Kinetics; Leukemia, Experimental; Leukemia, Myeloid, Acute; Macrophage-1 Antigen; Mice; Receptors, Fc; Swine; Time Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

In the absence of serum, growth of ML-1 human myeloblastic leukemia cells is induced by the insulin-like growth factor-1 (IGF1) together with transferrin (Tf), whereas monocytic differentiation is initiated by the transforming growth factor-beta (TGF-beta) in combination with Tf. Initiation of growth was followed by the rapid release of arachidonic acid (AA), hydroxyeicosatetraenoic acids (HETEs) and phospholipids into the culture medium. In contrast, induction of differentiation occurred without the release of these lipids beyond the level present in control. Inhibitors of enzymes involved in the formation of AA and of HETEs, including phospholipase A2 and lipoxygenases, caused interference with growth but not with differentiation, and an inhibitor of the cyclooxygenase path affected neither growth nor differentiation. These results indicate that the initiation of ML-1 cell growth but not of cell differentiation is dependent upon the increased formation of AA and its derivatives formed primarily via the lipoxygenase path.

Topics: Arachidonic Acid; Caffeic Acids; Cell Differentiation; Cell Division; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; Insulin-Like Growth Factor I; Leukemia, Myeloid, Acute; Lipoxygenase Inhibitors; Masoprocol; Phospholipids; Transferrin; Transforming Growth Factor beta; Tritium; Tumor Cells, Cultured

Recently, both Bcl-2, which promotes cell survival, and Bax, which promotes cell death, have been implicated as major players in the control of apoptotic pathways, and it has been suggested that the ratio of Bcl-2 and Bax protein controls the relative susceptibility of cells to death stimuli. We have used M1 myeloid leukemia cells and genetically engineered M1 variants as a model system to study apoptosis induced by two distinct apoptotic stimuli. This includes apoptosis induced by activation of wild type p53 function of a temperature sensitive p53 transgene expressed in M1 cells, which do not express endogenous p53, and apoptosis induced by TGF beta 1. It is shown that the kinetics of apoptosis induced by p53 is more rapid than apoptosis induced by TGF beta 1. It is also shown that ectopic expression of Bcl-2, at levels which blocked TGF beta 1-induced apoptosis of M1 cells, delayed, but did not block, p53-induced apoptosis. Both p53 and TGF beta 1 down-regulated endogenous Bcl-2 expression, but only p53 up-regulated Bax expression, where bax has been identified as a p53 immediate early response gene. Thus, the p53-mediated up-regulation of Bax may provide at least a partial explanation for the more rapid rate of apoptosis induced by p53 compared to by TGF beta 1, as well as for the ineffectiveness of ectopoic Bcl-2 to abrogate p53-mediated apoptosis. These findings provide first insights to the molecular mechanisms which mediate p53-induced apoptosis, identifying bax and bcl-2 as p53 regulated genes, and serve as a paradigm of how the intracellular balance of Bcl-2 to Bax is differentially altered by distinct death stimuli.

Topics: Apoptosis; bcl-2-Associated X Protein; Cells, Cultured; Genes, p53; Humans; Leukemia, Myeloid, Acute; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Up-Regulation

Ten patients with acute leukemia after primary myelodysplastic syndrome (MDS-AL) were examined to clarify the biologic nature of the leukemic cells in comparison with that of de novo acute myelocytic leukemia (AML). The morphologic and cytochemical features of the leukemic cells from all these patients corresponded well to those of de novo AML, and they were diagnosed with MDS-AML. Phenotypically, the frequent expression of the lymphocyte activation antigens, CD25 and CD30, was characteristic in MDS-AML. The in vitro response of MDS-AML cells to various growth factors was similar to that of de novo AML cells. Transforming growth factor beta 1 (TGF beta 1) suppressed growth factor-dependent colony formation by normal bone marrow cells, MDS bone marrow cells, and de novo AML cells, but did not inhibit colony formation by MDS-AML cells. The number of TGF beta 1 high-affinity binding sites of MDS-AML samples (< 5 to 47 sites/cell) was markedly lower than that in de novo AML samples (120 to 221 sites/cell). Our results indicate that the reduced TGF beta 1 may represent disregulation of the proliferation system in MDS-AML cells. This is thought to occur during the MDS phase, and may be related to the poorer response shown to conventional chemotherapy of AML.

Topics: Adult; Aged; Antigens, CD; Antigens, Neoplasm; Female; Genotype; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-3; Ki-1 Antigen; Leukemia, Myeloid, Acute; Male; Middle Aged; Myelodysplastic Syndromes; Phenotype; Receptors, Cell Surface; Receptors, Interleukin-2; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Blast cells, obtained from patients with acute myelogenous leukemia (AML), that express surface binding sites for human stem cell factor (SCF) respond proliferatively upon exposure to this molecule. In the presence of human transforming growth factor-beta 1 (TGF-beta 1) the capacity of SCF to augment the proliferative state of AML blasts was, however, almost completely abolished. This inhibitory action of TGF-beta 1 could be reversed by a neutralizing anti-TGF-beta 1 antibody. Studies on the mechanism of TGF-beta 1 inhibition of SCF-induced proliferation of AML blasts revealed that TGF-beta 1 treatment of these cells was associated with down-regulation of SCF receptor surface expression, as detected with a specific monoclonal antibody, which appeared to be preferentially due to an acceleration of decay of mRNA for the c-kit proto-oncogene encoding the SCF receptor, without an effect on the overall transcriptional activity of the c-kit gene. Direct evidence to prove the importance of c-kit down-regulation in the inhibitory effect of TGF-beta 1 on AML growth came also from experiments demonstrating that signal transduction of SCF could be significantly diminished in the presence of TGF-beta 1, as demonstrated by measuring c-kit kinase-associated phosphorylation of target proteins.

Topics: Cell Division; Down-Regulation; Hematopoietic Cell Growth Factors; Humans; Leukemia, Myeloid, Acute; Proto-Oncogene Mas; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-kit; Recombinant Proteins; RNA, Messenger; Stem Cell Factor; Transforming Growth Factor beta; Tumor Cells, Cultured

Because limited studies examined effects of transforming growth factor (TGF) beta 1 on growth of human acute myelogenous leukemia (AML) cells, we used factor-dependent and primary AML cells to assess TGF-beta 1 effects on human AML cell growth. OCI-AML1 cells were growth inhibited by TGF-beta 1 regardless of which growth factor was used as a stimulus. In contrast, AML-193 cells were resistant to TGF-beta 1 when grown with or without growth factors. UCSD/AML1 cells were sensitive to TGF-beta 1 inhibition when grown with most cytokines but were relatively resistant to TGF-beta 1 in the presence of macrophage colony-stimulating factor (M-CSF). Although cells grown from 5 of 6 AML patients were inhibited by TGF-beta 1, cells from 1 AML patient were growth stimulated by TGF-beta 1 in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, or mast cell growth factor (kit ligand). Thus, 3 growth patterns with TGF-beta 1 were observed: (a) sensitivity to growth inhibition; (b) resistance; and (c) factor-dependent resistance. Further studies showed that AML-193 and UCSD/AML1 cells expressed type II TGF-beta 1 receptors and that ability of TGF-beta 1 to decrease GM-CSF receptors did not correlate with growth inhibition. AML-193 cells and UCSD/AML1 cells grown with M-CSF could be propagated in 1 ng/ml TGF-beta 1, but UCSD/AML1 cells grown with GM-CSF and TGF-beta 1 died. Morphology and agarose gel analysis of DNA showed UCSD/AML1 cells underwent apoptosis when grown with GM-CSF and TGF-beta 1 but not with M-CSF and TGF-beta 1. Similar studies of OCI-AML1 cells showed that TGF-beta 1 induced apoptosis of cells grown in 5637 bladder cell-conditioned medium or GM-CSF. These studies indicate that human AML cells exhibit heterogeneous growth responses to TGF-beta 1 and that some effects of TGF-beta 1 on myeloid cells occur through programmed cell death.

Topics: Apoptosis; Cell Division; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-3; Leukemia, Myeloid, Acute; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Stem Cell Assay

When added to RPMI-1640 medium containing fetal bovine serum, DNA-directed antineoplastic agents such as cytosine arabinoside (araC), daunorubicin, and actinomycin D induce the monocytic differentiation of ML-1 human myeloblastic leukemia cell populations to an extent that depends upon both drug and serum concentration. Differentiation is not induced in the absence of serum or when antibodies to tumor necrosis factor-alpha and transforming growth factor-beta are added to the cultures, indicating that these serum-contained cytokines participate in initiating the differentiation process. The drug- and cytokine-dependent cell maturation, which results in the inhibition of leukemic cell growth, is achieved at much lower concentrations of drug than is required for growth-inhibition through drug-mediated cell kill. RNA- and protein-targeted agents cannot replace DNA-specific agents in the process of differentiation-induction. The DNA-specific agents render the leukemic cells responsive to the low concentrations of differentiation-inducing cytokines that are present in serum, causing them to mature, and subsequently, to cease growth. This sensitization may be a component of the clinically selective action of DNA-specific antitumor agents.

Topics: Animals; Antineoplastic Agents; Cattle; Cell Differentiation; Cytarabine; Cytokines; Dactinomycin; Daunorubicin; DNA, Neoplasm; Drug Synergism; Humans; Leukemia, Myeloid, Acute; Monocytes; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The effects that three different growth inhibitory cytokines exert on expression and function of members of the Jun family were studied in this work. M1 myeloblastic cells were chosen for this purpose because of their high growth sensitivity to interleukin 6 (IL-6), transforming growth factor beta 1 and alpha- and beta-interferons. It is reported here that IL-6 elevated the junB and c-jun mRNA levels and induced the formation of a novel DNA-protein complex with high sequence specificity to 12-O-tetradecanoylphorbol-13-acetate response element (TRE) oligonucleotides. This IL-6 induced TRE binding complex was abolished by anti-Jun specific antibodies and was efficiently competed by an oligonucleotide that comprises the mouse homologue of a previously described human c-myc negative DNA element. It persisted in cells for at least 48 h after IL-6 treatment and failed to be induced by alpha- and beta-interferons or by transforming growth factor beta 1, which affected differently the pattern of jun mRNA expression. To further explore regulatory and functional aspects of this induced TRE binding activity, an IL-6 resistant M1 clone was isolated and further analyzed. This clone carried a postreceptor deficiency that abrogated completely the growth inhibitory responses to IL-6 but did not interfere with the induction of two differentiation related cell surface markers. Interestingly, the IL-6 resistant clone had lost two molecular responses to IL-6, induction of TRE binding activity and suppression of the c-myc gene. The data correlate the IL-6 induced AP1 activity with the suppression of c-myc and growth inhibition.

Topics: Animals; Base Sequence; Cell Differentiation; Cell Division; Clone Cells; DNA-Binding Proteins; Genes, jun; Interferon Type I; Interleukin-6; Leukemia, Myeloid, Acute; Mice; Molecular Sequence Data; RNA, Messenger; Tetradecanoylphorbol Acetate; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

The possible involvement of protein kinase C in transducing the growth suppressive signals of interferons was studies in this work in two different hematopoietic cell lines. Chronic exposure of human Burkitt lymphoma and mouse M1 myeloblastic cell lines to phorbol myristate acetate (PMA), reduced by more than 90% the PKC protein levels and enzymatic activity in cell extracts. The depletion of PKC from cells abrogated the ability of IFN (alpha + beta) to arrest cell growth at the G0/G1 resting phase of the cell cycle. In contrast, other responses to IFN such as the induction of (2'-5') oligoadenylate synthetase gene, continued to take place at the same dose response pattern thus excluding the possibility that early targets in the pathway, such as the number or affinity of IFN cell surface receptors might be affected by PMA. The same prolonged treatment of M1 cells with PMA did not interfere with the ability of another cytokine, transforming growth factor beta (TGF-beta), to induce the normal type of G0/G1 arrest further supporting the specificity of the effect towards IFN responses. Unexpectedly, depletion of PKC from cells did not interfere with the negative effects of IFN on c-myc mRNA and protein expression in spite of the direct involvement of this molecular event in growth responses to IFN. The putative PKC dependent molecular event could therefore function either downstream to or in combination with the reduction in c-myc protein levels, providing a necessary but not a sufficient step to arrest cell cycle progression at the G0/G1 phase.

Topics: Animals; Burkitt Lymphoma; Cell Cycle; Cell Line; G1 Phase; Hematopoiesis; Humans; Interferons; Leukemia, Myeloid, Acute; Mice; Protein Kinase C; Proto-Oncogene Proteins c-myc; Resting Phase, Cell Cycle; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transforming Growth Factor beta

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.

Topics: Aged; Aged, 80 and over; Antibodies; Antigens, CD; Blast Crisis; Cell Division; Cells, Cultured; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Interleukin-1; Interleukin-3; Interleukin-6; Kinetics; Leukemia, Myeloid, Acute; Male; Middle Aged; Monocytes; Recombinant Proteins; Transforming Growth Factor beta